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Mutations of mitotic checkpoint genes in human cancers

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Abstract

Genetic instability was one of the first characteristics to be postulated to underlie neoplasia. Such genetic instability occurs in two different forms. In a small fraction of colorectal and some other cancers, defective repair of mismatched bases results in an increased mutation rate at the nucleotide level and consequent widespread microsatellite instability. In most colorectal cancers, and probably in many other cancer types, a chromosomal instability (CIN) leading to an abnormal chromosome number (aneuploidy) is observed. The physiological and molecular bases of this pervasive abnormality are unknown. Here we show that CIN is consistently associated with the loss of function of a mitotic checkpoint. Moreover, in some cancers displaying CIN the loss of this checkpoint was associated with the mutational inactivation of a human homologue of the yeast BUB1 gene; BUB1 controls mitotic checkpoints and chromosome segregation in yeast. The normal mitotic checkpoints of cells displaying microsatellite instability become defective upon transfer of mutant hBUB1 alleles from either of two CIN cancers.

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... An alternative hypothesis suggests that mutation of mitosis genes causes aneuploidy. Three such mutant genes have so far been identified; two of these are thought to control centrosome replication, i.e., mutant p53 [Fukasawa et al., 1996] and an over-expressed kinase SKT15 [Zhou et al., 1998], and one is thought to be a ''mitotic checkpoint gene'' [Lengauer et al., 1997;Cahill et al., 1998]. However, the mutant p53 was found in less than 50% [Lengauer et al., 1997] and the mutated checkpoint gene in only 11% of aneuploid colon cancers [Cahill et al., 1998]. ...
... Three such mutant genes have so far been identified; two of these are thought to control centrosome replication, i.e., mutant p53 [Fukasawa et al., 1996] and an over-expressed kinase SKT15 [Zhou et al., 1998], and one is thought to be a ''mitotic checkpoint gene'' [Lengauer et al., 1997;Cahill et al., 1998]. However, the mutant p53 was found in less than 50% [Lengauer et al., 1997] and the mutated checkpoint gene in only 11% of aneuploid colon cancers [Cahill et al., 1998]. Likewise, the mutant kinase was found in only 12% of primary breast cancers whereas presumably all cancers were aneuploid because they carried "six or more [kinase] signals" [Zhou et al., 1998]. ...
... 4. DNA of cancers rendered aneuploid by somatic mutation should be able to convert normal diploid cells to aneuploid cells via aneuploidy genes, because such mutations are reportedly dominant [Lengauer et al., 1997]. But animals carrying mutated ''mitotic checkpoint genes,'' such as p53 [Cahill et al., 1998] in their germlines are viable (see above) and thus not aneuploid, although the cells of some of these animals are at a relatively high risk of aneuploidy (see Conclusions) [Kim et al., 1993;Purdie et al., 1994;Bouffler et al., 1995]. As yet all transfections that have generated aneuploidy in a dominant fashion have done so by artificially unbalancing the dosage of normal, un-mutated mitosis genes [Futcher and Carbon, 1986;Burke et al., 1989;Mayer and Aguilera, 1990]. 5. ...
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The many complex phenotypes of cancer have all been attributed to “somatic mutation.” These phenotypes include anaplasia, autonomous growth, metastasis, abnormal cell morphology, DNA indices ranging from 0.5 to over 2, clonal origin but unstable and non‐clonal karyotypes and phenotypes, abnormal centrosome numbers, immortality in vitro and in transplantation, spontaneous progression of malignancy, as well as the exceedingly slow kinetics from carcinogen to carcinogenesis of many months to decades. However, it has yet to be determined whether this mutation is aneuploidy, an abnormal number of chromosomes, or gene mutation. A century ago, Boveri proposed cancer is caused by aneuploidy, because it correlates with cancer and because it generates “pathological” phenotypes in sea urchins. But half a century later, when cancers were found to be non‐clonal for aneuploidy, but clonal for somatic gene mutations, this hypothesis was abandoned. As a result aneuploidy is now generally viewed as a consequence, and mutated genes as a cause of cancer although, (1) many carcinogens do not mutate genes, (2) there is no functional proof that mutant genes cause cancer, and (3) mutation is fast but carcinogenesis is exceedingly slow. Intrigued by the enormous mutagenic potential of aneuploidy, we undertook biochemical and biological analyses of aneuploidy and gene mutation, which show that aneuploidy is probably the only mutation that can explain all aspects of carcinogenesis. On this basis we can now offer a coherent two‐stage mechanism of carcinogenesis. In stage one, carcinogens cause aneuploidy, either by fragmenting chromosomes or by damaging the spindle apparatus. In stage two, ever new and eventually tumorigenic karyotypes evolve autocatalytically because aneuploidy destabilizes the karyotype, ie. causes genetic instability. Thus, cancer cells derive their unique and complex phenotypes from random chromosome number mutation, a process that is similar to regrouping assembly lines of a car factory and is analogous to speciation. The slow kinetics of carcinogenesis reflects the low probability of generating by random chromosome reassortments a karyotype that surpasses the viability of a normal cell, similar again to natural speciation. There is correlative and functional proof of principle: (1) solid cancers are aneuploid; (2) genotoxic and non‐genotoxic carcinogens cause aneuploidy; (3) the biochemical phenotypes of cells are severely altered by aneuploidy affecting the dosage of thousands of genes, but are virtually un‐altered by mutations of known hypothetical oncogenes and tumor suppressor genes; (4) aneuploidy immortalizes cells; (5) non‐cancerous aneuploidy generates abnormal phenotypes in all species tested, e.g., Down syndrome; (6) the degrees of aneuploidies are proportional to the degrees of abnormalities in non‐cancerous and cancerous cells; (7) polyploidy also varies biological phenotypes; (8) variation of the numbers of chromosomes is the basis of speciation. Thus, aneuploidy falls within the definition of speciation, and cancer is a species of its own. The aneuploidy hypothesis offers new prospects of cancer prevention and therapy. Cell Motil. Cytoskeleton 47:81–107, 2000. © 2000 Wiley‐Liss, Inc.
... The BUBl protein kinase is a mitotic checkpoint protein involved in monitoring spindle assembly (Roberts et al., 1994 ;Cahill et al., 1998 ;Ouyang et al., 1998). The spindle assembly checkpoint is a surveillance mechanism which monitors the proper assembly of the mitotic spindle and is essential for ensuring the accurate segregation of chrom osomes, thus preventing any gain or loss of genetic material (aneuploidy). ...
... (Hoyt et al., 1991 ;Li and Murray, 1991), and the M psl gene (Weiss and Winey, 1996). The spindle checkpoint machinery is highly conserved, as homologues of these genes have now been identified in many organisms, including mice and humans (Li and Benezra, 1996 ;Taylor and McKeon, 1997 ;Cahill et al., 1998 ;Ouyang et al., 1998 ;Taylor et al., 1998 ;Martinez-Exposito et al., 1999). ...
... Abnormal checkpoint genes may be responsible for the genetic instability observed in various human cancers. In one report, two colorectal tumour cell lines that show high rates of aneuploidy, were shown to carry mutant alleles of the BUBl gene (Cahill et al., 1998). W hen these mutant versions of the BU Bl gene were expressed exogenously, the cells showed disrupted mitotic checkpoint control. ...
Thesis
The SV40 large T antigen is a viral oncogene able to immortalize mammalian cells in culture, and to occasionally transform them to tumourigenicity. It can also cause genomic instability by inducing chromosome aberrations and aneuploidy. T antigen is a multifunctional protein which interacts with a number of cellular proteins. Studies have previously shown that the critical activities required for these functions map to the N-terminus of the protein. The N-terminus carries the pRb binding activity and the J domain, but data suggests that there may be additional proteins binding in this region. To isolate such proteins, the yeast two-hybrid approach was chosen, and the N-terminus of T antigen fused to the LexA DNA binding domain was used as a bait. In order to optimize the screen, different versions of the system were setup, by cloning T antigen into an N-terminal fusion vector, two C-terminal fusion vectors and an inducible C-terminal fusion vector. A HeLa library and two mouse embryonic libraries carrying cDNAs fused either to the B42 or the GAL4 activation domains were screened. A number of proteins were identified as potential interactors with the N-terminus of T antigen in yeast. One of these proteins was the Bub1 mitotic checkpoint protein, a member of the family of proteins that monitor the assembly of the mitotic spindle. The interaction of Bub1 with T antigen was investigated in mammalian cells. T antigen was found to co-immunoprecipitate with endogenous Bub1 from extracts of rat embryo fibroblasts conditionally immortalized with T antigen, as well as from NIH 3T3 cells ectopically expressing T antigen. The interaction of T antigen with Bub3, another checkpoint protein of the same family and a binding partner of Bub1, was also demonstrated. In addition, T antigen was found to co-localize with Bub1 in the nuclei of early prophase cells. To investigate the functional significance of this interaction, FACS analysis was used to show that expression of T antigen in cells treated with nocodazole (a microtubule disrupting agent) makes them more refractory to the spindle assembly checkpoint. Moreover, kinase assays indicated that T antigen expression affects the kinase activity of Bub1. The T antigen/Bub1 interaction suggests a novel role for T antigen and provides a new insight into its ability to cause aneuploidy. Finally, a preliminary investigation of two other proteins isolated from the yeast two-hybrid screens, Hsp40 and β-tubulin, indicated that these proteins may also co-localize with T antigen in mammalian cells.
... It is a type of genomic instability that has been introduced recently to the field of human cancer biology. 3,4 Recently chromosomal instability has been described in many human dysplastic lesions, and it has been proposed as a marker of progression to cancer and considered as a primary event in neoplastic transformation. [5][6][7][8][9][10][11][12][13][14][15] Whether chromosomal instability in HPIN can provide additional predictive information for early cancer progression is still unknown. ...
... mosome alterations occurring at each cell generation. 3,4 Chromosomal instability is thought to arise as a result of aberrations in mitotic machinery of chromosome constitution, leading to excessive numeric chromosomal changes. 3,4,27 Chromosomal instability is usually manifested in a form of numeric chromosomal changes of 1 or more chromosomes. ...
... 3,4 Chromosomal instability is thought to arise as a result of aberrations in mitotic machinery of chromosome constitution, leading to excessive numeric chromosomal changes. 3,4,27 Chromosomal instability is usually manifested in a form of numeric chromosomal changes of 1 or more chromosomes. In this study we found the presence of chromosomal aneusomies in 5 of 20 (25%) cases of men with HPIN who subsequently developed prostate carcinoma compared with 2 of 24 (8.3%) cases of the men with HPIN who did not develop prostate carcinoma (P ϭ .1). ...
Article
Context.—High-grade prostate intraepithelial neoplasia (HPIN) is the most likely precursor of prostate cancer. The condition of many patients with a diagnosis of HPIN in prostate needle core biopsy could, if left untreated, progress to invasive cancer. Currently there is no available clinical, immunohistochemical, or morphologic criteria that are predictive of this progression. Objective.—To determine whether chromosomal instability in these precursor lesions could increase their predictive value for cancer detection. Design.—Dual-color interphase fluorescence in situ hybridization analysis was performed on archived prostate needle core biopsies from 54 patients with initial diagnosis of isolated HPIN and follow-up of 3 years or more. We used commercially available centromere probes for chromosomes 4, 7, 8, and 10. We had interpretable results in 44 patients as follows: (1) group A: 24 HPIN patients with persistent HPIN and/or benign lesions in the follow-up biopsies, and (2) group B: 20 HPIN patients with progression to prostate carcinoma. Results.—Twenty-five percent of the patients in group B displayed numeric chromosomal aberrations. Only 8.3% of the patients from group A had chromosomal abnormalities (P = .1). The observed overall chromosomal changes in HPIN were higher than those in normal or hyperplastic epithelium, with a statistically significant difference (P < .05). All aberrations were detected in the form of chromosomal gain. Overall, the commonest aberration was gain of chromosome 8, followed by gains of chromosomes 7 and 10. Conclusion.—These results indicated that although no single numeric chromosomal abnormality could be assigned as a predictor of HPIN progression to carcinoma, the overall level of numeric chromosomal abnormalities shows a trend of elevation in HPIN patients whose condition subsequently progressed to carcinoma.
... Yeast cells defective in the mitotic checkpoint mechanism lose chromosomes at elevated rates and are hypersensitive to mitotic-spindle inhibitors [33,34]. In humans, although mutations in known spindle checkpoint genes are extremely rare [1,2,[11][12][13]20,23,24,35,36,38], several studies have shown evidence that mutations and/or reduced levels of mitotic checkpoint proteins can cause checkpoint malfunction and chromosomal instability (CIN), and thereby contribute to tumor formation [10,16,21,33]. A few studies have shown that non-mutational silencing of human BUB1 and BUBR1 genes is more common than mutational inactivation, contributing to the development of aneuploidy in human colorectal cancers [25,29]. ...
... Furthermore, no copy number gains were detected at 15q15 (BUBR1) and no genomic losses were found at 7p22 (MAD1), so mechanisms other than copy number changes must be operative. No alternative mechanisms of upregulation of these three genes have been described [2,6,7,13,23,26,35] and MAD1 promoter hypermethylation seems to be an uncommon mechanism of expression downregulation in kidney carcinomas or other tumor types [13]. ...
Article
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Background : A defective mitotic checkpoint has been proposed to contribute to chromosomal instability (CIN). We have previously shown that expression changes of the mitotic arrest deficiency (MAD) gene family plays a role in renal cell cancer (RCC) characterized by numerical chromosomal changes, namely papillary and chromophobe carcinomas, but nothing is known about the expression of mitotic checkpoint genes in the clear cell histotype (ccRCC). Methods : We analyzed the mRNA expression levels of the major mitotic checkpoint genes of the budding uninhibited by benzimidazole family ( BUB1 , BUBR1 , BUB3 ) and of the MAD gene family ( MAD1 , MAD2L1 , MAD2L2 ) by real-time quantitative PCR in 39 ccRCC and in 36 normal kidney tissue samples.We have additionally analyzed these tumors by comparative genomic hybridization (CGH) in order to evaluate the relationship between mitotic checkpoint defects and the pattern of chromosome changes in this subset of RCC. Results : BUB1 , BUBR1 , MAD1 and MAD2L1 showed significant expression differences in tumor tissue compared to controls ( BUB1 , BUBR1 and MAD2L1 were overexpressed, whereas MAD1 was underexpressed). Overexpression of BUB1 and BUBR1 was significantly correlated with the number of genomic copy number changes ( p < 0.001 for both genes) and with Furhman grade of the tumors ( p = 0.006 and p = 0.005, respectively). Conclusions : We conclude that BUB1 and BUBR1 overexpression plays a role in cytogenetic and morphologic progression of ccRCC.
... Complex genomic changes that arise in tumors are a consequence of Chromosomal Instability (CIN), which leads to numerical [(N)-CIN] as well as structural chromosomal instability [(S)-CIN] [1]. The increased levels of aneuploidy and structural complexity in these tumors indicate errors in DNA repair, mitotic segregation and cell cycle checkpoints [2,3] and may cause (N)-CIN. Structural rearrangements emerge by anomalous DNA repair pathways that cause abnormalities in both homologous and non-homologous end-joining of double-stranded DNA [4,5]. ...
... (S)-CIN may also appear through telomere-mediated events, where decisively short telomeres get identified as DNA breaks capable of recombining (either homologously or nonhomologously) when DNA-repair pathways get compromised and leads to activation of telomerase [6]. The mechanism leading to aneuploidy is distinct from structural changes and aneuploidy arises by disruptions in cell cycle checkpoints and errors in mitotic segregation [2,7]. ...
Preprint
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Background: Complex genomic changes that arise in tumors are a consequence of chromosomal instability. In tumor cells genomic aberrations disrupt core signaling pathways involving various genes, thus delineating of signaling pathways can help understand the pathogenesis of cancer. The bioinformatics tools can further help in identifying networks of interactions between the genes to get a greater biological context of all genes affected by chromosomal instability. Results: Karyotypic analyses was done in 150 clinically confirmed breast cancer patients and 150 age and gender matched healthy controls after 72 hours Peripheral lymphocyte culturing and GTG-banding. Karyotypic analyses found more frequent chromosomal aberrations (structural and numerical) on chromosome 1, 2, 3, 4, 5, 8, 9, 17 and X. The data of genes harbored by chromosomal regions showing increased aberration frequency was retrieved from online databases. Pathway analysis on a set of genes that were not linked together revealed that genes HDAC3, NCOA1, NLRC4, COL1A1, RARA, WWTR1, and BRCA1 were enriched in the RNA Polymerase II Transcription pathway which is involved in recruitment, initiation, elongation and dissociation during transcription. Conclusion: The chromosomal instability analysis in a non-target in cancer patients thus can be used to identify genes and decipher the pathway involved in tumorigenesis. The bioinformatics approach can help in identifying aberrant genes in transcription pathways and their relation with breast cancer progression.
... 18 Likewise, DNA sequencing analysis for the mitotic checkpoint gene budding uninhibited by benzimidazole 1 (BUB1 gene) rarely identified any meaningful mutations in human cancer samples, while in experimental models, BUB1 gene can cause significantly elevated CIN. 19,20 Based on the above results and various other reports, 11 it is now widely accepted that genomic instability in many sporadic human cancers is not due to the inactivation of caretaker genes. In fact, so far, various DNA mutation studies in multiple sporadic cancers suggested that more than 69% of cancers did not harbor mutations in caretaker genes. ...
... However, in a more predominant form of cancer, sporadic cancers, all the reported DNA sequencing studies fail to identify frequently mutated genes among known or predicted DNA repair and mitotic checkpoint gene families. [17][18][19][20] While we cannot rule out unlikely possibility that mutations from unknown/uncharacterized caretaker genes are not discovered yet, the infrequent rate of mutations in caretaker genes in sporadic cancers are still an enigma. Moreover, mutation frequency does not necessarily accompany frequency of inactivation of such gene products. ...
... Complex genomic changes that arise in tumors are a consequence of Chromosomal Instability (CIN), which leads to numerical [(N)-CIN] as well as structural chromosomal instability [(S)-CIN] [1]. The increased levels of aneuploidy and structural complexity in these tumors indicate errors in DNA repair, mitotic segregation and cell cycle checkpoints [2,3] and may cause (N)-CIN. Structural rearrangements emerge by anomalous DNA repair pathways that cause abnormalities in both homologous and non-homologous end-joining of doublestranded DNA [4,5]. ...
... (S)-CIN may also appear through telomere-mediated events, where decisively short telomeres get identified as DNA breaks capable of recombining (either homologously or nonhomologously) when DNArepair pathways get compromised and leads to activation of telomerase [6]. The mechanism leading to aneuploidy is distinct from structural changes and aneuploidy arises by disruptions in cell cycle checkpoints and errors in mitotic segregation [2,7]. ...
Article
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Background Complex genomic changes that arise in tumors are a consequence of chromosomal instability. In tumor cells genomic aberrations disrupt core signaling pathways involving various genes, thus delineating of signaling pathways can help understand the pathogenesis of cancer. The bioinformatics tools can further help in identifying networks of interactions between the genes to get a greater biological context of all genes affected by chromosomal instability. Methods Karyotypic analyses was done in 150 clinically confirmed breast cancer patients and 150 age and gender matched healthy controls after 72 h Peripheral lymphocyte culturing and GTG-banding. Reactome database from Cytoscape software version 3.7.1 was used to perform in-silico analysis (functional interaction and gene enrichment). Results Frequency of chromosomal aberrations (structural and numerical) was found to be significantly higher in patients as compared to controls. The genes harbored by chromosomal regions showing increased aberration frequency in patients were further analyzed in-silico. Pathway analysis on a set of genes that were not linked together revealed that genes HDAC3, NCOA1, NLRC4, COL1A1, RARA, WWTR1, and BRCA1 were enriched in the RNA Polymerase II Transcription pathway which is involved in recruitment, initiation, elongation and dissociation during transcription. Conclusion The current study employs the information inferred from chromosomal instability analysis in a non-target tissue for determining the genes and the pathways associated with breast cancer. These results can be further extrapolated by performing either mutation analysis in the genes/pathways deduced or expression analysis which can pinpoint the relevant functional impact of chromosomal instability.
... Molecules 2020, 25, 4632 2 of 41 by mutations in corresponding genes lead to cancer development and progression, which results in uncontrolled cell division [4]. Therefore, in order to stop such undesired cancer cell division, mitotic poisons can be applied. ...
Article
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Cancer is one of the greatest challenges of the modern medicine. Although much effort has been made in the development of novel cancer therapeutics, it still remains one of the most common causes of human death in the world, mainly in low and middle-income countries. According to the World Health Organization (WHO), cancer treatment services are not available in more then 70% of low-income countries (90% of high-income countries have them available), and also approximately 70% of cancer deaths are reported in low-income countries. Various approaches on how to combat cancer diseases have since been described, targeting cell division being among them. The so-called mitotic poisons are one of the cornerstones in cancer therapies. The idea that cancer cells usually divide almost uncontrolled and far more rapidly than normal cells have led us to think about such compounds that would take advantage of this difference and target the division of such cells. Many groups of such compounds with different modes of action have been reported so far. In this review article, the main approaches on how to target cancer cell mitosis are described, involving microtubule inhibition, targeting aurora and polo-like kinases and kinesins inhibition. The main representatives of all groups of compounds are discussed and attention has also been paid to the presence and future of the clinical use of these compounds as well as their novel derivatives, reviewing the finished and ongoing clinical trials.
... Accordingly, the function of BUB1B is to ensure proper chromosome segregation by suppressing the onset of anaphase by inhibiting APC/C activation 13 . Given the critical role of BUB1B in mitotic checkpoint signaling and chromosome congression, impairment in BUB1B and SAC often results in aneuploidy and chromosomal instability, which can contribute to an increase in cancer incidence [14][15][16] . ...
Article
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Currently, the controversy regarding the expression profile and function of BUB1B in different malignancies still exist. In this project, we aimed to explore the role and molecular mechanism of BUB1B in the progression of extrahepatic cholangiocarcinoma (ECC). The expression levels of BUB1B in human ECC were evaluated by immunohistochemistry, western blot, and real-time PCR. The role and mechanism of BUB1B in CCA cell proliferation and invasion were investigated in both in vitro and in vivo functional studies. To indicate the clinical significance, a tissue microarray was performed on 113 ECC patients, followed by univariate and multivariate analyses. The expression of BUB1B was increased in both human CCA tissues and CCA cells. Results from loss-of-function and gain-of-function experiments suggested that the inhibition of BUB1B decreased the proliferation and invasiveness of CCA cells in vitro and in vivo, while overexpression of BUB1B achieved the opposite effect. Furthermore, the activation of c-Jun N-terminal kinase-c-Jun (JNK)-c-Jun pathway was regulated by BUB1B. BUB1B regulated the proliferation and invasiveness of CAA cells in a JNK-c-Jun-dependent manner. Clinically, ECC patients with BUB1B high expression had worse overall survival and recurrence-free survival than those with BUB1B low expression. Multivariate analysis identified that BUB1B was an independent predictor for postoperative recurrence and overall survival of ECC patients. In conclusion, BUB1B promoted ECC progression via JNK/c-Jun pathways. These findings suggested that BUB1B could be a potential therapeutic target and a biomarker for predicting prognosis for ECC patients.
... The mitotic checkpoint signaling is not an all-or-none response because the strength of the checkpoint response varies with the number of unattached kinetochores (Collin et al. 2013). Cahill et al. (1998) first reported that inactivating BUB1 mutations generated a weakened checkpoint response and caused chromosomal instability CIN in a subset of colon cancer cell lines. Lee et al. (1999) reported about acquired mutations in BUB1 and p53 genes and loss of spindle assembly checkpoint in tumors with BRCA2 deficiency from animals. ...
Article
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Loss of mitosis regulation is a common feature of malignant cells that leads to aberrant cell division with inaccurate chromosome segregation. The mitotic checkpoint is responsible for faithful transmission of genetic material to the progeny. Defects in this checkpoint, such as mutations and changes in gene expression, lead to abnormal chromosome content or aneuploidy that may facilitate cancer development. Furthermore, a defective checkpoint response is indicated in the development of drug resistance to microtubule poisons that are used in treatment of various blood and solid cancers for several decades. Mitotic slippage and senescence are important cell fates that occur even with an active mitotic checkpoint and are held responsible for the resistance. However, contradictory findings in both the scenarios of carcinogenesis and drug resistance have aroused questions on whether mitotic checkpoint defects are truly responsible for these dismal outcomes. Here, we discuss the possible contribution of the faulty checkpoint signaling in cancer development and drug resistance, followed by the latest research on this pathway for better outcomes in cancer treatment.
... Mitotic checkpoint serine/threonine-protein kinase BUB1 also known as BUB1, it is an enzyme that is encoded by the BUB1 gene in humans. [47,48] Disturbed mitotic checkpoints are a common feature of numerous human cancers. More exactly, mutations in the spindle checkpoint can result in chromosomal instability and aneuploidy, a feature exhibit in over 90% of all solid tumors. ...
Article
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Background: This study was carried out to identify potential key genes associated with the pathogenesis and prognosis of breast cancer (BC). Methods: Seven GEO datasets (GSE24124, GSE32641, GSE36295, GSE42568, GSE53752, GSE70947, GSE109169) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between BC and normal breast tissue samples were screened by an integrated analysis of multiple gene expression profile datasets. Hub genes related to the pathogenesis and prognosis of BC were verified by employing protein-protein interaction (PPI) network. Results: Ten hub genes with high degree were identified, including CDK1, CDC20, CCNA2, CCNB1, CCNB2, BUB1, BUB1B, CDCA8, KIF11, and TOP2A. Lastly, the Kaplan-Meier plotter (KM plotter) online database demonstrated that higher expression levels of these genes were related to lower overall survival. Experimental validation showed that all 10 hub genes had the same expression trend as predicted. Conclusion: The findings of this research would provide some directive significance for further investigating the diagnostic and prognostic biomarkers to facilitate the molecular targeting therapy of BC, which could be used as a new biomarker for diagnosis and to guide the combination medicine of BC.
... Although the biological importance of CIN in cancer has been recognized, the molecular basis of CIN in cancers remains unclear, as CIN results from a heterogeneous mechanism. Multiple genetic alterations contribute to CIN, such as genes involving DNA damage and repair [14], mitotic checkpoint [15], chromosome condensation and segregation from mutational inactivation of STAG2 [16,17], and possibly sister chromatid cohesion (hSecurin) [17,18]. Some studies have shown conflicting results [17,18], so it is challenging to unify the possible mechanisms into one general mechanism so as to explain CIN in cancers. ...
Article
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Immune checkpoint inhibitors (ICIs) have become the standard of care in various cancers, although their predictive tools have not yet completely developed. Here, we aimed to exam the role of 70-gene chromosomal instability signature (CIN70) in cancers, and its association with previous predictors, tumor mutation burden (TMB), and microsatellite instability (MSI), for patients undergoing ICIs, as well as the possible predictive value for ICIs. We examined the association of CIN70 with TMB and MSI, as well as the impact of these biomarkers on the survival of 33 cancer cohorts from The Cancer Genome Atlas (TCGA) databank. The predictive value of the ICIs of CIN70 in previously published reports was also validated. Using the TCGA dataset, CIN70 scores were frequently (either positively or negatively) associated with TMB, but were only significantly associated with MSI status in three types of cancer. In addition, our current study showed that all TMB, MSI, and CIN70 had their own prognostic values for survival in patients with various cancers, and that they could be cancer type-specific. In two validation cohorts (melanoma by Hugo et al. and urothelial cancer by Snyder et al.), no significant difference of CIN70 scores was found between responders and non-responders (p-value = 0.226 and 0.108, respectively). In addition, no overall survival difference was noted between patients with a high CIN70 and those with a low CIN70 (p-value = 0.106 and 0.222, respectively). In conclusion, the current study, through a comprehensive bioinformatics analysis, demonstrated a correlation between CIN70 and TMB, but CIN70 is not the predictor for cancer patients undergoing ICIs. Future prospective studies are warranted to validate these findings.
... For instance, Mad2 overexpression induces chromosomal instability (e.g. aneuploidy) (Cahill et al. 1998), one type of genomic stability, in KRAS-driven lung cancer. Although removing 1 3 KRAS results in remission, Mad2-induced genomic instability activates alternative signaling pathways, including ERK, AKT and STAT3, which release the dependency of cancer cells on KRAS and lead to a relapse (Sotillo et al. 2010). ...
Article
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Cancer cells evolve throughout disease progression and tumor relapse. Such evolution is a dynamic process resulting in genotypic and phenotypic cellular changes, conferring a high level of cell plasticity. Data derived from next-generation sequencing strategies have implicated that cancer cell plasticity could be driven by genetic changes induced by genomic instability during cancer evolution. Understanding the mechanisms of how genomic instability promotes mutagenesis and cancer cell plasticity could thus be critical avenue for cancer prevention and intervention. In this review, we discuss the relationships between cancer cell plasticity, genomic instability and mutagenesis during cancer evolution. We offer our insight and opinion on therapeutic strategies in this rapidly progressing research field.
... /179994 doi: bioRxiv preprint 2007 and several studies suggest that kinase defective BUB1 plays a role in tumorigenesis (Kops et al. 2005). For example, four out of 19 colorectal cancer cell lines with CIN have mutations in BUB1 (Cahill et al. 1998). Lymphoid leukemia and lymphoma cells also show deletions in the coding region of BUB1 (Ru et al. 2002). ...
Preprint
Chromosomal instability (CIN) is a hallmark of cancer cells. Spindle Assembly Checkpoint (SAC) proteins such as Bub1 monitor errors in chromosome segregation and cause cell cycle delay to prevent CIN. Altered expression of BUBl is observed in several tumor samples and cancer cell lines which display CIN. Caffeic Acid Phenethyl Ester (CAPE) which is an active component of propolis obtained from bee hives has anti-inflammatory antioxidant and anticarcinogenic properties. We used budding yeast S. cerevisiae as a model organism to investigate the molecular mechanism by which CAPE can inhibit the growth of cells with high levels of CIN. Here we show that CAPE leads to growth inhibition of bub1 Δ strains. CAPE treatment suppressed chromosome mis-segregation in bub1Δ strain possibly due to apoptosis of chromosomally instable bub1 Δ cells. We propose that CAPE may serve as a potential therapeutic agent for treatment of BUB1 deficient cancers and other cancers that exhibit CIN.
... BUB1B codes for a kinase controlling spindle checkpoint function: BUB1B delays the onset of anaphase by acting at the kinetochore and inhibiting the anaphase-promoting complex/cyclosome; this mechanism ensures proper chromosome segregation prior to cell division (Cahill et al., 1998;Taylor et al., 1998;Perera et al., 2007). In humans, deleterious biallelic mutations (missense or frameshift) which inactivate the BUB1B kinase domain are associated with a Premature Chromatid Separation Trait, which leads to Mosaic Variegated Aneuploidy Syndrome (Limwongse et al., 1999). ...
Article
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Spermatogenesis is a cell differentiation process that ensures the production of fertilizing sperm, which ultimately fuse with an egg to form a zygote. Normal spermatogenesis relies on Sertoli cells, which preserve cell junctions while providing nutrients for mitosis and meiosis of male germ cells. Several genes regulate normal spermatogenesis, some of which are not exclusively expressed in the testis and control multiple physiological processes in an organism. Loss-of-function mutations in some of these genes result in spermatogenesis and sperm functionality defects, potentially leading to the insurgence of rare genetic disorders. To identify genetic intersections between spermatogenesis and rare diseases, we screened public archives of human genetic conditions available on the Genetic and Rare Diseases Information Center (GARD), the Online Mendelian Inheritance in Man (OMIM), and the Clinical Variant (ClinVar), and after an extensive literature search, we identified 22 distinct genes associated with 21 rare genetic conditions and defective spermatogenesis or sperm function. These protein-coding genes regulate Sertoli cell development and function during spermatogenesis, checkpoint signaling pathways at meiosis, cellular organization and shape definition during spermiogenesis, sperm motility, and capacitation at fertilization. A number of these genes regulate folliculogenesis and oogenesis as well. For each gene, we review the genotype–phenotype association together with associative or causative polymorphisms in humans, and provide a description of the shared molecular mechanisms that regulate gametogenesis and fertilization obtained in transgenic animal models.
... Early studies reported that BRCA2 deficient mice have intact G2/M in response to DNA damage, seems that BRCA2 doesn't control this cell cycle checkpoint. However, further studies demonstrated that BRCA2 deficient mice had defective spindle assembly checkpoint, acquired mutations in the components of the mitotic checkpoint, such as p53, Bubl, and Mad3L, and had defective mitotic checkpoints, all providing evidence for the role of BRCA2 in G2/M regulation [119,120]. In contrary to BRCA1 which is involved in both initial and the maintenance of G2/M checkpoint, BRCA2 appears to be more important for the maintenance of this cell cycle [79,121]. ...
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Background: DNA repair pathways, cell cycle arrest checkpoints, and cell death induction are present in cells to process DNA damage and prevent genomic instability caused by various extrinsic and intrinsic ionizing factors. Mutations in the genes involved in these pathways enhances the ionizing radiation sensitivity, reduces the individual's capacity to repair DNA damages, and subsequently increases susceptibility to tumorigenesis. Body: BRCA1 and BRCA2 are two highly penetrant genes involved in the inherited breast cancer and contribute to different DNA damage pathways and cell cycle and apoptosis cascades. Mutations in these genes have been associated with hypersensitivity and genetic instability as well as manifesting severe radiotherapy complications in breast cancer patients. The genomic instability and DNA repair capacity of breast cancer patients with BRCA1/2 mutations have been analyzed in different studies using a variety of assays, including micronucleus assay, comet assay, chromosomal assay, colony-forming assay, γ -H2AX and 53BP1 biomarkers, and fluorescence in situ hybridization. The majority of studies confirmed the enhanced spontaneous & radiation-induced radiosensitivity of breast cancer patients compared to healthy controls. Using G2 micronucleus assay and G2 chromosomal assay, most studies have reported the lymphocyte of healthy carriers with BRCA1 mutation are hypersensitive to invitro ionizing radiation compared to non-carriers without a history of breast cancer. However, it seems this approach is not likely to be useful to distinguish the BRCA carriers from non-carrier with familial history of breast cancer. Conclusion: In overall, breast cancer patients are more radiosensitive compared to healthy control; however, inconsistent results exist about the ability of current radiosensitive techniques in screening BRCA1/2 carriers or those susceptible to radiotherapy complications. Therefore, developing further radiosensitivity assay is still warranted to evaluate the DNA repair capacity of individuals with BRCA1/2 mutations and serve as a predictive factor for increased risk of cancer mainly in the relatives of breast cancer patients. Moreover, it can provide more evidence about who is susceptible to manifest severe complication after radiotherapy.
... Circulating cell free DNA is a noninvasive and sensitive biomarker, and a number of methods have been employed for its measurement in plasma and serum including quantitative polymerase chain reaction (PCR) using PicoGreen assay [7], real-time quantitative PCR, or immunological methods such as enzyme-linked immunosorbent assay (ELISA) [8] and quantitative, multiplex PCR for circulating nuclear or mitochondrial DNA [9]. Circulating cell free DNA may be used for the early diagnosis of primary breast and pancreatic cancers [10,11] as well as for monitoring of tumor burden following treatment [12], residual disease in patients with specific cancer subtypes after surgery [13], and resistance to treatment [14]. ...
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Background: Cell free circulating DNA (cfcDNA) is a promising diagnostic tool for prostate cancer (PCa). This study aimed to measure the cfcDNA concentration and integrity in PCa patients using quantitative polymerase chain reaction (qPCR) analysis. This study also assessed the correlation between these molecular biomarkers with total prostate-specific antigen (PSA), Gleason score, prostate volume, and age. Methods: Eleven PCa patients and 9 persons with benign prostatic hyperplasia (BPH) were recruited. Blood samples were collected before prostate biopsy and plasma quantified by qPCR amplification of the ALU 115 DNA sequence, with the ratio of ALU 247 to ALU 115 reflecting cfcDNA integrity. Results: There were no significant differences in median, interquartile range (IQR) cfcDNA concentration or cfcDNA integrity between the patients with PCa (47.9 (214.93) ng/mL; 0.61 (0.49)) and persons with BPH (41.5 (55.13) ng/mL, p = 0.382; 0.67 (0.45), p = 0.342). A weakly positive correlation exists between cfcDNA concentration and total PSA (r = 0.200, p = 0.555) but not with age or Gleason score in PCa patients. Conclusion: cfcDNA concentration was relatively nonsignificantly higher in PCa patients in comparison to persons with BPH, whereas cfcDNA integrity was similar in both groups. Though limited in sample size, this study shows that cfcDNA concentration may be a potentially valuable noninvasive biomarker for the diagnosis of PCa.
... As for other mitotic checkpoint proteins, BUB1 abnormality is associated with various types of cancers. BUB1 mutations are only occasionally found in cancer [16][17][18], but BUB1 overexpression is a frequent phenomenon that is associated with high proliferative activity of tumor cells and a poor clinical outcome in various solid cancers [18][19][20][21][22][23][24][25][26]. However, the functional involvement of BUB1 in the pathophysiology of MM is unknown. ...
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Chromosome instability (CIN), the hallmarks of cancer, reflects ongoing chromosomal changes caused by chromosome segregation errors and results in whole chromosomal or segmental aneuploidy. In multiple myeloma (MM), CIN contributes to the acquisition of tumor heterogeneity, and thereby, to disease progression, drug resistance, and eventual treatment failure; however, the underlying mechanism of CIN in MM remains unclear. Faithful chromosomal segregation is tightly regulated by a series of mitotic checkpoint proteins, such as budding uninhibited by benzimidazoles 1 (BUB1). In this study, we found that BUB1 was overexpressed in patient-derived myeloma cells, and BUB1 expression was significantly higher in patients in an advanced stage compared to those in an early stage. This suggested the involvement of aberrant BUB1 overexpression in disease progression. In human myeloma-derived cell lines (HMCLs), BUB1 knockdown reduced the frequency of chromosome segregation errors in mitotic cells. In line with this, partial knockdown of BUB1 showed reduced variations in chromosome number compared to parent cells in HMCLs. Finally, BUB1 overexpression was found to promote the clonogenic potency of HMCLs. Collectively, these results suggested that enhanced BUB1 expression caused an increase in mitotic segregation errors and the resultant emergence of subclones with altered chromosome numbers and, thus, was involved in CIN in MM.
... The availability of BubR1 chemical probes will enable us to determine how LLPS-mediated formation of central spindle condensate controls chromosome stability in mitosis. Since BubR1 mutations are implicated in chromosome stability and tumorigenesis in gastrointestinal tracts (83,85), it would be of interest to see whether and how the coacervates formed by CENP-E and BubR1 are modulated by disease-associated mutations. ...
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Liquid-liquid phase separation (LLPS) of biomolecules drives the formation of subcellular compartments with distinct physicochemical properties. These compartments, free of lipid bilayers and therefore called membraneless organelles, include nucleoli, centrosomes, heterochromatin, and centromeres. These have emerged as a new paradigm to account for subcellular organization and cell fate decisions. Here we summarize recent studies linking LLPS to mitotic spindle, heterochromatin, and centromere assembly and their plasticity controls in the context of the cell division cycle, highlighting a functional role for phase behavior and material properties of proteins assembled onto heterochromatin, centromeres, and central spindles via LLPS. The techniques and tools for visualizing and harnessing membraneless organelle dynamics and plasticity in mitosis are also discussed, as is the potential for these discoveries to promote new research directions for investigating chromosome dynamics, plasticity, and inter-chromosome interactions in the decision-making process during mitosis.
... In addition, during DNA segregation, the NCRP 2020-All rights reserved. This electronic file was provided to a member of SC 1-26 Single user only, copying and networking prohibited 4.1 CANCER / 51 spindle assembly checkpoint ensures that all chromosomes are properly connected by the mitotic spindle (Cahill et al. 1998;Lengauer et al. 1998). ...
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The overall aim of this Report is to provide input for the development of biologically based dose-response (BBDR) models for radiation- induced cancers and circulatory disease that use an adverse outcome pathways and key-events approach for providing parameters for these models. These mechanistic data can be integrated with the most recent epidemiologic data to develop overall doseresponse curves for radiation-induced adverse health outcomes. This integration of the findings from radiation biology and epidemiology will enhance the risk assessment process by reducing uncertainties in estimated risk following exposure to low doses and low dose rates of ionizing radiation. For many decades the basis for setting radiation protection guidance for exposure to low absorbed doses and low absorbed-dose rates of ionizing radiation has been the estimation of the risk of radiation-induced cancer. In addition, there is ongoing discussion concerning risks of radiation-induced noncancer effects1 (particularly circulatory disease). The estimates for radiation-induced cancer have been derived primarily from exposure to higher doses and higher dose rates of ionizing radiation and assumptions on how to extrapolate to low doses and low dose rates. For the purpose of this Report, for low linear-energy transfer (LET) radiation, a low absorbed dose is <100 mGy delivered acutely, and a low absorbeddose rate is <5 mGy h–1 for any accumulated absorbed dose (NCRP 2015). This Report addresses the conclusions and recommendations from three previous National Council on Radiation Protection and Measurements (NCRP) reports and commentaries on the topic of the risks of adverse health outcomes at low doses and low dose rates of ionizing radiation (NCRP 2012, 2015, 2018a). In this context, the present Report proposes a path forward to enhance the estimation of risk at low doses and low dose rates. Such a modified approach is needed to supplement the information that can be obtained from the conduct of even large epidemiologic studies such as the One Million U.S. Workers and Veterans Study of Low-Dose Radiation Health Effects (million U.S. workers and veterans study) 1For this Report the term noncancer is restricted to somatic noncancer outcomes and does not include heritable effects. (Bouville et al. 2015; Boice et al. 2019), the International Nuclear Workers Study (Leuraud et al. 2015; Richardson et al. 2015), the European pooled study of radiation-induced cancer from pediatric computed tomography (Bernier et al. 2019), or other low-dose pooling studies (Lubin et al. 2017; Little, Kitahara et al. 2018). This Report presents such an approach based upon the integration of data from epidemiology and radiation biology. An essential component of the integration process is the use of BBDR models with parameters being developed from analyzing adverse outcome pathways and their associated key events. In principle, an adverse outcome pathway is the series of necessary steps that result in an initial molecular event leading to an adverse health outcome (for this Report, either cancer or circulatory disease). Definitions of adverse outcome pathways and key events are given in Section 2 and can be found also in recent reviews (Edwards et al. 2016; Preston 2017). Also, when considering mechanistic data underlying the induction of adverse health outcomes, it is important to distinguish between potential bioindicators and biomarkers of these outcomes. A bioindicator is a cellular alteration that is on a critical pathway to the disease endpoint itself (i.e., necessary, but not by itself sufficient for the endpoint), such as a specific mutation in a target cell that is associated with tumor formation. Thus, a bioindicator can be perceived as informing on the shape of the dose-response curve for the disease outcome or on cancer frequency itself, and therefore, is equivalent to a key event. A biomarker is a biological phenotype [e.g., chromosome alteration, deoxyribonucleic acid (DNA) adduct, gene expression change, specific metabolite] that can be used to indicate a response to an exposure at the cell or tissue level. In this regard, a biomarker is generally a measure of the potential for development of an adverse health outcome such as cancer (e.g., a predictor of exposure level). This Report expands upon this general approach of adverse outcome pathways, key events, and BBDR models to enhance the process of low-dose, low dose-rate risk estimation. The arrangement of this Report for the application of this general approach is: here is what we know, here is what we need to know, and this is how we can obtain the necessary knowledge. A synopsis of Sections 2 through 7 is given below. Section 2 (Introduction) provides an overview of current approaches to radiation risk assessment, the associated uncertainties and possible ways forward for enhancing the estimation of risks of cancer and circulatory disease at low doses and low dose rates. Section 3 (Epidemiology, Biosamples and Biomarkers: Cancer and Circulatory Disease) presents a review of the radiation epidemiologic studies for which biomarker data or biological samples were used. For noncancer effects it was clear that the only adverse health outcome for which significant data from radiation biology are available for use in BBDR models is circulatory disease and so this forms the basis for the discussion on noncancer effects. There are a large number of radiation epidemiologic studies available that are very informative for estimating risks at higher doses but that can only be used with a fairly high degree of uncertainty for predicting low-dose risks. A review of the main radiation epidemiologic studies has been provided in NCRP Commentary No. 27 (NCRP 2018a). Section 3.1 briefly describes the major epidemiologic radiation studies with associated biosamples that potentially can be employed to conduct investigations of bioindicators of the pathogenesis of radiation-induced cancer and other health endpoints. While none of the current investigations has yet been able to identify definitive bioindicators, there are several suggestions of biomarkers that merit confirmation through further investigations and might be informative in the absence of more definitive bioindicator studies. The details and references for these studies are provided in Section 3.1.3. Section 3.2 indicates that it is likely that bioindicators of radiation- induced noncancer effects at low doses will be restricted to circulatory disease and so this is the sole topic reviewed for biomarkers associated with noncancer responses. With current knowledge, substantive biomarker information is only available in two major radiation studies: the Japanese atomic-bomb survivors, and the Mayak Production Association workers (Mayak workers), although little use has been made of this latter population in analyses to date. In summary, there is a paucity of radiation-specific bioindicators of cancer and circulatory disease and a relative lack of radiation- specific biomarkers predictive of adverse health outcomes. Thus, it is necessary to consider the mechanisms of formation of cancers and circulatory disease, especially for radiation-induced responses, to aid with the identification of bioindicators of adverse health outcomes and to a lesser extent, biomarkers of association with an adverse health outcome. Section 4 (Radiation-Induced Biological Effects Related to Cancer and Circulatory Disease) reviews the underlying mechanisms of carcinogenesis and circulatory disease with the aim of identifying potential bioindicators of the adverse health outcome, and if possible radiation-associated bioindicators of such responses. There has been an increased understanding of the underlying mechanisms of human diseases as a result of new molecular, cellular and computational approaches, further enhanced by informative experimental animal systems that model human disease. To a lesser extent such approaches have been used to better understand the etiology of radiation-induced diseases. There is a description of the types of studies that have identified pathways and potential key events in the carcinogenesis process (Section 4.1). While currently there are no fully validated bioindicators or biomarkers of radiation-induced cancer, there is a substantial and increasing body of knowledge on radiation-induced cancer mechanisms, particularly in experimental animal systems. Quantification of inflammation and generation of persistently elevated reactive oxygen species (ROS) holds promise as a further bioindicator that is also recognized as an enabling hallmark of cancer in the context of Hanahan and Weinberg (2011). In addition, cell-survival parameters can be of importance in mechanistic models of carcinogenesis. The use of data from experimental animal systems provides opportunities to demonstrate the added value of building and applying mechanistic models of radiation-induced cancer. There are additional opportunities to apply similar models in some human radiation-induced cancers, most notably thyroid, where some work utilizing knowledge of the CLIP2 marker is already available. The incorporation of quantitative mechanistic data into appropriate cancer models (discussed in Sections 5 and 6) is likely to increase the precision of estimated risks, particularly at low-dose levels and so continued efforts to identify and validate bioindicators of radiation- induced cancers will assist in refining risk estimation. Section 4.2 outlines the biology of circulatory disease, a significant radiation-induced noncancer disease2 and the one which currently offers the best opportunity for bioindicator identification given the mechanistic data already available. The complex inflammatory processes underlying most major types of circulatory disease are reviewed, specifically those associated with atherosclerosis. The possible ways that low-dose radiation exposure and other 2NCRP (2018a) stated that radiation-induced cardiovascular disease (a circulatory disease) remains an area where further investigation is necessary. Although there is evidence that cardiovascular disease may be a factor at exposures lower than previously estimated, that evidence was not yet sufficient to allow for development of an approach to including cardiovascular disease in NCRP’s overall system of radiation protection published in NCRP (2018b). biological stressors might affect the circulatory system are also reviewed. While it is not possible yet to identify bioindicators of radiation-induced circulatory disease, it appears feasible to build upon the rapidly increasing knowledge of the mechanisms of formation of circulatory disease to develop adverse outcome pathways and at least some of the associated key events. Section 5 (Biologically Based Dose-Response Models) assesses biomathematical models of chronic disease, especially those for cancer and circulatory disease (with particular emphasis in circulatory disease on models of atherosclerosis). First, general material outlining the overall goals of biomathematical models is presented, followed by discussion of modeling considerations, particularly application of specific models using human, animal or cell data to cancer and circulatory disease. Biologically based modeling of radiation- induced cancers of the breast, colon, lung, and thyroid gland have been conducted. After considerations of some general features of BBDR models of cancer development in Section 5.1, a number of BBDR models and their application to various human and animal datasets are presented in Section 5.2. Despite some shortcomings (e.g., the fact that different models might explain the available data using different mechanistic assumptions), multiple pathway models are considered a promising conceptual approach to developing a general model framework for the complex process of carcinogenesis in various tissues. In certain cases, multiple pathway models may allow predictions that can be validated against experimental data. Circulatory disease models are considered in Section 5.3. These are less well developed than those that have been constructed to model cancer. A number of candidate models of atherosclerosis are considered. Atherosclerosis is the disease process underlying the main types of circulatory disease, specifically ischemic heart disease (IHD) and stroke, which is thought to have a largely inflammatory etiology. A number of atherosclerosis models, which share certain features, have been proposed for these inflammatory processes, specifically the adhesion and transport of monocytes through the epithelial cell layer, and diffusion through the intima. However, it is not yet clear what the radiation-associated mechanisms may be for most types of circulatory disease. Having identified the types of BBDR models that could possibly be used to enhance the estimation of low-dose, low dose-rate radiation adverse health outcomes, it is necessary to determine whether there is a generalized model that can be used for: all radiationinduced cancer types, or circulatory disease as a class. It was concluded that it would be unlikely that a single model structure could be used for describing cancer and circulatory disease. Also, it appears likely that there may be different responses even for different types of circulatory disease. The concept of a generalized model is discussed in Section 6 (Proposed Generalized Model Framework of Cancer and Circulatory Disease). It is proposed that a form of multistage clonal expansion model would be appropriate for integrating data from epidemiology and radiation biology for estimating low-dose, low dose-rate cancer risk. The parameters for such a model structure are proposed to be developed from an adverse outcome pathways and key-events approach. In such an approach the key events are considered to be bioindicators of the adverse health outcome itself. In support of this proposal to utilize generalized multistage clonal expansion models, there has been considerable recent discussion on the use of such parameterized models for environmental chemicals (OECD 2020). The Organization for Economic Co-operation and Development (OECD 2020) website provides a considerable amount of information on developing adverse outcome pathways and their use in risk assessment and ultimately in risk management practice. This general approach is also described and applied in the research program of the U.S. Environmental Protection Agency (EPA 2018). A description of biologically detailed models of specific cancers that have been applied with some levels of success is provided to indicate the viability of the use of BBDR models for estimating adverse health outcomes at low doses and low dose rates. While not definitive at this time, the approach certainly has a real likelihood of being successful. Section 7 (Research Needs) provides specific examples of research activities, both large and small that are designed for developing adverse outcome pathways and their associated key events. These include epidemiologic, human sample, laboratory animal, cellular, and molecular studies. Such research activities include investigating some general but critical responses, in order to derive greater insight into the parameters of most importance for further model development. Currently, one can envisage the following to be of high relevance: • target cell population numbers and characteristics; • survival parameters for these populations after radiation exposure; • target gene(s) critical for pathogenesis and their mutation or epimutation frequency as a function of radiation dose; • proliferation characteristics in normal and mutation-carrying cell populations; and • timing and frequency of acquisition of further mutational events in key genes and the impact of these on survival and proliferation characteristics. For enhanced model development, it is necessary to more fully identify the mechanisms of cancer development in response to radiation. The following are of importance in this regard: • Mechanisms in the development of a radiation-induced disease may differ from those in sporadic disease. • Does radiation initiate or accelerate the same processes that lead to sporadic disease, or are distinct molecular pathways involved? • BBDR models have the potential to address such questions if appropriate bioindicators become available for specific types of cancer or other diseases. • For transcriptomics, proteomics, metabolomics and epigenomics, adequate BBDR models ideally might require measurements at several time points because the profiles of phenotypic alterations may differ by stage in the pathogenesis of a disease. Clearly, the overarching need is the furthering of research targeted at the underlying mechanisms of radiation-induced adverse health outcomes (cancer and noncancer disease) leading to the identification of truly informative bioindicators of the apical endpoint (i.e., the adverse health outcome). The framework for such an approach can be the characterization of adverse outcome pathways for specific outcomes and the identification of key events from the initial event to adverse health outcome. In this context, a key-event or informative bioindicator is a true surrogate for the adverse health outcome. This approach will require the integration of data from epidemiology and radiation biology to maximize the information for estimating low-dose responses for adverse health outcomes. A particularly important result will be the ability to better describe the form of the dose-response curve for different types of radiation- induced cancer, for example, and thereby avoid the need to rely on application of the linear-nonthreshold (LNT) model without sufficient biological substantiation. A concerted effort will be needed; this is going to require a well-defined and quite extensive research effort. The need for this effort is recognized by many in the risk assessment and risk management arena.
... Interestingly in colorectal cancer cell lines exhibiting chromosomal instability BU Bl mutations have been found, suggesting that abrogation of the checkpoint can lead to the development of chromosomal instability (Cahill, D. P. et al. 1998). ...
Thesis
The biological progression of breast cancer is uncertain. One hypothesis suggests that as invasive ductal breast tumours grow they dedifferentiate, i.e. they evolve from well-differentiated grade I tumours to poorly differentiated grade III tumours over time. The work presented here addresses the pathological hypothesis of dedifferentiation by taking a novel genetic approach. CGH was used to screen the entire genomes of 90 invasive ductal breast carcinomas stratified by grade (40 grade I and 50 grade III tumours). The different grades of breast tumour showed distinct quantitative and qualitative differences. Specifically, the genetic changes found could not support the dedifferentiation hypothesis for the majority of grade I breast carcinomas. These differences were investigated in a subsequent molecular genetic study that provides further evidence against dedifferentiation. The molecular cytogenetic findings have lead to several other lines of enquiry. First, the pattern of genetic changes seen in grade I tumours is very similar to that seen in lobular breast carcinomas, thus leading to the hypothesis that at the genetic level these morphologically distinct tumours may actually be very similar. In both these tumour types, the most frequent change, often occurring with only a few other genetic changes, is loss of 16q. The target gene on 16q known to be important in lobular breast tumorigenesis is E-cadherin (CDHl), therefore this candidate gene was tested in grade I ductal breast tumours. However, the findings suggest that in ductal breast carcinoma CDHl is not the target gene. Therefore, despite the apparent genetic similarity, lobular and grade I ductal breast tumours are genetically different. Second, a number of regions of amplification have been revealed in both grades of tumour; some of these are novel changes. Refined mapping of two amplicons has been performed, which will be discussed. Taken together, these findings integrate molecular cytogenetic, genetic and pathological approaches to provide new insights into the biology of breast tumour progression.
... KEGG and GO analyses suggested key genes could regulate tumor stemness by affecting cell cycle. BUB1, BUB1B, and MAD2L1 played a central role in mitosis, they could regulate the spindle checkpoint activity, and delay the onset of anaphase ensuring proper chromosome alignment and segregation [30][31][32][33][34]. BUB1 mutations could cause chromosomal instability, DNA damage, and increase the risk of colon cancer [33,35,36]. ...
Preprint
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Background: Cancer stem cells are hidden tumor cells with the potential for self-renewal and multi-differentiation, which are the principal cause of tumor heterogeneity, metastasis and drug resistance. However, the key genes that maintain cell stemness of colon adenocarcinoma (COAD) are still unknown. Our study was designed to identify key genes associated with the stemness of COAD cell. Methods: RNA sequencing data and clinical information were downloaded from The Cancer Genome Atlas (TCGA). Weighted gene coexpression network analysis (WGCNA) was used to recognize key genes based on mRNAsi. Next, the gene expression data downloaded from Gene Expression Omnibus (GEO), Oncomine, and Gene Expression Profiling Integrative Analysis (GEPIA) were used to demonstrate the expression of key genes. Results: The mRNAsi score of COAD tissue was obviously superior than that of normal tissue. N stages, M stages and overall survival were highly related to mRNAsi. WGCNA showed that the green module was positively related to mRNAsi. We obtained 27 genes closely related to each other at the transcriptional and protein levels. The main biological functions of key genes were organelle fission, chromosomal region, and protein serine/threonine kinase activity. The enriched signaling pathway were cell cycle, progesterone−mediated oocyte maturation, human T−cell leukemia virus 1 infection, oocyte meiosis, DNA replication, cellular senescence, viral carcinogenesis, and p53 signaling pathway. The key genes were highly expressed in COAD and various cancers, which was validated multiple databases. Conclusions: 27 key genes were well correlated with maintaining stemness of COAD, which were significantly related to cell cycle events. The key genes may be therapeutic targets for inhibiting the properties of COAD stem cells.
... Besides the MCC, several auxiliary proteins contribute to SAC activation, including kinases (BUB1, MPS1 and Aurora B) [50][51][52][53], the microtubule motor protein centromere protein E (CENP-E) [54] and the ROD-ZW10-ZWILCH (RZZ) complex [55]. Similar to AURKA, overexpression or mutational inactivation of SAC components is frequently observed in cancers with CIN, suggesting a critical role of SAC in preventing CIN [56,57]. In our study, the expression of several MCC proteins and SAC components, including BUB1B and MAD2L1, was increased in SMAD4-deficient CRC and pancreatic cancer cells. ...
Article
Full-text available
SMAD4 loss-of-function mutations have been frequently observed in colorectal cancer (CRC) and are recognized as a drug target for therapeutic exploitation. In this study, we performed a synthetic lethal drug screening with SMAD4-isogenic CRC cells and found that aurora kinase A (AURKA) inhibition is synthetic lethal with SMAD4 loss. Inhibition of AURKA selectively inhibited the growth of SMAD4−/− CRC in vitro and in vivo. Mechanistically, SMAD4 negatively regulated AURKA level, resulting in the significant elevation of AURKA in SMAD4−/− CRC cells. Inhibition of AURKA induced G2/M cell cycle delay in SMAD4+/+ CRC cells, but induced apoptosis in SMAD4−/− CRC cells. We further observed that a high level of AURKA in SMAD4−/− CRC cells led to abnormal mitotic spindles, leading to cellular aneuploidy. Moreover, SMAD4−/− CRC cells expressed high levels of spindle assembly checkpoint (SAC) proteins, suggesting the hyperactivation of SAC. The silencing of key SAC proteins significantly rescued the AURKA inhibition-induced cell death in SMAD4−/− cells, suggesting that SMAD4−/− CRC cells are hyper-dependent on AURKA activity for mitotic exit and survival during SAC hyperactivation. This study presents a unique synthetic lethal interaction between SMAD4 and AURKA and suggests that AURKA could be a potential drug target in SMAD4-deficient CRC.
... Aneuploidy, a common feature in most cancer cells, is caused by an increased rate of chromosome mis-segregation tightly associated with mitotic errors (Holland and Cleveland 2012;Santaguida and Amon 2015;Funk et al. 2016). Bub1 mutations, including deletions and point mutations, as well as differential Bub1 gene expression have been implicated in carcinogenesis (Cahill et al. 1998;Ohshima et al. 2000;Hernando et al. 2001;Ru et al. 2002;Shichiri et al. 2002;Klebig et al. 2009;Wang et al. 2015). Bub1 overexpression in mice resulted in aneuploidy and tumor formation likely mediated through Aurora B hyperactivation (Ricke et al. 2011). ...
Article
Full-text available
Accurate chromosome segregation is required for cell survival and organismal development. During mitosis, the spindle assembly checkpoint acts as a safeguard to maintain the high fidelity of mitotic chromosome segregation by monitoring the attachment of kinetochores to the mitotic spindle. Bub1 is a conserved kinase critical for the spindle assembly checkpoint. Bub1 also facilitates chromosome alignment and contributes to the regulation of mitotic duration. Here, focusing on the spindle assembly checkpoint and on chromosome alignment, we summarize the primary literature on Bub1, discussing its structure and functional domains, as well its regulation and roles in mitosis. In addition, we discuss recent evidence for roles of Bub1 beyond mitosis regulation in TGFβ signaling and telomere replication. Finally, we discuss the involvement of Bub1 in human diseases, especially in cancer, and the potential of using Bub1 as a drug target for therapeutic applications.
... When SAC is malfunctioning, cells without proper spindle attachments can bypass anaphase checkpoints and divide [1]. The inactivation of the entire mitotic checkpoint can generate chromosome mis-segregation leading to CIN or cell death [67,68] in various cancers [69][70][71]. ...
Article
Full-text available
Chromosomal instability (CIN), the increasing rate in which cells acquire new chromosomal alterations, is one of the hallmarks of cancer. Many studies highlighted CIN as an important mechanism in the origin, progression, and relapse of acute myeloid leukemia (AML). The ambivalent feature of CIN as a cancer-promoting or cancer-suppressing mechanism might explain the prognostic variability. The latter, however, is described in very few studies. This review highlights the important CIN mechanisms in AML, showing that CIN signatures can occur largely in all the three major AML types (de novo AML, secondary-AML, and therapy-related-AML). CIN features in AML could also be age-related and reflect the heterogeneity of the disease. Although most of these abnormalities show an adverse prognostic value, they also offer a strong new perspective on personalized therapy approaches, which goes beyond assessing CIN in vitro in patient tumor samples to predict prognosis. Current and emerging AML therapies are exploring CIN to improve AML treatment, which includes blocking CIN or increasing CIN beyond the limit threshold to induce cell death. We argue that the characterization of CIN features, not included yet in the routine diagnostic of AML patients, might provide a better stratification of patients and be extended to a more personalized therapeutic approach.
... However, this seems an unlikely scenario. Indeed, while SAC genes are frequently mutated in various solid tumors including colon, breast, prostate and lung [66][67][68][69][70][71][72], a survey of four MB datasets included in the cBioPortal [4,[73][74][75] revealed a lower-than-expected mutation rate in BUB1, BUB1B, BUB3, MAD1L1, MAD2L1, CDC20 (the maximal rate of inactivating mutations was 0.3%, as in the case on BUB1). A similarly low mutation frequency (0,7%) was found for CENPE, which is known as a functional component of the SAC [76]. ...
Article
Full-text available
Medulloblastoma (MB) is the most frequent brain tumor in children. The standard treatment consists in surgery, followed by radiotherapy and chemotherapy. These therapies are only partially effective since many patients still die and those who survive suffer from neurological and endocrine disorders. Therefore, more effective therapies are needed. Primary microcephaly (MCPH) is a rare disorder caused by mutations in 25 different genes. Centromere-associated protein E (CENPE) heterozygous mutations cause the MCPH13 syndrome. As for other MCPH genes, CENPE is required for normal proliferation and survival of neural progenitors. Since there is evidence that MB shares many molecular features with neural progenitors, we hypothesized that CENPE could be an effective target for MB treatment. In ONS-76 and DAOY cells, CENPE knockdown induced mitotic defects and apoptosis. Moreover, CENPE depletion induced endogenous DNA damage accumulation, activating TP53 or TP73 as well as cell death signaling pathways. To consolidate CENPE as a target for MB treatment, we tested GSK923295, an allosteric inhibitor already in clinical trial for other cancer types. GSK923295, induced effects similar to CENPE depletion with higher penetrance, at low nM levels, suggesting that CENPE’s inhibition could be a therapeutic strategy for MB treatment.
... Deficiencies in SAC function have been widely assumed to underlie chromosomal instability in cancer cells. 1 Challenging this initial hypothesis, however, cancer genomic and epigenomic analyses fail to identify general alterations in essential SAC effectors. Moreover, cancer cells are found to be largely proficient in SAC function, challenging the idea that SAC defects contribute to chromosomal instability. ...
Article
The spindle-assembly checkpoint facilitates mitotic fidelity by delaying anaphase onset in response to microtubule vacancy at kinetochores. Following microtubule attachment, kinetochores receive microtubule-derived force, which causes kinetochores to undergo repetitive cycles of deformation; this phenomenon is referred to as kinetochore stretching. The nature of the forces and the relevance relating this deformation are not well understood. Here, we show that kinetochore stretching occurs within a framework of single end-on attached kinetochores, irrespective of microtubule poleward pulling force. An experimental method to conditionally interfere with the stretching allowed us to determine that kinetochore stretching comprises an essential process of checkpoint silencing by promoting PP1 phosphatase recruitment after the establishment of end-on attachments and removal of the majority of checkpoint-activating kinase Mps1 from kinetochores. Remarkably, we found that a lower frequency of kinetochore stretching largely correlates with a prolonged metaphase in cancer cell lines with chromosomal instability. Perturbation of kinetochore stretching and checkpoint silencing in chromosomally stable cells produced anaphase bridges, which can be alleviated by reducing chromosome-loaded cohesin. These observations indicate that kinetochore stretching-mediated checkpoint silencing provides an unanticipated etiology underlying chromosomal instability and underscores the importance of a rapid metaphase-to-anaphase transition in sustaining mitotic fidelity.
... Thus, changes (i.e., increases in cell-to-cell heterogeneity) in nuclear areas and micronucleus formation are used to identify putative CIN genes [5,62,63] that can be subsequently validated with mitotic chromosome enumeration ( Figure 4B). HCT116 cells were again selected, as they are karyotypically stable and have been employed extensively in similar CIN-based studies [20,40,62,64,65]. As predicted, USP22 silencing induced significant increases (siUSP22-Pool, 2.3-fold; siUSP22-2, 1.9-fold; siUSP22-3, 4.0-fold) in median micronucleus formation relative to siControl ( Figure 4C; Table S4). ...
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Chromosome instability (CIN) is an enabling feature of oncogenesis associated with poor patient outcomes, whose genetic determinants remain largely unknown. As mitotic chromatin compaction defects can compromise the accuracy of chromosome segregation into daughter cells and drive CIN, characterizing the molecular mechanisms ensuring accurate chromatin compaction may identify novel CIN genes. In vitro, histone H2B monoubiquitination at lysine 120 (H2Bub1) impairs chromatin compaction, while in vivo H2Bub1 is rapidly depleted from chromatin upon entry into mitosis, suggesting that H2Bub1 removal may be a pre-requisite for mitotic fidelity. The deubiquitinating enzyme USP22 catalyzes H2Bub1 removal in interphase and may also be required for H2Bub1 removal in early mitosis to maintain chromosome stability. In this study, we demonstrate that siRNA-mediated USP22 depletion increases H2Bub1 levels in early mitosis and induces CIN phenotypes associated with mitotic chromatin compaction defects revealed by super-resolution microscopy. Moreover, USP22-knockout models exhibit continuously changing chromosome complements over time. These data identify mitotic removal of H2Bub1 as a critical determinant of chromatin compaction and faithful chromosome segregation. We further demonstrate that USP22 is a CIN gene, indicating that USP22 deletions, which are frequent in many tumor types, may drive genetic heterogeneity and contribute to cancer pathogenesis.
... Cancer cells are known to possess a huge amount of genomic instability including aneuploidy (Lengauer et al., 1998, Rajagopalan andLengauer 2004). Aneuploidy usually occurs due to a weakened mitotic checkpoint (Cahill et al., 1998, Rajagopalan et al., 2004. Thus, the increased amount of aneuploidy in SSEA1 + cells as compared to SSEA1cells might be due to the increased proliferation as measured by EdU incorporation. ...
Thesis
High Grade Serous Ovarian Adenocarcinoma (HGSOC) is a highly aggressive disease with poor prognosis and the leading cause of gynecological tumor-related deaths. The poor prognosis is related to the fact that already during early stages tumor cells start to spread into the peritoneum. This metastatic spread and the colonization of organs located within the peritoneum are the biggest problems with regard to therapy of HGSOC. Tumors often consist of a functionally heterogeneous population of cancer cells with distinct features. Subsets of tumor cell populations are able to promote tumor progression, metastatic spread and colonization, as well as outgrowth of tumor cells at distant organs. Therefore, the identification and targeting of so-called tumor-initiating cells is crucial. Knowledge about the intrinsic features of tumor-initiating cells and targeting them may ultimately lead to tumor regression and improved patient survival. However, no conclusive evidence about markers for a tumor-initiating population has been provided so far and even less is understood regarding the molecular mechanisms driving tumor-initiating cancer cell populations in HGSOC. My work shows that Stage Specific Embryonic Antigen 1 negative (SSEA1-) cells are enriched for tumor-initiating abilities in human HGSOC. Furthermore, SSEA1- cells can give rise to both SSEA1- and SSEA1+ cells whereas SSEA1+ cells only give rise to SSEA1+ cells demonstrating a hierarchical organization with SSEA1- cells being on top. Gene expression profiling demonstrated an enrichment of the transcription factor SAM-pointed ETS domaincontaining factor (SPDEF) in SSEA1- cells. Lentiviral knockdown of SPDEF impaired in vivo tumor growth and in vitro colony formation, whereas overexpression of SPDEF resulted in increased colony formation in vitro and tumor formation in vivo. Strikingly, also SSEA1+ cells acquired the capacity to initiate tumors in vivo and form colonies in vitro after SPDEF expression was re-introduced. I also show, that SPDEF negatively regulates the expression of the transcription factor Forkhead box protein A2 (FOXA2). FOXA2 overexpression resulted in decreased tumor-promoting capacity in an in vivo tumor formation assay. Based on these results, I propose that the transcriptional programs modulated by SPDEF, as well as those genes changed due to suppressed FOXA2 target gene transcription, lead to increased survival, clonogenicity and stemness of SSEA1- SPDEFhigh FOXA2low cells and may therefore promote ovarian cancer tumor initiation and metastatic spread. In summary, the data I generated indicate that SSEA1- cells represent a cellular subpopulation with increased tumor-initiating ability in HGSOC. These cells express higher levels of SPDEF, which exerts its tumorigenic potential by suppressing FOXA2 expression. Developing SPDEF inhibitors might be promising to target the SSEA1- tumor-initiating population and might ultimately lead to tumor regression and improved patient survival.
... BubR1, which is a regulator of genomic integrity, may lie at the interface of aging and cancer. BubR1 is a putative serine-threonine protein kinase with a variety of critical cellular functions, most of which are largely mitotic [2,3]. It is a component of the spindle assembly checkpoint (SAC) where its function is strictly regulated to ensure proper chromosome segregation through its involvement in coordinating kinetochore-microtubule interactions, chromosome migration and alignment, and anaphase inhibition [4][5][6]. ...
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BubR1 is an essential component of the spindle assembly checkpoint (SAC) during mitosis where it functions to prevent anaphase onset to ensure proper chromosome alignment and kinetochore-microtubule attachment. Loss or mutation of BubR1 results in aneuploidy that precedes various potential pathologies, including cancer and mosaic variegated aneuploidy (MVA). BubR1 is also progressively downregulated with age and has been shown to be directly involved in the aging process through suppression of cellular senescence. Post-translational modifications, including but not limited to phosphorylation, acetylation, and ubiquitination, play a critical role in the temporal and spatial regulation of BubR1 function. In this review, we discuss the currently characterized post-translational modifications to BubR1, the enzymes involved, and the biological consequences to BubR1 functionality and implications in diseases associated with BubR1. Understanding the molecular mechanisms promoting these modifications and their roles in regulating BubR1 is important for our current understanding and future studies of BubR1 in maintaining genomic integrity as well as in aging and cancer.
... but it is found out that mutations in mitotic checkpoint gene, such as BUB1, are clearly linked with the CIN phenotype in colon cancer, but they uncommon in lung cancer [35,42] . ...
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In the past, few decades cancer has become a worldwide problem to mankind. Lung cancer is the most life-threatening among all cancer types. Non-small cell lung cancer (NSCLC) is the main reason for approximately 80% to 90% of deaths. Lack of early detection and incompetent conventional therapies is the leading cause for poor prognosis and overall survival rate of lung cancer patients. Immense progress in the field of nanotechnology and nanomedicine has given inspiration to the development of an alternative strategy in the treatment of lung cancer. The unique physicochemical properties of the nanoparticles likeability to cross the different biological barriers, effectiveness in delivering hydrophobic drugs which are difficult to incorporate in the body, and targeting in the particular disease sites have given rise to enormous advantages for nanoparticulate systems for the early diagnosis and active delivery of drugs for a better treatment for lung cancer. Recently, many formulations of nanocarriers like lipid-based, polymeric and branched polymeric, metal-based, magnetic, and mesoporous silica are being used in this treatment. Innovative strategies have been employed to utilize the multicomponent, three-dimensional structure of nanoparticles and modify it and construct a new structure moiety that has multifunctional capabilities. Developing such designs permits simultaneous drug delivery of chemotherapeutics as well as anticancer gene therapies to site-specific targets. In lung cancer, nanoparticle-based therapeutics is now breaking the ground in the diagnosis, imaging, screening, and treatment of primary and metastatic tumors. This review emphasizes the pathogenesis of lung cancer and its treatment by nanotechnology.
... Mutations in the BUB1 gene cause chromosome missegregation during CRC progression [68]. However, the role of BUB1 in CRC development remains poorly understood. ...
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Every year, more than a million individuals are diagnosed with colorectal cancer (CRC) across the world. Certain lifestyle and genetic factors are known to drive the high incidence and mortality rates in some groups of individuals. The presence of enormous amounts of reactive oxygen species is implicated for the on-set and carcinogenesis, and oxidant scavengers are thought to be important in CRC therapy. In this review, we focus on the ethnicity-based CRC disparities in the U.S., the negative effects of oxidative stress and apoptosis, and gene regulation in CRC carcinogenesis. We also highlight the use of antioxidants for CRC treatment, along with screening for certain regulatory genetic elements and oxidative stress indicators as potential biomarkers to determine the CRC risk and progression.
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Cancer is a group of diseases in which cells divide continuously and excessively. Cell division is tightly regulated by multiple evolutionarily conserved cell cycle control mechanisms, to ensure the production of two genetically identical cells. Cell cycle checkpoints operate as DNA surveillance mechanisms that prevent the accumulation and propagation of genetic errors during cell division. Checkpoints can delay cell cycle progression or, in response to irreparable DNA damage, induce cell cycle exit or cell death. Cancer-associated mutations that perturb cell cycle control allow continuous cell division chiefly by compromising the ability of cells to exit the cell cycle. Continuous rounds of division, however, create increased reliance on other cell cycle control mechanisms to prevent catastrophic levels of damage and maintain cell viability. New detailed insights into cell cycle control mechanisms and their role in cancer reveal how these dependencies can be best exploited in cancer treatment.
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Background: Complex genomic changes that arise in tumors are a consequence of chromosomal instability. In tumor cells genomic aberrations disrupt core signaling pathways involving various genes, thus delineating of signaling pathways can help understand the pathogenesis of cancer. The bioinformatics tools can further help in identifying networks of interactions between the genes to get a greater biological context of all genes affected by chromosomal instability. Methods: Karyotypic analyses was done in 150 clinically confirmed breast cancer patients and 150 age and gender matched healthy controls after 72 hours Peripheral lymphocyte culturing and GTG-banding. Reactome database from Cytoscape software version 3.7.1 was used to perform. in-silico analysis (functional interaction and gene enrichment). Results: Frequency of chromosomal aberrations (structural and numerical) was found to be significantly higher in patients as compared to controls. The genes harbored by chromosomal regions showing increased aberration frequency in patients were further analyzed in-silico. Pathway analysis on a set of genes that were not linked together revealed that genes HDAC3, NCOA1, NLRC4, COL1A1, RARA, WWTR1, and BRCA1 were enriched in the RNA Polymerase II Transcription pathway which is involved in recruitment, initiation, elongation and dissociation during transcription. Conclusion: The current study employs the information inferred from chromosomal instability analysis in a non-target tissue for determining the genes and the pathways associated with breast cancer. These results can be further extrapolated by performing either mutation analysis in the genes/pathways deduced or expression analysis which can pinpoint the relevant functional impact of chromosomal instability.
Article
Lung adenocarcinoma (LUAD) is one of the most common causes of cancer death in men. BUB1B (BUB1 mitotic checkpoint serine/threonine kinase B) has been reported to contribute to the initiation and development of several cancers. Here, we aimed to explore the potential role of BUB1B in LUAD. We found BUB1B was upregulated in LUAD, suggesting its potential role as a biomarker for LUAD diagnosis. Significantly, LUAD patients with high BUB1B expression had a shorter survival time than those with low BUB1B expression. Knocking-out BUB1B resulted in suppression of cell proliferation, migration, and invasion in vitro, and inhibition of tumor growth in the xenograft experiment. Further analysis revealed that BUB1B regulates glycolysis in LUAD and interacting with ZNF143 in LUAD cells. The interaction was demonstrated by silencing ZNF143, which led to a decrease in proliferation, migration, and invasion in LUAD cells, whereas overexpressing BUB1B had the opposite effects. Our study suggested that the ZNF143/BUB1B axis plays a pivotal role in LUAD progression, which might be a potential target for LUAD management.
Article
The spindle assembly checkpoint (SAC) is a surveillance mechanism that prevents unequal segregation of chromosomes during mitosis. Abnormalities in the SAC are associated with chromosome instability and resultant aneuploidy. This study was performed to evaluate the SAC competence in canine malignant melanoma (CMM) using four aneuploid cell lines (CMeC1, CMeC2, KMeC, and LMeC). After treatment with nocodazole, a microtubule disrupting agent, CMeC1, KMeC, and LMeC cells were arrested in M phase, whereas CMeC2 cells were not arrested, and progressed into the next cell cycle phase without cytokinesis. Chromosome spread analysis revealed a significantly increased rate of premature sister chromatid separation in CMeC2 cells. Expression of the phosphorylated form of the SAC regulator, monopolar spindle 1 (Mps1), was lower in CMeC2 cells than in the other CMM cell lines. These results indicate that the SAC is defective in CMeC2 cells, which may partially explain aneuploidy in CMM. Thus, CMeC2 cells may be useful for further studies of the SAC mechanism in CMM and in determining the relationship between SAC incompetence and aneuploidy.
Chapter
DNA repair pathways, such as base excision repair, nucleotide excision repair, mismatch repair, nonhomologous end joining and homology directed repair, play a critical role in maintaining genomic integrity. A deficiency of these pathways may therefore lead to genomic instability which is a hallmark of almost all types of human malignancies. Various types of genomic instability have been characterized in tumor cells, including chromosomal instability (CIN) such as copy number variation and aneuploidy, microsatellite instability (MSI or MIN) manifested by the expansion or contraction of repeated microsatellite sequences and clonal base-pair mutations, including small-scale insertions, deletions, transversions and point mutations in oncogenes and tumor suppressor genes which play an important role in the initiation of tumorigenesis. This chapter describes the current understanding of the defects of DNA repair pathways in the development of cancer as well as DNA repair pathway targeting as a potential strategy for cancer therapy.
Article
Centromere structure and function are defined by the epigenetic modification of histones at centromeric and pericentromeric chromatin. The constitutive heterochromatin found at pericentromeric regions is highly enriched for H3K9me3 and H4K20me3. Although mis-expression of the methyltransferase enzymes that regulate these marks, Suv39 and Suv420, is common in disease, the consequences of such changes are not well understood. Our data show that increased centromere localization of Suv39 and Suv420 suppresses centromere transcription and compromises localization of the mitotic kinase Aurora B, decreasing microtubule dynamics and compromising chromosome alignment and segregation. We find that inhibition of Suv420 methyltransferase activity partially restores Aurora B localization to centromeres and that restoration of the Aurora B-containing chromosomal passenger complex to the centromere is sufficient to suppress mitotic errors that result when Suv420 and H4K20me3 is enriched at centromeres. Consistent with a role for Suv39 and Suv420 in negatively regulating Aurora B, high expression of these enzymes corresponds with increased sensitivity to Aurora kinase inhibition in human cancer cells, suggesting that increased H3K9 and H4K20 methylation may be an underappreciated source of chromosome mis-segregation in cancer. This article has an associated First Person interview with the first author of the paper.
Chapter
Eukaryotic cell division is divided into several phases and each of these phases has their own control mechanisms. Failure of any of these control mechanisms may lead to development of errors which may be propagated to up-coming generations leading to development of carcinogenic phenotype. Therefore, cell cycle has become an attractive target in anticancer research which is mainly focused on dealing with the regulators and checkpoints involved in the progression of cell cycle. The major components involved in controlling the cell cycle are cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CDKIs). Apart from these, an efficient DNA repair system and the proper assembly of spindle fibers also contribute to smooth progression of cell cycle. Therefore, in addition to the great dependency of anticancer research on cyclins, CDKs, and CDKIs, DNA repair system and assembly of spindle fiber also contribute to the foundation of anticancer research. In this chapter, we describe cell cycle and its importance in anticancer research, the clinical studies based on cell cycle to curb neoplastic development, and approaches used in anti-tumor research to counter cancer progression.
Article
The nuclear pore protein NUP62 localizes to spindle poles in mitosis and plays a role in maintaining centrosome homeostasis. In this study, we found that NUP62-depleted cells exhibited a defective spindle assembly checkpoint (SAC) and that depletion of NUP62 caused a slight decrease in MAD2 protein levels after nocodazole treatment. However, depletion of NUP62 did not cause a failure in kinetochore localization of the SAC proteins BUBR1, MAD1, and MAD2 in prometaphase. NUP62 depletion slightly prolonged mitotic timing but did not affect cell doubling time. In addition, NUP62 depletion caused a SAC defect and induced aneuploidy in human neural stem cells. Furthermore, overexpression of NUP62Q391P, a mutant protein causing autosomal recessive infantile bilateral striatal necrosis, resulted in a defect in the SAC, indicating that the amino acid residue Q391 in NUP62 is crucial for its effect on the SAC. Overall, we conclude that NUP62 maintains the SAC downstream of kinetochores and thereby ensures maintenance of chromosomal stability.
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Accumulating evidence has demonstrated that gene alterations play a crucial role in LUAD development, progression, and prognosis. The current study aimed to identify the hub genes associated with LUAD. In the present study, we used TCGA database to screen the hub genes. Then, we validated the results by GEO datasets. Finally, we used cBioPortal, UALCAN, qRT-PCR, HPA database, TCGA database, and Kaplan-Meier plotter database to estimate the gene mutation, gene transcription, protein expression, clinical features of hub genes in patients with LUAD. A total of 5,930 DEGs were screened out in TCGA database. Enrichment analysis revealed that DEGs were involved in the transcriptional misregulation in cancer, viral carcinogenesis, cAMP signaling pathway, calcium signaling pathway, and ECM-receptor interaction. The combining results of MCODE and CytoHubba showed that ADCY8, ADRB2, CALCA, GCG, GNGT1, and NPSR1 were hub genes. Then, we verified the above results by GSE118370, GSE136043, and GSE140797 datasets. Compared with normal lung tissues, the expression level of ADCY8 and ADRB2 were lower in LUAD tissues, but the expression level of CALCA, GCG, GNGT1, and NPSR1 were higher. In the prognosis analyses, the low expression of ADCY8 and ADRB2 and the high expression of CALCA, GCG, GNGT1, and NPSR1 were correlated with poor OS and poor PFS. The significant differences in the relationship of the expression of 6 hub genes and clinical features were observed. In conclusion, 6 hub genes will not only contribute to elucidating the pathogenesis of LUAD, and may be potential therapeutic targets for LUAD.
Chapter
The act of counting objects is a difficult and underappreciated task. Many of the entrenched misconceptions in science come from sloppy counting protocols. Knowing the number and diversity of data objects (i.e., how many classes of objects are present in the data set and how many members belong to each of those classes) tells us a great deal about the nature of biological data. This chapter teaches us that the process of counting biological objects (e.g., organisms, species, genes, proteins, and variants thereof) will often reveal or clarify profound biological mysteries. Examples will include why there are at least 50 million species of living organisms on earth; the significance of having just a handful of species belonging to the monotremes, while there are many thousands of species of beetles; why there is only a small number of general classes of body plans; what we learn by comparing the number of herbivorous mammals to the number of carnivorous mammals; acquired diseases are more common than genetic diseases why rare diseases are biologically, not just numerically, different from common diseases.
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Cancer immunotherapy based on carbon-quantum-dots (CQDs) has proven effective. The CQDs composited cancer cell (CM-cancer) exhibit robust customized immunogenicity, which can recruit macrophages and dendritic cells to effectively deliver the cancer antigens into lymph nodes to activate CD8+ T cells, and eventually leads to an anti-cancer immune response all over the body. The CM-cancer is not only able to inhibit primary tumors, but also clearly eliminate metastatic tumors. Our research demonstrates a promising personalized cancer immunotheraputic technology for potential clinical applications.
Article
The MAD2-dependent spindle checkpoint blocks anaphase until all chromosomes have achieved successful bipolar attachment to the mitotic spindle. The DNA damage and DNA replication checkpoints block anaphase in response to DNA lesions that may include single-stranded DNA and stalled replication forks. Many of the same conditions that activate the DNA damage and DNA replication checkpoints also activated the spindle checkpoint. The mad2Δ mutation partially relieved the arrest responses of cells to mutations affecting the replication proteins Mcm3p and Pol1p. Thus a previously unrecognized aspect of spindle checkpoint function may be to protect cells from defects in DNA replication. Furthermore, in cells lacking either the DNA damage or the DNA replication checkpoints, the spindle checkpoint contributed to the arrest responses of cells to the DNA-damaging agent methyl methanesulfonate, the replication inhibitor hydroxyurea, and mutations affecting Mcm2p and Orc2p. Thus the spindle checkpoint was sensitive to a wider range of chromosomal perturbations than previously recognized. Finally, the DNA replication checkpoint did not contribute to the arrests of cells in response to mutations affecting ORC, Mcm proteins, or DNA polymerase δ. Thus the specificity of this checkpoint may be more limited than previously recognized.
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Background: Cyanidin-3-O-glucoside (cyan) exhibits antioxidant and anticancer properties. The cell cycle proteins and antimitotic drugs might be promising therapeutic targets in hepatocellular carcinoma. Aim: To investigate the effect of cyan administration on cell cycle in hepatic precancerous lesion (PCL) induced by diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) in Wistar rats. Methods: In vivo, DEN/2-AAF-induced hepatic PCL, rats were treated with three doses of cyan (10, 15, and 20 mg/kg/d, for four consecutive days per week for 16 wk). Blood and liver tissue samples were collected for measurement of the followings; alpha fetoprotein (AFP) liver function and RNA panel differential expression was evaluated via real time polymerase chain reaction. Histopathological examination of liver sections stained with H&E and immunohistochemical study using glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies were assessed. Results: Cyan administration mitigated the effect of DEN/2-AFF induced PCL, decreased AFP levels, and improved liver function. Remarkably, treatment with cyan dose dependently decreased the long non-coding RNA MALAT1 and tubulin gamma 1 mRNA expressions and increased the levels of miR-125b, all of which are involved in cell cycle and mitotic spindle assembly. Of note, cyan decreased GSTP foci percent area and PCNA positively stained nuclei. Conclusion: Our results indicated that cyan could be used as a potential therapeutic agent to inhibit liver carcinogenesis in rat model via modulation of cell cycle.
Thesis
Tumor recurrence is one of the major obstacles to overcome in the future to improve the overall survival of patients with colon cancer. High rates and patterns of therapeutic failure seen in patients are consistent with a steady accumulation of drug-resistant cancer stem cells (CSCs). Here, we demonstrate that the nuclear receptor PXR (NR1I2) acts as a key regulator of colon CSC chemoresistance and of their ability to generate post-treatment tumor relapse. We first determined that the enrichment of PXR paralleled that of CSC markers upon treatment of colon cancer cells with the standard of care chemotherapy. We found that PXR was highly expressed in colorectal cancer cells displaying CSC markers and function and that it was instrumental for the emergence of CSCs following chemotherapy in vitro and in vivo. mRNA profiling experiments in colon CSCs indicated that PXR transcriptionally controls a large network of genes including markers of stemness, genes involved in resistance to drug/apoptosis or migration/invasion. Finally, PXR down-regulation altered the survival and self-renewal of colon CSCs in vitro and hampered their capacity to resist chemotherapy in vivo, leading to significant delays of post-chemotherapy tumor relapse. Clinically, we observed that patients bearing tumors with high PXR expression display a lower probability of recurrence-free survival, while a molecular signature associated with poor progression-free survival was specifically enriched in PXRoverexpression LS174T cells and was decreased in patient-derived CSC cells after PXR depletion. This study strongly suggests that targeting PXR may represent a novel treatment strategy to prevent drug resistance and recurrence through the sensitization of CSCs to standard chemotherapy. Taken together, our data strongly suggest that PXR plays an instrumental role in the so-called "intrinsic" pan-resistance of CSCs against therapy. Keywords: Colon Cancer, PXR, Drug resistance, Tumor Recurrence
Book
L’analyse des informations scientifiques récentes sur la nécessité d’évaluer, à l’aide des tests in vitro ou in vivo, la toxicité des substances endogènes et/ou exogènes a été réalisée plus spécifiquement dans trois domaines de la toxicologie, à savoir la génotoxicité, la cytotoxicité et la perturbation endocrinienne. Cette synthèse a permis de mieux cerner les difficultés techniques liées à la réalisation d’essais toxicologiques, les migrats ne représentant que de faibles quantités de produits, non-identifiés ou partiellement identifiés. Concernant l’évaluation de la cytotoxicité, une batterie de tests semble indispensable, afin de couvrir différents mécanismes d’action (survie ou développement cellulaire, altérations fonctionnelles, morphologiques, etc.). Cependant, leur intérêt peut paraître limité du fait leur manque de représentativité des effets in vivo. L’évaluation de la toxicité cellulaire par des tests serait en corrélation avec la réglementation actuelle des produits consommés et il est souhaitable qu’au moins un test de mutations géniques et un test d’aberrations chromosomiques soient effectués en premier lieu. L’évaluation du potentiel de perturbation endocrinienne ne peut être réalisée qu’au moyen d’une batterie de tests in vitro et in vivo. En l’état actuel, ces tests ne paraissent pas suffisamment validés pour être pris en compte dans le cadre de l’évaluation de la cytotoxicité. Il serait cependant nécessaire de suivre l’avancée des recherches dans ce domaine. Ce livre est destiné davantage aux étudiants et aux scientifiques impliqués dans le domaine de la toxicologie afin de mieux comprendre et pour une bonne maitrise du mode d’action des métabolites toxiques au niveau des organites cellulaires, ainsi que les différents tests appliqués pour confirmer la cytotoxicité des xénobiotiques
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Germline pathogenic mutations in BReast CAncer (BRCA1) genes are thought to drive normal fallopian tube epithelial (FTE) cell transformation to high-grade serous ovarian cancer. No human models capture the sequence of events for disease initiation and progression. Here, we generate induced pluripotent stem cells (iPSCs) from healthy individuals and young ovarian cancer patients with germline pathogenic BRCA1 mutations (BRCA1mut). Following differentiation into FTE organoids, BRCA1mut lines exhibit cellular abnormalities consistent with neoplastic transformation compared to controls. BRCA1mut organoids show an increased production of cancer-specific proteins and survival following transplantation into mice. Organoids from women with the most aggressive ovarian cancer show the greatest pathology, indicating the potential value to predict clinical severity prior to disease onset. These human FTE organoids from BRCA1mut carriers provide a faithful physiological in vitro model of FTE lesion generation and early carcinogenesis. This platform can be used for personalized mechanistic and drug screening studies.
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Previous studies have suggested that expression of p53 in cancer cells can result in either growth arrest or apoptosis. Accordingly, expression of p53 in a series of colorectal cancer cell lines yielded growth arrest in some lines (A-lines) and apoptosis in others (D-lines). To investigate the basis of this difference, we evaluated the role of p21WAF1/Cip1, a known mediator of p53-induced growth arrest. Inactivation of p21 by homologous recombination converted an A-line to a D-line, suggesting that p21 could protect cells from apoptosis. However, examination of p53-induced p21 expression in naturally occurring D-lines and A-lines demonstrated that the induction of p21 could not account for the differential response to p53. Moreover, when a D-line was fused to an A-line, the resulting hybrid cells underwent apoptosis in response to p53, indicating that the apoptosis pathway was dominant over the growth arrest pathway. Therefore, the apoptotic response to p53 in colorectal cancer cells is modulated by at least two factors: p21-mediated growth arrest that can protect cells from apoptosis in A-cells, and trans-acting factors in D-cells that can overcome this protection, resulting in cell death.
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Much excitement has recently been generated by the discovery of the Smad genes, encoding proteins that transduce signals from the transforming growth factor beta family of cytokines. Here, we report the completion of cloning of the six known human Smads, providing novel sequences for Smad5 and Smad6. Previously, Smad4 and Smad2 were shown to be mutated in human cancers. However, analysis of the other four Smad genes revealed no mutations in a total of 167 tumors, including those from colon, breast, lung, and pancreas. These results suggest that the various Smad genes have different functions and demonstrate that mutations in these four genes do not, in general, account for the widespread resistance to transforming growth factor beta that is found in human tumors.
Article
The TGF@type H receptor(RI!) was found to be mutatedwithin a polyadeninetract in 100of 111(90%) colorectal cancers with microsatellite instability. Other polyadenine tracts of similar length were mutated in these samplesbut not as frequentlyas RI!. In most cases, the polyadeninetract mutationsaffectedboth allelesofRI!, and in four tumors heterozygousfor the polyadenine mutations, three had additional mutations that were expected to inactivate the other RH allele. These genetic data support the idea that RU behaves like a tumor suppressor during CR cancer development and is a critical target of inactivation in mismatch repair-deficient tumors.
Article
THE genomes of all eukaryotes contain tracts of DNA in which a single base or a small number of bases is repeated. Expansions of such tracts have been associated with several human disorders including the fragile X syndrome1. In addition, simple repeats are unstable in certain forms of colorectal cancer, suggesting a defect in DNA replication or repair2-4. We show here that mutations in any three yeast genes involved in DNA mismatch repair (PMS1, MLH1 and MSH2) lead to 100- to 700-fold increases in tract instability, whereas mutations that eliminate the proof-reading function of DNA polymerases have little effect. The meiotic stability of the tracts is similar to the mitotic stability. These results suggest that tract instability is associated with DNA poly-merases slipping during replication, and that some types of colo-rectal cancer may reflect mutations in genes involved in DNA mismatch repair.
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We have investigated the feedback control that prevents cells with incompletely assembled spindles from leaving mitosis. We isolated budding yeast mutants sensitive to the anti-microtubule drug benomyl. Mitotic arrest-deficient (mad) mutants are the subclass of benomyl-sensitive mutants in which the completion of mitosis is not delayed in the presence of benomyl and that die as a consequence of their premature exit from mitosis. A number of properties of the mad mutants indicate that they are defective in the feedback control over the exit from mitosis: their killing by benomyl requires passage through mitosis; their benomyl sensitivity can be suppressed by an independent method for delaying the exit from mitosis; they have normal microtubules; and they have increased frequencies of chromosome loss. We cloned MAD2, which encodes a putative calcium-binding protein whose disruption is lethal. We discuss the role of feedback controls in coordinating events in the cell cycle.
Article
Publisher Summary To help cope with the rapidly expanding protein kinase family, a database of the catalytic domain amino acid sequences has been established. This database would be a useful resource for the initial classification of novel protein kinases and for other studies that require extensive sequence comparisons. The catalytic domain database is updated frequently and often includes new sequences before they can be found in the Genbank/EMBL/PIR resources. More importantly, the availability of this large group of sequences in a single file saves investigators from the tedious task of collecting them themselves. This chapter describes the current makeup of the catalytic domain database and present two examples of its use: analysis and graphic display of conserved catalytic domain residues using conservation plots and classification of protein kinases by phylogenetic mapping. A prerequisite for both of these tasks is a multiple sequence alignment. The protein kinase catalytic domain database file can be obtained electronically over Internet using the standard network file transfer program (FTP). The database file PKINASES.IG contains brief descriptions of the kinases and references for the sequences. One hundred and seventeen distinct sequences had been entered by mid-February, 1990. Seventy-five of these are taken from protein-serine/threonine kinases and 42 from protein-tyrosine kinases. Sixty-eight of the sequences are from vertebrate species, 24 from yeasts (both budding and fission), 18 from Drosophila, two from nematode, and one each from Aplysia , Aspergillus , Hydra , bean plant ( Phaseolus ), and avian erythroblastosis virus S13. 43 of the 68 current vertebrate entries are taken from human sources and twenty two of the remaining vertebrate sequences derive from four other mammals: bovine, rabbit, rat, and mouse.
Article
The TGF beta type II receptor (RII) was found to be mutated within a polyadenine tract in 100 of 111 (90%) colorectal cancers with microsatellite instability. Other polyadenine tracts of similar length were mutated in these samples but not as frequently as RII. In most cases, the polyadenine tract mutations affected both alleles of RII, and in four tumors heterozygous for the polyadenine mutations, three had additional mutations that were expected to inactivate the other RII allele. These genetic data support the idea that RII behaves like a tumor suppressor during CR cancer development and is a critical target of inactivation in mismatch repair-deficient tumors.
Article
Microsatellite instability has been observed in both sporadic and hereditary forms of colorectal cancer. In the hereditary form, this instability is generally due to germline mutations in mismatch repair (MMR) genes. However, only one in ten patients with sporadic tumours exhibiting microsatellite instability had a detectable germline mutation. Moreover, only three of seven sporadic tumour cell lines with microsatellite instability had mutations in a MMR gene, and these mutations could occur somatically. These results demonstrate that tumours can acquire somatic mutations that presumably do not directly affect cell growth but result only in genetic instability. They also suggest that many sporadic tumours with microsatellite instability have alterations in genes other than the four now known to participate in MMR.
Article
A series of 80 microclone probes derived from the chromosomal region 1p36 was used to isolate corresponding clones from the ICRF human P1 library (see Francis et al., this issue). Hybridization screenings were performed using probe pools on high-density filter grids. A total of 87 P1 clones specific for 1p36 were isolated. This large-scale approach allowed a detailed evaluation of the complexity, quality, and utility of this library. The isolated P1 clones were used both for size determination by pulsed-field gel electrophoresis and as probes for fluorescence in situ hybridization (FISH) analysis. FISH of P1 clones is shown to be both easy and efficient to perform on metaphase chromosomes and interphase nuclei. This observation is expected to reveal new avenues for diagnosis of disease-related chromosomal changes. The use of P1 clones as a tool in clinical and tumor interphase cytogenetics is discussed and compared with FISH data of other long insert clones such as cosmids and YAC clones.
Article
Checkpoints help in the prevention of genetic damage by giving cells time to repair damaged structures before proceeding in the cell cycle. Genetic analyses in budding and fission yeast have identified a large number of cell cycle checkpoint genes. Several of these encode proteins related to components of other signal transduction pathways, including protein kinases, lipid kinases, and 14-3-3 proteins. In fission yeast, checkpoints play an important role in keeping cells from entering mitosis before they pass Start.
Article
Normal cell multiplication requires that the events of mitosis occur in a carefully ordered fashion. Cells employ checkpoints to prevent cycle progression until some prerequisite step has been completed. To explore the mechanisms of checkpoint enforcement, we previously screened for mutants of Saccharomyces cerevisiae which are unable to recover from a transient treatment with a benzimidazole-related microtubule inhibitor because they fail to inhibit subsequent cell cycle steps. Two of the identified genes, BUB2 and BUB3, have been cloned and described (M. A. Hoyt, L. Totis, and B. T. Roberts, Cell 66:507-517, 1991). Here we present the characterization of the BUB1 gene and its product. Genetic evidence was obtained suggesting that Bub1 and Bub3 are mutually dependent for function, and immunoprecipitation experiments demonstrated a physical association between the two. Sequence analysis of BUB1 revealed a domain with similarity to protein kinases. In vitro experiments confirmed that Bub1 possesses kinase activity; Bub1 was able to autophosphorylate and to catalyze phosphorylation of Bub3. In addition, overproduced Bub1 was found to localize to the cell nucleus.
Article
Colorectal cancer occurs in approximately 150,000 people each year in the United States. Prognostic assessment influences the treatment of patients with colorectal cancer, including decisions about adjuvant therapy. We evaluated chromosome 18q allelic loss, a genetic event associated with tumor progression, as a prognostic marker for this disease. We developed procedures to examine the status of chromosome 18q with microsatellite markers and DNA from formalin-fixed, paraffin-embedded tumors. Allelic loss of chromosome 18q was assessed in 145 consecutively resected stage II or III colorectal carcinomas. Among patients with stage II disease, the five-year survival rate was 93 percent in those whose tumor had no evidence of allelic loss of chromosome 18q and 54 percent in those with allelic loss; among patients with stage III disease, survival was 52 and 38 percent, respectively. The overall estimated hazard ratio for death in patients whose tumor had chromosome 18q allelic loss was 2.83 (P = 0.008) according to univariate analysis. Furthermore, chromosome 18q allelic loss remained a strong predictive factor (hazard ratio for death, 2.46; 95 percent confidence interval, 1.06 to 5.71; P = 0.036) after adjustment for all other evaluated factors, including tumor differentiation, vein invasion, and TNM stage. The status of chromosome 18q has strong prognostic value in patients with stage II colorectal cancer. The prognosis in patients with stage II cancer and chromosome 18q allelic loss is similar to that in patients with stage III cancer, who are thought to benefit from adjuvant therapy. In contrast, patients with stage II disease who do not have chromosome 18q allelic loss in their tumor have a survival rate similar to that of patients with stage I disease and may not require additional therapy.
Article
The genomes of all eukaryotes contain tracts of DNA in which a single base or a small number of bases is repeated. Expansions of such tracts have been associated with several human disorders including the fragile X syndrome. In addition, simple repeats are unstable in certain forms of colorectal cancer, suggesting a defect in DNA replication or repair. We show here that mutations in any three yeast genes involved in DNA mismatch repair (PMS1, MLH1 and MSH2) lead to 100- to 700-fold increases in tract instability, whereas mutations that eliminate the proof-reading function of DNA polymerases have little effect. The meiotic stability of the tracts is similar to the mitotic stability. These results suggest that tract instability is associated with DNA polymerases slipping during replication, and that some types of colorectal cancer may reflect mutations in genes involved in DNA mismatch repair.
Article
DNA mismatch-repair systems exist that repair mispaired bases formed during DNA replication, genetic recombination and as a result of damage to DNA. Some components of these systems are conserved in prokaryotes and eukaryotes. Genetic defects in mismatch-repair genes play an important role in common cancer-susceptibility syndromes and sporadic cancers.
Article
Precise coordination of the S and M phases of the eukaryotic cell cycle is critical not only for normal cell division, but also for effective growth arrest under conditions of stress. When damaged, a cell must communicate signals to both the mitotic and DNA synthesis machineries so that a mitotic block is not followed by an extra S phase, or vice versa. The biochemical mechanisms regulating this coordination, termed checkpoints, have been identified in lower eukaryotes, but are largely unknown in mammalian cells. Here we show that p21 WAF1/CIP1, the prototype inhibitor of cyclin-dependent kinases (CDKs), is required for this coordination in human cells. In the absence of p21, DNA-damaged cells arrest in a G2-like state, but then undergo additional S phases without intervening normal mitoses. They thereby acquire grossly deformed, polyploid nuclei and subsequently die through apoptosis. Perhaps not by coincidence, the DNA-damaging agents that can cause S/M uncoupling are used in the clinic to kill cancer cells preferentially.
Article
The spindle assembly checkpoint keeps cells with defective spindles from initiating chromosome segregation. The protein kinase Mps1 phosphorylates the yeast protein Mad1p when this checkpoint is activated, and the overexpression of Mps1p induces modification of Mad1p and arrests wild-type yeast cells in mitosis with morphologically normal spindles. Spindle assembly checkpoint mutants overexpressing Mps1p pass through mitosis without delay and can produce viable progeny, which demonstrates that the arrest of wild-type cells results from inappropriate activation of the checkpoint in cells whose spindle is fully functional. Ectopic activation of cell-cycle checkpoints might be used to exploit the differences in checkpoint status between normal and tumor cells and thus improve the selectivity of chemotherapy.
Article
In Saccharomyces cerevisiae, MAD2 is required for mitotic arrest if the spindle assembly is perturbed. The human homolog of MAD2 was isolated and shown to be a necessary component of the mitotic checkpoint in HeLa cells by antibody electroporation experiments. Human, or Homo sapiens, MAD2 (hsMAD2) was localized at the kinetochore after chromosome condensation but was no longer observed at the kinetochore in metaphase, suggesting that MAD2 might monitor the completeness of the spindle-kinetochore attachment. Finally, T47D, a human breast tumor cell line that is sensitive to taxol and nocodazole, had reduced MAD2 expression and failed to arrest in mitosis after nocodazole treatment. Thus, defects in the mitotic checkpoint may contribute to the sensitivity of certain tumors to mitotic spindle inhibitors.
Article
Saccharomyces cerevisiae cells containing one or more abnormal kinetochores delay anaphase entry. The delay can be produced by using centromere DNA mutations present in single-copy or kinetochore protein mutations. This observation is strikingly similar to the preanaphase delay or arrest exhibited in animal cells that experience spontaneous or induced failures in bipolar attachment of one or more chromosomes and may reveal the existence of a conserved surveillance pathway that monitors the state of chromosome attachment to the spindle before anaphase. We find that three genes (MAD2, BUB1, and BUB2) that are required for the spindle assembly checkpoint in budding yeast (defined by antimicrotubule drug-induced arrest or delay) are also required in the establishment and/or maintenance of kinetochore-induced delays. This was tested in strains in which the delays were generated by limited function of a mutant kinetochore protein (ctf13-30) or by the presence of a single-copy centromere DNA mutation (CDEII delta 31). Whereas the MAD2 and BUB1 genes were absolutely required for delay, loss of BUB2 function resulted in a partial delay defect, and we suggest that BUB2 is required for delay maintenance. The inability of mad2-1 and bub1 delta mutants to execute kinetochore-induced delay is correlated with striking increases in chromosome missegregation, indicating that the delay does indeed have a role in chromosome transmission fidelity. Our results also indicated that the yeast RAD9 gene, necessary for DNA damage-induced arrest, had no role in the kinetochore-induced delays. We conclude that abnormal kinetochore structures induce preanaphase delay by activating the same functions that have defined the spindle assembly checkpoint in budding yeast.
Article
The authors are grateful to the members of their laboratories for their contributions to the reviewed studies and for their critical reading of the manuscript and to F. Giardiello and S. Hamilton for photographs of colorectal lesions. The authors are supported by Public Health Service grants CA 43460, CA 57345, and CA 62924. B. V. is an Investigator of the Howard Hughes Medical Institute.
Article
It might now seem obvious that the mechanisms regulating cell division would be found to be a highly conserved feature of eukaryotic cells. This was less clear 20 years ago when the pioneering genetic studies of the cell cycle were initiated. This article presents one view as to what lies at the heart of the budding yeast cell cycle. It is written on the premise that most of the key players, such as cyclin-dependent kinases, the anaphase-promoting complex, the origin recognition complex, Cdc6p and Mcm proteins, were performing similar functions in the common ancestor of yeast and man. Ideas about the budding yeast cell cycle might, therefore, have universal significance for other eukaryotic cells.
Article
This review focuses on the genomic instability underlying the microsatellite mutator phenotype (MMP) pathway for cancer. MMP was discovered by the application of DNA fingerprinting by Arbitrarily Primed PCR (AP-PCR) to the analysis of somatic genetic alterations in colon tumors. The unbiased nature of AP-PCR permitted to infer, from the mobility shifts observed in some fingerprint bands, the accumulation by a subset of colon tumors of hundreds of thousands of somatic mutations in simple repeated sequences or microsatellites. We deduced that this enormous agglomeration of clonal mutations was due to the previous occurrence of 'mutator mutations' in DNA replication or repair factors leading to a decreased fidelity of replication. These mutator mutations appeared to be the remote cause for the development of these MMP tumors, whose existence unmistakably validated the hypothesis of 'cancer as a mutator phenotype'. Since these original observations, rapid progress has occurred in the field. The mutator mutations were identified as those occurring in members of the DNA mismatch repair gene family, which are also associated with hereditary non-polyposis colorectal cancer (HN-PCC). In this review I discuss the experimental approach that allowed the discovery of MMP and the features of the genomic instability of these tumors. I also review recent developments that affect the understanding of the role of the mismatch repair mutator mutations in the unfolding of MMP during carcinogenesis.
Article
When cells divide, the chromosomes must be delivered flawlessly to the daughter cells. Missing or extra chromosomes can result in birth defects and cancer. Chance events are the starting point for chromosome delivery, which makes the process prone to error. Errors are avoided by diverse uses of mechanical tension from mitotic forces. Tension stabilizes the proper chromosome configuration, controls a cell cycle checkpoint, and changes chromosome chemistry.
Article
We would like to thank members of the Hartwell laboratory, especially Eric Foss, members of the Seattle project, Jim Roberts, Andrew Murray, and an annonymous reviewer for helpful comments on the manuscript. A. G. P. was supported by a Merck Distinguished Fellow Award and an MSTP training grant from the NIH. D. P. T. was supported by a fellowship from the Jane Coffin Childs Memorial Fund and an NIH training program in Cancer Research CA09437. L. H. H. is a Research Professor of the American Cancer Society.
Article
It has long been considered that genetic instability is an integral component of human neoplasia. In a small fraction of tumours, mismatch repair deficiency leads to a microsatellite instability at the nucleotide sequence level. In other tumours, an abnormal chromosome number (aneuploidy) has suggested an instability, but the nature and magnitude of the postulated instability is a matter of conjecture. We show here that colorectal tumours without microsatellite instability exhibit a striking defect in chromosome segregation, resulting in gains or losses in excess of 10(-2) per chromosome per generation. This form of chromosomal instability reflected a continuing cellular defect that persisted throughout the lifetime of the tumour cell and was not simply related to chromosome number. While microsatellite instability is a recessive trait, chromosomal instability appeared to be dominant. These data indicate that persistent genetic instability may be critical for the development of all colorectal cancers, and that such instability can arise through two distinct pathways.
Article
The mitotic checkpoint ensures proper chromosome segregation by delaying anaphase until chromosomes are aligned on the spindle. Following prolonged spindle damage, however, cells eventually exit mitosis and undergo apoptosis. We show here that a murine homolog of the yeast mitotic checkpoint gene BUB1 localizes to the kinetochore during mitosis. By expressing a dominant-negative mutant, we show that mBub1 is not only required for the checkpoint response to spindle damage, but acts in the timing of a normal mitosis. In addition, when mBub1 function is compromised, cells escape apoptosis and continue cell cycle progression, despite leaving mitosis with a disrupted spindle. These data demonstrate a role for kinetochore-associated mBub1 in regulating exit from mitosis, and suggest functional links between the mitotic checkpoint and subsequent apoptotic events in G1.
Article
The discovery of anticancer drugs is now driven by the numerous molecular alterations identified in tumor cells over the past decade. To exploit these alterations, it is necessary to understand how they define a molecular context that allows increased sensitivity to particular compounds. Traditional genetic approaches together with the new wealth of genomic information for both human and model organisms open up strategies by which drugs can be profiled for their ability to selectively kill cells in a molecular context that matches those found in tumors. Similarly, it may be possible to identify and validate new targets for drugs that would selectively kill tumor cells with a particular molecular context. This article outlines some of the ways that yeast genetics can be used to streamline anticancer drug discovery.
Article
The spindle assembly checkpoint modulates the timing of anaphase initiation in mitotic cells containing improperly aligned chromosomes and increases the probability of successful delivery of a euploid chromosome set to each daughter cell. We have characterized cDNA sequences from several organisms with highly significant predicted protein sequence homologies to Saccharomyces cerevisiae Bub1p, a protein required for function of the spindle assembly checkpoint in budding yeast. The localization of mouse and human orthologs is in agreement with known conservation of synteny. Mouse backcross mapping data indicate that the murine gene resides on chromosome 2 near IL1A, 73 cM from the mouse centromere. Radiation hybrid mapping data indicate that the human locus exhibits linkage to microsatellite marker D2S176, which is located within 10 cM of human IL1A. Multiple-tissue Northern analysis indicates conservation of expression pattern in mouse and human with markedly high mRNA levels in testis. Northern analysis of two different spindle assembly checkpoint protein gene products from human, BUB1 and MAD2, reveals an expression pattern with common tissue distribution consistent with roles in a common pathway. In addition, we demonstrate that an mRNA found to accumulate in a rat fibroblast cell transformation system encodes rat BUB1, and we find that rat BUB1 mRNA accumulation correlates with the proliferation status of cells in culture.
Defectsin acellcyclecheckpointmaybe responsiblefor the genomicinstability of cancer cells
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Mammalian BUB1 protein kinases: map positions and in vivo expression G2 delay (data not shown). FACS analysis showed that the cells had two copies of the DNA, showing that Ca 2+ causes severe cell-cycle delay in G2 rather than in earlier stages
  • F Pangilinan
Pangilinan, F. et al. Mammalian BUB1 protein kinases: map positions and in vivo expression. Genomics 46, 379–388 (1997). G2 delay (data not shown). FACS analysis showed that the cells had two copies of the DNA, showing that Ca 2+ causes severe cell-cycle delay in G2 rather than in earlier stages (Fig. 1).