Active pRb Research Articles

Chromatin composition alterations and the critical role of MeCP2 for epigenetic silencing of progesterone receptor-B gene in endometrial cancers.

To understand the epigenetic mechanism underlying the PR-B gene silencing in endometrial cancer (EC) cells, we compared the chromatin composition between transcriptionally active and silenced PR-B genes in EC cell lines and cancer tissues. Chromatin Immunoprecipitation (ChIP) assay was performed to measure MBD occupancy and histone acetylation/methylation in transcriptionally active and silenced PR-B genes. PR-B-positive/-negative, as well as epigenetic inhibitor-treated/-untreated EC cells were used as study models. Real-time polymerase chain reaction (PCR) and Western blot analysis were applied to measure the mRNA and protein levels of PR-B, MBD, and histones. A close association among PR-B methylation, MBD binding and PR-B gene silencing was observed. Treatment with epigenetic inhibitors led to dynamic changes in the PR-B chromatin composition and gene expression. Increased H3/H4 acetylation and H3-K4 methylation, and decreased H3-K9 methylation were found to be associated with re-activation of silenced PR-B genes. MeCP2 knockdown resulted in a decreased MeCP2 binding to PR-B genes and an increased PR-B expression. ChIP analysis of MeCP2 binding to PR-B genes in the PR-B-positive/-negative EC samples confirmed the significant role of MeCP2 in PR-B silencing. PR-B gene expression is regulated by a concerted action of epigenetic factors including DNA methylation, MBD binding, and histone modifications. MeCP2 occupancy of PR-B genes plays a critical role in PR-B gene silencing. These findings enriched our knowledge of the epigenetic regulation of PR-B expression in EC, and suggested that the epigenetic re-activation of PR-B could be explored as a potential strategy to sensitize the PR-B-negative endometrial cancers to progestational therapy.

Microrna-155 Controls The RB/E2F Axis In Normal and Malignant B Cells Via The Non-Canonical TGFβ1-SMAD5 Signaling Module

MicroRNA-155 (miR-155) plays pleiotropic roles in the biology of normal and malignant B cells. MiR-155 knockout (KO) mice have fewer germinal center B cells, while overexpression of this miRNA is associated with aggressive DLBCL. Although several miR-155 targets have been identified, a mechanism that unifies the features of loss and gain of miR-155 function in normal and malignant cells remains to be described. In B cells, TGFβ signals are suppressive indicating that deregulation of this pathway may interfere with the developmental regulation of lymphocytes and contribute to the pathogenesis of B cell malignancies. Earlier, we described the direct targeting of the transcription factor SMAD5 by miR-155, and uncovered the presence of non-canonical signaling model in B cell lymphomas whereby TGFβ1, a cytokine that typically activates SMAD2/3, phosphorylated SMAD5. Herein, we used the miR-155 KO mice and genetically modified DLBCL cell lines to investigate which downstream effectors of TGFβ signals are disrupted by the miR-155/SMAD5 interaction, thus shedding light on the phenotypes associates with miR-155 loss and gain of function.We confirmed the phosphorylation of SMAD5 by TGFβ1 in DLBCL cell lines, and demonstrated for the first time that this non-canonical signal is also present in untransformed normal mature B cells. We stably expressed miR-155 in the TGFβ1-responsive DLBCL cell lines Ly7, Ly18 and DHL5, and readily detected suppression of SMAD5, but not of other SMADs. TGFβ1 cytostatic activities include up-regulation of p15 and p21, which are primarily found in the context of SMAD2/3 activation. However, we found that stable expression of miR-155, and downregulation of SMAD5, significantly limited TGFβ1-dependent induction of both p15 and p21 in DLBCL. TGFβ1-mediated upregulation of p15 and p21 limits the activity of cyclin/CDK complexes, enriches for hypophosphorylated (active) RB, and promotes cell cycle arrest. We measured the effects of miR-155 in this process, and found that the accumulation of hypophosphorylated RB following TGFβ1 exposure was blunted in miR-155 expressing cells, resulting in an impaired G0/G1 arrest. The impact of miR-155 on TGFβ1 activity was also detectable by directly measuring the phosphorylation levels of RB's Ser780 residue. Active pRB blocks cell cycle progression at least in part by binding to and inhibiting the E2F family of transcriptional regulators. Thus, we performed co-immunoprecipitation experiments and quantified the levels of RB-bound E2F1. In these assays, following TGFβ1 exposure we found a markedly decreased pRB-E2F1 complex formation in miR-155 expressing cells when compared to their controls. In agreement with these data, DLBCL cell lines expressing miR-155 displayed higher levels of free E2F1. Together, these data suggested the existence of a miR-155-SMAD5-p15/p21 axis that regulates TGFβ1 effects towards RB and E2F in DLBCL. To confirm the specific role of each component in this circuit, we used an RNAi strategy to transiently or stably knockdown (KD) SMAD5, p15 or p21 in our DLBCL models. In control RNAi cells, exposure to TGFβ1 led to decrease in RB phosphorylation, whereas these effects were abrogated upon KD of each of these genes, resulting in accumulation of hyperphosphorylated RB, a phenocopy of miR-155 expression. To define if the interplay between miR-155/SMAD5 and RB was also present in non-malignant cells, we purified mature B lymphocytes from miR-155 WT and KO mice. Examination of four pairs of mice, showed a higher expression of SMAD5 in cells from miR-155 KO than WT mice. In addition, TGFβ1-mediated suppression of phospho-RB was consistently more pronounced in miR-155 KO than in WT B cells, which resulted in a significantly higher G0/G1 arrest in cells lacking this miRNA. Of note, in absence of TGFβ1 there was no significant difference in cell cycle profile of mature B cells from miR-155 WT and KO mice. We concluded that an unrestrained TGFβ activity, secondary to SMAD5 upregulation, may help explain the deficient germinal center B cells formation found in miR-155 KO mice.Together, our findings demonstrate that miR-155 overexpression is a novel model for deregulation of the lymphomagenic RB/E2F axis, and define an unsuspected role for the non-canonical TGFβ1 activation of SMAD5 in the developmental regulation of mature B cells. Disclosures:No relevant conflicts of interest to declare.

A transcriptionally active pRb–E2F1–P/CAF signaling pathway is central to TGFβ-mediated apoptosis

Transforming growth factor-β (TGFβ) modulates the expression of multiple apoptotic target genes; however, a common and central signaling pathway, acting downstream of TGFβ and leading to cell death, has yet to be uncovered. Here, we show that TGFβ-induced apoptosis in cancer cells requires the transcription factor E2F1 (E2 promoter-binding factor 1). Using the E2F1 knockout mouse model, we also found E2F1 to be required for TGFβ-mediated apoptosis in normal cells. Moreover, we found TGFβ to increase E2F1 protein stability, acting at the post-translational level. We further investigated the molecular mechanisms by which E2F1 contributes to TGFβ-mediated apoptosis and found that TGFβ treatment led to the formation of a transcriptionally active E2F1–pRb–P/CAF complex on multiple TGFβ pro-apoptotic target gene promoters, thereby activating their transcription. Together, our findings define a novel process of gene activation by the TGFβ-E2F1 signaling axis and highlight E2F1 as a central mediator of the TGFβ apoptotic program.

PI3K a Selective Inhibitor Induces Growth Inhibition In Mantle Cell Lymphoma

AbstractAbstract 1812Deregulation of the phosphatidylinositol 3-kinase (PI3K)-mediated signaling plays an important role in the development of cell proliferation of mantle cell lymphoma (MCL).The PI3K pathway activation in MCL has been shown to result from constitutive B cell receptor (BCR) activation which is directly mediated by the Class IA PI3K p110 isoforms (a, β, and d). However, their relative contribution in MCL is not fully understood. In this study, the activity and molecular mechanisms of isoform-selective PI3K inhibitors which target different isoforms of the p110-kDa subunit has been investigated. We utilized the isoform-selective PI3K inhibitors; PI3-Ka inhibitor IV (p110a), TGX115 (p110b), IC87114 (p110d) and the non-specific PI3K inhibitor LY294002 (all inhibitors were purchased commercially).The p110a and p110d but not p110b isoform protein expression was detected in all tested MCL cell lines (Granta 519, JVM-2, Z138, Jeko-1, MINO). PI3-Ka inhibitor IV as well as non-specific PI3K inhibitor LY294002 induced cell growth inhibition with dose-dependent manner (IC50 at 48 hrs; PI3-Ka inhibitor IV: 17.5 μM for Granta 519, 14.3 μM for Jeko-1, 16.5 μM for Z138, LY294002: 14.8 μM for Granta 519, 19.4 μM for Jeko-1, 15.0 μM for Z138, MTT test). However, neither IC87114 nor TGX115 showed significant cell growth inhibition up to 40mM. Low dose of PI3-Ka inhibitor IV (5 μM) or LY294002 (5 μM) induced G0/G1 cell cycle arrest (increase of G0/G1 phase: PI3-Ka inhibitor IV 17.9 % for Granta 519, 28.2 % for Jeko-1, LY294002 19.3 % for Granta 519, 14.5 % for Jeko-1), and the higher dose (10 μM) increased apoptosis(specific apoptosis: PI3-Ka inhibitor IV 10.8 % for Granta 519, 15.3 % for Jeko-1, LY294002 13.6 % for Granta 519, 19.6 % for Jeko-1). No induction of cell cycle arrest/apoptosis by IC87114 or TGX115 treatment was observed. We then tried to assess the inhibition of PI3K/Akt signaling activation by p110a and p110d inhibitors. PI3-Ka inhibitor IV (10 μM) completely diminished phosphorylated (p-) Akt in all cell lines analyzed. Further investigation with 1–10 μM PI3-Ka inhibitor IV or IC87114 in Granta 519 and Jeko-1 cells declared that 1 μM PI3-Ka inhibitor IV almost diminished p-Akt and p-S6rp in both cells. The phosphorylation level of other PI3K/Akt signaling downstream substrates, GSK3-b and 4E-BP1, were down-regulated in dose dependent manner. Recently, GSK3-b kinase has been shown to negatively regulate cell cycle progression through Cyclin D1 repression in MCL. We observed that PI3-Ka inhibitor IV decreased Cyclin D1 expression and active pRb which are responsible for G0/G1 cell cycle arrest. The treatment with IC87114 (10 μM) performed moderate decrease of p-Akt, p-S6rp, and p-4E-BP, while no change in the levels of p-GSK3-b, Cyclin D1, or p-pRb was observed in both Granta 519 and Jeko-1 cells. We also tested whether the combination of PI3-Ka inhibitor IV or IC87114 with the proteasome inhibitor bortezomib induces synergistic cytotoxicity in MCL. No synergistic anti-proliferative effect was observed in any of the MCL cell lines analyzed. These findings demonstrate that p110a may be the responsible Class IA PI3K isoform for the development of MCL cell proliferation, and p110a isoform-selective PI3K inhibitor but not p110d or p110b inhibitors may provide a better therapeutic index relative to pan-PI3K inhibitors. Disclosures:No relevant conflicts of interest to declare.

Induction of cell cycle arrest and apoptosis by 5-fluorouracil in laryngeal cancer cells containing HPV16 E6 and E7 oncoproteins

ObjectiveTo investigate the cytotoxic effect of 5-fluorouracil (5Fu) on HPV16-associated laryngeal cancer cells. Design and methodsCytotoxicity assay and TUNEL assay were used to assess the effect of 5Fu on laryngeal cancer cells transfected with HPV16 E6 or E7. Results5Fu induced apoptosis in the cells either with or without HPV16 in a dose- and time-dependent manner. 5Fu caused the accumulation of active pRb and p21WAF1/CIP1, together with an increase in Bak and Bax expression and a decrease in Bcl-2 levels in all the transfected cells. G1/S phase cell cycle arrest was associated with the antiproliferation activity of 5Fu. 5Fu also presented some effects on the E6 and E7 oncoproteins. ConclusionsHPV16 E6 and E7 oncoproteins do not prevent 5Fu-medicated apoptosis and G1/S cell arrest in laryngeal cancers. The sensitivity of 5Fu treatment is associated with the decrease of Bcl-2 and/or the increase in Bak and p21WAFI/CIP1.

A Functional Genetic Screen Identifies TFE3 as a Gene That Confers Resistance to the Anti-proliferative Effects of the Retinoblastoma Protein and Transforming Growth Factor-β

A Functional Genetic Screen Identifies TFE3 as a Gene That Confers Resistance to the Anti-proliferative Effects of the Retinoblastoma Protein and Transforming Growth Factor-β

A Confederacy of Kinases: Cdk2 and Cdk4 Conspire to Control Embryonic Cell Proliferation

The mouse embryo is surprisingly resistant to loss of individual cyclins or cyclin-dependent kinases. In a recent issue of Developmental Cell, Berthet et al. (2006) describe collaboration of Cdk2 and Cdk4 in embryogenesis that is revealed only upon their simultaneous loss, resulting in inappropriate activation of the retinoblastoma protein and embryonic lethality.

Up-Regulation of the Progesterone Receptor (PR)-C Isoform in Laboring Myometrium by Activation of Nuclear Factor-κB May Contribute to the Onset of Labor through Inhibition of PR Function

Progesterone acting via the progesterone receptor (PR) plays a critical role in maintaining uterine quiescence during pregnancy. In the present study, we tested the hypothesis that the transactivating capability of the PR is down-regulated in the myometrium at term by a change in uterine PR isoform ratio resulting from local activation of the nuclear factor (NF)-kappaB pathway. Overexpression of the truncated PR-C isoform in human myometrial cells inhibited PR-B transactivation. Expression of PR isoforms, PR-A, PR-B, and PR-C, was characterized by immunoblotting and quantitative PCR (Q-PCR) in fundal and lower uterine segment myometrium from pregnant women in labor and not in labor and in the pregnant mouse uterus during late gestation. We observed a marked increase in levels of PR-C and transcriptionally active PR-B specifically in fundal myometrium of women in labor. In pregnant mouse uterus, levels of PR-B and PR-C also increased between 15 days post coitum and term, whereas expression of PR-A was dramatically up-regulated at 19 days post coitum. In studies of uterine tissues of mice injected intraamniotically with surfactant protein A and of human myometrial and T47D breast cancer cells in culture, up-regulation of PR isoform expression was observed in response to activation of the NF-kappaB pathway. Chromatin immunoprecipitation analysis revealed IL-1beta induced binding of NF-kappaB to the PR promoter. Collectively, these findings suggest that up-regulation of inhibitory PR isoform expression by NF-kappaB activation in both laboring human fundus and pregnant mouse uterus near term may inhibit PR transactivation and thereby lead to a loss of uterine quiescence and the onset of labor.

Inhibition of hypoxia-induced apoptosis by modulation of retinoblastoma protein-dependent signaling in cardiomyocytes.

Apoptotic cell death is an important mode of cell loss contributing to heart dysfunction. To analyze the importance of the E2F-dependent regulation of gene transcription in cardiomyocyte apoptosis, the function of cell cycle factors impinging on the retinoblastoma protein (pRb)/E2F pathway was investigated. In isolated neonatal ventricular myocytes, apoptotic cell death induced by hypoxia (deferoxamine, 100 micro mol/L) specifically activated cyclin-dependent kinases (cdks) 2 and 3. Apoptotic cell death was inhibited by ectopic expression of cdk inhibitors p21(CIP) and p27(KIP1) but not p16(INK4). In addition, apoptosis was also abrogated by forced expression of kinase dead mutant proteins of cdk2/3 but not of cdk4/6. Introduction of cdk inhibitors or dominant-negative cdk2/3 blocked pRb hyperphosphorylation and abrogated E2F-dependent gene transcription, including that of the E2F-responsive genes of proapoptotic caspase 3 and caspase 7. Moreover, introduction of constitutively active pRb and transcriptionally inert mutant E2F1/DP1 efficiently protected cardiomyocytes from apoptosis. In conclusion, these data demonstrate that cdk-specific inactivation of pRb and the subsequent activation of E2F-dependent gene transcription are required for cardiomyocyte apoptosis.

A key role of the hSNF5/INI1 tumour suppressor in the control of the G1-S transition of the cell cycle.

The hSNF5/INI1 gene encodes a member of the SWI/SNF chromatin remodelling complexes. It was recently identified as a tumour suppressor gene mutated in sporadic and hereditary Malignant Rhabdoid Tumours (MRT). However, the role of hSNF5/INI1 loss-of-function in tumour development is still unknown. Here, we show that the ectopic expression of wild-type hSNF5/INI1, but not that of truncated versions, leads to a cell cycle arrest by inhibiting the entry into S phase of MRT cells. This G1 arrest is associated with down-regulation of a subset of E2F targets including cyclin A, E2F1 and CDC6. This arrest can be reverted by coexpression of cyclin D1, cyclin E or viral E1A, whereas it cannot be counteracted by pRB-binding deficient E1A mutants. Moreover, hSNF5/INI1 is not able to arrest cells lacking a functional pRB. These observations suggest that the hSNF5/INI1-induced G1 arrest is dependent upon the presence of a functional pRB. However, the observation that a constitutively active pRB can efficiently arrest MRT cells indicates that hSNF5/INI1, at the difference of the ATPase subunits of the SWI/SNF complex, is dispensable for pRB function. Altogether, these data show that hSNF5/INI1 is a potent regulator of the entry into S phase, an effect that may account for its tumour suppressor role.

Phosphorylation of pRb is required for HGF-induced muscle cell proliferation and is p27kip1-dependent.

Hepatocyte growth factor (HGF) plays a crucial role in the differentiation of skeletal muscle cells, where a process in which the retinoblastoma protein (pRb) has been implicated. We addressed the role of pRb in HGF-mediated effects on the proliferation and differentiation of adult skeletal muscle myoblasts. HGF shifted pRb to its hyperphosphorylation forms and increased the transactivation of E2F1, a transcription factor required for S phase entry. A constitutively active pRb mutant blocked HGF-dependent pRb phosphorylation and transactivation of E2F1 and increased cell proliferation. Accordingly, this mutant reversed the inhibitory effects of HGF on the expression of the cyclin-dependent kinase (CDK) inhibitor p27 and myogenic differentiation markers. HGF-mediated pRb phosphorylation was reversed by ectopic expression of p27, but neither the myogenic regulatory factor, MEF2, nor the myogenic inhibitory protein Twist had that effect. These results suggest that in response to HGF signaling, there is a decrease in p27 expression that results in an accumulation of hyperphosphorylated Rb protein, and subsequent progression of myoblasts into the G1 phase of the cell cycle.

Phosphorylation-dependent and -independent functions of p130 cooperate to evoke a sustained G1 block.

The retinoblastoma (pRb)-related p130 pocket protein is a regulator of cell growth and differentiation, and a candidate tumour suppressor. Both pRb and p130 operate through interactions with cellular proteins, including the E2F transcription factors. While such interactions are controlled by phosphorylation of multiple sites of pRb, regulation of p130 remains poorly understood. We now identify 22 in vivo phosphorylation sites of p130, targeted by diverse kinases, and present evidence for three cyclin-dependent kinase 4(6) [Cdk4(6)] specific phosphorylations, which appear critical for controlling the growth-restraining activity of p130. When expressed in U2OS cells, the phosphorylation-deficient mutant p130(Delta)(CDK4), in which the Cdk4 specific sites were mutated to alanine residues, imposed a more sustained G1 arrest than a constitutively active pRb(Delta)(CDK), known to repress all cellular E2F activity. Experiments using p130(Delta)(Cdk4) and another phosphorylation-deficient mutant, p130(PM19A), with 19 phosphorylation sites mutated, revealed that the p130-imposed G1 block reflects cooperative growth-suppressive effects of phosphorylation-regulated E2F binding and phosphorylation-independent sequestration of cyclin E(A)-Cdk2 through the N-terminal cyclin binding motif of p130.

Reversal of an antiestrogen-mediated cell cycle arrest of MCF-7 cells by viral tumor antigens requires the retinoblastoma protein-binding domain

Proliferation of MCF-7 cells is estrogen dependent and antiestrogen sensitive. In the absence of estrogens or presence of antiestrogens MCF-7 cells arrest in the G1 phase of the cell cycle, and this arrest is associated with an accumulation of the active, hypophosphorylated form of the retinoblastoma protein (pRb). Because active pRb negatively regulates passage from G1 to S phase, this suggests that pRb is a crucial target of estrogen action, and that its inactivation might lead to antiestrogen resistance. We tested this hypothesis by expressing viral tumor antigens (T antigens), which bind and inactivate pRb, in MCF-7 cells, and determining the effects on cell proliferation in the presence of antiestrogens. The results of these experiments demonstrate that T antigen expression confers antiestrogen resistance to MCF-7 cells. Using a panel of mutant T antigens, we further demonstrate that the pRb-binding, but not the p53 binding domain is required to confer antiestrogen resistance. Thus, pRb is an important target of estrogen action, and its inactivation can contribute to the development of antiestrogen resistance.

Inhibition of S-phase progression by adeno-associated virus Rep78 protein is mediated by hypophosphorylated pRb.

Adeno-associated virus (AAV) has an antiproliferative action on cells. We investigated the effect of the AAV replication proteins (Rep) on the cell division cycle using retroviral vectors. Rep78 and Rep68 inhibited the growth of primary, immortalized and transformed cells, while Rep52 and Rep40 did not. Rep68 induced cell cycle arrest in phases G(1) and G(2), with elevated CDK inhibitor p21 and reduced cyclin E-, A- and B1-associated kinase activity. Rep78-expressing cells were also impaired in S-phase progression and accumu lated almost exclusively with hypophosphorylated retinoblastoma protein (pRb). The differences between Rep78 and Rep68 were mapped to the C-terminal zinc finger domain of Rep78. Rep78-induced S-phase arrest could be bypassed by adenoviral E1A or papillomaviral E7 proteins but not by E1A or E7 mutants unable to bind pRb. Rb(-/-) primary mouse embryonic fibroblasts displayed a strongly reduced S-phase arrest when challenged with Rep78, compared with matched Rb(+/+) controls. These results suggest that physiological levels of active pRb can interfere with S-phase progression. We propose that the AAV Rep78 protein arrests cells within S-phase by a novel mechanism involving the ectopic accumulation of active pRb.

Involvement of Myc activity in a G(1)/S-promoting mechanism parallel to the pRb/E2F pathway.

The retinoblastoma protein (pRb)/E2F pathway regulates commitment of mammalian cells to replicate DNA. On the other hand, mitogen-stimulated cells deprived of E2F activity can still maintain physiologically relevant levels of cyclin E-dependent kinase activity and gradually enter S phase, suggesting the existence of a DNA synthesis-inducing mechanism parallel to the pRb/E2F axis. Here we show that regulatable ectopic expression of cyclin E or transcriptionally active Myc can rapidly induce DNA synthesis in U2OS-derived cell lines whose E2F activity is blocked by a constitutively active pRb (pRbDeltacdk) mutant. The effect of Myc is associated with Cdc25A phosphatase and cyclin E-CDK2 kinase activation and abolished by antagonizing Myc activity with the dominant-negative (dn) MadMyc chimera. Moreover, while abrogation of either endogenous E2F or Myc activity only delays and lowers DNA synthesis in synchronized U2OS cells or rat diploid fibroblasts, concomitant neutralization of both abolishes it. Whereas ectopic Myc and E2F1 rescue the G(1)/S delay caused by pRbDeltacdk (or dnDP1) and MadMyc, respectively, cyclin E or Cdc25A can restore DNA replication even in cells concomitantly exposed to pRbDeltacdk and MadMyc. However, coexpression of dnCDK2 neutralizes all of these rescuing effects. Finally, proper transcription of cyclin E and Cdc25A at the G(1)/S transition requires both Myc and E2F activities, and subthreshold levels of ectopic cyclin E and Cdc25A synergistically restore DNA synthesis in cells with silenced Myc and E2F activities. These results suggest that Myc controls a G(1)/S-promoting mechanism regulating cyclin E-CDK2 in parallel to the "classical" pRb/E2F pathway.

TGFbeta-induced growth inhibition involves cell cycle inhibitor p21 and pRb independent from p15 expression.

It is generally assumed that TGFbeta induces cell cycle arrest through the cooperative action of cell cycle inhibitors p15, p27 and p21. Here, we found that several pancreatic carcinoma cell lines exert TGFbeta-induced negative growth control in spite of the loss of p15 and p16 expression. In these cell lines, TGFbeta-induced growth control correlates with the upregulation of the p21 protein and active pRb expression. Conversely, cells without p21 and/or pRb expression are resistant to TGFbeta -induced growth inhibition. Moreover, overexpression of p21 in the p21-deficient cell line Panc Tu1 leads to growth arrest. Thus, TGFbeta-induced growth control correlates with p21 expression and pRb status independent of p15 and/or p16 expression.

Expression of G1 cyclins and cyclin-dependent kinase-2 activity during terminal differentiation of cultured human trophoblast.

Cyclin-dependent kinases (Cdks) and their cyclin partners regulate mammalian cell proliferation and withdrawal from the cell cycle and, as such, control differentiation in many tissues. Studies were undertaken to examine the roles of cell cycle proteins in differentiating cytotrophoblasts. Cyclin E gene and protein expression was down-regulated after 24 h in cultured trophoblasts. Cdk2-associated kinase activity was decreased after 96 h in culture as was the amount of cyclin E in complexes with Cdk2; however, levels of the Cdk inhibitor, p27Kip1, were significantly increased. In freshly isolated trophoblasts and in 24-h cultures, the retinoblastoma gene product (pRb) was found in both the active and inactive forms, yet only hypophosphorylated, active pRb was present in syncytiotrophoblast. Thus, inactivation of Cdk2 through cyclin E down-regulation and increased p27Kip1 expression leads to an accumulation of active pRb in syncytiotrophoblast. Prevention of entry into S phase by hypophosphorylated pRb may allow trophoblasts to respond to signals that potentiate differentiation. Our studies suggest that regulation of G1-phase Cdk activity may be involved in the terminal differentiation process of cytotrophoblasts.

Altered G1 phase regulation in osteosarcoma.

Alterations in the normal cell cycle lead to abnormal cell proliferation and to tumor development. To explore the role of the cyclin D/Cdk4 complex and the retinoblastoma protein (pRb) in the growth and spread of osteoblastic osteosarcoma (OS), 40 tumor samples were selected. In 17 of these cases, lung metastases occurred during follow-up. Expression of pRb, cyclin D1 and its catalytic subunit, Cdk4, was studied by immunohistochemistry and immunoblotting. As controls, non-neoplastic tissues surrounding the tumor were used. The expression level and pattern were compared to clinical outcome. Cdk4 was over-expressed in 80% of OS, independently of clinical outcome, and showed an intense and uniform distribution in tumor cells compared to normal cells. However, co-immunoprecipitation of Cdk4 with cyclin D1 revealed low levels of cyclin D/Cdk4 complex; 20 of 40 OS examined had a negative or minimal immunostaining for active pRb. The probability of relapse was significantly higher in pRb-negative than in the -positive patients (p < 0.05). The ratio of unphosphorylated/hyperphosphorylated pRb was lower in relapsed patients than in patients with no evident disease, though the difference was not statistically significant. High levels of pRb/cyclin D1 were found in all samples exhibiting functional pRb expression. Our results show that G1 phase deregulation is involved in formation and development of OS. The expression levels of both pRb and cyclin D1 had a clear correlation with clinical outcome, suggesting that these parameters could be used as prognostic markers.

Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb.

In cycling cells, the retinoblastoma protein (pRb) is un- and/or hypo-phosphorylated in early G1 and becomes hyper-phosphorylated in late G1. The role of hypo-phosphorylation and identity of the relevant kinase(s) remains unknown. We show here that hypo-phosphorylated pRb associates with E2F in vivo and is therefore active. Increasing the intracellular concentration of the Cdk4/6 specific inhibitor p15(INK4b) by transforming growth factor beta treatment of keratinocytes results in G1 arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb. Conversely, p15(INK4b)-independent transforming growth factor beta-mediated G1 arrest of hepatocellular carcinoma cells results in loss of Cdk2 kinase activity with continued Cdk6 kinase activity and pRb remains only hypo-phosphorylated. Introduction of the Cdk4/6 inhibitor p16(INK4a) protein into cells by fusion to a protein transduction domain also prevents pRb hypo-phosphorylation with an increase in unphosphorylated pRb. We conclude that cyclin D:Cdk4/6 complexes hypo-phosphorylate pRb in early G1 allowing continued E2F binding.

Involvement of pRB family in TGF beta-dependent epithelial cell hypertrophy.

Although renal hypertrophy is often associated with the progressive loss of renal function, the mechanism of hypertrophy is poorly understood. In both primary cultures of rabbit proximal tubules and NRK-52E cells (a renal epithelial cell line), transforming growth factor beta 1 (TGF beta) converted epidermal growth factor (EGF)-induced hyperplasia into hypertrophy. TGF beta did not affect EGF-induced increases in c-fos mRNA abundance or cyclin E protein abundance, but inhibited EGF-induced entry into S, G2, and M phases. EGF alone increased the amount of hyperphosphorylated (inactive) pRB; TGF beta blocked EGF-induced pRB phosphorylation, maintaining pRB in the active form. To determine the importance of active pRB in TGF beta-induced hypertrophy, NRK-52E cells were infected with SV40 large T antigen (which inactivates pRB and related proteins and p53), HPV16 E6 (which degrades p53), HPV16 E7 (which binds and inactivates pRB and related proteins), or both HPV16 E6 and E7. In SV40 large T antigen expressing clones, the magnitude of EGF + TGF beta-induced hypertrophy was inhibited and was inversely related to the magnitude of SV40 large T antigen expression. In the HPV16-infected cells, EGF + TGF beta-induced hypertrophy was inhibited in E7- and E6E7-expressing, but not E6-expressing cells. These results suggest a requirement for active pRB in the development of EGF + TGF beta-induced renal epithelial cell hypertrophy. We suggest a model of renal cell hypertrophy mediated by EGF-induced entry into the cell cycle with TGF beta-induced blockade at G1/S, the latter due to maintained activity of pRB or a related protein.