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Expression of the Rho-family GTPase gene RHOF in lymphocyte subsets and malignant lymphomas
British Journal of Haematology
Abstract
We have studied the expression of RHOF, a member of the Rho-GTPase family, in an array of lymphoid cells and tissues. Previous microarray studies demonstrated RHOF upregulation in a subset of transformed follicular lymphomas. Real-time quantitative polymerase chain reaction evaluated RHOF expression in lymphocyte subpopulations, and normal and malignant lymphoid tissue. Cells and tissues of B-cell origin expressed higher RHOF levels than their T-cell counterparts. Neoplastic cells and tissues of B-cell origin expressed higher levels of RHOF than their benign cellular counterparts. Relatively elevated levels of RHOF were seen in lymphomas derived from germinal centre origin.
... In contrast, Rho proteins are only rarely mutated in tumours, whereas their expression and/or activity are frequently altered. For example, several Rho GTPases are upregulated in some human tumours, including RhoA, RhoC, Rac1, Rac2, Rac3, Cdc42, Wrch2/RhoV and RhoF [11][12][13]. ...
... Overexpression of RhoF induce the generation of abundant long actin-rich Cdc42-independent filopodia and a moderate increase in stress fibers, but the relevance of these changes for cell migration and physiology is unknown [73]. Interestingly, RhoF is upregulated in malignant B-cell lymphomas [13]. Whether this overexpression leads to a change in adhesion or migration of lymphoma cells is not yet known. ...
Rho GTPases contribute to multiple cellular processes that could affect cancer progression, including cytoskeletal dynamics, cell cycle progression, transcriptional regulation, cell survival and vesicle trafficking. In vitro several Rho GTPases have oncogenic activity and/or can promote cancer cell invasion, and this correlates with increased expression and activity in a variety of cancers. Conversely, other family members appear to act as tumour suppressors and are deleted, mutated or downregulated in some cancers. Genetic models are starting to provide new information on how Rho GTPases affect cancer development and progression. Here, we discuss how Rho GTPases could contribute to different steps of cancer progression, including proliferation, survival, invasion and metastasis.
... RHOF, a member of the Rho GT3 family, was a risk factor in the scoring formula and has been involved in regulating various cell functions, such as cell adhesion and remodeling of pseudopods [24]. Launce G et al. showed that B-cellderived tumor cells and tissues expressed higher levels of RHOF than benign cells [25]. Li et al. found that RHOF is upregulated in hepatocellular carcinoma and plays a key role in promoting cancer cell migration, invasion, and EMT [26]. ...
Limited studies have explored novel pancreatic cancer (PC) subtypes or prognostic biomarkers based on the altered activity of relevant signaling pathway gene sets. Here, we employed non-negative matrix factorization (NMF) to identify three immune subtypes of PC based on C7 immunologic signature gene set activity in PC and normal samples. Cluster 1, the immune-inflamed subtype, showed a higher response rate to immune checkpoint blockade (ICB) and had the lowest tumor immune dysfunction and exclusion (TIDE) scores. Cluster 2, the immune-excluded subtype, exhibited strong associations with stromal activation, characterized by elevated expression levels of transforming growth factor (TGF)-β, cell adhesion, extracellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT) related genes. Cluster 3, the immune-desert subtype, displayed limited immune activity. For prognostic prediction, we developed an immune-related prognostic risk model (IRPM) based on four immune-related prognostic genes in pancreatic cancer, RHOF, CEP250, TSC1, and KIF20B. The IRPM demonstrated excellent prognostic efficacy and successful validation in an external cohort. Notably, the key gene in the prognostic model, RHOF, exerted significant influence on the proliferation, migration, and invasion of pancreatic cancer cells through in vitro experiments. Furthermore, we conducted a comprehensive analysis of somatic mutational landscapes and immune landscapes in PC patients with different IRPM risk scores. Our findings accurately stratified patients based on their immune microenvironment and predicted immunotherapy responses, offering valuable insights for clinicians in developing more targeted clinical strategies.
... RhoF has also been shown to maintain normal B cell function. Overexpression of RhoF has been observed in malignant B cells and is believed to promote transformation (Gouw et al., 2005). The subcellular localization of RhoD is important because it plays a role in endosomal trafficking and Golgi homeostasis (Blom et al., 2015). ...
The non-receptor tyrosine kinase effector of Cdc42, ACK, has been identified as an oncogene. Aberrant expression and mutations of ACK have been found in many types of cancer, such as prostate, breast and pancreatic. Clinically, ACK mutants or overexpression correlates with poor prognosis. Recent studies have uncovered substrates for ACK and shown their downstream signalling pathways can contribute to cancer progression. However, whether ACK can be used as a therapeutic target in cancer remains a debatable issue. It is likely that the ACK signalling network is yet to be completely elucidated. To understand whether ACK is an effective, as well as a safe target for cancer therapy, the Owen/Mott group performed a yeast-2-hybrid screen to identify new binding partners of ACK. 14 novel proteins that interact with ACK were identified, one of which was a small G protein regulator Vav3, the least-studied member of Vav-family proteins. The first part of this project characterised the interactions between ACK and Vav-family proteins in human cells. ACK was shown not only to bind to Vav3, but also to Vav1 and Vav2. From mutagenesis studies, it was shown that ACK binds Vav2 differently to Vav1 and Vav3. Further experiments indicated that the interaction with Vav1 and Vav3 stabilized ACK protein levels, an outcome that could underpin the oncogenic properties of ACK and the situation found in many cancers. For Vav2, it was shown that ACK phosphorylates Tyr142 and directs Vav2 GEF activity towards the small G protein Cdc42, resulting in an increase of HEK293T cell proliferation. Knockdown of Vav2 significantly reduced the level of activated Cdc42 and decreased the proliferative rate in ACK-transformed cells. Finally, the ACK-Vav2-Cdc42 pathway was shown to promote cell migration in an androgen-independent prostate cancer cell line. Taken together, this project has identified a new potential player in ACK-driven oncogenesis.
... accessed on 19 October 2021), and the resili ency of the loggerhead immune system contributes to loggerhead longevity. Previou sly, RHOF has been implicated in normal B-cell function and is upregulated in malig nant lymphoid tissue [57]. SHISA6 regulates stem cell differentiation in spermatogon ia by inhibition of Wnt [58]. ...
Background: Digital transcriptomics is rapidly emerging as a powerful new technology for modelling the environmental dynamics of the adaptive landscape in diverse lineages. This is particularly valuable in taxa such as turtles and tortoises (order Testudines) which contain a large fraction of endangered species at risk due to anthropogenic impacts on the environment, including pollution, overharvest, habitat degradation, and climate change. Sea turtles (family Cheloniidae) in particular invite a genomics-enabled approach to investigating their remarkable portfolio of adaptive evolution. The sex of the endangered loggerhead sea turtle (Caretta caretta) is subject to temperature-dependent sex determination (TSD), a mechanism by which exposure to temperatures during embryonic development irreversibly determines sex. Higher temperatures produce mainly female turtles and lower temperatures produce mainly male turtles. Incubation temperature can have long term effects on the immunity, migratory ability, and ultimately longevity of hatchlings. We perform RNA-seq differential expression analysis to investigate tissue- and temperature-specific gene expression within brain (n = 7) and gonadal (n = 4) tissue of male and female loggerhead hatchlings. Results: We assemble tissue- and temperature-specific transcriptomes and identify differentially expressed genes relevant to sexual development and life history traits of broad adaptive interest to turtles and other amniotic species. We summarize interactions among differentially expressed genes by producing network visualizations, and highlight shared biological pathways related to migration, immunity, and longevity reported in the avian and reptile literature. Conclusions: The measurement of tissue- and temperature-specific global gene expression of an endangered, flagship species such as the loggerhead sea turtle (Caretta caretta) reveals the genomic basis for potential resiliency and is crucial to future management and conservation strategies with attention to changing climates. Brain and gonadal tissue collected from experimentally reared loggerhead male and female hatchlings comprise an exceedingly rare dataset that permits the identification of genes enriched in functions related to sexual development, immunity, longevity, and migratory behavior and will serve as a large, new genomic resource for the investigation of genotype–phenotype relationships in amniotes.
... For example, the TRAF family member-associated NF-KB activator (TANK) modulates NF-kB activation through binding with TRAF and TBK1 proteins [37,38]. The RHOF, representative of the Rho GTPase family implicated in tumorigenesis by regulating cytoskeleton's dynamic [39], has a potential role in germinal center formation [40] and has recently been involved in NF-kB regulation [41]. The SDCBP gene encodes a PDZ domain-containing protein, involved in exosome biogenesis [42] and Rho GTPase family regulation [43], and participates in NF-KB activation in melanoma [44]. ...
B-cell receptor (BCR) signaling is crucial for the pathophysiology of most mature B-cell lymphomas/leukemias and has emerged as a therapeutic target whose effectiveness remains limited by the occurrence of mutations. Therefore, deciphering the cellular program activated downstream this pathway has become of paramount importance for the development of innovative therapies. Using an original ex vivo model of BCR-induced proliferation of chronic lymphocytic leukemia cells, we generated 108 temporal transcriptional and proteomic profiles from 1 h up to 4 days after BCR activation. This dataset revealed a structured temporal response composed of 13,065 transcripts and 4027 proteins, comprising a leukemic proliferative signature consisting of 430 genes and 374 proteins. Mathematical modeling of this complex cellular response further highlighted a transcriptional network driven by 14 early genes linked to proteins involved in cell proliferation. This group includes expected genes (EGR1/2, NF-kB) and genes involved in NF-kB signaling modulation (TANK, ROHF) and immune evasion (KMO, IL4I1) that have not yet been associated with leukemic cells proliferation. Our study unveils the BCR-activated proliferative genetic program in primary leukemic cells. This approach combining temporal measurements with modeling allows identifying new putative targets for innovative therapy of lymphoid malignancies and also cancers dependent on ligand–receptor interactions.
... We uncovered 195 up-and 428 down-regulated genes in rituximab-resistant DLCBL cell lines compared to sensitive cell lines ( Figure 2B), suggesting that there are large gene expression differences inherent to rituximab resistance. When Gene Ontology (GO) analysis for differentially expressed genes (both up-and downregulated genes combined) was performed, several key signaling pathways known to be involved in tumor development were enriched ( Figure 2C); most notably the GTPase signaling pathway [93,94] (Figure 2D), confirming the intrinsic differences in DLBCL cell lines compared. Table S1). ...
Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Although significant progress has been made in recent years to treat DLBCL patients, 30%–40% of the patients eventually relapse or are refractory to first line treatment, calling for better therapeutic strategies for DLBCL. Long non-coding RNAs (lncRNAs) have emerged as a highly diverse group of non-protein coding transcripts with intriguing molecular functions in human disease, including cancer. Here, we review the current understanding of lncRNAs in the pathogenesis and progression of DLBCL to provide an overview of the field. As the current knowledge of lncRNAs in DLBCL is still in its infancy, we provide molecular signatures of lncRNAs in DLBCL cell lines to assist further lncRNA research in DLBCL.
... It is not ubiquitously expressed, unlike RhoA, Rac1 and Cdc42, but can be found in MKs and platelets 84,97,157 and has been shown to also drive filopodia formation 58,130 . High expression levels have also been found in malignant B-cell lymphoma and were hypothesized to promote malignant transformation 158 . ...
This work focuses on megakaryocyte physiology with a special interest in the description of the localization of megakaryocytes in the bone marrow in mice single-deficient of the small Rho GTPase RhoA or double-deficient for RhoA and Cdc42. RhoA knock-out mice revealed intraluminal presence of megakaryocytes in bone marrow sinusoids. In a next step, potential aggravation, attenuation or preservation of this phenotype was studied in related mouse strains and also in the setting of platelet depletion and blockage of important megakaryocyte and platelet glycoprotein receptors in order to understand underlying singling pathways. A second part of this thesis studied the role of RhoF in filopodia formation and scrutinized RhoF deficient mice with regard to platelet activation and degranulation.
... It has been reported that this interaction occurs through epigenetic silencing due to mutation (Serra et al., 2014). After transmission of the signal to TF TCF3 with DNA migration through RHOF, which is modified by methylation and acetylation (Gouw et al., 2005), it upregulates target gene NFKBIL1 to activate inflammation and immune response and inhibit the development of CRC (Atzei et al., 2010;Mcallister et al., 2014;Taniue et al., 2016). ...
Colorectal cancer (CRC) is the third most commonly diagnosed type of cancer worldwide. The mechanisms leading to the progression of CRC are involved in both genetic and epigenetic regulations. In this study, we applied systems biology methods to identify potential biomarkers and conduct drug discovery in a computational approach. Using big database mining, we constructed a candidate protein-protein interaction network and a candidate gene regulatory network, combining them into a genome-wide genetic and epigenetic network (GWGEN). With the assistance of system identification and model selection approaches, we obtain real GWGENs for early-stage, mid-stage, and late-stage CRC. Subsequently, we extracted core GWGENs for each stage of CRC from their real GWGENs through a principal network projection method, and projected them to the Kyoto Encyclopedia of Genes and Genomes pathways for further analysis. Finally, we compared these core pathways resulting in different molecular mechanisms in each stage of CRC and identified carcinogenic biomarkers for the design of multiple-molecule drugs to prevent the progression of CRC. Based on the identified gene expression signatures, we suggested potential compounds combined with known CRC drugs to prevent the progression of CRC with querying Connectivity Map (CMap).
... For example, neoplastic cells and transformed lymphomas exhibit elevated RHOF expression compared with their benign counterparts. 30 It has also been mentioned that proteins of the RHOF subgroup have unique abilities relating to the regulation of dynamic cytoskeletal reorganization. Our study provides new data indicating that RHOF-modulated EMT is involved in counteracting drug treatment in PC cells. ...
The highly refractory nature of pancreatic cancer (PC) to chemotherapeutic drugs is one of the key reasons contributing to the poor prognosis of this disease. MicroRNAs (miRNAs) are key regulators of gene expression and have been implicated in a variety of processes from cancer development through to drug resistance. Herein, through miRNA profiling of gemcitabine-resistant (GR) and parental PANC-1 cell lines, we found a consistent reduction of miR-3656 in GR PANC-1 cells. miR-3656 overexpression enhanced the antitumor effect of gemcitabine, whereas silencing of miR-3656 resulted in the opposite effect. By performing mechanistic studies using both in vitro and in vivo models, we found that miR-3656 could target RHOF, a member of the Rho subfamily of small GTPases, and regulate the EMT process. Moreover, enforced EMT progression via TWIST1 overexpression compromised the chemotherapy-enhancing effects of miR-3656. Finally, we found significantly lower levels of miR-3656 and higher levels of RHOF in PC tissues compared with adjacent noncancerous pancreatic tissues, and this was also associated with poor PC patients’ prognosis. Taken together, our results suggest that the miR-3656/RHOF/EMT axis is an important factor involved in regulating GR in PC, and highlights the potential of novel miR-3656-based clinical modalities as a therapeutic approach in PC patients.
... It controlled filopodia formation, through diaphanous-related formin mDia2, and acted as an alternative trigger for actin stress fibers formation in epithelial cells, via diaphanous-related formin mDia1 [21,22]. Rif was also shown to play pivotal roles in other physiological processes, such as dentritic spine formation, early-stage mycosis fungoides, and follicular lymphoma development [23][24][25]. ...
Members of the well-known semaphorin family of proteins can induce both repulsive and attractive signaling in neural network formation and their cytoskeletal effects are mediated in part by small guanosine 5’-triphosphatase (GTPases). The aim of this study was to investigate the cellular role of Rif GTPase in the neurotrophin-induced neurite outgrowth. By using PC12 cells which are known to cease dividing and begin to show neurite outgrowth responding to nerve growth factor (NGF), we found that semaphorin 6A was as effective as nerve growth factor at stimulating neurite outgrowth in PC12 cells, and that its neurotrophic effect was transmitted through signaling by mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K). We further found that neurotrophin-induced neurite formation in PC12 cells could be partially mediated by inhibition of Rif GTPase activity downstream of MAPKs and PI3K signaling. In conclusion, we newly identified Rif as a regulator of the cytoskeletal rearrangement mediated by semaphorins.
... Among these, RAC1 and RHOA are already present in the Cancer Gene Census [33], adding confidence to the hypothesis that also RHOF might play a role in cancer. Moreover, relatively elevated levels of RHOF were observed in lymphomas derived from the germinal centre [34]. ...
The increasing availability of resequencing data has led to a better understanding of the most important genes in cancer development. Nevertheless, the mutational landscape of many tumor types is heterogeneous and encompasses a long tail of potential driver genes that are systematically excluded by currently available methods due to the low frequency of their mutations. We developed LowMACA (Low frequency Mutations Analysis via Consensus Alignment), a method that combines the mutations of various proteins sharing the same functional domains to identify conserved residues that harbor clustered mutations in multiple sequence alignments. LowMACA is designed to visualize and statistically assess potential driver genes through the identification of their mutational hotspots.
We analyzed the Ras superfamily exploiting the known driver mutations of the trio K-N-HRAS, identifying new putative driver mutations and genes belonging to less known members of the Rho, Rab and Rheb subfamilies. Furthermore, we applied the same concept to a list of known and candidate driver genes, and observed that low confidence genes show similar patterns of mutation compared to high confidence genes of the same protein family.
LowMACA is a software for the identification of gain-of-function mutations in putative oncogenic families, increasing the amount of information on functional domains and their possible role in cancer. In this context LowMACA emphasizes the role of genes mutated at low frequency otherwise undetectable by classical single gene analysis.
LowMACA is an R package available at http://www.bioconductor.org/packages/release/bioc/html/LowMACA.html. It is also available as a GUI standalone downloadable at: https://cgsb.genomics.iit.it/wiki/projects/LowMACA
... Following activation, Rho GTPases trigger a signaling cascade to direct a variety of cellular responses (41)(42)(43). Rho GTPases have been reported to contribute to the majority of steps of cancer initiation and progression, including unlimited proliferation potential and evasion from apoptosis, and several Rho GTPases are upregulated in certain types of human tumor, including RhoA, RhoC, Rac1 (44,45). The upregulated Rho GTPases exert their pro-oncogenic effects via the stimulation of cell cycle progression and regulation of gene transcription (46). ...
Hypoxic conditions regulate several metabolic enzymes and transcription factors that are involved in cancer, ischemia and pulmonary diseases. The Ras homolog (Rho) family, including Rho member A (RhoA), is involved in reorganization of the actin cytoskeleton, cell migration and in the regulation of apoptosis and gene transcription. The aim of the present study was to investigate the expression of hypoxia‑inducible factor (HIF)‑α and the activity of RhoA in PC12 neuroblastoma cells under hypoxic conditions. The upregulation of HIF‑α and RhoA by hypoxia was determined using reverse transcription‑quantitative polymerase chain reaction and western blot assays, cell apoptosis was analyzed using flow cytometry, and the activity of caspase 3 was examined using a western blot assay and caspase 3 activity assay kit. The PC12 cells were induced to apoptosis following exposure to hypoxia, and exhibited increased expression of HIF‑α and increased mRNA and protein expression levels of RhoA. The overexpression of HIF‑α attenuated the hypoxia‑induced apoptosis of the PC12 cells. In addition, RhoA knockdown using small interfering RNA abrogated the antagonism of HIF‑1α towards hypoxia‑induced apoptosis. The results of the present study confirmed the protective role of HIF‑1α and RhoA in hypoxia‑induced PC12 cell apoptosis, and that the upregulation of HIF‑1α by hypoxia is RhoA‑dependent.
... Our understanding of endogenous Rif is limited, although it is expressed at higher levels in malignant lymphoma cells, compared with normal B-lymphocytes, suggesting Rif overexpression promotes malignant transformation [78]. Using recombinant Rho protein affinity purification-mass spectrometry (AP-MS) our group has identified interacting protein networks for Rif in platelets and confirmed some of these interactions by immunoblotting (Goggs, Mellor and Poole, unpublished observations). ...
Rho GTPases are critical for platelet function. Although the roles of RhoA, Rac and Cdc42 are characterized, platelets express other Rho GTPases, whose activities are less well understood. This review summarizes our understanding of the roles of platelet Rho GTPases and focuses particularly on the functions of Rif and RhoG. In human platelets, Rif interacts with cytoskeleton regulators including formins mDia1 and mDia3, whereas RhoG binds SNARE-complex proteins and cytoskeletal regulators ELMO and DOCK1. Knockout mouse studies suggest that Rif plays no critical functions in platelets, likely due to functional overlap with other Rho GTPases. In contrast, RhoG is essential for normal granule secretion downstream of the collagen receptor GPVI. The central defect in RhoG-/- platelets is reduced dense granule secretion, which impedes integrin activation and aggregation and limits platelet recruitment to growing thrombi under shear, translating into reduced thrombus formation in vivo. Potential avenues for future work on Rho GTPases in platelets are also highlighted, including identification of the key regulator for platelet filopodia formation and investigation of the role of the many Rho GTPase regulators in platelet function in both health and disease.
... In contrast, Rho proteins expression and/or activity are frequently altered in a variety of human cancers. For instance RhoA, RhoE, RhoC, RhoF Rac1, Rac2, Rac3, Cdc42 and Wrch2/RhoV are frequently overexpressed in many types of cancers (Faried, et al., 2005;Gomez del Pulgar, Benitah, Valeron, Espina, & Lacal, 2005;Gouw, Reading, Jenson, Lim, & Elenitoba-Johnson, 2005;Islam, et al., 2009;X. R. Li, et al., 2006;Varker, Phelps, King, & Williams, 2003;C. ...
The cholesterol biosynthesis pathway, also known as the mevalonate (MVA) pathway, is an essential cellular pathway that is involved in diverse cell functions. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) is the rate-limiting step in cholesterol biosynthesis and catalyzes the conversion of HMG-CoA to MVA.
Given its role in cholesterol and isoprenoid biosynthesis, the regulation of HMGCR has been intensely investigated. Because all cells require a steady supply of MVA, both the sterol (i.e. cholesterol) and non-sterol (i.e. isoprenoid) products of MVA metabolism exert coordinated feedback regulation on HMGCR through different mechanisms. The proper functioning of HMGCR as the proximal enzyme in the MVA pathway is essential under both normal physiologic conditions and in many diseases given its role in cell cycle pathways and cell proliferation, cholesterol biosynthesis and metabolism, cell cytoskeletal dynamics and stability, cell membrane structure and fluidity, mitochondrial function, proliferation, and cell fate.
The blockbuster statin drugs (‘statins’) directly bind to and inhibit HMGCR, and their use for the past thirty years has revolutionized the treatment of hypercholesterolemia and cardiovascular diseases, in particular coronary heart disease. Initially thought to exert their effects through cholesterol reduction, recent evidence indicates that statins also have pleiotropic immunomodulatory properties independent of cholesterol lowering.
In this review we will focus on the therapeutic applications and mechanisms involved in the MVA cascade including Rho GTPase and Rho kinase (ROCK) signaling, statin inhibition of HMGCR, geranylgeranyltransferase (GGTase) inhibition, and farnesyltransferase (FTase) inhibition in cardiovascular disease, pulmonary diseases (e.g. asthma and chronic obstructive pulmonary disease (COPD), and cancer.
... There are multiple Rho proteins that are upregulated in several human tumor types including RhoA, 15,16 RhoC, 17 Rac1, 15,16 Rac2, 18 Rac3, 19 Cdc-42, 15,16 Wrch-1, 20 and RhoF. 21 For example, overexpression of Rac1 was detected in breast, 16 lung, 22 oral squamous cell 23 testicular, 24 and gastric carcinoma. 25 RhoA is overexpressed in breast, 16 colon, 16 lung, 16 gastric, 25 head and neck, 26 bladder, 27 and testicular carcinomas. ...
The Rho family of GTPases (members of the Ras superfamily) are best known for their roles in regulating cytoskeletal dynamics. It is also well established that misregulation of Rho proteins contributes to tumorigenesis and metastasis. Unlike Ras proteins, which are frequently mutated in cancer (around 30%), Rho proteins themselves are generally not found to be mutated in cancer. Rather, misregulation of Rho activity in cancer was thought to occur by overexpression of these proteins or by misregulation of molecules that control Rho activity, such as activation or overexpression of GEFs and inactivation or loss of GAPs or GDIs. Recent studies, enabled by next-generation tumor exome sequencing, report activating point mutations in Rho GTPases as driver mutations in melanoma, as well as breast, and head and neck cancers. The Rac1(P29L) mutation identified in these tumor studies was previously identified by our lab as an activating Rac mutation in C. elegans neuronal development, highlighting the conserved nature of this mutation. Furthermore, this finding supports the relevance of studying Rho GTPases in model organisms such as C. elegans to study the mechanisms that underlie carcinogenesis. This review will describe the recent findings that report activating Rho mutations in various cancer types, moving Rho GTPases from molecules misregulated in cancer to mutagenic targets that drive tumorigenesis.
... Previous work has suggested that Rif has a role in lymphocyte immune functions. Some studies demonstrated that Rif is up-regulated in a subset of transformed FLs (follicular lymphomas), and a significant percentage of FLs transformed to an aggressive DLBCL (diffuse large Bcell lymphoma) [27]. Other research has revealed that Rif is deleted in early-stage mycosis fungoides, which is the most frequent form of cutaneous T-cell lymphoma [28]. ...
The Rif GTPase is a recent addition to small Rho GTPase family; it shares low homology with other members in the family and evolutionarily parallels with the development of vertebrates. Rif has the conserved Rho GTPase domain structures and cycles between a GDP-bound inactive form and a GTP-bound active form. In its active form, Rif signals through multiple downstream effectors. In the present review, our aim is to summarize the current information about the Rif effectors and how Rif remodels actin cytoskeleton in many aspects.
... RHOF is involved in actin filament organization by generating filopodial structures (Pellegrin and Mellor, 2005). Its underexpression has recently been found in T cell lymphomas compared with B cell lymphoma tissues, but there are no data in CTCL (Gouw et al., 2005). ...
The etiology of mycosis fungoides (MF), the most frequent form of cutaneous T cell lymphoma (CTCL), is poorly understood. No specific genetic aberration has been detected, especially in early-stage disease, possibly due to the clinical and histological heterogeneity of patient series and to the different sources of malignant cells (skin, blood, or lymph node) included in most studies. Frozen skin biopsies from 16 patients with early-stage MF were studied using array-based comparative genomic hybridization. A DNA pool from healthy donors was used as the reference. Results demonstrated recurrent loss of 19, 7p22.1-p22.3, 7q11.1-q11.23, 9q34.12, 12q24.31, and 16q22.3-q23.1, and gain of 8q22.3-q23.1 and 21q22.12. The 12q24.31 region was recurrently deleted in 7/16 patients. Real-time PCR investigation for deletion of genes BCL7A, SMAC/DIABLO, and RHOF-three tumor suppressor genes with a putative role in hematological malignancies-demonstrated that they were deleted in 9, 10, and 13 cases, respectively. The identified genomic alterations and individual genes could yield important insights into the early steps of MF pathogenesis.
... These include NF-kB, 18 BCL-11A, 19 BALassociated protein, 20 and RhoF. 21 Importantly, many proteins previously unknown to be expressed in tFL such as ribosomal S6 kinase, Wnt2 protein, betacatenin like protein1, and the eta (Z) isoform of protein kinase C (PKC) were identified in the FFPE sample. ...
There exists a need for robust approaches for tandem mass spectrometry (MS/MS)-based identification of proteins in formalin-fixed paraffin-embedded (FFPE) material. We demonstrate herein the identification of proteins in FFPE material using enzymatic cleavage for extraction of peptides from the FFPE specimen and liquid chromatography (LC) followed by MS/MS. We identified 324 proteins from a 3-year-old FFPE cell-block of a human lymphoma cell line. The identified proteins were assigned to the membrane, cytosol and nucleus, with diverse cellular functions. The results were comparable to those obtained with lysates from a fresh specimen of the lymphoma cell line. Western blotting analysis and immunofluorescence microscopy confirmed the expression of selected proteins. The functional significance of one protein (PKC eta) was validated using a PKC inhibitory peptide which resulted in lymphoma cell death in vitro. The ability to identify proteins from FFPE specimens has significant implications for MS/MS-based proteomics of vast repositories of archival primary tissue samples for disease-related discovery research.
Atypical Rho GTPases are a subtype of the Rho GTPase family that are involved in diverse cellular processes. The typical Rho GTPases, led by RhoA, Rac1 and Cdc42, have been well studied, while relative studies on atypical Rho GTPases are relatively still limited and have great exploration potential. With the increase in studies, current evidence suggests that atypical Rho GTPases regulate multiple biological processes and play important roles in the occurrence and development of human cancers. Therefore, this review mainly discusses the molecular basis of atypical Rho GTPases and their roles in cancer. We summarize the sequence characteristics, subcellular localization and biological functions of each atypical Rho GTPase. Moreover, we review the recent advances and potential mechanisms of atypical Rho GTPases in the development of multiple cancers. A comprehensive understanding and extensive exploration of the biological functions of atypical Rho GTPases and their molecular mechanisms in tumors will provide important insights into the pathophysiology of tumors and the development of cancer therapeutic strategies.
Rho GTPases are involved in multiple human malignancies and diverse biological functions. However, the patterns and prognostic significance of the expression of RhoD subfamily in acute myeloid leukemia (AML) remain unknown. Here, we detected the expressions of RhoD subfamily genes in AML on the basis of several published datasets and analyzed the survival of RhoD subfamily across the TCGA profiles and in a GEO series. We found that the expression of RhoF, but not RhoD, increased in AML patients in TCGA and GEO (all P<0.001); the survival analysis of two independent cohorts demonstrated that higher RhoF expression was significantly associated with poorer overall survival (OS) (P<0.001), whereas RhoD expression had no significant effect on OS in patients with AML (P>0.05); the subgroup analysis showed that high RhoF expression was correlated with poor 1-, 3-, and 5-year OS (P<0.05 for all); upregulated RhoF expression had a more significant prognostic value for OS in the younger patients (age<60), the intensive chemotherapy group, and wild-type groups (IDH1, NRAS, and TP53) (P<0.05 for all). Multivariate analysis indicated high RhoF expression as a strongly independent unfavorable prognostic factor for OS in patients without transplantation (P<0.05). Furthermore, a higher RhoF expression was closely associated with an older age, intermediate-/poor-risk cytogenetics and mutations in IDH1, NRAS, and TP53. RhoF expression was negatively correlated with BM blasts (P=0.020) and WBC (P=0.003). These findings suggest that high RhoF expression is associated with worsening OS in AML patients and is a potential therapeutic target for the treatment of AML.
Consumption of a high-fat diet has been associated with an increased risk of developing colorectal cancer (CRC). However, the effects of the interaction between dietary fat content and the aryl hydrocarbon receptor (AhR) on colorectal carcinogenesis remain unclear. Mainly known for its role in xenobiotic metabolism, AhR has been identified as an important regulator for maintaining intestinal epithelial homeostasis. Although previous research using whole body AhR knockout mice has revealed an increased incidence of colon and cecal tumors, the unique role of AhR activity in intestinal epithelial cells (IECs) and modifying effects of fat content in the diet at different stages of sporadic CRC development are yet to be elucidated. In the present study, we have examined the effects of a high-fat diet on IEC-specific AhR knockout mice in a model of sporadic CRC. Although loss of AhR activity in IECs significantly induced the development of premalignant lesions, in a separate experiment, no significant changes in colon mass incidence were observed. Moreover, consumption of a high-fat diet promoted cell proliferation in crypts at the premalignant colon cancer lesion stage and colon mass multiplicity as well as β-catenin expression and nuclear localization in actively proliferating cells in colon masses. Our data demonstrate the modifying effects of high-fat diet and AhR deletion in IECs on tumor initiation and progression.
NEW & NOTEWORTHY Through the use of an intestinal-specific aryl hydrocarbon receptor (AhR) knockout mouse model, this study demonstrates that the expression of AhR in intestinal epithelial cells is required to reduce the formation of premalignant colon cancer lesions. Furthermore, consumption of a high-fat diet and the loss of AhR in intestinal epithelial cells influences the development of colorectal cancer at various stages.
Background:
Gene signatures for predicting the outcome of lung squamous cell carcinoma (LUSC) have been employed for many years. However, various signatures have been applied in clinical practice. Therefore, in this study, we aimed to filter out an effective LUSC prognostic gene signature by simultaneously integrating mRNA and miRNA.
Methods:
First, based on data from the Cancer Genome Atlas (TCGA), mRNAs, and miRNAs that were related to overall survival (OS) of LUSC were obtained by least absolute shrinkage and selection operator (LASSO). Subsequently, the predicting effect was tested by time-dependent receiver operating characteristic (ROC) curve analysis and Kaplan-Meier survival analysis. Next, related clinical indexes were added to evaluate the efficiency of the selected gene signatures. Finally, validation and comparison using three independent gene signatures were performed using data from the Gene Expression Omnibus (GEO).
Results:
Our data showed that the prognostic index (PI) contained 17 mRNAs and one miRNA. According to the best normalized cut-off of PI (0.0247), the hazard radio (HR) of the PI was 3.40 (with a 95% confidence interval (CI) of [2.33, 4.96]). Moreover, when clinical factors were introduced, the PI was still the most significant index. In addition, only two GO terms with a p-value of less than 0.05 were reported. In addition, validation implied that using our 18-gene signature, only HR (1.36 [1.01,1.83]) was significant when compared with the other three groups of gene biomarkers.
Conclusions:
The 18-gene signature selected based on data from the TCGA database had an effective prognostic value of LUSC patients.
The Rho GTPases were discovered more than 30 years ago, and they were for a long time considered to follow simple cycling between GDP-bound and GTP-bound conformations, as for the Ras subfamily of small GTPases. The Rho GTPases consist of 20 members, but at least 10 of these do not follow this classical GTPase cycle. Thus, based on their kinetic properties, these Rho GTPases can instead be classified as atypical. Some of these atypical Rho GTPases do not hydrolyze GTP, and some have significantly increased intrinsic GDP/GTP exchange activity. This review focuses on this latter category of atypical Rho GTPases, the so-called ‘fast-cycling’ Rho GTPases. The different members of these fast-cycling atypical Rho GTPases are described in more detail here, along with their potential regulatory mechanisms. Finally, some insights are provided into the involvement of the atypical Rho GTPases in human pathologies.
Lipids include sterols, mono- and diglycerides, phospholipids, among others. They operate as structural components of cell membranes (Vol. 1 – Chap. 7. Plasma Membrane; Tables 2.1 and 2.2), contributors of energy metabolism, participants of intracellular transport (Vol. 1 – Chap. 9. Intracellular Transport), and signaling molecules.
Protein tyrosine kinases (PTK), i.e., enzymes that catalyze the phosphorylation of Tyr residues of proteins. are mainly associated with growth factor signaling. They actually modulate multiple cellular events, such as differentiation, growth, metabolism, and apoptosis. On the other hand, protein serine/ threonine kinases are principally related to second messengers, such as cyclic nucleotides cAMP (Sect. 11.1) and cGMP (Sect. 11.2), lipidic and related mediators diacylglycerol and inositol trisphosphate (Chap. 2), and calmodulin (Sect. 11.5.3).
Mitogen-activated protein kinase (MAPK) modules are commonly used to transduce signals aimed at regulating cell fate (cell differentiation, proliferation, senescence, and death) and inflammation. They mediate responses to extracellular signals, such as hormones, growth factors, and cytokines, as well as stresses and intercellular interactions.
Protein phosphorylation is a common, reversible, post-translational modification of proteins (Vols.1 – Chap. 5. Protein Synthesis and 3 – Chap. 1. Signal Transduction). Except pseudophosphatases, phosphatases removes a phosphate group from their substrates. They thus antagonize kinases that attach phosphate groups to Ser, Thr, and Tyr residues in the same substrates using ATP. Phosphorylated residues SerP, ThrP, and TyrP account for about 86, 12, and 2% of the phosphoproteome, respectively. Mammalian genomes encode about 100 protein Tyr kinases and phosphatases, and about 100 and about 25 protein Ser/Thr kinases and phosphatases.
Guanosine triphosphatases intervene in: (1) signal transduction from the intracellular edge of the plasma membrane and intracellular domain of transmembrane receptors; (2) protein synthesis at the ribosome (Vol. 1 – Chap. 5. Protein Synthesis); (3) control of cell division (Vol. 2 – Chap. 2. Cell Growth and Proliferation); (4) proper protein folding; (5) translocation of proteins through the membrane of the endoplasmic reticulum; and (6) vesicular transport within the cell (Vol. 1 – Chap. 9. Intracellular Transport).
Other important signaling mediators include: (1) endogenous, gaseous, diffusible messengers, or gasotransmitters, such as carbon monoxide (CO; Sect. 10.2), nitric oxide (NO; Sect. 10.3), and hydrogen sulfide (H2S; Sect. 10.4), in addition to oxygen and carbon dioxide, as well as several reactive oxygen (ROS) and nitrogen species (RNS; Sect. 10.6), that act as intra- and intercellular regulators; (2) membrane-bound enzymatic complexes such as the reduced form of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase or NOx; Sect. 10.5); (3) some coregulators, such as (protein kinase) A-kinase-anchoring proteins (AKAP; Sect. 10.8.4) and annexins (Sect. 10.8.5); and (4) transcription factors involved in stress response, such as the proteic complex nuclear factor κ light-chain-enhancer of activated B cells (NFκB; Sect. 10.9.1), the heterodimer hypoxia-inducible factors (HIF; Sect. 10.9.2), and members of the Forkhead box (Fox; Sect. 10.9.3) and P53 families (Sect. 10.9.4; Table 10.1).
Numerous protein serine/threonine kinases operate in signal transduction, either as initiating nodes, such as receptor (RSTK; Vol. 3 – Chap. 8. Receptor Kinases), or as intermediate nodes of signaling cascades, non-receptor (intracellular; NRSTK) protein Ser/Thr kinases. Typically, second messengers activate protein Ser/Thr kinases, whereas extracellular signals stimulate protein Tyr kinases (Chap. 4). For example, IκB kinases (IKK) of IκB inhibitors of nuclear factor-κB are serine kinases (Sect. 10.9.1).
Multiple signaling processes are characterized by bursts of calcium ions that moves to specific locations for optimal activation of cell activity. Intracellular calcium regulates numerous protein functions in tiny cellular domains ( V1.2 channels form signaling clusters in plasmalemmal nanodomains. Fluorescence microscopy is aimed at imaging in real time communication within and between cells. Calcium fluxes through Ca\({}^{++}\) channels and gap junctions can be imaged once constitutive proteins (e.g., connexin) have been tagged with tetracysteines [1429].
Dual-specificity kinases (DSK; or Ser/Thr/Tyr kinases) phosphorylate their substrates on serine, threonine, and/or tyrosine residues. Dual-specificity kinases intervene in the regulation of cell growth, differentiation, and apoptosis. Dual-specificity kinases include, at least, members of the superfamily of mitogen-activated protein kinases as well as those of the family of glycogen synthase kinases.
Among kinases (or phosphotransferases) that transfer phosphate groups from donor molecules such as ATP or GTP to specific substrates include protein kinases that modify the activity of their proteic substrates. Kinases are used to transmit signals in cells. Other types of kinases act on amino acids, lipids, carbohydrates, and nucleotides, that are then used in signaling pathways or cell metabolism.
The pro-oncogenic role of RhoA has been well identified in other cancers, but rarely in cervical cancer (CC), one of the main causes of cancer-related death in women. In the present study, we identified the overexpression of RhoA and its downstream effectors, ROCK-1 and ROCK-II, in CC specimens using western blotting. Then, we determined the effect of RhoA on the proliferation and migration of Hela cells, one of CC cell lines, by upregulating or downregulating the RhoA expression in Hela cells. We found that there was an overexpression of RhoA, ROCK-I/II in CC, which was associated with the progression of CC. And we confirmed that RhoA promoted the proliferation and migration of CC cells. In conclusion, we found a positive correlation among RhoA with the progression of CC by in vivo and in vitro evidences. A high RhoA expression in CC may predict a high metastatic potential of CC.
Abstract The Rho GTPases are essential regulators of basic cellular processes, including cell migration, cell contraction and cell division. Most studies still involve just the three canonical members, RhoA, Rac1 and Cdc42, although the Rho GTPases comprise at least 20 members. The aim of this review is to highlight some of the recent advances in our knowledge regarding the lessstudied Rho members, with the focus on RhoD and Rif. The phenotypic alterations to cell behaviour that are triggered by RhoD and Rif suggest that they have unique impacts on cytoskeletal dynamics that distinguish them from the well-studied members of the Rho GTPases. In addition, RhoD has a role in the regulation of intracellular transport of vesicles. Taken together, the available data indicate that RhoD and Rif have functions as master regulators in the integration of cytoskeletal reorganisation and membrane trafficking.
The Rif and RhoD proteins belong to the Rho subfamily of small GTPases. Rif and RhoD have for too long remained in the shadows of the better known Rho GTPases Cdc42, Rac1 and RhoA. With this review article, our aim is to provide the currently available information regarding Rif and RhoD. Taken together, the data available to date indicate that Rif and RhoD have unique roles in the regulation of actin dynamics, and that RhoD can link actin reorganisation to endosomal vesicle transport.
Cancer is a complex genetic disease, resulting from defects of multiple genes. Development of microarray techniques makes it possible to survey the whole genome and detect genes that have influential impacts on the progression of cancer. Statistical analysis of cancer microarray data is challenging because of the high dimensionality and cluster nature of gene expressions. Here, clusters are composed of genes with coordinated pathological functions and/or correlated expressions. In this article, we consider cancer studies where censored survival endpoint is measured along with microarray gene expressions. We propose a hybrid clustering approach, which uses both pathological pathway information retrieved from KEGG and statistical correlations of gene expressions, to construct gene clusters. Cancer survival time is modeled as a linear function of gene expressions. We adopt the clustering threshold gradient directed regularization (CTGDR) method for simultaneous gene cluster selection, within-cluster gene selection, and predictive model building. Analysis of two lymphoma studies shows that the proposed approach - which is composed of the hybrid gene clustering, linear regression model for survival, and clustering regularized estimation with CTGDR - can effectively identify gene clusters and genes within selected clusters that have satisfactory predictive power for censored cancer survival outcomes.
Background:
Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem.
Results:
We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data.
Conclusions:
The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.
Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem.
We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data.
The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.
Follicular lymphoma (FL) is the most common form of low-grade non-Hodgkin's lymphoma. Transformation to diffuse large B cell lymphoma (DLBCL) is an important cause of mortality. Using cDNA microarray analysis we identified 113 transformation-associated genes whose expression differed consistently between serial clonally related samples of FL and DLBCL occurring within the same individual. Quantitative RT-PCR validated the microarray results and assigned blinded independent group of 20 FLs, 20 DLBCLs, and five transformed lymphoma-derived cell lines with 100%, 70%, and 100% accuracy, respectively. Notably, growth factor cytokine receptors and p38beta-mitogen-activated protein kinase (MAPK) were differentially expressed in the DLBCLs. Immunohistochemistry of another blinded set of samples demonstrated expression of phosphorylated p38MAPK in 6/6 DLBCLs and 1/5 FLs, but not in benign germinal centers. SB203580 an inhibitor of p38MAPK specifically induced caspase-3-mediated apoptosis in t(14;18)+/p38MAPK+-transformed FL-derived cell lines. Lymphoma growth was also inhibited in SB203580-treated NOD-SCID mice. Our results implicate p38MAPK dysregulation in FL transformation and suggest that molecular targeting of specific elements within this pathway should be explored for transformed FL therapy.
Tumor progression involves the transition from normal to malignant cells, through a series of cumulative alterations. During this process, invasive and migratory properties are acquired, enabling cells to metastasize (reach and grow in tissues far from their origin). Numerous cellular changes take place during epithelial malignancy, and disruption of E-cadherin based cell-cell adhesion is a major event. The small Rho GTPases (Rho, Rac and Cdc42) have been implicated in multiple steps during cellular transformation, including alterations on the adhesion status of the tumor cells. This review focuses on recent in vivo evidence that implicates RhoGTPases in epithelial tumor progression. In addition, we discuss different hypotheses to explain disruption of cadherin-mediated cell-cell adhesion, directly or indirectly, through activation of Rho GTPases. Understanding the molecular mechanism of how cadherin adhesion and RhoGTPases interplay in normal cells and how this balance is altered during cellular transformation will provide clues as to how to interfere with tumor progression.
Small GTPases of the Rho family have a critical role in controlling cell morphology, motility and adhesion through dynamic regulation of the actin cytoskeleton [1,2]. Individual Rho GTPases have been shown to regulate distinct components of the cytoskeletal architecture; RhoA stimulates the bundling of actin filaments into stress fibres [3], Rac reorganises actin to produce membrane sheets or lamellipodia [4] and Cdc42 causes the formation of thin, actin-rich surface projections called filopodia [5]. We have isolated a new Rho-family GTPase, Rif (Rho in filopodia), and shown that it represents an alternative signalling route to the generation of filopodial structures. Coordinated regulation of Rho-family GTPases can be used to generate more complicated actin rearrangements, such as those underlying cell migration [6]. In addition to inducing filopodia, Rif functions cooperatively with Cdc42 and Rac to generate additional structures, increasing the diversity of actin-based morphology.
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
Transformation by oncogenic Ras requires the function of the Rho family GTPases. We find that Ras-transformed cells have elevated levels of RhoA-GTP, which functions to inhibit the expression of the cell cycle inhibitor p21/Waf1. These high levels of Rho-GTP are not a direct consequence of Ras signalling but are selected for in response to sustained ERK-MAP kinase signalling. While the elevated levels of Rho-GTP control the level of p21/Waf, they no longer regulate the formation of actin stress fibres in transformed cells. We show that the sustained ERK-MAP kinase signalling resulting from transformation by oncogenic Ras down-regulates ROCK1 and Rho-kinase, two Rho effectors required for actin stress fibre formation. The repression of Rho- dependent stress fibre formation by ERK-MAP kinase signalling contributes to the increased motility of Ras-transformed fibroblasts. Overexpression of the ROCK target LIM kinase restores actin stress fibres and inhibits the motility of Ras-transformed fibroblasts. We propose a model in which Ras and Rho signalling pathways cross-talk to promote signalling pathways favouring transformation.
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way
to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform,
provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification
of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters
into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to
a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated
only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model
needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA
integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler
PCR using the established mathematical model.
Cellular movement is central to invasion. The Rho family of small GTPases co-ordinate the cytoskeletal and adhesion modelling within cells that is crucial for normal migratory responses. Consequently, Rho proteins, and their regulators and effectors, are targets for subversion during oncogenic transformation and tumour development. Recent findings have thrown light on how actin regulators may be linked to oncogenesis and the development of cancer.
Cyclin D1 overexpression is a valuable marker for the diagnosis of mantle cell lymphoma (MCL). We used a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) method to quantify levels of cyclin D1, CD20, and cyclophilin A mRNA in manually microdissected, paraffin-embedded tissue sections using an ABI 7700 qRT-PCR system. The study group included 21 cases of MCL and 37 cases of other types of B-cell non-Hodgkin's lymphoma. Cyclin D1 mRNA copy number was normalized to CD20 and cyclophilin A mRNA and evaluated statistically by analysis of variance. The relative cyclin D1 levels were similar whether normalized to CD20 or cyclophilin A, indicating that CD20 levels are stable and can be used as a B-cell-specific normalizer. Statistically significant differences were found in the median levels of cyclin D1 mRNA (expressed as % CD20 mRNA) among cases of MCL (87.6), small lymphocytic lymphoma (9.9), follicular lymphoma (2.4), diffuse large B-cell lymphoma (5.9), marginal zone B-cell lymphoma (39.8), and Burkitt lymphoma (7.1) (P < 0.05). We conclude that qRT-PCR can be used to quantify cyclin D1 mRNA levels in archival tissue sections. Normalization of cyclin D1 to a B-cell-specific marker more accurately reflects overexpression by MCL than other methods that normalize using constitutively expressed mRNA species.
The RAS oncogenes were identified almost 20 years ago. Since then, we have learnt that they are members of a large family of small GTPases that bind GTP and hydrolyse it to GDP. This is then exchanged for GTP and the cycle is repeated. The switching between these two states regulates a wide range of cellular processes. A branch of the RAS family--the RHO proteins--is also involved in cancer, but what is the role of these proteins and would they make good therapeutic targets?
Analysis of global gene expression with DNA microarrays has great potential to improve the understanding of tumorigenesis, advance tumor diagnosis and classification, and affect cancer treatment. Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma. However, we now realize that the disease is extremely heterogeneous. This review summarizes the progress in understanding DLBCL that has been made as a result of the application of gene expression profiling. (C)2003 The Japanese Society of Hematology.
RHO-GTPases and cancer
- E Sahai
- C J Marshall
Sahai, E. & Marshall, C.J. (2002) RHO-GTPases and cancer. Nature
Reviews Cancer, 2, 133-142.