p27Kip1 is required for PTEN-induced G1 growth arrest

The tumor suppressor PTEN is one of the most commonly inactivated genes in human cancer. Glioblastoma multiforme cells harboring mutant PTEN have abnormally high levels of 3' phosphoinositides and elevated protein kinase B activity. Expression of wild-type PTEN in glioma cells, containing endogenous mutant PTEN, reduces 3' phosphoinositides levels, inhibits PKB activity, and induces G1 cell cycle arrest. We investigated the mechanism of the PTEN-induced growth arrest in glioma cell lines. Expression of PTEN is associated with increased expression of p27Kip1, decreased expression of cyclins A and D3, inhibition of cdk2 activity, and dephosphorylation of pRb. Inactivation of p53, by the human papilloma virus E6 oncoprotein, does not prevent PTEN-induced G1 arrest, implying that p53 is not required for G1 arrest. In contrast, p27Kip1 antisense oligonucleotides abrogated the growth arrest induced by PTEN. Furthermore, blocking p27Kip1 expression prevented the PTEN-induced reduction of cyclin-dependent kinase 2 activity, indicating that p27Kip1 functions upstream of cyclin-dependent kinase 2 in the PTEN regulatory cascade. These results implicate p27Kip1 as a critical mediator of PTEN-induced G1 arrest.     

Role of LXCXE motif-dependent interactions in the activity of the retinoblastoma protein

Cell cycle control by pRb requires the integrity of the pocket domain, which is a region necessary for interactions with a variety of proteins, including E2F and LXCXE-motif containing proteins. Through knowledge of the crystal structure of pRb we have prepared a panel of pRb mutant derivatives in which a cluster of lysine residues that demark the LXCXE peptide binding domain were systematically mutated. One of the mutant derivatives, Rb6A, exhibits significantly reduced LXCXE-dependent interactions with HPV E7, cyclinD1 and HDAC2, but retained LXCXE-independent binding to E2F. Consistent with these results, Rb6A could down-regulate E2F-1-dependent activation of different E2F responsive promoters, but was compromised in Rb-dependent repression. Most importantly, Rb6A retained wild-type growth arrest activity, and colony forming activity similar to wild-type pRb. It is compatible with these results that directly targeting HDAC2 to E2F responsive promoters as an E2F/HDAC hybrid protein failed to effect cell cycle arrest. These results suggest that LXCXE-dependent interactions are not essential for pRb to exert growth arrest.     

Interaction between HIV-1 Tat and pRb2/p130: a possible mechanism in the pathogenesis of AIDS-related neoplasms

Tat protein is an early nonstructural protein necessary for virus replication, which is secreted by infected cells and taken up by uninfected cells. Extensive evidence indicates that Tat may be a cofactor in the development of AIDS-related neoplasms. The molecular mechanism underlying Tat's oncogenic activity may include deregulation of cellular genes. Among these genes, it has recently been shown that pRb2/p130 oncosuppressor protein is one of the targets in the interaction between HIV gene product Tat and host proteins. However, whether the HIV-1 gene product Tat may inactivate the oncosuppressive function of pRb2/p130 has not yet been elucidated. Here, we show that mRNA levels of pRb2/p130 increase in the presence of Tat, whereas no change in the phosphorylation status of pRb2/p130 is observed. In addition, Tat can inhibit the growth control activity exerted by pRb2/p130 in the T98G cell line. Finally, Tat does not compete with E2F-4 in binding to pRb2/p130. The interaction between Tat and pRb2/p130 seems to result in the deregulation of the control exerted by pRb2/p130 on the cell cycle. Taken together, these results open a window on the role of pRb2/p130 in AIDS-related oncogenesis.     

Inositol hexaphosphate inhibits growth,and induces G1 arrest and apoptotic death of prostate carcinoma DU145 cells: modulation of CDKI-CDK-cyclin and pRb-related protein-E2F complexes

Cancer chemopreventive effects of inositol hexaphosphate (IP6), a dietary constituent, have been demonstrated against a variety of experimental tumors, however, limited studies have been done against prostate cancer (PCA), and molecular mechanisms are not well defined. In the present study, we investigated the growth inhibitory effect and associated mechanisms of IP6 in advanced human PCA cells. Advanced human prostate carcinoma DU145 cells were used to study the anticancer effect of IP6. Flow cytometric analysis was performed for cell cycle progression and apoptosis studies. Western immunoblotting, immunoprecipitation and kinase assay were performed to investigate the involvement of G1 cell cycle regulators and their interplay, and end point markers of apoptosis. A significant dose- as well as time-dependent growth inhibition was observed in IP6-treated cells, which was associated with an increase in G1 arrest. IP6 strongly increased the expression of CDKIs (cyclin-dependent kinase inhibitors), Cip1/p21 and Kip1/p27, without any noticeable changes in G1 CDKs and cyclins, except a slight increase in cyclin D2. IP6 inhibited kinase activities associated with CDK2, 4 and 6, and cyclin E and D1. Further studies showed the increased binding of Kip1/p27 and Cip1/p21 with cyclin D1 and E. In down-stream of CDKI-CDK/cyclin cascade, IP6 increased hypophosphorylated levels of Rb-related proteins, pRb/p107 and pRb2/p130, and moderately decreased E2F4 but increased its binding to both pRb/p107 and pRb2/p130. At higher doses and longer treatment times, IP6 caused a marked increase in apoptosis, which was accompanied by increased levels of cleaved PARP and active caspase 3. IP6 modulates CDKI-CDK-cyclin complex, and decreases CDK-cyclin kinase activity, possibly leading to hypophosphorylation of Rb-related proteins and an increased sequestration of E2F4. Higher doses of IP6 could induce apoptosis and that might involve caspases activation. These molecular alterations provide an insight into IP6-caused growth inhibition, G1 arrest and apoptotic death of human prostate carcinoma DU145 cells.     

Identification of a novel activity of human papillomavirus type 16 E6 protein in deregulating the G1/S transition

In this study we show that E6 of human papillomavirus has the ability to deregulate the cell cycle G1/S transition. In rodent immortalized fibroblasts (NIH3T3) serum deprivation or over-expression of the cyclin-dependent kinase inhibitors, p16(INK4a) or p27(KIP1), leads to G1 cell cycle arrest. HPV16 E6 overcomes the antiproliferative signals, gaining the ability to drive serum-deprived and p16(INK4a) or p27(KIP1) over-expressing cells into S phase. E6 protein from the benign HPV type 1 displays a similar activity to HPV16 E6 to deregulate the G1/S transition. Thus, this activity appears to be conserved between E6 proteins from non-oncogenic and oncogenic HPV types. Furthermore, we show that HPV16 E6 is not able to circumvent a G1 arrest imposed by pRb mutant in which all CDK phosphorylation sites have been mutated. These data indicate that the viral protein acts upstream of pRb and its mechanism in promoting cell cycle progression is dependent on pRb phosphorylation. In summary, this study describes a novel biological function of HPV E6 and shows that the S phase entry, required for viral DNA replication, is not exclusively controlled by E7, but that E6 also is involved in this event.     

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.     

p16INK4a, but not constitutively active pRb, can impose a sustained G1 arrest: molecular mechanisms and implications for oncogenesis.

p16ink4 and pRb, two components of a key G1/S regulatory pathway, and tumor suppressors commonly targeted in oncogenesis, are among the candidates for gene therapy of cancer. Wild-type p16 and a constitutively active pRb(delta cdk) mutant both blocked G1 in short-term experiments, but only p16 imposed a sustained G1 arrest. Unexpectedly, cells conditionally exposed to pRb(delta cdk) entered S phase after 2 days, followed by endoreduplication between days 4-6. The distinct phenotypes evoked by p16 vs pRb(delta cdk) appear mediated by cyclin E/CDK2 which, while active in the pRb(delta cdk)-expressing cells, became rapidly inhibited through restructuring diverse cyclin/CDK/p21 complexes by p16. These results provide novel insights into the roles of p16, pRb and cyclin E in G1/S control and multistep oncogenesis, with implications for gene therapy strategies.     

Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase delta (p125)

The retinoblastoma gene product (pRb) interacts with many cellular proteins to function in the control of cell division, differentiation, and apoptosis. Several pRb binding proteins complex with pRb through an amino acid sequence called the LXCXE motif. The catalytic subunit of DNA polymerase delta (p125) contains a LXCXE motif. To further study the biochemical function of this polymerase, we sought to determine if p125 interacts with pRb. Experiments using GST-pRb fusion proteins showed that p125 from breast epithelial (MCF10A) cell extracts associates with pRb. In addition, GST-p125 fusion proteins bound pRb from the same cell extracts. The pRb that associated with GST-p125 was largely unphosphorylated. Coimmunoprecipitation experiments using cell cycle synchronized cells revealed that p125 and pRb form a complex predominantly during G1 phase, the phase during which pRb is mostly unphosphorylated. In vitro phosphorylation of GST-pRb by the cyclin dependent kinases reduced the ability of p125 to associate with GST-pRh. Addition of the LXCXE containing protein SV40 large T antigen to GST-pRb blocks the ability of p125 to associate with pRb, suggesting that it may be through a LXCXE sequence by which p125 interacts with pRb. Finally, in vitro polymerase assays demonstrate that GST-pRb fusion protein stimulates DNA polymerase delta activity.     

Cytokine response gene 6 induces p21 and regulates both cell growth and arrest

Cytokine response gene #6 (CR6), cloned from interleukin 2-stimulated T lymphocytes, is homologous to GADD45 and MyD118, genes which promote cell cycle arrest and apoptosis. To determine how this gene family could possibly mediate both cell survival/proliferation and cell cycle arrest/death, transfectants were generated so that the genes could be expressed ectopically, independently from their normal inducing agents. In cycling retinoblastoma protein-negative (pRb-) cells, ectopic CR6 expression blocked G2/M transition, but did not prevent G1/S transition so that endoreduplication resulted. By comparison, when CR6, GADD45, and MyD118 genes were expressed ectopically in proliferating pRb+ cells, either G1/S or G2/M transition was effectively blocked, so that there was no endoreduplication. Consistent with these findings, in proliferating pRb-cells, ectopic expression of CR6 promoted the expression of both G1 and G2/M cyclins. By comparison, in pRb+ cells, the expression of G1 cyclins was increased, while expression of the mitotic cyclins was decreased. However, in pRb+ cells, cyclin-dependent kinase activities associated with both G1 and G2/M cyclins were decreased. Moreover, ectopic expression of all three genes resulted in the expression of the CKI, p21, both in pRb- and pRb+ cells. The physiologic induction of CR6 expression by IL2 in quiescent normal human T cells occurs transiently in the first half of G1, coordinately with the expression of p21. Therefore, this gene family regulates G1 and G2, and promotes either cell growth or arrest by a common mechanism.     

Expression of cell-cycle-regulated proteins pRb2/p130, p107, p27(kip1), p53, mdm-2, and Ki-67 (MIB-1) in prostatic gland adenocarcinoma.

PURPOSE: The quest for prognostic molecular markers in prostatic carcinoma is still in progress. Many proteins have already been screened by immunohistochemistry with the aim to find the most reliable indicator of progressive disease. In this study, we evaluated the expression of pRb2/p130, p107, p27(kip1), p53, mdm-2, and Ki-67 (MIB-1) by immunohistochemistry in 24 prostate carcinomas compared with the paired expression of normal prostates. EXPERIMENTAL DESIGN: Expression of the different proteins in normal and pathological specimens was evaluated by the Wilcoxon test. A matrix of correlation (Spearman coefficient) was used to evaluate the possible association in expression among the different proteins. Logistic regression analysis was used to test the multivariable prognostic value of the levels of protein expression for the probability of disease development. RESULTS: p53 and Ki-67 (MIB-1) showed a higher expression in cancer than in normal tissue (P = 0.006 and <0.001, respectively). pRb2/p130, p107, and p27(kip1) showed an overall lower expression in cancer, but the difference between cytoplasmic and nuclear expression was always higher for cancer (Ps, from <0.001 to 0.016). mdm-2 expression was lower in cancer, but the difference between cytoplasmic and nuclear expression was not significant (P = 0.571) when compared with that in normal tissue. A positive correlation between p27 and pRb2/p130 levels expressed, in normal and cancer counterparts in the same sample, as the difference between cytoplasmic and nuclear protein concentrations (P = 0.045) was found. Additionally, p107 expression showed an inverse correlation with Ki-67 (MIB-1) expression in the most aggressive tumors (P = 0.046). Logistic regression output showed that Ki-67 (MIB-1) and pRb2/p130 (expressed as differences between cytoplasmic and nuclear concentrations) were the variables associated with a higher risk of cancer. The highest value was reported for Ki-67 (MIB-1) (odds ratio, 2.11), followed by pRb2/p130 (odds ratio, 1.01). pRb2/p130 alone was associated with a sensitivity (rate of cases having a posterior probability of disease >/=0.5) of 61% with a false positive rate of 22%. Ki-67 (MIB-1) alone yielded a sensitivity of 69% and a false positive rate of 14%. The combined model (Ki-67 + pRb2/p130) yielded a sensitivity of 83% with a false positive rate of 17%. Interestingly, one specimen in which we also found a high-grade prostatic intraepithelial neoplasia showed the progressive loss of pRb2/p130 from normal prostatic cells to prostatic intraepithelial neoplasia cells, suggesting that in prostatic cancer, lack of expression of the tumor suppressor gene pRb2/p130 could be involved in the progression of the disease, from an early stage. CONCLUSIONS: This study showed that all of the proteins but mdm-2 were expressed at a different rate in normal and pathological prostate specimens. Multivariate analysis showed that pRb2/p130 and p107 may be involved in the pathogenesis and progression of prostate cancers, and that the expression of the retinoblastoma-related protein pRb2/p130 along with Ki-67 (MIB-1), expressed as differences between cytoplasmic and nuclear concentrations, could be considered new parameters to be evaluated in discriminating patients at a higher risk for prostate cancer.     

The initiation of UV-induced G(1) arrest in human cells is independent of the p53/p21/pRb pathway but can be attenuated through expression of the HPV E7 oncoprotein.

It is well established that the initiation of G(1) arrest in cultured cells exposed to ionizing radiation (IR) is fully dependent upon the p53/p21waf1/pRb signaling cascade. However, the extent to which this pathway regulates G(1) arrest following exposure to UV is less clear. Here we demonstrate that primary human fibroblasts from either skin or lung, in which p53 has been functionally inactivated through expression of the human papillomavirus E6 oncoprotein, each undergoes a prolonged G(1) arrest upon UV irradiation. This same phenomenon is also observed for UV-exposed human tumor cell strains that are genetically deficient for p53, p21waf1 and/or pRb. Furthermore, for the isogenic wild-type counterparts of these primary and tumor cell strains, the onset of UV-induced G(1) arrest precedes any increase in the ratio of hypo- to hyper-phosphorylated pRb and virtually the entire period of growth arrest occurs in the absence of p21waf1 induction. The above data on UV-treated cells are in contrast to the expected situation for IR, for which G(1) arrest is abolished in all deficient cell lines, and, in the wild-type counterparts, correlates precisely with p21waf1 induction and an increase in the ratio of hypo- to hyper-phosphorylated pRb. Remarkably, it was observed that both IR- and UV-induced G(1) arrest are significantly attenuated in primary fibroblasts expressing the human papillomavirus E7 oncoprotein, which functionally inactivates pRb in addition to many other cellular proteins. Our findings conclusively demonstrate that the p53/p21/pRb cascade is not essential for the initiation of G(1) arrest in UV-exposed human cells and, furthermore, indicate the involvement in this process of any among a number of human papillomavirus E7-interacting cellular proteins.