Biology

The retinoblastoma gene product (pRb) interacts with many cellular proteins to function in the control of cell division, dierentiation, and apoptosis. Several pRb binding proteins complex with pRb through an amino acid sequence called the LXCXE motif. The catalytic subunit of DNA polymerase d (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 G 1 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-pRb. 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 d activity. Oncogene (2000) 19, 5464 ± 5470.

C57BL/GJ mice were treated with N-methylnitrosourea (NMU) and the evolution of leukemic T-cells clones into frank thymic lymphomas was followed in 42 animals using serology of T-cell markers, rearrangements of the T-cell receptor genes y, /II and 82 and detection of carcinogen-induced Ki-ras mutations and trisomy of chromosome 15. During the latent period, multiple populations of T-cell clones were present in the thymus, many contained trisomy 15, but few had detectable Ki-ras mutations. Since most frank lymphomas consisted of a single T-cell clone with both a mutation of Ki-ras and trisomy 15, the results imply that these two events are critical for the evolution of T-cell clones from the preleukemic phase to a more malignant disease stage. Progression to frank lymphomas is coincident with changes in the expression pattern of the T-cell growth factor interleukin-2 receptor, which may play a role in the selection, expansion and thymus-independent growth of a T-cell clone.

In this study, we determined the effects of the cyclin-dependent kinase (cdk) inhibitors, olomoucine and roscovitine, on the circadian rhythm of optic nerve impulse activity recorded from the eye of the marine snail Bulla gouldiana. 2. We found that olomoucine lengthened period and altered circadian phase in a dose-dependent manner without appreciably affecting gene transcription or translation. We also found that the more specific cdk inhibitor, roscovitine, was approximately 10-fold more effective in lengthening circadian period, while the inactive analogue, iso-olomoucine, was ineffective. 3. The current results, along with previous results from our laboratory, are consistent with the hypothesis that the biochemical mechanism responsible for generating the ocular circadian rhythm in B. gouldiana is related to the biochemical mechanism that regulates the eukaryotic cell division cycle, i.e., by modulation of the activity of protein kinases belonging to the cdk family.

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 ...

Global climate change and anthropogenic activities are threatening the future survival of coral reef ecosystems. The ability of reef-building zooxanthellate coral to survive these stressors may be determined through fundamental differences within their symbiotic dinoflagellates (Symbiodinium sp.). We define the in vitro apoptotic response of 2 evolutionarily distant Symbiodinium sp., subtypes B2 and C1, to determine the synergistic effects of disease and temperature on cell viability using flow cytometry. The putative yellow band disease (YBD) consortium of Vibrio spp. bacteria and temperature (33°C) had a positive synergistic effect on C1 apoptosis, while B2 displayed increased apoptosis to elevated temperature (29 and 33°C), the Vibrio consortium, and a lone virulent strain of V. alginolyticus, but no synergistic effects. Additionally, heat shock protein 60 expression revealed differential cell-mediated temperature sensitivity between subtypes via western blotting. This result mar...

The recent finding that the Retinoblastoma protein (Rb) is able to regulate apoptosis in a non-transcriptional manner directly at the mitochondria by interaction with the pro-apoptotic protein Bax prompted this investigation of the complex formed between Rb and Bax. Because the function of Rb in the cellular processes of proliferation, apoptosis, senescence and differentiation is regulated by phosphorylation we endeavored to elucidate the phosphorylation status of Rb with respect to its association with Bax and its role in apoptosis. In this study we found that Rb phosphorylated on at least 4 C-terminal phosphorylation sites (S608, S795, S807/S811, and T821) is present at the mitochondria under non-stressed cellular conditions. An in vitro binding assay showed that Bax binds to Rb phosphorylated at S807/S811 in 3 cancer cell types. Physiologically relevant association between Bax and Rb phosphorylated on S807/S811 was demonstrated by reciprocal co-immunoprecipitation experiments usi...

The phosphorylation state of Retinoblastoma protein (Rb) plays a role in cell proliferation and apoptosis. Within cells, cyclin dependent kinases (cdks) phosphorylate Rb in response to growth stimulatory signals, whereas protein phosphatase 1 (PP1) dephosphorylates Rb when cells stop proliferating or undergo apoptosis in response to antiproliferative or stress signals. Stimulation of PP1 activity via siRNA mediated knockdown of its interacting protein PNUTS (Phosphatase Nuclear Targeting Subunit) leads to Rb dephosphorylation and apoptosis in cancer cells. We utilized two separate methods to modulate the phosphorylation state of Rb in cancer cells. Kinase activity toward Rb is inhibited by the clinically relevant cdk inhibitor, Roscovitine. In addition, siRNA mediated PNUTS knockdown stimulates phosphatase activity toward Rb. Either of these treatments in cancer cells causes a 2-fold stimulation of apoptosis. When activation of phosphatase activity is combined with inhibition of cdk activity toward Rb, however, cells exhibit a 4-fold increase in apoptosis. The mechanism by which PNUTS knockdown mediated PP1 activation leads to apoptosis was determined to be dependent on the activity of the transcription factor E2F1. The Rb phosphorylation profiles resulting from each treatment were analyzed and found to be similar but not identical. In addition, the two treatments differentially effect the expression of bcl-2 family proteins. Thus inhibition of cdk activity and activation of PP1 activity toward pRb are functionally distinct processes that together increase the apoptotic effect in cells.

Protein phosphatase type 1 catalytic subunit (PP1c) is a serine/threonine phosphatase involved in the dephosphorylation of many proteins in eukaryotic cells. It associates with several known targeting or regulatory subunits that directly regulate PP1c activity toward specific substrates. The recently identified Phosphatase Nuclear Targeting Subunit (PNUTS) binds to PP1c and inhibits PP1 activity toward phosphorylase a. One of the substrates of PP1c has been shown to be the cell cycle regulatory protein, Retinoblastoma (pRb). In this study, we show that PNUTS dissociates from PP1c under mildly hypoxic cell growth conditions that lead to an increase of PP1c activity toward pRb. We developed an assay that measures pRb-directed PP1c activity and show that a GST-PNUTS fusion protein inhibits phosphatase activity toward pRb when using PP1c from cell lysates, GST-PP1c, or purified PP1c. These studies suggest that PNUTS is involved in the regulation of PP1c activity toward pRb.

Immunoprecipitated retinoblastoma protein from HL60 cells migrated as a series of bands during electrophoresis. The heterogeneity appeared to be generated by phosphorylation of the retinoblastoma protein. Treatment of the cells with the phorbol ester, tetradecanoyl phorbol acetate (TPA), resulted in both a loss of the heterogeneity of the pRB species and a significant decrease in the level of pRB phosphorylation. These changes accompanied differentiation of the HL60 cells into macrophages. Treatment of the cells with dibutyryl cAMP also resulted in dephosphorylation of pRB as well as cell cycle arrest, although no recognizable differentiation occurred. These results are consistent with a model in which TPA and dibutyryl cAMP dependent pathways can activate pRB by altering its phosphorylation.Key words: retinoblastoma, cell cycle, differentiation, leukemia.

There is abundant evidence that Retinoblastoma (Rb) activity is important in the control of cell proliferation and apoptosis. Reversible phosphorylation of the Rb protein that is carried out by cyclin dependent kinases and Protein phosphatase 1 (PP1) regulates its functions. A PP1 interacting protein, PNUTS (Phosphatase Nuclear Targeting Subunit) is proposed to be a regulator of Rb phosphorylation. In this study, PNUTS knockdown in MCF7, SKA and HCT116 cancer cells causes a reduction in viability due to increased apoptosis. However, normal cells (MCF10A breast and CCD-18Co colon) do not exhibit reduced viability when PNUTS expression is diminished. PNUTS knockdown has no effect in Rb-null Saos-2 cells. However, when Rb is stably expressed in Saos-2 cells, PNUTS knockdown reduces cell number. Knockdown of PNUTS in p53-/-HCT116 cells indicates that p53 is dispensable for the induction of apoptosis. Loss of PNUTS expression results in increased Rb-phosphatase activity and Rb dephosphorylation. E2F1 dissociates from Rb in cells depleted of PNUTS and the resulting apoptosis is dependent on caspase-8. These results indicate that Rb phosphorylation state can be manipulated by targeting Rb phosphatase activity and suggest that PNUTS may be a potential target for therapeutic pro-apoptotic strategies. Results PNUTS RNAi effects cell number in cancer cells but not in nontransformed cells. Based on previous in vitro studies suggesting that PNUTS inhibited Retinoblastoma-directed PP1 activity, 13 and recent data suggesting that PNUTS plays a role in controlling cell death in response to cellular stress, 32 we used RNA interference to determine the effect of PNUTS knockdown on cell proliferation and cell death. Oligonucleotides were designed and utilized as described

Steroidogenesis-inducing protein (SIP) is a novel growth factor isolated from human ovarian follicular¯uid. 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¯ow 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 twofold. These studies further characterize the mitogenic activity of SIP at the molecular level and suggest that this protein may be an effective biological response modi®er for ovarian carcinoma cells.

Exposure of CV-1P cells to hypoxic conditions causes cell proliferation inhibition concomitant with the accumulation of pRb in the hypophosphorylated, growth suppressive form. This is in part due to inhibition of pRb-directed cdk4 and cdk2 activity. In this study we attempted to elucidate the mechanism by which cdk4 is inactivated under hypoxic conditions. After 18 h of hypoxia, CV-1P cells are inhibited from progressing from G 1 phase into the S phase of the cell cycle. This occurs in conjunction with dephosphorylation of serine-795, which is a putative substrate of cdk4. The amounts of cdk4, cdk6, and the D type cyclins are not affected by 18 h of hypoxia. The levels of cdki p16, p18, p19, and p57 under aerobic or hypoxic conditions were analyzed and although the levels of most cdki are unaffected by hypoxic conditions, the level of p16 increases significantly by 18 h of hypoxia. The mechanism by which cdk4 activity is inhibited under hypoxic conditions may be mediated through p16 association with cdk4. Immunoprecipitation analysis shows that p16 binds to cdk4 under hypoxic conditions but does not in cells maintained under aerobic conditions. Thus p16 may be involved in hypoxia-induced growth inhibition.

The tumor suppressor Retinoblastoma (Rb) protein is highly phosphorylated in cancer cells largely due to the overexpression of cyclins or the loss of expression of cyclin dependent kinase inhibitors (cdki). Hyperphosphorylation of Rb promotes proliferation, and plays a role in the regulation of apoptosis. Recently, inhibition of cyclin dependent activity toward Rb has been identified as a strategy that has shown clinical efficacy. We utilized a method to induce phosphatase activity toward Rb in cells by shRNA silencing of PNUTS (Phosphatase Nuclear Targeting Subunit) that regulates PP1-mediated dephosphorylation of Rb. In this study, the effect of Rb dephosphorylation on the epithelial to mesenchymal transition (EMT) was determined. The EMT transition is observed in cancer cells that have acquired invasive characteristics. In breast cancer cells grown in 3D Matrigel cultures, MCF7 cells undergo apoptosis in response to Rb dephosphorylation, whereas MDA-MB-231 and Hs578T cells exhibit a reduction in the EMT. Cells devoid of phosphorylated Rb (nontransformed MCF10A and Rb-null MDA-MB-468) lacked any response to PNUTS depletion, showing the effect is Rb-dependent. In addition, these studies showed that Rb dephosphorylation in 3D Matrigel cultures of highly invasive HT1080 cells led to the inhibition of the EMT. Furthermore we observed association between dephosphorylated Rb with ZEB1, a zinc-finger E-box-binding transcription factor that regulates expression of E-and N-cadherins. Finally Rb dephosphorylation led to inhibition of ZEB1 transcriptional activity, this data supports the notion that Rb dephosphorylation modulates the EMT. These studies suggest targeting Rb phosphorylation in mesenchymal cancer cells may decrease invasiveness.