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ABSTRACT: We recently reported that homologous chromosomes make contact at the sites of double-strand breaks (DSBs) induced by ionizing radiation (IR) and the restriction endonuclease I-PpoI in G 0/G 1-phase somatic human cells. The contact involves short segments of homologous chromosomes and is centered on a DSB that occurs in a gene; contact does not occur at a DSB in intergenic DNA. Contact between homologous chromosomes is abrogated by inhibition of transcription and requires the kinase activity of ATM, but not DNA-PK. Here, we report additional insights into the mechanism underlying this novel phenomenon. We identify four patterns of homologous chromosome contact, and show that contact between homologous arms, but not centrosomes, is induced by IR. Significantly, we demonstrate that contact is induced by IR in non-proliferating, G 0-phase human cells derived from tissue explants. Finally, we show that contact between homologous chromosomes is detectable as early as 5 min after IR. These results point to the existence of a mechanism that rapidly localizes homologous chromosome arms at sites of DSBs in genes in G 0-phase human cells.
Cell cycle (Georgetown, Tex.) 01/2013; 12(4). · 5.36 Impact Factor
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ABSTRACT: It has been well established that genes participating in oncogenic rearrangements are non-randomly positioned and frequently close to each other in human cell nuclei. However, the actual distance between these fusion partners has never been determined. The phenomenon of fluorescence resonance energy transfer (FRET) is observed when a donor fluorophore is close (<10 nm) to transfer some of it energy to an acceptor fluorophore. The aim of this study was to validate the use of FRET on directly labeled DNA molecules to assess the frequency of positioning at <10 nm distances between genes known to be involved in rearrangement and to correlate it with their probability to undergo rearrangement. In the validation experiments, the frequency of FRET-sensitized emission (SE) was found to be 93-96% between probes for the immediately adjacent chromosomal regions as compared to 0.1-0.2% between probes for the random loci located on large linear separation. Further, we found that the frequency of FRET-SE between four pairs of genes that form rearrangements in thyroid cancer was 5% for RET and CCDC6, 4% for RET and NCOA4, 2% for BRAF and AKAP9, and 2% for NTRK1 and TPR. Moreover, the frequency with which FRET was observed showed strong correlation (r = 0.9871) with the prevalence of respective rearrangements in thyroid cancer. Our findings demonstrate that FRET can be used as a technique to analyze proximity between specific DNA regions and that the frequency of gene positioning at distances allowing FRET correlates with their probability to undergo chromosomal rearrangements. © 2012 Wiley Periodicals, Inc.
Genes Chromosomes and Cancer 08/2012; 51(11):1037-44. · 3.31 Impact Factor
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ABSTRACT: Double-strand DNA breaks (DSBs) are continuously induced in cells by endogenously generated free radicals and exogenous genotoxic agents such as ionizing radiation. DSBs activate the kinase activity in sensor proteins such as ATM and DNA-PK, initiating a complex DNA damage response that coordinates various DNA repair pathways to restore genomic integrity. In this study, we report the unexpected finding that homologous chromosomes contact each other at the sites of DSBs induced by either radiation or the endonuclease I-PpoI in human somatic cells. Contact involves short segments of homologous chromosomes and is centered on a DSB in active genes but does not occur at I-PpoI sites in intergenic DNA. I-PpoI-induced contact between homologous genes is abrogated by the transcriptional inhibitors actinomycin D and α-amanitin and requires the kinase activity of ATM but not DNA-PK. Our findings provide documentation of a common transcription-related and ATM kinase-dependent mechanism that induces contact between allelic regions of homologous chromosomes at sites of DSBs in human somatic cells.
Proceedings of the National Academy of Sciences 05/2012; 109(24):9454-9. · 9.68 Impact Factor
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ABSTRACT: Ionizing radiation (IR) exposure increases the risk of thyroid cancer and other cancer types. Chromosomal rearrangements, such as RET/PTC, are characteristic features of radiation-associated thyroid cancer and can be induced by radiation in vitro. IR causes double-strand breaks (DSBs), suggesting that such damage leads to RET/PTC, but the rearrangement mechanism has not been established. To study the mechanism, we explored the possibility of inducing RET/PTC by electroporation of restriction endonucleases (REs) into HTori-3 human thyroid cells. We used five REs, which induced DSB in a dose-dependent manner similar to that seen with IR. Although all but one RE caused DSB in one or more of the three genes involved in RET/PTC, rearrangement was detected only in cells electroporated with either PvuII (25 and 100 U) or StuI (100 and 250 U). The predominant rearrangement type was RET/PTC3, which is characteristic of human thyroid cancer arising early after Chernobyl-related radioactive iodine exposure. Both enzymes that produced RET/PTC had restriction sites only in one of the two fusion partner genes. Moreover, the two enzymes that produced RET/PTC had restriction sites present in clusters, which was not the case for RE that failed to induce RET/PTC. In summary, we establish a model of DSB induction by RE and report for the first time the formation of carcinogenic chromosomal rearrangements, predominantly RET/PTC3, as a result of DSB produced by RE. Our data also raise a possibility that RET/PTC rearrangement can be initiated by a complex DSB that is induced in one of the fusion partner genes.
Endocrine Related Cancer 02/2012; 19(3):271-81. · 4.36 Impact Factor
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ABSTRACT: Rat and mouse have been widely used to estimate the radiation risk and tumorigenic effects of radiation with extrapolating the findings to humans. RET/PTC is a characteristic genetic alteration frequently found in radiation-induced thyroid cancer in human populations. Recently, nuclear architecture and spatial proximity between recombinogenic genes have been implicated as important factors in the generation of RET/PTC and other chromosomal rearrangements in human cells. However, it is unknown whether the nuclear architecture in rodent thyroid cells is similar to that of human thyroid cells. The aim of this study was to test whether the proximity effects that are observed between loci involved in RET/PTC rearrangements in humans are conserved across different species.
Using 3D fixation, fluorescence in situ hybridization, and confocal microscopy, we compared the distance between genes involved in RET/PTC rearrangement in normal thyroid cells from humans, mice, and rats.
While in humans, RET, NCOA4, and H4 are all located on the same chromosome (10q), in rodents these genes are located on separate chromosomes. In mouse, RET is located on chromosome 6F1, NCOA4 on 14B, and H4 on 10B5.3. In rat, RET is on chromosome 4q42, NCOA4 on 16p16, and H4 (TST1) on 9q36. We further observed that in human thyroid cells, mean distance between genes involved in two most common types of RET/PTC, that is, RET and NCOA4 (partners of RET/PTC3) and RET and H4 (partners of RET/PTC1), was 1.08±0.04 and 1.24±0.05 μm, respectively. In mouse thyroid cells, these distances were 3.21±0.1 and 3.43±0.1 μm, and in rat cells the values were 3.37±0.1 and 3.87±0.1 μm (p<0.001). Moreover, we found that in contrast to human thyroid cells, in rodent cells these genes were randomly positioned with respect to each other.
The differences in nuclear architecture and spatial positioning of genes involved in RET/PTC rearrangements between human and rodent thyroid cells raise a concern about suitability of animal models for assessing RET/PTC-driven thyroid carcinogenesis in humans.
Thyroid: official journal of the American Thyroid Association 12/2011; 21(12):1331-7. · 2.60 Impact Factor
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ABSTRACT: Ionizing radiation is a well-known mutagen and a risk factor for thyroid cancer. MicroRNAs (miRNAs) play an important role in the regulation of gene expression on post-transcriptional level and are dysregulated in thyroid cancer. The goal of this study was to investigate the effects of acute exposure to 1 and 10 Gy of γ-irradiation on miRNA expression in normal human thyroid cells.
Expression of 319 miRNAs was studied in primary cultures of normal human thyroid cells 4 and 24 hours postirradiation using a miRNA expression array with further confirmation of miRNAs expression by reverse transcription-polymerase chain reaction.
We identified 30 miRNAs that were unregulated or downregulated more than twofold after irradiation as compared to nonirradiated thyroid cells, with no significant difference found between 1 and 10 Gy of radiation. Four distinct patterns of miRNA expression change were observed: miRNAs downregulated at 4 hours and returned to normal levels at 24 hours, miRNAs upregulated at 4 hours and returned to normal levels at 24 hours, and miRNAs either upregulated or downregulated at both time points. No dysregulation of miRNAs known to occur in thyroid cancer was observed.
Acute exposure of thyroid cells to γ-radiation results in several specific patterns of miRNA response. It appears that alteration in miRNA expression seen 4-24 hours after irradiation has no direct association with carcinogenesis. However, it is likely to affect other cell functions, such as DNA repair.
Thyroid: official journal of the American Thyroid Association 02/2011; 21(3):261-6. · 2.60 Impact Factor
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ABSTRACT: Thyroid cancer is the most common type of endocrine malignancy, encompassing tumors with various levels of invasive growth and aggressiveness. Rap1GAP, a Rap1 GTPase-activating protein, inhibits the RAS superfamily protein Rap1 by facilitating hydrolysis of GTP to GDP. In this study, we analyzed 197 thyroid tumor samples and showed that Rap1GAP was frequently lost or downregulated in various types of tumors, particularly in the most invasive and aggressive forms of thyroid cancer. The downregulation was due to promoter hypermethylation and/or loss of heterozygosity, found in the majority of thyroid tumors. Treatment with demethylating agent 5-aza-deoxycytidine and/or histone deacetylation inhibitor trichostatin A induced gene reexpression in thyroid cells. A genetic polymorphism, Y609C, was seen in 7% of thyroid tumors but was not related to gene downregulation. Loss of Rap1GAP expression correlated with tumor invasiveness but not with specific mutations activating the mitogen-activated protein kinase pathway. Rap1GAP downregulation was required in vitro for cell migration and Matrigel invasion. Recovery of Rap1GAP expression inhibited thyroid cell proliferation and colony formation. Overall, our findings indicate that epigenetic or genetic loss of Rap1GAP is very common in thyroid cancer, where these events are sufficient to promote cell proliferation and invasion.
Cancer Research 02/2010; 70(4):1389-97. · 7.86 Impact Factor
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ABSTRACT: Poorly differentiated carcinomas represent an aggressive group of thyroid tumors with controversial classification placement and poorly understood pathogenesis. Molecular data in this group of tumors are extremely heterogeneous, possibly reflecting different inclusion criteria. Recently homogeneous diagnostic criteria have been proposed by our group (Turin proposal) that need to be complemented by detailed molecular characterization.
The objective of the study was to define a comprehensive molecular typing of poorly differentiated thyroid carcinomas classified following homogeneous diagnostic criteria.
Sixty-five cases of poorly differentiated carcinoma selected following the Turin proposal have been screened for N-, K-, H-RAS, BRAF, RET/PTC1 and 3, and PAX8/PPARgamma mutations-rearrangements using alternative techniques and in two different laboratories. Molecular data were compared with clinical pathological parameters and survival by univariate and multivariate analysis.
RAS mutations in codon 61 were by far the most common genetic alteration in poorly differentiated carcinomas (23% of cases), with all mutation in NRAS except one in the HRAS gene. A single BRAF mutation was found in a poorly differentiated carcinoma with a residual component of a tall cell variant of papillary carcinoma. No KRAS, RET/PTC, or PAX8/PPARgamma genetic alteration was detected. In this series, the presence of RAS mutations was a unique negative prognostic parameter at multivariate analysis.
The present study demonstrates that strictly classified poorly differentiated carcinomas are genetically homogeneous, RAS mutations being the almost exclusive genetic event. Moreover, the detection of RAS mutations might be clinically relevant for the prognostic stratification of these tumors.
The Journal of clinical endocrinology and metabolism 10/2009; 94(12):4735-41. · 6.50 Impact Factor
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ABSTRACT: Thyroid cancer, and its most common type, papillary carcinoma, frequently have chromosomal rearrangements and therefore represent a good model for the understanding of mechanisms of chromosomal rearrangements in solid tumors. Several types of rearrangement known to occur in thyroid cancer, including RET/PTC, NTRK1 and BRAF/AKAP9, are more common in radiation-associated thyroid tumors and RET/PTC can be induced experimentally by exposing human thyroid cells to ionizing radiation. In this review, the molecular mechanisms of generation of RET/PTC and other chromosomal rearrangements are discussed, with the emphasis on the role of nuclear architecture and interphase gene proximity in the generation of intrachromosomal rearrangements in thyroid cells.
Molecular and Cellular Endocrinology 09/2009; 321(1):36-43. · 4.19 Impact Factor
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ABSTRACT: BRAF gene mutations are identified in about 45% of papillary thyroid carcinomas (PTC) and represent the most common genetic event in this tumor. Here, we report a case of PTC, solid variant, with a complex BRAF mutation that involves one nucleotide substitution, C1796T, and a CTT triplet insertion, 1798_1799insCTT, located on the same allele. This mutation leads to the replacement of a threonine with an isoleucine, T599I, and replacement of a valine with an alanine and a leucine, V600delinsAL. This mutation was identified both in the preoperative fine needle aspirate sample and in the surgical specimen after total thyroidectomy. Other rare BRAF mutations in PTC are reviewed.
Endocrine Pathology 05/2009; 20(2):122-6. · 1.36 Impact Factor
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ABSTRACT: RET/PTC rearrangements have been reported in papillary thyroid carcinomas with variable frequency in studies that used different detection methods.
Our objective was to determine the role of different detection methods and tumor genetic heterogeneity on RET/PTC detection.
Sixty-five papillary carcinomas were analyzed for RET/PTC1 and RET/PTC3 using five detection methods: standard-sensitivity RT-PCR, high-sensitivity RT-PCR, real-time LightCycler RT-PCR, Southern blot analysis, and fluorescence in situ hybridization.
RET/PTC rearrangements were detected by standard-sensitivity RT-PCR in 14 tumors. High-sensitivity RT-PCR detected RET/PTC in all of these and in 12 additional cases, where the levels of expression corresponded to one to five positive cells. Real-time LightCycler RT-PCR detected RET/PTC in 12 and Southern blot analysis in 11 tumors. By fluorescence in situ hybridization, 14 tumors were positive, including nine cases with 50-86% positive cells and five cases with 17-35% positive cells. Overall, nine (14%) tumors harbored clonal rearrangements, which were present in the majority of tumor cells and detected by all five methods. Five (8%) cases had subclonal rearrangements present in a smaller portion of tumor cells and detected by most methods. Twelve (18%) tumors had nonclonal RET/PTC that were detected only by high-sensitivity RT-PCR. No other mutations were found in tumors harboring clonal RET/PTC, whereas 60% of tumors with subclonal and 42% of tumors with nonclonal RET/PTC harbored additional mutations.
Our data suggest that broad variability in the reported prevalence of RET/PTC rearrangement is at least in part a result of the use of different detection methods and tumor genetic heterogeneity.
Journal of Clinical Endocrinology & Metabolism 10/2006; 91(9):3603-10. · 6.50 Impact Factor
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ABSTRACT: Papillary carcinoma is the most common type of thyroid malignancy. It has been recently shown that these tumors commonly have one of three genetic alterations: BRAF point mutations, RET/PTC rearrangements, or RAS point mutations. In this study, we analyze the relationship between these alterations and the microscopic features of papillary carcinomas, their clinical features, and prognostic characteristics. Ninety-seven papillary carcinomas were studied; in all cases, frozen tissue was available for nucleic acid extraction. Of 96 unselected cases, 42% were positive for BRAF, 18% for RET/PTC, and 15% for RAS mutations. Morphologic features were evaluated in detail in 61 cases and 6 characteristic nuclear features and 3 additional microscopic features were assessed quantitatively. At least 4 nuclear features were found in each tumor, with nuclear pseudoinclusions being the least frequent finding in all mutation groups. BRAF mutations were associated with older patient age, typical papillary appearance or the tall cell variant, a higher rate of extrathyroidal extension, and more advanced tumor stage at presentation. RET/PTC rearrangements presented at younger age and had predominantly typical papillary histology, frequent psammoma bodies, and a high rate of lymph node metastases. Tumors with RAS mutations were exclusively the follicular variant of papillary carcinoma and correlated with significantly less prominent nuclear features and low rate of lymph node metastases. These findings demonstrate that BRAF, RET/PTC, and RAS mutations are associated with distinct microscopic, clinical, and biologic features of thyroid papillary carcinomas.
American Journal of Surgical Pathology 03/2006; 30(2):216-22. · 4.35 Impact Factor
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ABSTRACT: Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1-8 of the AKAP9 gene and exons 9-18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.
Journal of Clinical Investigation 02/2005; 115(1):94-101. · 15.39 Impact Factor
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Marina N Nikiforova,
Raffaele Ciampi,
Giuliana Salvatore,
Massimo Santoro, Manoj Gandhi,
Jeffrey A Knauf,
Gerry A Thomas,
Stephen Jeremiah,
Tatyana I Bogdanova,
Mykola D Tronko,
James A Fagin,
Yuri E Nikiforov
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ABSTRACT: Point mutations of the BRAF gene have been recently described with high prevalence in papillary thyroid carcinomas. However, this molecular alteration has not been studied in radiation-induced thyroid tumors. We analyzed the prevalence of BRAF point mutations and RET/PTC rearrangements in 55 post-Chernobyl papillary carcinomas, compared with 82 sporadic papillary carcinomas. Radiation-induced tumors demonstrated a low prevalence (4%) of BRAF point mutations and high prevalence (58%) of RET/PTC rearrangements. Sporadic papillary carcinomas revealed a clearly distinct pattern, with 37% of tumors harboring BRAF mutations and 20% RET/PTC rearrangements. These results demonstrate a significant difference in the molecular genetic profile of sporadic and radiation-induced thyroid tumors.
Cancer Letters 07/2004; 209(1):1-6. · 4.24 Impact Factor
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Marina N Nikiforova,
Edna T Kimura, Manoj Gandhi,
Paul W Biddinger,
Jeffrey A Knauf,
Fulvio Basolo,
Zhaowen Zhu,
Riccardo Giannini,
Giuliana Salvatore,
Alfredo Fusco,
Massimo Santoro,
James A Fagin,
Yuri E Nikiforov
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ABSTRACT: Activating point mutations of the BRAF gene have been recently reported in papillary thyroid carcinomas. In this study, we analyzed 320 thyroid tumors and six anaplastic carcinoma cell lines and detected BRAF mutations in 45 (38%) papillary carcinomas, two (13%) poorly-differentiated carcinomas, three (10%) anaplastic carcinomas, and five (83%) thyroid anaplastic carcinoma cell lines but not in follicular, Hürthle cell, and medullary carcinomas, follicular and Hürthle cell adenomas, or benign hyperplastic nodules. All mutations involved a T-->A transversion at nucleotide 1796. In papillary carcinomas, BRAF mutations were associated with older age, classic papillary carcinoma or tall cell variant histology, extrathyroidal extension, and more frequent presentation at stages III and IV. All BRAF-positive poorly differentiated and anaplastic carcinomas contained areas of preexisting papillary carcinoma, and mutation was present in both the well-differentiated and dedifferentiated components. These data indicate that BRAF mutations are restricted to papillary carcinomas and poorly differentiated and anaplastic carcinomas arising from papillary carcinomas. They are associated with distinct phenotypical and biological properties of papillary carcinomas and may participate in progression to poorly differentiated and anaplastic carcinomas.
Journal of Clinical Endocrinology & Metabolism 11/2003; 88(11):5399-404. · 6.50 Impact Factor
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ABSTRACT: The follicular variant (FV) of papillary thyroid carcinoma is characterized by a follicular growth pattern and cytologic features of papillary carcinoma. ret/PTC rearrangements are common in classic papillary thyroid carcinoma (PTC) and PAX8-PPAR gamma and ras mutations in follicular thyroid carcinoma. Their prevalence in FV has not been established. We studied these genetic alterations and clinical-pathologic features in 30 FV cases and compared those with 46 non-FV papillary carcinomas. FV cases revealed 1 ret/PTC rearrangement (3%) and 13 ras mutations (43%). Non-FV cases harbored 13 ret/PTC (28%) (P = .006) and no ras mutations (P = .0002). No PAX8-PPAR gamma was found in either group. FV cases demonstrated a significantly higher prevalence of tumor encapsulation, angiovascular invasion, and poorly differentiated areas and a lower rate of lymph node metastases. These data indicate that the FV of papillary carcinoma has a distinct set of molecular alterations and is characterized by a high frequency of ras point mutations.
American Journal of Clinical Pathology 08/2003; 120(1):71-7. · 2.60 Impact Factor
