Rb Tumor Suppressor Pathways Research Articles

Human fibroblasts require the Rb family of tumor suppressors, but not p53, for PML-induced senescence.

Cellular senescence is a permanent cell cycle arrest that can be triggered by a variety of stresses including short telomeres and activated oncogenes. Promyelocytic leukemia protein (PML) is a central component of the senescence response, and is able to trigger the process when overexpressed in human diploid fibroblasts (HDFs). Senescence induced by PML in HDFs is characterized by a modest increase in p53 levels and activity, the accumulation of hypophosphorylated Rb and a reduced expression of E2F-dependent genes. To dissect the p53 and Rb family requirements for PML-induced senescence, we used the oncoproteins E6 and E7 from human papillomavirus type 16. We found that the coexpression of E6 and E7 inhibited the growth arrest and senescence induced by PML. In addition, these viral oncoproteins blocked the formation of PML bodies and excluded both p53 and Rb from PML bodies. Expression of dominant-negative p53 alone failed to block PML-induced senescence and expression of E6 only delayed the process. On the other hand, expression of E7 was sufficient to block PML-induced senescence, while an E7 mutant unable to bind Rb did not. Together, these data indicate that PML-induced senescence engages the Rb tumor-suppressor pathway predominantly.

Flavopiridol Induces Mitochondrial-Mediated Apoptosis in Murine Glioma GL261 Cells via Release of Cytochrome c and Apoptosis Inducing Factor

Glioblastoma (GBM) remains one of the most challenging solid cancers to treat due to its highly proliferative, angiogenic and invasive nature. Over 80% of adult high-grade astrocytomas show inactivation of the Rb tumor suppressor pathway. Therefore, one possible therapeutic strategy would be to directly modulate cyclin dependent kinase (CDK) activity resulting in inhibition of Rb phosphorylation and cell cycle progression. The small molecule CDK inhibitor, flavopiridol, has demonstrated antitumor activity in human xenograft models and is currently in clinical trials showing efficacy in patients with advanced disease. We have developed an experimental animal model using the murine glioma GL261 cells as a novel in vivo system to screen potential therapeutic agents for GBM. Results of in vitro testing demonstrate that flavopiridol has several relevant clinical characteristics such as its ability to: 1. inhibit cell growth; 2. inhibit cell migration;3. decrease expression of CDK inhibitor cyclin D1, CDK4 and p21; 4. induce apoptosis in cells with high levels of p27 expression; and5. decrease the expression of the anti-apoptotic protein Bcl-2. The mechanism by which flavopiridol induces apoptosis is mitochondrial-mediated. We demonstrate by electron microscopy and immunohistochemistry that drug treatment induces mitochondrial damage that was accompanied by the release of cytochrome c into the cytosol together with the translocation of apoptosis inducing factor (AIF) into the nucleus. This finding in murine glioma cells differs markedly from the mechanism of flavopiridol-induced apoptosis cell death reported by us for human glioma cells (Alonso et al., Mol Cancer Ther 2003; 2:139) where drug treatment induced a caspase- and cytochrome c-independent pathway in the absence of detectable damage to mitochondria. In apoptotic human glioma cells only translocation of AIF into the nucleus occurred. Thus, the same drug induces apoptosis inkills different types of glioma cells by different mitochondrial-dependent pathways.

The INK4a/ARF locus and melanoma.

Inactivation of the INK4a/ARF (or CDKN2a) locus is a common and critical genetic event in the development of human and mouse melanoma. This locus engages the Rb and p53 tumor suppressor pathways through its capacity to encode two distinct gene products, p16(INK4a) and p14(ARF). This review highlights the body of evidence supporting a role for both p16(INK4a) and p14(ARF) in the suppression of melanoma, and speculates as to why this locus is preferentially targeted in this tumor type. In addition, the potential importance of these two pathways in mediating UV-induced melanoma genesis will be addressed via genetic and molecular evidence in the mouse.

TP53 and liver carcinogenesis.

Primary hepatocellular carcinoma (HCC) is one of the most common malignancies and has the fourth highest mortality rate worldwide. The major risk factors, including chronic infections with the hepatitis B or C virus, are exposure to dietary aflatoxin B1(AFB1), vinyl chloride, or alcohol consumption. Southern China and sub-Saharan Africa have the highest dietary AFB1 exposure, making it and hepatitis B virus (HBV) the major causes of cancer mortality in these geographic areas. Recent studies have discovered genetic and epigenetic changes involved in the molecular pathogenesis of HCC, including somatic mutations in the p53 tumor suppressor gene (TP53). AFB1 induces typical G:C to T:A transversions at the third base in codon 249 of p53. Chronic active hepatitis B and C (HCV) infection, and further inflammatory and oxyradical disorders including Wilson disease (WD) or hemochromatosis, generate reactive oxygen/nitrogen species that can damage DNA and mutate the p53 gene. The X gene of HBV (HBx) is the most common open reading frame integrated into the host genome in HCC. The integrated HBx is frequently mutated and has a diminished ability to function as a transcriptional cotransactivator and to activate the NF-kappa B pathway. However, the mutant HBx proteins still retain their ability to bind to and abrogate p53-mediated apoptosis. In summary, both viruses and chemicals are implicated in the etiology and molecular pathogenesis of HCC. The resultant molecular changes in the ras and Wnt signal-transduction pathways, and the p53 and Rb tumor suppressor pathways significantly contribute to liver carcinogenesis

Pathogenetic and biologic significance of TP14ARF alterations in nonsmall cell lung carcinoma

The INK4a/ ARF locus on human chromosome band 9p21 carries two tumor suppressor genes, TP14 ARF and TP16 INK4a, and both are frequently inactivated in nonsmall cell lung carcinoma (NSCLC). TP14 ARF and TP16 INK4a play important roles in the TP53 and RB tumor suppressor pathways, respectively. To elucidate the genetic and epigenetic status of the TP14 ARF and TP16 INK4 a genes in NSCLC, we comprehensively analyzed mutations, homozygous deletions, methylations in the CpG regions, and expression of the TP14 ARF and TP16 INK4a genes in 31 NSCLC cell lines. TP16 INK4a (84%) was inactivated more frequently than TP14 ARF (55%). Moreover, p16 INK4a was inactivated in all 17 cell lines with TP14 ARF inactivation. Three cell lines with base substitutions in exon 2 resulted in missense mutations of TP16 INK4a but silent mutations of TP14 ARF. There was a case of mutation in exon 1α unique to TP16 INK4a, but not a mutation in exon 1β unique to TP14 ARF. The TP16 INK4a gene was methylated in 6 cell lines, but the TP14 ARF gene was not methylated in any cell line. Unlike a mutually exclusive relationship for inactivation between TP16 INK4a and RB, TP14 ARF and TP53 did not show such a relationship ( P = 0.61, Fisher exact test). Thus, the present results indicate the TP16 INK4a gene to be the primary target of INK4a/ ARF locus alterations. Transient TP14 ARF expression induced G1 arrest in the cells with wild-type TP53, but not in the cells with mutated TP53. Thus, the pathogenetic and biologic significance of TP14 ARF inactivation is different between NSCLC cells with wild-type TP53 and those with mutated TP53.

Altered expression of rb and p53 in uveal melanomas following plaque radiotherapy

PURPOSE : To examine the expression of proteins in the Rb and p53 tumor suppressor pathways in uveal melanomas following plaque radiotherapy.METHODS : Immunohistochemistry and cell culture studies. Immunohistochemistry for Rb, p16, cyclin D1, p53, HDM2, and Bcl-2 was performed on twelve eyes containing posterior uveal melanomas that were enucleated following plaque radiotherapy. Cell culture studies were performed in three cases.RESULTS : The irradiated eyes were enucleated for radiation complications (five cases), local tumor recurrence (three cases), and other reasons (four cases). On histopathologic examination, all cases showed evidence of tumor cell loss. However, residual tumor cells were present in all cases, including those that were clinically regressed. Residual cells from three of the clinically regressed cases were cultured and demonstrated minimal cell division, marked cell death, and extensive chromosomal damage. Strong p53 staining was observed in six cases (50%) and was significantly associated with recent radiotherapy (P = .04). Abnormal cytoplasmic staining for Rb was observed in four cases (33%).CONCLUSIONS : Plaque radiotherapy of uveal melanomas induces DNA damage, inhibits cell division, and promotes cell death. These changes may be due, at least in part, to induction of p53, which activates genes involved in both cell cycle arrest and apoptosis. Plaque radiotherapy can also cause alterations in the expression of Rb, but the significance of this finding will require further study.

Telomerase and human tumorigenesis

Human cancer cells, unlike their normal counterparts, have shed the molecular restraints to limited cell growth and are immortal. Exactly how cancer cells manage this at the molecular level is beginning to be understood. Human cells must overcome two barriers to cellular proliferation. The first barrier, referred to as senescence, minimally involves the p53 and Rb tumor-suppressor pathways. Inactivation of these pathways results in some extension of lifespan. However, inactivation of these pathways is insufficient for immortalization. As normal cells undergo repeated rounds of DNA replication, their telomeres shorten due to the inability of traditional DNA polymerases to completely replicate the end of the chromosomal DNA. This shortening continues until the cells reach a second proliferative block referred to as crisis, which is characterized by chromosomal instability, end-to-end fusions, and cell death. Stabilization of the telomeric DNA through either telomerase activation or the activation of the alternative mechanism of telomere maintenance (ALT) is essential if the cells are to survive and proliferate indefinitely. Conversely, loss of telomere stabilization by an already-immortalized cell results in loss of immortality and cell death. Together this indicates that telomere maintenance is a critical component of immortality. In this review we attempt to describe our current understanding of the role of telomere maintenance in senescence, crisis, and tumorigenesis.

Deregulation of the Rb and p53 Pathways in Uveal Melanoma

Uveal melanoma is the most common primary eye cancer, yet its molecular pathogenesis is poorly understood. In this study, we investigated the immunohistochemical expression of proteins in the Rb and p53 tumor suppressor pathways in 33 uveal melanomas from enucleated eyes. Strong nuclear staining for Rb was present in most tumors. However, a few cases displayed weak nuclear staining and strong cytoplasmic staining (possibly indicating Rb mutation), and this aberrant staining correlated strongly with failed radiotherapy or thermotherapy before enucleation. Staining for cyclin D1 was positive in most tumors and was associated with advanced age and larger tumor size, which are both poor prognostic factors. Generally, immunostaining for p53 was weak (suggesting a lack of p53 mutations), although p53 positivity correlated strongly with staining for phosphorylated Rb, supporting the notion that inappropriate phosphorylation of Rb can induce p53. Strong immunostaining for MDM2, which can functionally block p53 activity, was observed in most tumors and correlated significantly with female sex. Strong cytoplasmic staining was observed for Bcl2, which can inhibit both p53-dependent and -independent apoptosis. We conclude that Rb and p53 are mutated infrequently in uveal melanoma, but their respective pathways may be functionally inactivated.

Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways.

Most cervical carcinomas express high-risk human papillomaviruses (HPVs) E6 and E7 proteins, which neutralize cellular tumor suppressor function. To determine the consequences of removing the E6 and E7 proteins from cervical cancer cells, we infected HeLa cells, a cervical carcinoma cell line that contains HPV18 DNA, with a recombinant virus that expresses the bovine papillomavirus E2 protein. Expression of the E2 protein resulted in rapid repression of HPV E6 and E7 expression, followed approximately 12 h later by profound inhibition of cellular DNA synthesis. Shortly after E6/E7 repression, there was dramatic posttranscriptional induction of p53. Two p53-responsive genes, mdm2 and p21, were induced with slightly slower kinetics than p53 and appeared to be functional, as assessed by inhibition of cyclin-dependent kinase activity and p53 destabilization. There was also dramatic posttranscriptional induction of p105(Rb) and p107 after E6/E7 repression, followed shortly thereafter by induction of p130. By 24 h after infection, only hypophosphorylated p105(Rb) was detectable and transcription of several Rb/E2F-regulated genes was dramatically repressed. Constitutive expression of the HPV16 E6/E7 genes alleviated E2-induced growth inhibition and impaired activation of the Rb pathway and repression of E2F-responsive genes. This dynamic response strongly suggests that the p53 and Rb tumor suppressor pathways are intact in HeLa cells and that repression of HPV E6 and E7 mobilizes these pathways in an orderly fashion to deliver growth inhibitory signals to the cells. Strikingly, the major alterations in the cell cycle machinery underlying cervical carcinogenesis can be reversed by repression of the endogenous HPV oncogenes.

E2F-Rb complexes assemble and inhibit cdc25A transcription in cervical carcinoma cells following repression of human papillomavirus oncogene expression.

Expression of the bovine papillomavirus E2 protein in cervical carcinoma cells represses expression of integrated human papillomavirus (HPV) E6/E7 oncogenes, followed by repression of the cdc25A gene and other cellular genes required for cell cycle progression, resulting in dramatic growth arrest. To explore the mechanism of repression of cell cycle genes in cervical carcinoma cells following E6/E7 repression, we analyzed regulation of the cdc25A promoter, which contains two consensus E2F binding sites and a consensus E2 binding site. The wild-type E2 protein inhibited expression of a luciferase gene linked to the cdc25A promoter in HT-3 cervical carcinoma cells. Mutation of the distal E2F binding site in the cdc25A promoter abolished E2-induced repression, whereas mutation of the proximal E2F site or the E2 site had no effect. None of these mutations affected the activity of the promoter in the absence of E2 expression. Expression of the E2 protein also led to posttranscriptional increase in the level of E2F4, p105(Rb), and p130 and induced the formation of nuclear E2F4-p130 and E2F4-p105(Rb) complexes. This resulted in marked rearrangement of the protein complexes that formed at the distal E2F site in the cdc25A promoter, including the replacement of free E2F complexes with E2F4-p105(Rb) complexes. These experiments indicated that repression of E2F-responsive promoters following HPV E6/E7 repression was mediated by activation of the Rb tumor suppressor pathway and the assembly of repressing E2F4-Rb DNA binding complexes. Importantly, these experiments revealed that HPV-induced alterations in E2F transcription complexes that occur during cervical carcinogenesis are reversed by repression of HPV E6/E7 expression.

PML is induced by oncogenicrasand promotes premature senescence

Oncogenic ras provokes a senescent-like arrest in human diploid fibroblasts involving the Rb and p53 tumor suppressor pathways. To further characterize this response, we compared gene expression patterns between ras-arrested and quiescent IMR90 fibroblasts. One of the genes up-regulated during ras-induced arrest was promyelocytic leukemia (PML) protein, a potential tumor suppressor that encodes a component of nuclear structures known as promyelocytic oncogenic domains (PODs). PML levels increased during both ras-induced arrest and replicative senescence, leading to a dramatic increase in the size and number of PODs. Forced PML expression was sufficient to promote premature senescence. Like oncogenic ras, PML increased the levels of p16, hypophosphorylated Rb, phosphoserine-15 p53, and expression of p53 transcriptional targets. The fraction of Rb and p53 that colocalized with PML markedly increased during ras-induced arrest, and expression of PML alone forced p53 to the PODs. E1A abolished PML-induced arrest and prevented PML induction and p53 phosphorylation in response to oncogenic ras. These results imply that PML acts with Rb and p53 to promote ras-induced senescence and provide new insights into PML regulation and activity.

Preferential inactivation of the p53 tumor suppressor pathway and lack of EGFR amplification distinguish de novo high grade pediatric astrocytomas from de novo adult astrocytomas.

Classification of high grade astrocytomas of children into genetic subtypes similar to the adult remains to be defined. Here we report an extensive characterization of 29 high grade pediatric astrocytomas, 7 WHO grade III and 22 WHO grade IV, for genetic alterations frequently observed in high grade adult astrocytomas occurring in either the p53/MDM2/p14ARF or Rb/CDK4/p16INK4a tumor suppressor pathways. In addition, we have assessed the contribution of EGFR overexpression and amplification and LOH for chromosome 10, two genetic alterations commonly associated with the development of de novo adult glioblastoma for their roles in the development of de novo astrocytomas of childhood. Our results suggest two major differences in the genetic pathway(s) leading to the formation of de novo high grade astrocytomas in children compared with those of the adult. Our findings show preferential inactivation of the p53 tumor suppressor pathway in >95% of pediatric astrocytomas versus inactivation of the Rb tumor suppressor pathway in <25% of the same tumors. In addition, de novo high grade pediatric astrocytomas lack amplification of the EGFR gene compared with EGFR amplification in one-third of adult glioblastomas. Since drug treatments and gene therapy strategies exploit specific genetic alterations in tumor cells, our findings have important implications for the future development of treatments for high grade pediatric astrocytomas.

Disruption of the G1/S transition in human papillomavirus type 16 E7-expressing human cells is associated with altered regulation of cyclin E.

The development of neoplasia frequently involves inactivation of the p53 and retinoblastoma (Rb) tumor suppressor pathways and disruption of cell cycle checkpoints that monitor the integrity of replication and cell division. The human papillomavirus type 16 (HPV-16) oncoproteins, E6 and E7, have been shown to bind p53 and Rb, respectively. To further delineate the mechanisms by which E6 and E7 affect cell cycle control, we examined various aspects of the cell cycle machinery. The low-risk HPV-6 E6 and E7 proteins did not cause any significant change in the levels of cell cycle proteins analyzed. HPV-16 E6 resulted in very low levels of p53 and p21 and globally elevated cyclin-dependent kinase (CDK) activity. In contrast, HPV-16 E7 had a profound effect on several aspects of the cell cycle machinery. A number of cyclins and CDKs were elevated, and despite the elevation of the levels of at least two CDK inhibitors, p21 and p16, CDK activity was globally increased. Most strikingly, cyclin E expression was deregulated both transcriptionally and posttranscriptionally and persisted at high levels in S and G2/M. Transit through G1 was shortened by the premature activation of cyclin E-associated kinase activity. Elevation of cyclin E levels required both the CR1 and CR2 domains of E7. These data suggest that cyclin E may be a critical target of HPV-16 E7 in the disruption of G1/S cell cycle progression and that the ability of E7 to regulate cyclin E involves activities in addition to the release of E2F.

Immunohistochemical analysis of the p16INK4 cyclin-dependent kinase inhibitor in malignant mesothelioma.

The identification in 1994 of the CDKN2 gene as a target for mutations in a wide range of human cancers, including malignant mesothelioma, has been controversial because subsequent studies have detected a lower frequency of CDKN2 gene mutations in primary tumors than in cultured cell lines. These reports raised the hypothesis that another gene, distinct from CDKN2, might be the target of the chromosome 9p21 deletions frequently observed in these tumors. To address whether inactivation of CDKN2 function is an essential event in the etiology of malignant mesothelioma, we examined p16INK4 protein expression in primary thoracic mesotheliomas, in nonmalignant pleural tissues, and in independent mesothelioma cell lines. We also studied the growth rate of tumor cell lines following stable transfection of CDKN2 gene. Retinoblastoma (Rb) and p16INK4 protein expression was determined by immunohistochemical analysis from archival paraffin specimens of 12 primary thoracic mesotheliomas and a nonmalignant pleural biopsy specimen. In addition, protein immunoblot analysis for Rb and p16INK4 expression was conducted on 15 independent mesothelioma cell lines, and the ability of a transfected CDKN2 gene to suppress the growth of the mesothelioma cell lines H2373 and H2461 in vitro was examined. We demonstrated abnormal p16INK4 expression in 12 of 12 primary mesothelioma specimens and in 15 of 15 mesothelioma cell lines. All tumor specimens and the tumor cell lines showed expression of wild-type Rb protein. In addition, we have confirmed the ability of a transfected CDKN2 gene to suppress growth of two independent mesothelioma cell lines. Immunohistochemical analysis of the p16INK4 gene product is feasible in archival biopsy samples. With this analysis, CDKN2 gene inactivation can be determined in tumors that are contaminated with nonmalignant cells. Furthermore, since loss of p16INK4 protein expression can result from both genetic (gene mutations) and epigenetic (abnormal DNA hypermethylation) mechanisms, as we and others have shown recently, examination of protein expression is a highly sensitive method for analyzing the CDKN2 status in large numbers of tumor samples. This study suggests that inactivation of the CDKN2 gene is an essential step in the etiology of malignant mesotheliomas. Defining the role of the p16INK4:Rb tumor suppressor pathway and its immediate downstream substrates will be an important goal in designing future therapeutic strategies.