P53-related Protein Research Articles

Over-expression of p73β results in apoptotic death of post-mitotic hNT neurons

The p53-related p73 protein is an important mediator of apoptosis, development and tumorigenesis. Previously, we showed that over-expression of the p73β isoform induced apoptosis in proliferating neuronal cells; however, the study did not address the effect of p73 in post-mitotic neurons. To address this question, we used post-mitotic hNT neurons, which have been used as a model of human central nervous system neurons. We found that over-expression of p73β in hNT neurons resulted in apoptosis and an increase in the expression of p57 Kip2 and Bax, but no increase in p53 expression. These results suggest that apoptosis of post-mitotic neurons induced by p73β may involve these mediators. Understanding the regulation of p73 expression will be important for understanding the development of the nervous system and may have implications for the treatment of neurological diseases.

P53: link to the past, bridge to the future

Although the p53 tumor suppressor was identified nearly three decades ago and plays a pivotal role in human cancer, its complexity continues to surprise the research community. Indeed, it was only recently discovered that p53 belongs to a multigene family that also includes p63 and p73 (Kaghad et al. 1997; Schmale and Bamberger 1997). Both p63 and p73 express an array of isoforms as a result of multiple promoter usage and alternative splicing (Yang et al. 1998; Zaika et al. 2002). This complexity within the p53 gene family has, until now, been considered a unique feature of p63 and p73. In this issue, Bourdon et al. (2005) use currently available molecular techniques to revisit the structure of p53, the founding member of this gene family. This work reveals that like the p53-related genes p63 and p73, p53 also makes use of several promoters and undergoes alternative splicing to generate multiple isoforms. Thus, a comparison of p53-related genes reveals strikingly similar traits among family members that produce a plethora of related but functionally diverse proteins that add further complexity to the role of p53-related proteins in development, cancer, and aging

Detecting p53 family proteins in haemocytic leukemia cells ofMytilus edulisfrom Pictou Harbour, Nova Scotia, Canada

Evaluating patterns of expression of p53-related proteins in cells is a novel approach in defining environmentally linked diseases. We have examined the induction of haemocytic leukemia in Mytilus edulis by municipal and industrial contaminants in Pictou Harbour, Nova Scotia, Canada. We used a murine monoclonal antibody, 1E10, as a diagnostic reagent to detect leukemic cells. We first characterized the reactivity of 1E10 with both normal and leukemic Mytilus haemocytes by confocal microscopy. We then compared p53 gene family expression (p53, p63–p73, and p97) in normal versus leukemic haemocytes using a panel of monoclonal and polyclonal antibodies to p53 family proteins. The immunochemical data demonstrate that haemocytic leukemia cells of M. edulis differentially express p63–p73 and p97–p120 proteins. We subsequently used 1E10 to diagnose haemocytic leukemia in 500 M. edulis previously deployed 6 months earlier in Pictou Harbour. In the field, Mytilus caged near untreated municipal wastewater and bleached kraft pulpmill effluents have a significantly greater chance of developing haemocytic leukemia than do mussels exposed to reference sites.

P63 deficiency activates a program of cellular senescence and leads to accelerated aging

The p53 tumor suppressor plays a key role in organismal aging. A cellular mechanism postulated to drive the aging process is cellular senescence, mediated in part by p53. Although senescent cells accumulate in elderly individuals, most studies have relied on correlating in vitro senescence assays with in vivo phenotypes of aging. Here, using two different mouse models in which the p53-related protein p63 is compromised, we demonstrate that cellular senescence and organismal aging are intimately linked and that these processes are mediated by p63 loss. We found that p63(+/-) mice have a shortened life span and display features of accelerated aging. Both germline and somatically induced p63 deficiency activates widespread cellular senescence with enhanced expression of senescent markers SA-beta-gal, PML, and p16(INK4a). Using an inducible tissue-specific p63 conditional model, we further show that p63 deficiency induces cellular senescence and causes accelerated aging phenotypes in the adult. Our results thus suggest a causative link between cellular senescence and aging in vivo, and demonstrate that p63 deficiency accelerates this process.

Association of the human papillomavirus type 16 E7 oncoprotein with the 600-kDa retinoblastoma protein-associated factor, p600

The human papillomavirus type 16 (HPV-16) E7 gene encodes a multifunctional oncoprotein that can subvert multiple cellular regulatory pathways. The best-known cellular targets of the HPV-16 E7 oncoprotein are the retinoblastoma tumor suppressor protein pRB and the related pocket proteins p107 and p130. However, there is ample evidence that E7 has additional cellular targets that contribute to its transforming potential. We isolated HPV-16 E7 associated cellular protein complexes by tandem affinity purification and mass spectrometry and identified the 600-kDa retinoblastoma protein associated factor, p600, as a cellular target of E7. Association of E7 with p600 is independent of the pocket proteins and is mediated through the N terminal E7 domain, which is related to conserved region 1 of the adenovirus E1A protein and importantly contributes to cellular transformation independent of pRB binding. Depletion of p600 protein levels by RNA interference substantially decreased anchorage-independent growth in HPV-positive and -negative human cancer cells. Therefore, p600 is a cellular target of E7 that regulates cellular pathways that contribute to anchorage-independent growth and cellular transformation.

A Role of p73 in Mitotic Exit

The p53-related p73 proteins regulate developmental processes, cell growth, and DNA damage response. p73 function is regulated by post-translational modifications and protein-protein interactions. At the G2/M transition, p73 is phosphorylated at Thr-86 by the p34cdc2/cyclin B complex; this is associated with its exclusion from condensed chromosomes and loss of DNA binding and transcriptional activation ability. Here we showed that p73 hypo-phosphorylated species reappear during mitotic exit, concomitant with p73 relocalization to telophase nuclei and recovered ability to activate transcription. Functional knock-out of p73 gene expression by small interfering RNAs (siRNAs) alters mitotic progression, yielding an increase of ana-telophase cells, the accumulation of aberrant late mitotic figures, and the appearance of abnormalities in the subsequent interphase. This p73 activity at the M-to-G1 transition is mediated by its transactivating function because expression of the transcription dominant negative mutant p73DD induces the same mitotic exit phenotype. We also found that the cyclin-dependent kinase inhibitor Kip2/p57 gene is a specific target of p73 regulation during mitotic exit and re-entry into G1. Both knock-out of p73 gene expression by siRNAs and abrogation of p73-dependent transcription by the p73DD mutant abrogate Kip2/p57 increase at the M-to-G1 transition. Moreover, similar abnormalities (e.g. delay in late mitotic stages with the accumulation of aberrant ana-telophase figures, and abnormalities in the following interphase) are observed in cultures in which the expression of Kip2/p57 is abrogated by siRNAs. These results identify a novel p73-Kip2/p57 pathway that coordinates mitotic exit and transition to G1.

Array CGH analysisin neoadjuvant chemotherapy for breast cancer

786 Background: To assess a potential and common pattern of genetic alterations in Korean women’s breast cancer correlated with clinicopathologic parameters and to assess the potential genes which may be helpful to predict the response of neoadjuvant chemotherapy, we analyzed 13 tumors from breast cancer patients by microarray-based CGH. Methods: Array CGH(including 350 cancer-related genes and 1,090 STS) was performed with fresh frozen tumor tissue from the breast cancer patients. Ten cases of them received neoadjuvant chemotherapy(cyclophosphamide 600 mg/m2, adriamycin 50 mg/m2(CA); cyclophosphamide 600 mg/m2, adriamycin 50 mg/m2, 5-FU 500 mg/m2(CAF); cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, 5-FU 500 mg/m2(CMF); CA and Taxol; Nabelvine), 2 cases adjuvant chemotherapy and 1 case had no history of chemotherapy. Immunohistochemical study for ER, PR, p53, and Her-2 were also done. Results: In array CGH analysis, the patients showed typical imbalances for breast cancer; gain of 8q(84.6%), 11q13(69.2%), 16p(84.6%), 22q(69.2%), 3p(61.5%) and losses of 1p(61.5%), 18q(38.5%) and 2q(38.5%). Other recurrent imbalance of newly recognized pattern for breast cancer was gain of 1p(92.3%). One patient, died within 5 months with pulmonary metastasis after diagnosis, displayed gains of 20q13, 8q and losses of 13q21, 18p and 18q which were known as chromosomal changes related with adverse prognosis in previous reports. Genetic alterations in the “progress” group after neoadjuvant chemotherapy showed gain of MAFG(v-maf musculoaponeurotic fibrosarcoma oncogene homolog G; 17q25) which was related to cellular stress response and loss of DCC(18q21.3) whose function was induction of apoptosis. Genetic alterations in the “stable” and “partial remission” group were gain of p73(1p36), which was p53-related protein, and activated by chemotherapy and thereafter contributed to the reduction of the tumor size. Conclusions: These data suggest that gains of 1p might be a newly identified CGH finings which characterize the Korean women breast cancer. These findings suggest that loss of DCC and gain of MAFG and p73 might be a possible predictor for the response of neoadjuvant chemotherapy. No significant financial relationships to disclose.

Rb Enhances p160/SRC Coactivator-dependent Activity of Nuclear Receptors and Hormone Responsiveness

The retinoblastoma tumor suppressor protein (Rb) is best known as a repressor of genes involved in cell cycle progression. Rb has also been implicated in activation of transcription, in particular by nuclear receptors (NRs) and by differentiation-related transcription factors, but the relevance of this activity is unclear. We show that Rb and the related proteins p107 and p130 enhance the activity of NRs related to NGFI-B (Nur factors) through direct interactions with NGFI-B and SRC-2. Although recruitment of SRC/p160 coactivators to the NGFI-B AF1 domain is independent of Rb, its presence enhances SRC-dependent transcription. Rb potentiation of SRC coactivators is exerted on a subset (Nur factors, hepatocyte nuclear factor-4 (HNF-4), SF-1, and ER) but not all NRs. The levels of Rb-related proteins modulate hormone responsiveness of the NGFI-B-dependent pituitary proopiomelanocortin gene and HNF-4-dependent transcription during enterocyte differentiation. Increased Rb expression upon cell differentiation may promote differentiated functions, at least in part, by potentiation of NR activity.

Expression of Cell Cycle–Regulated Proteins pRB2/p130, p107, E2F4, p27, and pCNA in Salivary Gland Tumors: Prognostic and Diagnostic Implications

The retinoblastoma family consists of the tumor suppressor nuclear phosphoprotein pRb/p105 and related proteins p107 and pRb2/p130. Recent immunohistochemical studies of the retinoblastoma family of proteins in lung and endometrial cancer and choroidal melanomas show a tight inverse correlation between the histologic grading in the most aggressive tumor types and pRb2/p130 expression. This led us to investigate the role of pRb2/p130 in salivary tumors. We studied the expression of pRb2/p130, p107, E2F4, p27, and PcNA by immunohistochemistry in a panel of 44 salivary gland tumors. We found a direct correlation between the cytoplasmic expression of pRb2/p130 and tumor grading and the presence of metastasis that was highly statistically significant (P < 0.001). Additionally, increased cytoplasmic pRb2/p130 expression was significantly correlated with a decreased probability of survival (P < 0.001). Interestingly, p107 nuclear expression showed a strong direct correlation when compared with the same variables. pRb2/p130 showed the highest percentage of undetectable nuclear levels in the specimens examined and the tightest inverse correlation (P < 0.0001) with both the histologic grading and pCNA expression in malignant salivary tumors. Additionally, E2F4 showed an identical localization pattern as to that of pRb2/p130. These data suggests an important role for pRb2/p130 in the pathogenesis and progression of certain salivary gland cancers.

P73-Dependent Apoptosis through Death Receptor: Impairment by Human Cytomegalovirus Infection

The discovery of p73, a p53-related protein with various isotypes resulting from different promoter usage or splicing events, provided new insights into regulation of neurogenesis and tumorigenesis. Among p73 isoforms described thus far, TA-truncated molecules (DeltaN) appeared as key proteins according to their antagonistic activity against transcription factor activity of p53 family members. We previously showed that infection by human cytomegalovirus (HCMV) induced drug resistance and altered p53- and p73-dependent apoptosis of infected cells through accumulation of DeltaN-p73alpha. In accordance with the ability of p53 to induce apoptosis through death receptors, we asked whether p73 activation could compensate for p53 deficiency. We showed that p73 transcriptional activity sensitized cells to apoptosis through death receptors in a caspase-dependent pathway. Expression of the death-inducing signaling complex (DISC) proteins was unchanged, whereas p73 activation through either cisplatin treatment or ectopic overexpression induced up-regulation of Fas transcription and expression at cell surface. According to its ability to flood cells with DeltaN-p73alpha, HCMV inhibited p73-dependent Fas-mediated apoptosis, gaining an additional trick to favor its survival in the host cell. Owing to the involvement of p53- and p73-dependent death receptor signaling in development of the central nervous system, immune surveillance of neural cells, and sensitivity of tumors to drugs, our previous and present data prompt us to consider stabilization of DeltaN-p73alpha by HCMV as a possible mechanism in impairment of embryogenesis and in tumorigenesis.

TAp63γ can substitute for p53 in inducing expression of the maspin tumor suppressor

Maspin is a Class II tumor suppressor protein and plays a role in tumor growth by inhibiting cellular invasion and motility. It is a member of the serpin family of protease inhibitors and has been shown to reduce angiogenesis. Maspin gene expression can be upregulated by the tumor suppressor p53. We tested 7 p53-related proteins of the p63 and p73 families for their ability to induce maspin expression. The p63 splice form TAp63gamma can substitute for p53 in activating the maspin promoter. TAp63gamma activates the promoter through the same consensus site as p53. In the DLD-1 colorectal adenocarcinoma cell line, harboring a tet-off regulated transgene, induction of TAp63gamma leads to an upregulation of maspin mRNA from the chromosomal gene. With a short lag phase also maspin protein levels are elevated after induced TAp63gamma expression. To assess a potential function of p63-dependent maspin upregulation in tumors we followed expression of p53, p63 and maspin by immunohistochemistry in hepatocellular carcinomas. Two types of tumors with wild-type or mutant p53 were assayed. Interestingly, the majority of tumors expressing only a mutated and inactive p53 protein nonetheless stain positive for maspin, whereas these tumors were positive for p63 protein expression. In summary, we show that TAp63gamma can substitute for p53 in transcriptional activation of the maspin tumor suppressor gene. TAp63gamma employs the same DNA recognition site for this activation as p53. We observe expression patterns of p53, p63 and maspin proteins in tumor tissue that may indicate also a function of maspin induction by p63 in tumors.

A molecular basis for the efficacy of magnesium treatment following traumatic brain injury in rats.

Magnesium ions have been shown to be a promising treatment for brain lesions caused by traumatic brain injury (TBI), as well as for the associated acute neurodegeneration and progressive functional deficits. This study investigated the effects of magnesium on the expression of the cell death/survival related proteins following TBI. Male Sprague-Dawley (SD) rats (n = 66, 280-320 g body weight) were subjected to sham surgery alone (n = 14), or to the surgery followed by a lateral fluid percussion brain injury of moderate severity (n = 52, 2.4-2.7 atm). The injured rats were randomly treated with an intravenous bolus of magnesium chloride (n = 26, 125 micromol) or saline vehicle (n = 26). The coronal brain sections were quantitatively analyzed for cell apoptosis and the expression of p53-related proteins, Bcl-2, cyclin D1 and PCNA at 1, 2, and 4 days post-injury by immunohistochemistry or in situ hybridization. Tissue damage was observed primarily in the ipsilateral cortex of the injured region with the induction of apoptosis and p53 mRNA level at 2 days after TBI. The expression of p53 and responding proteins (p21(WAF1/CIP1), Mdm2 and Bax) showed a temporal pattern similar to the apoptotic events in the time course experiments. They were induced in the early time points of days 1-2, decreasing by day 4 after TBI. In contrast, the expression of the cell survival related proteins - Bcl-2, cyclin D1, and PCNA - was most significant at day 4 post-injury, when the rate of apoptosis decreased. Magnesium treatment resulted in a reduction in apoptosis and expression of p53-related proteins. However, it had only a slight additive effect on the expression of the survival related proteins in the same time-course. These results provide a molecular basis for the efficiency of magnesium in treating TBI-induced tissue damage.

End-stage differentiation of neutrophil granulocytes in vivo is accompanied by up-regulation of p27kip1 and down-regulation of CDK2, CDK4, and CDK6.

The in vivo expression profiles of cell-cycle proteins regulating G1-to-S-phase transition were determined in three neutrophil precursor populations from human bone marrow: myeloblasts (MBs) and promyelocytes (PMs); myelocytes (MCs) and metamyelocytes (MMs); and band cells (BCs) and segmented neutrophil cells (SCs) and in mature polymorphonuclear neutrophils (PMNs) from peripheral blood. Complete cell-cycle arrest was observed in BCs/SCs and PMNs. Cyclins D1, D2, and D3 were found to be down-regulated during granulopoiesis, whereas a slight increase of cyclin E was seen. In contrast, cyclin-dependent kinase (CDK)2, -4, and -6 were down-regulated from the MC/MM stages and onward. The transcript levels of CDK2, -4, and -6 were concurrently down-regulated. As the only CDK inhibitor, p27kip1 protein and mRNA expression were up-regulated in MCs/MMs and reached peak levels in PMNs. Protein expression of retinoblastoma protein and the related pocket proteins p107 and p130 was down-regulated from the MC/MM stages and onward. This is the first report to describe expression levels of cell-cycle proteins during granulopoiesis in vivo, and it strongly contrasts the observations made in cell-culture systems in vitro.

Human papillomavirus type 16 E7 oncoprotein can induce abnormal centrosome duplication through a mechanism independent of inactivation of retinoblastoma protein family members.

The human papillomavirus type 16 (HPV-16) E7 oncoprotein rapidly induces centrosome duplication errors in primary human cells, thereby increasing the propensity for multipolar mitoses, which can lead to chromosome missegregation and aneuploidy. We analyzed a series of HPV-16 E7 mutants and demonstrate that this biological activity of the E7 oncoprotein is mediated by sequences encompassing the core pRB binding site but is independent of its ability to inactivate the retinoblastoma tumor suppressor protein pRB and the related pocket proteins p107 and p130. In addition, interaction of E7 with the S4 subunit of the 26S proteasome and dysregulation of cdc25A transcription are also dispensable for the induction of centrosome duplication errors. Consistent with these results, expression of HPV-16 E7 induces abnormal centrosome duplication in a cell line that lacks functional pRB and in mouse embryo fibroblasts that are deficient for pRB, p107, and p130. These results demonstrate that the molecular mechanism whereby HPV-16 E7 induces centrosome duplication errors is independent of its ability to inactivate pRB, p107, and p130 or to interact with the S4 proteasome subunit.

Apoptosis and cell cycle-related genes and proteins in classical Hodgkin lymphoma: application of tissue microarray technique.

The etiology and pathogenesis of Hodgkin lymphoma (HL) are not yet known. There are implications of genes involved in programmed cell death (apoptosis), and there have been repeated suggestions of an association with Epstein-Barr virus (EBV). The aim of this study was to investigate the protein expression patterns of key cell cycle-related genes, together with evidence of apoptosis and EBV status, in relation to clinical stage in HLs. A double immunohistochemical and in situ hybridization technique was used to detect the expression of bcl-2, p53, retinoblastoma (Rb), p21, Ki67 (MIB 1), and topoisomerase IIalpha (TopoIIalpha), together with latent membrane protein-1 and EBER for EBV status and TdT-mediated dUTP-FITC nick end-labeling (TUNEL) as a measure of apoptosis, on tissue microarray sections of 62 cases of classic HL (35 NS, 17 MC, 8 LR, and 2 LD). A panel of phenotypic markers was used to facilitate recognition of Hodgkin and Reed-Sternberg (H-RS) cells: CD3, CD20, CD30, CD15, and EMA. The H-RS cells of 62 classic Hodgkin lymphomas were bcl-2-positive in 35 cases (56.45%), p53-positive in 14 (22.58%), and positive for both EBV latent membrane protein-1 and EBER in 37 (59.68%); there was complete concordance of results for EBV by both procedures. No correlation was found between expression of bcl-2, p53, or EBV markers in H-RS cells and clinical stage (P > 0.05). Expression of Rb, Ki67, p21, and TopoIIalpha did, however, show significant differences with clinical stage. Expression of Rb and p21 in CD30-positive H-RS cells decreased with more advanced stage (P < 0.001). In contrast, Ki67 and ToPoIIalpha expression increased with later stage (P < 0.01). No correlation was found between expression of any of these markers in H-RS cells and the subtypes of nodular sclerosis HL, mixed cellularity HL, and LRHL (P > 0.05). TUNEL was found in the nonneoplastic cellular background in all cases and in H-RS cells in only 10 of 62 cases (16.12%) (8 nodular sclerosis HL, 1 mixed cellularity HL, and 1 LRHL). There was a significant correlation between high expression of bcl-2 and a low score by TUNEL (P < 0.05). These data are consistent with the notion that overexpression of bcl-2 may be linked to blockage of apoptosis-mediated death of H-RS cells in classic HL. Abnormal expression of p53-related protein may not play a major role in HL, because it is present in H-RS cells in only a minority of cases. Increased expression of Ki67 and TopoIIalpha by H-RS cells is significantly associated with advanced stage and may indicate aggressive disease. Adverse clinical outcome in HL also is associated with loss of Rb and p21 protein expression, consistent with the possible roles of Rb and p21 in inhibition of the growth of H-RS cells. Within the limitations of the methods used, almost two thirds of cases of HL provide evidence of an association with EBV. The tissue microarray technique is valuable not only for examination of large numbers of cases of a disease by a complex panel of markers but also potentially as a control for staining quality in immunohistochemistry and in situ hybridization.

Physical and functional interaction between HCV core protein and the different p73 isoforms.

Hepatitis C virus (HCV) core protein is a structural viral protein that packages the viral genomic RNA. In addition to this function, HCV core also modulates a number of cellular regulatory functions. In fact, HCV core protein has been found to modulate the expression of the cyclin-dependent inhibitor p21(WAF1/CIP1) and to promote both apoptosis and cell proliferation through its physical interaction with p53. Here, we studied the ability of HCV core to bind the p53-related p73 protein, its isoforms and its deletion mutants. We found that HCV core co-immunoprecipitated with p73 in HepG2 and SAOS-2 cells. Deletion mutational analysis of p73 indicates that the domain involved in HCV core binding is located between amino-acid residues 321-353. We also demonstrate that p73/core interaction results in the nuclear translocation of HCV core protein either in the presence of the p73 alpha or p73 beta tumor-suppressor proteins. In addition, the interaction with HCV core protein prevents p73 alpha, but not p73 beta dependent cell growth arrest in a p53-dependent manner. Our findings demonstrate that HCV core protein may directly influence the various p73 functions, thus playing a role in HCV pathogenesis.

Identification of CGI-121, a novel PRPK (p53-related protein kinase)-binding protein

PRPK (p53-related protein kinase) has been reported as a novel protein kinase which binds to the tumor suppressor protein p53 and induces phosphorylation of p53 at Ser 15. To identify novel binding partners of PRPK, we performed a yeast two-hybrid screening and isolated an expressed sequence tag CGI-121 by which a 20-kDa protein was encoded. We demonstrated the protein–protein interaction of CGI-121 with PRPK in vivo and in vitro. The protein expression of CGI-121 was observed in many cell lines and was immunocytochemically identified in both the nucleus and cytosol. Although PRPK interacted with both CGI-121 and p53, several attempts to demonstrate an association between CGI-121 and p53 were unsuccessful. In addition, coprecipitation of p53 using recombinant PRPK was inhibited by adding recombinant CGI-121 in vitro, suggesting that CGI-121 could act as a potent inhibitor of the binding of PRPK to p53.

Mutations in the Retinoblastoma-related Gene RB2 / p130 in Adult T-cell Leukaemia/Lymphoma

The retinoblastoma (Rb) family consists of the tumor suppressor Rb/p105 and related proteins p107 and Rb2/p130. Although the involvement of the RB / p105 gene in Adult T-cell leukaemia/lymphoma (ATL) has been studied, no mutational data is reported regarding the RB2 / p130 gene in ATL. We screened for mutations of the RB2 / p130 gene. Mutation was detected in 1 of 41 primary ATL sample. This is the first report describing mutation of the RB2 / p130 gene in ATL, suggesting that RB2/p130 may be involved in the development of ATL, and may behave as a tumor suppressor gene in T lymphocytes.

E2F Mediates Sustained G2 Arrest and Down-regulation of Stathmin and AIM-1 Expression in Response to Genotoxic Stress

Exposure of cells to genotoxic agents results in activation of checkpoint pathways leading to cell cycle arrest. These arrest pathways allow repair of damaged DNA before its replication and segregation, thus preventing accumulation of mutations. The tumor suppressor retinoblastoma (RB) is required for the G(1)/S checkpoint function. In addition, regulation of the G(2) checkpoint by the tumor suppressor p53 is RB-dependent. However, the molecular mechanism underlying the involvement of RB and its related proteins p107 and p130 in the G(2) checkpoint is not fully understood. We show here that sustained G(2)/M arrest induced by the genotoxic agent doxorubicin is E2F-dependent and involves a decrease in expression of two mitotic regulators, Stathmin and AIM-1. Abrogation of E2F function by dominant negative E2F abolishes the doxorubicin-induced down-regulation of Stathmin and AIM-1 and leads to premature exit from G(2). Expression of the E7 papilloma virus protein, which dissociates complexes containing E2F and RB family members, also prevents the down-regulation of these mitotic genes and leads to premature exit from G(2) after genotoxic stress. Furthermore, genotoxic stress increases the levels of nuclear E2F-4 and p130 as well as their in vivo binding to the Stathmin promoter. Thus, functional complexes containing E2F and RB family members appear to be essential for repressing expression of critical mitotic regulators and maintaining the G(2)/M checkpoint.

P21 Functions to Maintain Quiescence of p27-deficient Hepatocytes

Hepatocytes rarely proliferate in the healthy adult liver. We explored the roles of the cyclin kinase inhibitors p21 and p27 in maintaining hepatocyte quiescence. p27 is expressed throughout the wild-type liver, but the related protein p21 was not detected. However, p21 was detected in livers of p27-deficient mice. Increased p21 protein levels did not result from an increase in p21 mRNA expression, indicating that p21 expression is regulated post-transcriptionally. p21 protein levels increased in cultured primary hepatocytes treated with the proteasome inhibitor MG132 and cycloheximide, indicating that p21 expression is regulated at the level of protein stability in liver cells. Although increased expression of cyclin-dependent kinase (Cdk) 4, Cdk2, and proliferating cell nuclear antigen was detected in p27-deficient livers, increased hepatocyte proliferation was detected only in livers of mice deficient for both p21 and p27. In p27-deficient livers, p21 was found in complexes with Cdk2 and CdK4 and can compensate for the absence of p27. Our data indicate that cyclin kinase inhibitor activity is important for maintaining hepatocyte quiescence in the adult liver. Significant increases in p21 were detected in multiple tissues of mature p27-deficient mice compared with wild-type mice, suggesting that the ability of p21 to functionally substitute for p27 is not liver-specific.