Retinoblastoma Family Of Tumor Suppressors Research Articles

The anaphase-promoting complex/cyclosome co-activator, Cdh1, is a novel target of human papillomavirus 16 E7 oncoprotein in cervical oncogenesis.

The transforming properties of the high-risk human papillomavirus (HPV) E7 oncoprotein are indispensable for driving the virus life cycle and pathogenesis. Besides inactivation of the retinoblastoma family of tumor suppressors as part of its oncogenic endeavors, E7-mediated perturbations of eminent cell cycle regulators, checkpoint proteins and proto-oncogenes are considered to be the tricks of its transformative traits. However, many such critical interactions are still unknown. In the present study, we have identified the anaphase-promoting complex/cyclosome (APC) co-activator, Cdh1, as a novel interacting partner and a degradation target of E7. We found that HPV16 E7-induced inactivation of Cdh1 promoted abnormal accumulation of multiple Cdh1 substrates. Such a mode of deregulation possibly contributes to HPV-mediated cervical oncogenesis. Our mapping studies recognized the C-terminal zinc-finger motif of E7 to associate with Cdh1 and interfere with the timely degradation of FoxM1, a bona fide Cdh1 substrate and a potent oncogene. Importantly, the E7 mutant with impaired interaction with Cdh1 exhibited defects in its ability for overriding typical cell cycle transition and oncogenic transformation, thereby validating the functional and pathological significance of the E7-Cdh1 axis during cervical carcinoma progression. Altogether, the findings from our study discover a unique nexus between E7 and APC/C-Cdh1, thereby adding to our understanding of the mechanism of E7-induced carcinogenesis and provide a promising target for the management of cervical carcinoma.

Retinoblastoma‐independent regulation of cell proliferation and senescence by the p53–p21 axis in lamin A /C‐depleted cells

The expression of A-type lamin is downregulated in several cancers, and lamin defects are the cause of several diseases including a form of accelerated aging. We report that depletion of lamin A/C expression in normal human cells leads to a dramatic downregulation of the Rb family of tumor suppressors and a defect in cell proliferation. Lamin A/C-depleted cells exhibited a flat morphology and accumulated markers of cellular senescence. This senescent phenotype was accompanied by engagement of the p53 tumor suppressor and induction of the p53 target gene p21 and was prevented by small hairpin RNAs against p53, p21, or by the oncoprotein Mdm2. The expression of E2F target genes, normally required for cell cycle progression, was downregulated after lamin A/C depletion but restored after the inactivation of p53. A similar senescence response was observed in myoblasts from a patient with a lamin A mutation causing muscular dystrophy. We thus reveal a previously unnoticed mechanism of controlling cell cycle genes expression, which depends on p53 but does not require the retinoblastoma family of tumor suppressors and that can be relevant to understand the pathogenesis of laminopathies and perhaps aging.

PP2A interaction with Rb2/p130 mediates translocation of Rb2/p130 into the nucleus in all‐Trans retinoic acid‐treated ovarian carcinoma cells

One of the mechanisms by which all-trans retinoic acid (ATRA) has been shown to suppress the growth of CAOV3 ovarian carcinoma cells involves an increase in the accumulation of Rb2/p130 protein, a member of the retinoblastoma family of tumor suppressors. This increase in accumulation of RB2/p130 by ATRA results from increased stability of Rb2/p130 protein as a result of an increase in dephosphorylation of the protein by the serine/threonine phosphatase PP2A. We show that upon ATRA treatment, PP2A interacts with the Rb2/p130 C-terminus and specifically dephosphorylates two residues (S1080 and T1097) adjacent to NLS1 and NLS2 of Rb2/p130. Moreover, co-immunoprecipitation studies reveal that Rb2/p130 can form a complex with the nuclear transport proteins, importin α and importin β, binding to the same dephosphorylated NLS1 and NLS2 sites. Finally, mutation of S1080 and T1097 results in retension of Rb2/p130 in the cytoplasm. Our studies suggest that one mechanism by which ATRA treatment of CAOV3 cells induces G0/G1 arrest involves the recruitment of PP2A to the C-terminus of Rb2/p130, resulting in the dephosphorylation of the S1080 and T1097 adjacent to the NLS and the subsequent interaction of Rb2/p130 with importins leading to transport of the Rb2/p130 to the nucleus where it inhibits cell-cycle progression.

Expression of the E2F Family of Transcription Factors and Its Clinical Relevance in Ovarian Cancer

The E2F family of transcription factors plays a pivotal role in the regulation of cellular proliferation. On the basis of sequence homology and function, eight distinct members of E2F transcription factors (E2F-1 to E2F-8) have been distinguished to date. The regulation of E2F transcription factors is closely associated with the function of the retinoblastoma family of tumor suppressors (RB pathway). In the last decade various alterations of distinct components of the RB-E2F pathway were found to be associated with tumor progression. However, no data on the role of E2F family members are available in tumor biology of ovarian cancer. Here we describe an expression study of E2F transcription factors in various human ovarian cancer cell lines; its clinical relevance was examined in a training set of 77 ovarian cancer patients. Expression levels of E2F-1, E2F-2, and E2F-8 were elevated in all the ovarian cancer cell lines studied when compared with human peritoneal mesothelial cells (HPMCs). Interestingly, EGF treatment showed a time-dependent upregulation of the activating transcription factor E2F-3 and a simultaneous increase of DP-1, the heterodimeric partner of E2F-3. High expression of E2F-1, E2F-2, and E2F-8 was found to be associated with histopathologic grade 3 tumors and residual tumor over 2 cm in diameter after primary debulking surgery in ovarian cancer patients. Taken together, these data suggest that the proliferation-promoting E2F transcription factors E2F-1 and especially E2F-2 play a pivotal role in tumor biology of ovarian cancer and may be candidates for specific therapeutic targets.

Rb2/p130 and protein phosphatase 2A: key mediators of ovarian carcinoma cell growth suppression by all-trans retinoic acid

Despite a number of attempts to improve treatment of ovarian cancer, it remains the most common cause of death from gynecological cancers. Thus, it is very important to identify more effective drugs for treatment and prevention of ovarian cancer. All-trans-retinoic acid (ATRA) has been shown to arrest the growth of ovarian carcinoma cells in G0/G1 and to significantly elevate levels of Rb2/p130 protein, a member of the retinoblastoma family of tumor suppressors. As ATRA treatment leads to a significant increase in the amount of Rb2/p130 protein but not mRNA, the elevated levels of Rb2/p130 protein is likely the result of increased stability. In studies to elucidate the mechanism by which ATRA alters Rb2/p130 stability in ovarian cancer cells, it was determined that PP2A, a serine/threonine phosphatase, binds and dephosphorylates Rb2/p130. Dephosphorylated Rb2/p130 exhibits decreased ubiquitination and thus is not degraded by the proteasome. The sites at which PP2A catalytic subunit (PP2Ac) interacts with Rb2/p130 have been localized to the NLS in the C-terminus of Rb2/p130. These sites are also involved in the interaction of Rb/p130 with importin beta and importin alpha, members of the nuclear transport machinery. It is known that importin alpha recognizes a NLS on a target protein and importin beta binds the nuclear pore complex. Moreover, it has been shown that the binding of importin alpha to NLS significantly decreases with phosphorylation of NLS. In ATRA-treated ovarian carcinoma cells, PP2A binds to Rb2/p130 and dephosphorylates the NLS of Rb2/p130 leading to the interaction of importin alpha with Rb2/p130. Importin beta then binds to the importin alpha-Rb2/p130 complex, leading to the translocation of the Rb2/p130 to the nucleus where it acts to arrest ovarian cancer cells in G1 and suppress proliferation.

Human cytomegalovirus pp71: a new viral tool to probe the mechanisms of cell cycle progression and oncogenesis controlled by the retinoblastoma family of tumor suppressors.

The DNA tumor virus oncogenes (adenovirus E1A, simian virus 40 (SV40) large T antigen, and papillomavirus E7) have been instrumental in illuminating the molecules and mechanisms of cell cycle progression and carcinogenesis. However, since these multifunctional proteins target so many important cellular regulators, it is sometimes difficult to establish the functional importance of any individual interaction. Perhaps a herpesvirus protein, newly defined as a cell cycle regulator, can help address these issues. Like the DNA tumor virus proteins, the human cytomegalovirus (HCMV) pp71 protein contains a retinoblastoma protein (Rb) binding motif (LxCxD), and stimulates DNA synthesis in quiescent cells. Unlike E1A, T antigen, and E7, pp71 expression does not induce apoptosis, nor does it cooperate to transform primary cells. Determining how pp71 induces cell cycle progression without invoking apoptosis or leading to cellular transformation may help in defining the signals that ultimately lead to these processes.

Human cytomegalovirus pp71 stimulates cell cycle progression by inducing the proteasome-dependent degradation of the retinoblastoma family of tumor suppressors.

The oncoproteins of the DNA tumor viruses, adenovirus E1A, simian virus 40 T antigen, and papillomavirus E7, each interact with the retinoblastoma family of tumor suppressors, leading to cell cycle stimulation, apoptosis induction, and cellular transformation. These proteins utilize a conserved LXCXE motif, which is also found in cellular proteins, to target the retinoblastoma family. Here, we describe a herpesvirus protein that shares a subset of the properties of the DNA tumor virus oncoproteins but maintains important differences as well. The human cytomegalovirus pp71 protein employs an LXCXD motif to attack the retinoblastoma family members and induce DNA synthesis in quiescent cells. pp71 binds to and induces the degradation of the hypophosphorylated forms of the retinoblastoma protein and its family members p107 and p130 in a proteasome-dependent manner. However, pp71 does not induce apoptosis and fails to transform cells. Thus, the similarities and differences in comparison to E1A, T antigen, and E7 make pp71 an interesting new tool with which to further dissect the role of the retinoblastoma/E2F pathway in cellular growth control and carcinogenesis.

Purified JC Virus T and T′ Proteins Differentially Interact with the Retinoblastoma Family of Tumor Suppressor Proteins

The amino termini of polyomavirus T antigens contain LXCXE and J domains, which are necessary for binding and inactivating the retinoblastoma family of tumor suppressors. Both of these motifs are found in the JC virus (JCV) early proteins T135′, T136′, and T165′, leading to the suggestion that these recently discovered proteins complement the cell-cycle-deregulating function of the JCV large T antigen (TAg). To investigate this hypothesis, the three JCV T′ proteins were produced in a baculovirus expression system and purified by immunoaffinity chromatography. To facilitate purification, hybridomas that secrete antibodies recognizing amino-terminal epitopes of JCV early proteins were produced. Potential interactions between the early viral proteins and the cellular proteins pRB, p107, and p130 were investigated by incubating purified JCV TAg and T′ proteins with extracts of MOLT-4 cells, a human T cell line. The four viral proteins preferentially bound hypophosphorylated species of the cellular proteins and exhibited the highest binding affinity to p107 and the lowest affinity to pRB. TAg and T165′ bound more pRB and less p107 than did T135′ and T136′; T165′ also bound less p130 than the other three early proteins. Results of these in vitro interactions were compared to those obtained in vivo using POJ cells, a transformed human glial cell line that expresses JCV early proteins, relatively high levels of pRB and p107, and low levels of p130. Most of the pRB in POJ cells is hyperphosphorylated, and only a fraction of the hypophosphorylated form(s) of pRB is bound by the viral proteins. In contrast, only hypophosphorylated p130 is detected in the transformed cells, and most of this protein was found in complex with the viral proteins. Finally, nearly all of the p107 in POJ cells is bound by the JCV proteins.

Regulation of E2F: a family of transcription factors involved in proliferation control.

Members of the E2F family of transcription factors are key participants in orchestration of the cell cycle, cell growth arrest and apoptosis. Therefore, an understanding of the regulation of E2F activity is essential for an understanding of the control of cellular proliferation. E2F activity is regulated by the retinoblastoma family of tumor suppressors and by multiple other mechanisms. This review will describe our current knowledge of these mechanisms which together constitute a highly complex network by which the cell cycle and cellular proliferation can be controlled.