pRb Family Research Articles

Role of the interaction between large T antigen and Rb family members in the oncogenicity of JC virus

Human polyomaviruses (JC virus, BK virus and simian virus 40) are causative agents of some human diseases and, interestingly, are involved in processes of cell transformation and oncogenesis. These viruses need the cell cycle machinery of the host cell to complete their replication; so they evolved mechanisms that can interfere with the growth control of infected cells and force them into DNA replication. The retinoblastoma family of proteins (pRb), which includes pRb/p105, p107 and pRb2/p130, acts as one of the most important regulators of the G1/S transition of the cell cycle. Rb proteins represent an important target for viral oncoproteins. Early viral T antigens can bind all members of the pRb family, promoting the activation of the E2F family of transcription factors, thus inducing the expression of genes required for the entry to the S phase. The interaction between early viral antigens and cell cycle regulators represents an important mechanism through which viruses deregulate cell cycle and lead to cell transformation. In this review, we will discuss the effects of the interaction between large T antigen and Rb proteins in JC virus-mediated oncogenesis.

Differentiation and Injury-Repair Signals Modulate the Interaction of E2F and pRB Proteins with Novel Target Genes in Keratinocytes

E2F transcription factors are central to epidermal morphogenesis and regeneration afterinjury. The precise nature of E2F target genes involved in epidermal formation and repair has yetto be determined. Identification of these genes is essential to understand how E2F proteinsregulate fundamental aspects of epidermal homeostasis and transformation. We have conducted agenome-wide screen using CpG island microarray analysis to identify novel promoters bound byE2F3 and E2F5 in human keratinocytes. We further characterized several of these genes, anddetermined that multiple E2F and retinoblastoma (pRb) family proteins associate with them inexponentially proliferating cells. We also assessed the effect on E2F and pRb binding to thosegenes in response to differentiation induced by bone morphogenetic protein-6 (BMP-6), or toactivation of repair mechanisms induced by transforming growth factor-β (TGF-β). Thesestudies demonstrate promoter- and cytokine-specific changes in binding profiles of E2F and/orpRb family proteins. For example, E2F1, 3, 4 and p107 were recruited to the N-myc promoter incells treated with BMP-6, whereas E2F1, 3, 4, 5, p107 and p130 were bound to this promoter inthe presence of TGF-β. Functionally, these different interactions resulted in transcriptionalrepression by BMP-6 and TGF-β of the N-myc gene, via mechanisms that involved E2F bindingto the promoter and association with pRb-family proteins. Thus, multiple combinations of E2Fand pRb family proteins may associate with and transcriptionally regulate a given target promoterin response to differentiation and injury-repair stimuli in epidermal keratinocytes.

Cell cycle deregulation and loss of stem cell phenotype in the subventricular zone of TGF-β adaptor elf−/− mouse brain

The mammalian forebrain subependyma contains neural stem cells and other proliferating progenitor cells. Recent studies have shown the importance of TGF-β family members and their adaptor proteins in the inhibition of proliferation in the nervous system. Previously, we have demonstrated that TGF-β induces phosphorylation and association of ELF (embryonic liver fodrin) with Smad3 and Smad4 resulting in nuclear translocation. Elf −/− mice manifest abnormal neuronal differentiation, with loss of neuroepithelial progenitor cell phenotype in the subventricular zone (SVZ) with dramatic marginal cell hyperplasia and loss of nestin expression. Here, we have analyzed the expression of cell cycle-associated proteins cdk4, mdm2, p21, and pRb family members in the brain of elf −/− mice to verify the role of elf in the regulation of neural precursor cells in the mammalian brain. Increased proliferation in SVZ cells of the mutant mice coincided with higher levels of cdk4 and mdm2 expression. A lesser degree of apoptosis was observed in the mutant mice compared to the wild-type control. Elf −/− embryos showed elevated levels of hyperphosphorylated forms of pRb, p130 and p107 and decreased level of p21 compared to the wild-type control. These results establish a critical role for elf in the development of a SVZ neuroepithelial stem cell phenotype and regulation of neuroepithelial cell proliferation, suggesting that a mutation in the elf locus renders the cells susceptible to a faster entry into S phase of cell cycle and resistance to senescence and apoptotic stimuli.

Conditionally Activated E7 Proteins of High-Risk and Low-Risk Human Papillomaviruses Induce S Phase in Postmitotic, Differentiated Human Keratinocytes

The productive program of human papillomaviruses (HPVs) in epithelia is tightly linked to squamous differentiation. The E7 proteins of high-risk HPV genotypes efficiently inactivate the pRB family of proteins that control the cell cycle, triggering S phase in suprabasal keratinocytes. This ability has until now not been demonstrated for the low-risk HPV-6 or HPV-11 E7 proteins. An inducible system in which HPV-16 E7 is fused to the ligand binding domain of the human estrogen receptor (ER) was described by Smith-McCune et al. (K. Smith-McCune, D. Kalman, C. Robbins, S. Shivakumar, L. Yuschenkoff, and J. M. Bishop, Proc. Natl. Acad. Sci. USA 96:6999-7004, 1999). In the absence of hormone, E7ER is cytoplasmic, and upon addition of 17beta-estradiol, it translocates to the nucleus. Using organotypic epithelial raft cultures developed from primary human keratinocytes, we show that 16E7ER promotes either S-phase reentry or p21cip1 accumulation in differentiated keratinocytes in a stochastic manner as early as 6 h postinduction with 17beta-estradiol. A vector expressing the ER moiety alone had no effect. These observations prove unequivocally that the E7 protein drives S-phase reentry in postmitotic, differentiated keratinocytes rather than preventing S-phase exit while the cells ascend through the epithelium. HPV-11 E7ER and, much less efficiently, HPV-6 E7ER also promoted S-phase reentry by differentiated cells upon exposure to 17beta-estradiol. S-phase induction required the consensus pRB binding motif. We propose that the elevated nuclear levels of the low-risk HPV E7 protein afforded by the inducible system account for the positive results. These observations are entirely consistent with the fact that low-risk HPV genotypes replicate in the differentiated strata in patient specimens, as do the high-risk HPVs.

E6/E7 oncogenes increase and tumor suppressors decrease the proportion of self-renewing neural progenitor cells

Many if not most tissues need a controlled number of stem cells to maintain normal function. Cancer can be seen as a process of disturbed tissue homeostasis, in which too many cells have or acquire too primitive identity. Here we measured how oncogenes and tumour suppressors affect the differentiation capacity, proportion and characteristics of progenitor cells in a model tissue. Neural progenitor cells (NPCs) were exposed to human papilloma virus E6, E7 or E6/E7 oncogenes, which degrade tumour suppressors p53 and pRb family members, respectively. E6/E7-expressing or p53-/- NPCs were able to differentiate, but simultaneously retained high capacity for self-renewal, proliferation, ability to remain multipotent in conditions promoting differentiation and showed delayed cell cycle exit. These functions were mediated through p53 and pRb family, and involved MEK-ERK signalling. Decreased amount of p53 increased self-renewal and proliferation, whereas pRb affected only proliferation. Our results suggest that the oncogenes increase whereas p53 and pRb family tumour suppressors decrease the number and proportion of progenitor cells. These findings provide one explanation how oncogenes and tumour suppressors control tissue homeostasis and highlight their importance in stem cell self- renewal, linked both to cancer and life-long tissue turnover.

RBP1 Family Proteins Exhibit SUMOylation-Dependent Transcriptional Repression and Induce Cell Growth Inhibition Reminiscent of Senescence

The retinoblastoma binding protein 1 (RBP1) appears to be an important factor in the repression of E2F-dependent transcription by the retinoblastoma protein (pRB) family. The recent identification of the breast carcinoma associated antigen (BCAA) as an RBP1-like protein led us to investigate its biological properties and compare them to RBP1. Like RBP1, BCAA contains a carboxy-terminal R2 domain that elicits histone deacetylase (HDAC)-dependent transcriptional repression via interactions with the SAP30 subunit of the Sin3/HDAC complex. Each RBP1 family member also contains two HDAC-independent repression activities within a region termed R1, which can be subdivided into a SUMOylated moiety (R1sigma) and a predicted alpha-helical region (R1alpha). R1alpha is embedded within the ARID region and represses basal transcription only, whereas R1sigma represses both basal and activated transcription and depends on SUMOylation. Overexpression of either RBP1 or BCAA, but not the truncated BCAAMCF-7 isoform that is overexpressed in breast cancer cells, caused a profound inhibition of cell proliferation and induced expression of a senescence marker. In each case the presence of both R1 and R2 was necessary for suppression of cell growth, suggesting that both R1 and R2 transcriptional repression activities play a role in RBP1 family protein-mediated regulation of cellular proliferation.

Hsc70 Contacts Helix III of DnaJ‐like Domain from Polyomavirus T Antigens

The J domain of large T antigen, Hsc70, and its hydrolysis of ATP were found necessary for polyomavirus infection to release E2Fs from pRb family members. Hsc70's expected binding site on helix II of the DnaJ-like domain of T antigens is blocked in the crystal structure of a T antigen fragment bound to tumor suppressor pRb. We used NMR to find recognition sites for mammalian Hsc70 upon the J domain of murine polyomavirus T antigens (PyJ). The Hsc70 ATPase domain unexpectedly shifts most of the amide NMR peaks of the C-terminal end of helix III of PyJ. The Hsc70 ATPase domain buries the C-terminal end of helix III of PyJ, protecting it from an uncharged paramagnetic probe. This region corresponds to the QKRAA motif of orthodox J domains. Evolutionary trace analysis suggests helix III of J domains to be of wide importance to specificity for hsp70s. The NMR-detected Hsc70 contacts map to a PyJ surface homologous to a solvent-accessible surface of the SV40 T antigen fragment bound to pRb. This surface of helix III and the HPD loop may allow Hsc70 to form a ternary complex with T antigen and pRb, avoiding obstructions to helix II. The helix III docking site is consistent with large T antigen being able to free an E2F from pRb by positioning the substrate-binding domain of Hsc70 for direct contact with the E2F. NMR spectra show the pRb-binding region of E2F1 (aa243–437) to be unfolded in the absence of DP1. Hsc70 is known to bind unfolded proteins. In the absence of DP1, E2F1(aa243–437) acts as a substrate for Hsc70 in vitro, stimulating its ATPase activity in a manner dependent on PyJ from T antigen. If segments of an E2F are unstructured in vivo, it is conceivable that Hsc70, recruited by large T antigen to a complex with pRb, might act on the unstructured portion of the E2F when liberating it from the pRb family member.

The retinoblastoma protein is required for Ras-induced oncogenic transformation.

Most human cancers involve either mutational activation of the Ras oncogenic pathway and/or inactivation of the retinoblastoma tumor suppressor (RB) pathway. Paradoxically, tumors that harbor Ras mutations almost invariably retain expression of a wild-type pRB protein. We explain this phenomenon by demonstrating that Ras-induced oncogenic transformation surprisingly depends on functional pRB protein. Cells lacking pRB are less susceptible to the oncogenic actions of H-RasV12 than wild-type cells and activated Ras has an inhibitory effect on the proliferation of pRB-deficient human tumor cells. In addition, depletion of pRB from Ras-transformed murine cells or human tumor cells that harbor Ras pathway mutations inhibits their proliferation and anchorage-independent growth. In sharp contrast to pRB-/- 3T3 cells, fibroblasts deficient in other pRB family members (p107 and p130) are more susceptible to Ras-mediated transformation than wild-type 3T3 cells. Moreover, loss of pRB in tumor cells harboring a Ras mutation results in increased expression of p107, and overexpression of p107 but not pRB strongly inhibits proliferation of these tumor cells. Together, these findings suggest that pRB and p107 have distinct roles in Ras-mediated transformation and suggest a novel tumor-suppressive role for p107 in the context of activated Ras.

Requirement of E7 oncoprotein for viability of HeLa cells

Most human papillomavirus (HPV)-positive cervical cancers contain integrated copies of the viral genome in their chromosomes and express the viral oncoproteins E6 and E7. A virus-encoded transcription factor, E2, is known to repress E6/E7 expression in HPV-positive cancer cells, leading to growth inhibition, which indicates that E6/E7 is required for the survival of the cells. We found that the E2-mediated growth inhibition of HeLa cells, an HPV18-positive cancer cell line, was coupled with a reduction in telomerase activity, an effect which was rescued by the complementation of E7 expression, but not E6 expression, indicating that the cell viability and the telomerase activity in HeLa cells are maintained by an E7-associated function. Analysis of E7 mutants suggested that the binding to the pRB family of pocket proteins was involved in the ability of E7 to rescue the growth potential and telomerase activity inhibited by E2 expression. We also showed that the telomerase activity upregulated by E7 expression was determined by the hTERT promoter activity, and that c-Myc upregulation caused by pRB inactivation could account for the promoter activity. The activation of p53 and consequent accumulation of p21Cip1, which were triggered by the downregulation of E6, appeared not to be essential for the E2-mediated growth arrest.

Cytoplasmic and nuclear interaction between Rb family proteins and PAI-2: a physiological crosstalk in human corneal and conjunctival epithelial cells

Extracellular plasminogen activator inhibitor type-2 (PAI-2) is a potent inhibitor of urokinase-type plasminogen activator (u-PA) and also acts as a multifunctional protein. However, the biological activity of intracellular PAI-2, as well as its intracellular targets, until now remain an enigma. Here, we show that pRb2/p130 and Rb1/p105, but not p107, interact with PAI-2 in both the cytoplasm and nucleus of normal primary human corneal and conjunctival epithelial cells. We provided the first in vivo evidence that a specific fragment of the PAI-2 promoter is bound simultaneously by pRb2/ p130, PAI-2, E2F5, histone deacetylase 1 (HDAC1), DNA methyltransferase 1 (DNMT1), and histone methyltransferase (SUV39H1), in normal primary human corneal epithelial cells, and by pRb2/p130, PAI-2, E2F5, HDAC1, and DNMT1, in normal primary human conjunctiva epithelial cells. Our results strongly indicate a physiological interaction between pRb family members and PAI-2, suggesting the hypothesis that pRb2/p130 and PAI-2 may cooperate in modulating PAI-2 gene expression by chromatin remodeling, in normal corneal and conjunctival cells.

The E7 proteins of low- and high-risk human papillomaviruses share the ability to target the pRB family member p130 for degradation

High-risk human papillomaviruses (HPVs) (e.g., HPV-16) cause anogenital and head and neck cancers, and low-risk HPVs (e.g., HPV-6) cause benign hyperproliferative disease. The E7 protein of HPV-16 binds all retinoblastoma tumor suppressor protein (pRB) family members with higher affinity than HPV-6E7. The HPV-16 E7 protein has been reported to target pRB family members for degradation and to immortalize cells. Here we tested the hypothesis that the low-risk E7 protein has an intrinsic ability to decrease expression of pRB family members. First, we introduced a high-affinity pRB-binding site into HPV-6 E7 (6E7G22D) and showed that, in human foreskin keratinocytes, HPV-6 E7G22D decreased the level of pRB protein but not pRB mRNA. Second, we analyzed the ability of wild-type HPV-6 E7 to destabilize the other pRB family members, p107 and p130. HPV-6 E7, like HPV-16 E7, decreased the level of p130 protein. This decrease was inhibited by MG132, a proteasome inhibitor. Binding of HPV-6 E7 to p130 was necessary but not sufficient to decrease the level of p130. Furthermore, the destabilization of p130 correlated with a decrease in the expression of involucrin, a differentiation marker. We suggest that the shared activity of HPV-16 E7 and HPV-6 E7 to destabilize p130 and decrease or delay differentiation may be related to the role of E7 in the HPV life cycle. The added ability of HPV-16 E7 to regulate pRB and p107 may be related to oncogenic activity.

Negative Regulation of TSC1-TSC2 by Mammalian D-Type Cyclins

The metazoan cell cycle is driven by the timely and composite activities of cyclin-dependent kinases (CDKs). Among these, cyclin D- and cyclin E-dependent kinases phosphorylate the pRb family proteins during G(1) phase of the cell cycle and thereby advance cells beyond the restriction point. Increasing evidence suggests that cyclin D-dependent kinases might affect events other than Rb pathway-mediated entry into S phase, such as accumulation of cell mass. However, little is known about cyclin D activity toward Rb-independent pathway(s) or non-pRb substrates. In this article, we show that the tumor suppressor TSC2 is a cyclin D binding protein. Coexpression of cyclin D1-CDK4/6 in cultured cells leads to increased phosphorylation and decreased detection of both TSC2 and TSC1, and promotes the phosphorylation of the mTOR substrates, 4E-BP1 and S6K1, two key effectors of cell growth that are negatively regulated by the TSC1-TSC2 complex. At the cellular level, ectopic expression of cyclin D1 restores the cell size decrease caused by TSC1-TSC2 expression. Intriguingly, down-regulation of TSC proteins was also observed by the expression of a mutant cyclin D1 that is unable to bind to CDK4/6, or by the coexpression of cyclin D1 with either an INK4 inhibitor or with catalytically inactive CDK6, indicating that cyclin D may regulate TSC1-TSC2 independently of CDK4/6. Together, these observations suggest that mammalian D-type cyclins participate in cell growth control through negative regulation of TSC1-TSC2 function.

Pocket proteins p107 and p130 exhibit increased expression in macrophages during SIV encephalitis

Progression of HIV encephalitis (HIVE) is associated with neuronal damage and loss because of infiltration of infected and/or activated macrophages into the CNS. We have previously observed increased inactivation of the retinoblastoma susceptibility gene product (pRb) by phosphorylation in neurons and glia of HIVE and the simian model of HIVE (SIVE). To determine if other pRb family members are altered in response to increased macrophage-secreted factors, we investigated expression of pRb family members p107 and p130 in SIVE. Both p130 and p107 exhibited increased staining in macrophages, but not neurons, astrocytes or T-cells in SIVE. Increased p130 and p107 immunostaining was not limited to virally infected or PCNA-expressing macrophages. Most p107-positive staining was observed in perivascular macrophages, suggesting p107 may indicate macrophages at a specific stage of differentiation soon after migration. In contrast, cytoplasmic p130 was found in the majority of macrophages present in SIVE cases and may indicate activation as it was not seen in microglia in control CNS. These findings suggest that p107 and p130 are differentially expressed in CNS macrophage populations which may have multiple derivations and/or roles in lentiviral encephalitis.

Heterogeneous Tumor Evolution Initiated by Loss of pRb Function in a Preclinical Prostate Cancer Model

Because each change in the evolution of a cancer is predicated on the effects of previous events, a full understanding of selective changes and their effect on tumor progression can only be understood in the context of appropriate initiating events. Here, we define the effect of pRb function inactivation in prostate epithelium on both the initiation of prostate cancer and the establishment of selective pressures that lead to diminished Pten function and tumor evolution. Using genetically engineered mice, we show that inactivation of the pRb family proteins (Rb/p107/p130) induces epithelial proliferation and apoptosis and is sufficient to produce prostatic intraepithelial neoplasia (PIN) lesions. Over time, adenocarcinomas develop in all mice with no evidence of neuroendocrine tumors. Apoptosis is dependent on Pten function and not p53, unlike other epithelial cell types tested previously. Consequently, Pten hemizygosity reduces apoptosis by 50%, accelerating progression to adenocarcinomas with heterogeneous composition. Heterogeneity is associated with concurrent Pten haploinsufficiency and focal selective progression to complete Pten loss, which yields distinct tumor properties. Given that this analysis models the apparent timing of highly penetrant events in human prostate cancer, observed effects may recapitulate the natural evolution of prostate cancer development.

Murine γ-Herpesvirus 68 Latency Protein M2 Binds to Vav Signaling Proteins and Inhibits B-cell Receptor-induced Cell Cycle Arrest and Apoptosis in WEHI-231 B Cells

The MHV-68 latent protein, M2, does not have homology to any known viral or cellular proteins, and its function is unclear. To define the role played by M2 during MHV-68 latency as well as the molecular mechanism involved, we used M2 as bait to screen a yeast two-hybrid mouse B-cell cDNA library. Vav1 was identified as an M2-interacting protein in two independent screenings. Subsequent yeast two-hybrid interaction studies showed that M2 also binds to Vav2, but not Vav3, and that three "PXXP" motifs located at the C terminus of M2 are important for this interaction. The interactions between M2 and Vav proteins were also confirmed in vivo in 293T and WEHI-231 B-cells by co-immunoprecipitation assays. Rac1/GST-PAK "pull-down" experiments and Western blot analysis using a phospho-Vav antibody demonstrated that expression of M2 in WEHI-231 cells enhances Vav activity. We further showed in WEHI-231 cells that M2 expression promotes proliferation and survival and is associated with enhanced cyclin D2 and repressed p27(Kip1), p130, and Bim expression. Taken together, these experiments suggest that M2 might have an important role in disseminating the latent virus during the establishment and maintenance of latency by modulating B-cell receptor-mediated signaling events through Vav to promote B-cell activation, proliferation, and survival.

Pocket protein complexes are recruited to distinct targets in quiescent and proliferating cells.

Biochemical and genetic studies have determined that retinoblastoma protein (pRB) tumor suppressor family members have overlapping functions. However, these studies have largely failed to distinguish functional differences between the highly related p107 and p130 proteins. Moreover, most studies pertaining to the pRB family and its principal target, the E2F transcription factor, have focused on cells that have reinitiated a cell cycle from quiescence, although recent studies suggest that cycling cells exhibit layers of regulation distinct from mitogenically stimulated cells. Using genome-wide chromatin immunoprecipitation, we show that there are distinct classes of genes directly regulated by unique combinations of E2F4, p107, and p130, including a group of genes specifically regulated in cycling cells. These groups exhibit both distinct histone acetylation signatures and patterns of mammalian Sin3B corepressor recruitment. Our findings suggest that cell cycle-dependent repression results from recruitment of an unexpected array of diverse complexes and reveals specific differences between transcriptional regulation in cycling and quiescent cells. In addition, factor location analyses have, for the first time, allowed the identification of novel and specific targets of the highly related transcriptional regulators p107 and p130, suggesting new and distinct regulatory networks engaged by each protein in continuously cycling cells.

Simian Virus 40 Large T Antigen's Association with the CUL7 SCF Complex Contributes to Cellular Transformation

Simian virus 40 large T antigen (T Ag) is capable of immortalizing and transforming rodent cells. The transforming activity of T Ag is due in large part to perturbation of the tumor suppressor proteins p53 and the retinoblastoma (pRB) family members. Inactivation of these tumor suppressors may not be sufficient for T Ag-mediated cellular transformation. It has been shown that T Ag associates with an SCF-like complex that contains a member of the cullin family of E3 ubiquitin ligases, CUL7, as well as SKP1, RBX1, and an F-box protein, FBXW8. We identified T Ag residues 69 to 83 as required for T Ag binding to the CUL7 complex. We demonstrate that delta69-83 T Ag, while it lost its ability to associate with CUL7, retained binding to p53 and pRB family members. In the presence of CUL7, wild-type (WT) T Ag but not delta69-83 T Ag was able to induce proliferation of mouse embryo fibroblasts, an indication of cellular transformation. In contrast, WT and delta69-83 T Ag enabled mouse embryo fibroblasts to proliferate to similarly high densities in the absence of CUL7. Our data suggest that, in addition to p53 and the pRB family members, T Ag serves to bind to and inactivate the growth-suppressing properties of CUL7. In addition, these results imply that, at least in the presence of T Ag, CUL7 may function as a tumor suppressor.

Somatic Induction of Pten Loss in a Preclinical Astrocytoma Model Reveals Major Roles in Disease Progression and Avenues for Target Discovery and Validation

High-grade astrocytomas are invariably deadly and minimally responsive to therapy. Pten is frequently mutated in aggressive astrocytoma but not in low-grade astrocytoma. However, the Pten astrocytoma suppression mechanisms are unknown. Here we introduced conditional null alleles of Pten (Pten(loxp/loxp)) into a genetically engineered mouse astrocytoma model [TgG(deltaZ)T121] in which the pRb family proteins are inactivated specifically in astrocytes. Pten inactivation was induced by localized somatic retroviral (MSCV)-Cre delivery. Depletion of Pten function in adult astrocytoma cells alleviated the apoptosis evoked by pRb family protein inactivation and also induced tumor cell invasion. In primary astrocytes derived from TgG(deltaZ)T121; Pten(loxp/loxp) mice, Pten deficiency resulted in a marked increase in cell invasiveness that was suppressed by inhibitors of protein kinase C (PKC) or of PKC-zeta, specifically. Finally, focal induction of Pten deficiency in vivo promoted angiogenesis in affected brains. Thus, we show that Pten deficiency in pRb-deficient astrocytoma cells contributes to tumor progression via multiple mechanisms, including suppression of apoptosis, increased cell invasion, and angiogenesis, all of which are hallmarks of high-grade astrocytoma. These studies not only provide mechanistic insight into the role of Pten in astrocytoma suppression but also describe a valuable animal model for preclinical testing that is coupled with a primary cell-based system for target discovery and drug screening.

Intestinal Dysplasia Induced by Simian Virus 40 T Antigen Is Independent of p53

Transgenic mice expressing simian virus 40 large T antigen in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. Hyperplasia is dependent on T antigen binding to the retinoblastoma (pRb) family of tumor suppressor proteins. Mice expressing a truncated T antigen that inactivates the pRb-family, but is defective for binding p53, exhibit hyperplasia but do not progress to dysplasia. We hypothesized that the inhibition of the pRb family leads to entry of enterocytes into the cell cycle, resulting in hyperplasia, while inactivation of p53 is required for progression to dysplasia. Therefore, we examined T antigen/p53 complexes from the intestines of transgenic mice. We found that T antigen did not induce p53 stabilization, and we could not detect T antigen/p53 complexes in villus enterocytes. In contrast, T antigen expression led to a large increase in the levels of the cyclin-dependent kinase inhibitor p21. Furthermore, mice in which pRb was inactivated by a truncated T antigen in a p53 null background exhibited intestinal hyperplasia but no progression to dysplasia. These data indicate that loss of p53 function does not play a role in T antigen-induced dysplasia in the intestine. Rather, some unknown function of T antigen is essential for progression beyond hyperplasia.

Progesterone Inhibits the Estrogen-Induced Phosphoinositide 3-Kinase→AKT→GSK-3β→Cyclin D1→pRB Pathway to Block Uterine Epithelial Cell Proliferation

The mammalian cell cycle is regulated by the cyclin/cyclin-dependent kinase (CDK) phosphorylation of the retinoblastoma (pRB) family of proteins. Cyclin D1 with its CDK4/6 partners initiates the cell cycle and acts as the link between extracellular signals and the cell cycle machinery. Estradiol-17beta (E2) stimulates uterine epithelial cell proliferation, a process that is completely inhibited by pretreatment with progesterone (P4). Previously, we identified cyclin D1 localization as a key point of regulation in these cells with E2 causing its nuclear accumulation and P4 retaining it in the cytoplasm with the resultant inhibition of pRB phosphorylation. Here we show that E2 stimulates phosphoinositide 3-kinase to activate phosphokinase B/AKT to effect an inhibitory phosphorylation of glycogen synthase kinase (GSK-3beta). This pathway is suppressed by P4. Inhibition of the GSK-3beta activity in P4-treated uteri by the specific inhibitor, LiCl, reversed the nuclear accumulation of cyclin D1 and in doing so, caused pRB phosphorylation and the induction of downstream genes, proliferating cell nuclear antigen and Ki67. Conversely, inhibition of phosphoinositide 3 kinase by LY294002 or Wortmanin reversed the E2-induced GSK-3beta Ser9 inhibitory phosphorylation and blocked nuclear accumulation of cyclin D1. These data show the reciprocal actions of E2 and P4 on the phosphoinositide 3-kinase through to the GSK-3beta pathway that in turn regulates cyclin D1 localization and cell cycle progression. These data reveal a novel signaling pathway that links E2 and P4 action to growth factor-mediated signaling in the uterus.