Site-specific and temporally-regulated retinoblastoma protein dephosphorylation by protein phosphatase type 1.

pRb is dephosphorylated at mitotic exit by the type 1 serine/threonine protein phosphatases (PP1). Here we demonstrate for the first time that mitotic pRb dephosphorylation is a sequential, temporally-regulated event. We also provide evidence that the three mammalian isoforms of PP1, alpha, gamma-1, and delta, differ in their respective preferences for site-specific pRb dephosphorylation and that the mitotic and G(1) PP1-isoform counterparts exhibit differential activities towards mitotic pRb. Finally, the physiological relevance of the striking contrast between the patterns of Thr821 and Thr826 dephosphorylation, sites known to be important for disrupting binding of LXCXE-containing proteins to pRb, is addressed.

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.

Induced G2/M arrest and apoptosis in human epidermoid carcinoma cell lines by semisynthetic drug Ukrain.

Exposure of ME180 and A431 carcinoma cells to Ukrain (NSC-631570), a novel semisynthetic drug from Chelidonium majus L, results in cell growth inhibition which is concomitant with reversible G2/M cell cycle arrest and apoptosis at doses as low as 7 microM. In contrast, the same drug concentrations were not affective towards normal human keratinocytes. In order to investigate whether cell cycle control mechanisms are effected in response to Ukrain, we analyzed cell cycle distribution and levels of cyclins and cyclin-dependent kinases in drug treated carcinoma cells. We found alterations in levels of mitotic cyclins A and B1, and cyclin-dependent kinases CDK1 and CDK2, after treatment. We also observed an upregulation of CDK inhibitor p27 in both cancer cell lines which may lead to the G2/M cells accumulation.

The carboxyl-terminal region of the retinoblastoma protein binds non-competitively to protein phosphatase type 1alpha and inhibits catalytic activity.

pRB, a negative-growth regulatory protein, is a demonstrated substrate for type 1 serine/threonine protein phosphatases (PP1). In a recent report from this laboratory, we demonstrated that select forms of phosphorylated as well as hypophosphorylated pRB can be found complexed with the alpha-isotype of PP1 (PP1alpha). This complex can also be observed when PP1 is rendered catalytically dead by toxin inhibition. These data suggested to us that pRB may bind to PP1 at one or more sites other than the catalytically active one on the enzyme and that such binding may play a role other than bringing the substrate into contact with the enzyme to facilitate catalysis. To address this possibility we utilized a series of pRB deletion mutants and coprecipitation studies to map the pRB domain involved in binding to PP1. Together with competition assays using in vivo expression of SV40 T-antigen, we show here that the carboxyl-terminal region of pRB is both necessary and sufficient for physical interaction with PP1. Subsequent biochemical analyses demonstrated inhibition of PP1 catalytic activity toward the standard substrate phosphorylase a when this enzyme is bound to pRB containing this region. K(m) and V(max) calculations revealed that pRB binds to PP1 in a non-competitive manner. These data support the notion that pRB, in addition to being a substrate for PP1, also functions as a PP1 inhibitor. The significance of this finding with respect to the functional importance of this interaction is discussed.

Role of pRB dephosphorylation in cell cycle regulation.

pRB, the tumor suppressor product of the retinoblastoma susceptibility gene, is regarded as one of the key regulators of the cell cycle. This protein exerts its growth suppressive effect through its ability to bind and interact with a variety of cellular proteins. In turn, pRB binding and interacting ability is governed by its phosphorylation state. In recent years, this negative growth regulatory protein has captured a great deal of attention from investigators around the world due to its ability to modulate the activity of transcription regulatory proteins, enzymes which modify chromatin, and other cellular proteins which contribute to its complex role in mammalian cells. Hypophosphorylated pRB binds and sequesters transcription factors, most notably those of the E2F/DP family, inhibiting the transcription of genes required to traverse the G1 to S phase boundary. This cell cycle inhibitory function is abrogated when pRB undergoes phosphorylation mediated by cyclin/cdk complexes following cell stimulation by mitogens. Removal of these phosphates appears to be carried out by a multimeric complex of protein phosphatase type 1 (PP1) and noncatalytic regulatory subunits at the completion of mitosis. This dephosphorylation returns pRB to its active, growth suppressive state. While the mechanism of pRB phosphorylation has and continues to be extensively studied, dephosphorylation of pRB has received disproportionately less attention. The goal of this review is to revisit the role of pRB dephosphorylation in regulating the cell cycle. Emphasis will be placed on understanding the function and regulation of pRB during the cell cycle as well as our ever-expanding notions of pRB-PP1 interaction and the mechanism of pRB dephosphorylation at mitotic exit.

Induced apoptosis in human prostate cancer cell line LNCaP by Ukrain.

Exposure of LNCaP prostate cancer cells to Ukrain (NSC-631570), a novel semisynthetic drug from Chelidonium majus L., results in cell growth inhibition which is concomitant with apoptosis. After 24 h treatment with 3.5 microM of Ukrain as many as 73% cells were found in the G2/M phase. However, at higher drug concentrations (7 microM and 17.5 microM) the changes in cell phase distribution were less dramatic but cell accumulation in the G2/M phase was still evident. The rate of apoptotic cells rose steadily with increased drug concentration in a dose-dependent manner and reached 20% at a dosage of 17.5 microM. To investigate whether the cell cycle control mechanisms are affected in response to Ukrain, we analyzed the expression levels of some cyclins, cyclin-dependent kinases (CDK) and apoptosis-related proteins in drug treated cancer cells. Western blot experiments revealed alterations in levels of CDK1 and CDK2, after treatment. Up-regulation of the CDK inhibitor p27 was observed, which may lead to G2/M cell accumulation, but no substantial changes in expression of Bcl-2 and Bax proteins were found.

Molecular analysis of selected cell cycle regulatory proteins during aerobic and hypoxic maintenance of human ovarian carcinoma cells.

We have previously reported on the development of an in vitro model system for studying the effect of hypoxia on ovarian carcinoma cell proliferation and invasion (Krtolica and Ludlow, 1996). These data indicate that the cell division cycle is reversibly arrested during the G1 phase. Here, we have continued this study to include the proliferation properties of both aerobic and hypoxic human ovarian carcinoma cells at the molecular level. The growth suppressor product of the retinoblastoma susceptibility gene, pRB, appears to be functional in these cells as determined by SV40 T-antigen binding studies. Additional G1-to-S cell cycle regulatory proteins, cyclins D and E, cyclin-dependent kinases (cdks) 4 and 2, and cdk inhibitors p27 and p18, also appear to be intact based on their apparent molecular weights and cell cycle stage-specific abundance. During hypoxia, there is a decrease in abundance of cyclins D and E, with an increase in p27 abundance. cdk4 activity towards pRB and cdk2 activity towards histone H1 are also decreased. Co-precipitation studies revealed an increased amount of p27 complexing with cyclin E-cdk2 during hypoxia than during aerobic cell growth. In addition, pRB-directed phosphatase activity was found to be greater in hypoxic than aerobic cells. Taken together, a model is suggested to explain hypoxia-induced cell cycle arrest in SKA human ovarian carcinoma cells.

Binding of select forms of pRB to protein phosphatase type 1 independent of catalytic activity.

The product of the retinoblastoma susceptibility gene, pRB, is a demonstrated substrate for the type 1 serine/threonine protein phosphatases (PP1). Curiously, there has been a paucity of data supporting the idea that phosphorylated pRB can be found in a complex with PP1. To more fully characterize the association between these two proteins, we utilized a PP1-affinity chromatography approach to increase our ability to capture from mammalian cell lysate populations of pRB capable of binding to PP1. Western blot analysis of the bound proteins indicates that both faster migrating, hypophosphorylated pRB, as well as slower migrating, hyperphosphorylated pRB can bind. Phosphorylated pRB binding was confirmed by immunoprecipitation of eluted 32P-labeled pRB. In addition, Western blotting of eluted proteins with pRB phosphorylated-site-specific antibodies revealed select phosphorylated forms of pRB binding to PP1. Similar binding studies performed with toxin-inhibited PP1 indicate that catalytic activity of PP1 is not required for pRB binding. The significance of this finding with respect to the functional importance of this interaction is discussed.

Protein phosphatase type 1, the product of the retinoblastoma susceptibility gene, and cell cycle control.

Cell cycle regulation--three words which conjure in the minds of those conducting research in this area a myriad of proteins and biochemical pathways. In this examination, an overview of the mammalian cell cycle is presented with emphasis on the function of the negative growth regulatory protein, the product of the retinoblastoma susceptibility gene, pRb. Since the activity of this protein itself is regulated by phosphorylation on serine and threonine residues, more elaborate discussions on the enzymes involved in placing the phosphates on, and taking them off, are provided. The focus here is on the activity of the members of the type 1 class of serine/threonine phosphatases. More specifically, the role of PP1 in regulating cell cycle progression by dephosphorylating pRb during mitosis, thereby activating the growth suppressing function of pRb, is presented. Suggested avenues for further investigation regarding the functional significance, and ultimately the effect on cell cycle progression, of the complex between pRb and the type 1 phosphatases are also discussed.

CD44 splice variant expression in clear cell carcinoma of the ovary.

OBJECTIVE: CD44 is a surface glycoprotein widely distributed among different tissues. Malignant tumors may show a more complex pattern of CD44 expression, indicating a loss of splice control. The aim of our study is to investigate the expression of CD44 splice variants (CD44v) and its metastatic potential in clear cell carcinoma of the ovary. METHODS: Twenty-two cases of clear cell carcinoma of the ovary were evaluated for CD44 standard form (CD44s) and splice variants: -4v, -6v, and -9v expression by immunocytochemistry. RESULTS: Twenty-one primary ovarian tumors and 23 metastatic sites were available for evaluation. Eighteen of 21 (86%) of ovarian sections studied expressed CD44s; 15/21 (71%) expressed CD44-4v; 14/21 (67%) expressed CD44-6v; and 12/21 (57%) expressed CD44-9v. Of 23 metastatic sites evaluated, 87% expressed CD44s. In contrast, only 5 (22%) metastases had CD44-4v and CD44-6v expression and 8 (35%) had CD44-9v immunoreactivity. None of 10 normal contralateral ovaries expressed CD44s or any splice variants. In 2 cases we had tumor available from the primary surgery, and subsequent recurrences. Both recurrences showed decreased expression of CD44-4v and CD44-6v. CONCLUSIONS: Clear cell carcinoma of the ovary shows an abnormal pattern of CD44s expression and mRNA splicing when compared to the contralateral normal ovary in the same patient. Metastases of clear cell carcinoma show a downregulation in expression of some splice variants. Furthermore, we have data that suggest that as the tumors recur, CD44s and its isoforms are downregulated. Our results suggest that alternative mRNA splicing of CD44 may be important in the development of metastases from clear cell carcinoma of the ovary.

Hypoxia-induced pRB hypophosphorylation results from downregulation of CDK and upregulation of PP1 activities.

Exposure of CV-1P cells to hypoxic conditions results in reversible cell cycle arrest concomitant with accumulation of pRB in the hypophosphorylated, growth suppressive form. Similar to cell cycle arrest induced by serum starvation, we show here that hypoxia-induced arrest is accompanied by a decrease in pRB-directed CDK4 and CDK2 activities, lower cyclin D and E protein levels, and by an increase in p27 protein abundance. Immunoprecipitation studies reveal an increase in p27 association with cyclin E-CDK2 complexes. In contrast to cell cycle arrest induced by serum starvation, hypoxia increases PP1-mediated pRB dephosphorylation. These data reveal that synergy between decreased pRB-directed cyclin/CDK activity and increased pRB-directed phosphatase activity contribute towards inducing and maintaining pRB in its hypophosphorylated, growth suppressive state during hypoxia.

Rb binds c-Jun and activates transcription.

The retinoblastoma protein (Rb) acts as a critical cell-cycle regulator and loss of Rb function is associated with a variety of human cancer types. Here we report that Rb binds to members of the AP-1 family of transcription factors, including c-Jun, and stimulates c-Jun transcriptional activity from an AP-1 consensus sequence. The interaction involves the leucine zipper region of c-Jun and the B pocket of Rb as well as a C-terminal domain. We also present evidence that the complexes are found in terminally differentiating keratinocytes and cells entering the G1 phase of the cell cycle after release from serum starvation. The human papillomavirus type 16 E7 protein, which binds to both c-Jun and Rb, inhibits the ability of Rb to activate c-Jun. The results provide evidence of a role for Rb as a transcriptional activator in early G1 and as a potential modulator of c-Jun expression during keratinocyte differentiation.

Mitogenic activity of steroidogenesis-inducing protein (SIP) during hypoxic stress of human ovarian carcinoma cells.

Steroidogenesis-inducing protein (SIP) is a novel growth factor isolated from human ovarian follicular fluid. While the steroidogenic and mitogenic effects appear to be restricted towards gonadal cell types, we have recently demonstrated that SIP is also a potent mitogen for cell lines derived from ovarian surface epithelial carcinomas. Here, we demonstrate that SIP reverses hypoxia-induced cell proliferation arrest of the human ovarian carcinoma cell line SKA, as determined by flow cytometry and cell proliferation assays. Concomitant with this reversal of proliferation arrest is an increase in expression of cyclins D and E and a reduction in expression of the cyclin-dependent kinase inhibitor p27. Pretreatment of hypoxic SKA cells with SIP is also shown to increase Taxol sensitivity of these cells by two-fold. These studies further characterize the mitogenic activity of SIP at the molecular level and suggest that this protein may be an effective biological response modifier for ovarian carcinoma cells.

Regulation of human placental development by oxygen tension.

Cytotrophoblasts, specialized placental cells, proliferate early in pregnancy and then differentiate into tumor-like cells that establish blood flow to the placenta by invading the uterus and its vasculature. In this study, cytotrophoblasts cultured under hypoxic conditions (2 percent oxygen), mimicking the environment near the uterine surface before 10 weeks of gestation, continued proliferating and differentiated poorly. When cultured in 20 percent oxygen, mimicking the environment near uterine arterioles, the cells stopped proliferating and differentiated normally. Thus, oxygen tension determines whether cytotrophoblasts proliferate or invade, thereby regulating placental growth and cellular architecture.

Steroidogenesis-inducing protein, isolated from human ovarian follicular fluid, is a potent mitogen for cell lines derived from ovarian surface epithelial carcinomas.

The majority of human ovarian cancers originate from the surface epithelial (OSE) cells that surround the ovary. The incidence of OSE cancer is correlated with the number of ovulations that occur during fertile life. OSE cells remain quiescent but undergo rapid mitotic activity after ovulation to repair the wound. This increase in mitotic activity following each ovulation may give rise to mutations that make the OSE susceptible to malignant transformation. Steroidogenesis-inducing protein (SIP), a protein isolated from human follicular fluid obtained from hyperstimulated ovaries, is a potent mitogen for several gonadal cells. To investigate the possibility that SIP may be involved in the proliferation of OSE cells, we have studied its effects on DNA synthesis in seven cell lines derived from OSE carcinomas (HEY, MLS, SKA, OW-1, SAU, NIH:OVCAR-3, and Caov-3). The cells were cultured in serum-free medium in the presence of SIP for 18 hr, followed by incubation with [3H]thymidine for 4 hr. The radioactivity incorporated into the DNA was measured. SIP stimulated DNA synthesis in six of the cell lines. HEY, SKA, MLS, and OVCAR3 were most responsive to SIP. Interactions between SIP and other growth factors and cytokines known to be present in follicular fluid (EGF/TGFalpha, TGFbeta, FGF, IGF-1, IL-1beta, and TNFalpha) were also investigated in HEY and SKA cells. EGF/TGFalpha and IGF-1 potentiated the effects of SIP. TGFbeta had no effect on SIP, and/or EGF/TGFalpha stimulated DNA synthesis. Other growth factors which were tested in this study had no effect on DNA synthesis in SKA cells. Dibutyryl cyclic-AMP blocked the effects of SIP on DNA synthesis. We conclude that SIP is a potent mitogen for OSE cell lines and together with TGFalpha and IGF-1 may be involved in the proliferation of normal OSE cells after ovulation. Since SIP is obtained from the preovulatory follicle, it may represent a link between the number of ovulations and the increased incidence of OSE cancers.

Characterization of the mitotic phase pRb-directed protein phosphatase activity.

We have previously reported on the M-phase specific dephosphorylation of pRb and identified a type 1 serine/threonine protein phosphatase (PP1) as the enzyme mediating pRb dephosphorylation. In this report, we have characterized the pRb-directed phosphatase activity found in mitotic cells with respect to dose dependence and demonstrate that the pRb isoform conversion detected in vitro mirrors the pRb isoform conversion which occurs during mitosis of intact cells. Cell fractionation and PP1 catalytic subunit isolation studies support the notion that the pRb-directed phosphatase activity involves subpopulations of PP1 catalytic subunits. Coprecipitation studies revealed that PP1 can form a complex with hypophosphorylated pRb which was converted from the hyperphosphorylated form in mitotic cell extracts. Taken together with data from previous reports in the literature, a model for the regulation of PP1 activity towards pRb during mitotic exit is proposed.

High molecular weight protein phosphatase type 1 dephosphorylates the retinoblastoma protein.

pRb controls cell proliferation by restricting inappropriate entry of cells into the cell division cycle. As dephosphorylation of pRb during mitotic exit activates its growth suppressive function, identification of the protein phosphatase that dephosphorylates pRb, and characterization of the mechanism of its regulation, are essential to elucidating the mechanisms of cell growth control. By fractionating mitotic CV-1P cell extracts, we identify the protein phosphatase which dephosphorylates pRb as a type 1 serine/threonine phosphoprotein phosphatase (PP1). Molecular sizing analyses indicate that the catalytic enzyme (PP1c) is present in a high molecular weight complex, with a predicted molecular mass of 166 kDa. PP1-interacting proteins in the mitotic cell extracts are identified. Two PP1-interacting proteins (41 and 110 kDa) are shown to form distinct complexes with PP1c from fractions of separated mitotic cell extracts containing phosphorylase phosphatase activity. However, only the 110-kDa PP1-interacting protein is present in fractions containing pRb-directed phosphatase activity, identifying this protein as a putative activator of PP1 function toward pRb during mitosis.