Tumor suppressor gene p16/INK4A/CDKN2A‐dependent regulation into and out of the cell cycle in a spontaneous canine model of breast cancer

p16/INK4A/CDKN2A is an important tumor suppressor gene that arrests cell cycle in G1 phase inhibiting binding of CDK4/6 with cyclin D1, leaving the Rb tumor suppressor protein unphosphorylated and E2F bound and inactive. We hypothesized that p16 has a role in exit from cell cycle that becomes defective in cancer cells. Well characterized p16‐defective canine mammary cancer cell lines (CMT28, CMT27, and CMT12), derived stably p16‐transfected CMT cell clones (CMT27A, CMT27H, CMT28A, and CMT28F), and normal canine fibroblasts (NCF), were used to investigate expression of p16 after serum starvation into quiescence followed by re‐feeding to induce cell cycle re‐entry. The parental CMT cell lines used lack p16 expression either at the mRNA or protein expression levels, while p27 and other p16‐associated proteins, including CDK4, CDK6, cyclin D1, and Rb, were expressed. We have successfully demonstrated cell cycle arrest and relatively synchronous cell cycle re‐entry in parental CMT12, CMT28 and NCF cells as well as p16 transfected CMT27A, CMT27H, CMT28A, and CMT28F cells and confirmed this by ³H‐thymidine incorporation and flow cytometric analysis of cell cycle phase distribution. p16‐transfected CMT27A and CMT27H cells exited cell cycle post‐serum‐starvation in contrast to parental CMT27 cells. NCF, CMT27A, and CMT28F cells expressed upregulated levels of p27 and p16 mRNA, post‐serum starvation, as cells exited cell cycle and entered quiescence. Because quiescence and differentiation are associated with increased levels of p27, our data demonstrating that p16 was upregulated along with p27 during quiescence, suggests a potential role for p16 in maintaining these non‐proliferative states. J. Cell. Biochem. 114: 1355–1363, 2013. © 2012 Wiley Periodicals, Inc.     

Knockdown of CDK2AP1 in Primary Human Fibroblasts Induces p53 Dependent Senescence

Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a) no increase in senescence associated beta-galactosidase activity, (b) decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c) decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1 knockdown. Altogether, our results show that knockdown of CDK2AP1 in primary human fibroblasts reduced proliferation and induced premature senescence, with the observed phenotype being p53 dependent.     

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.     

Effects of modulators of protein phosphorylation on heme metabolism in human hepatic cells: induction of delta-aminolevulinic synthase mRNA and protein by okadaic acid

Effects of modulators of protein phosphorylation on delta-aminolevulinic acid (ALA) synthase and heme oxygenase-1 mRNA were analyzed in the human hepatic cell lines Huh-7 and HepG2 using a quantitative RNase protection assay. Okadaic acid was found to induce ALA synthase mRNA in a concentration-dependent fashion in both Huh-7 and HepG2 cells. The EC(50) for induction of ALA synthase mRNA in Huh-7 cells was 13.5 nM, with maximum increases occurring at okadaic acid concentrations of 25-50 nM. The EC(50) for induction of ALA synthase mRNA in HepG2 cells was 35.5 nM, with maximum increases occurring at okadaic acid concentrations of 50 nM. Concentration-dependent induction of ALA synthase mRNA paralleled the increase in ALA synthase protein. Maximum induction of ALA synthase was observed between 5 and 10 h post-treatment in both cell lines. Induction of ALA synthase mRNA in Huh-7 cells, but not HepG2 cells, was associated with an increase in ALA synthase mRNA stability. Okadaic acid also induced heme oxygenase-1 mRNA in both cell lines, but the magnitude of induction was only twofold, and was rapid and transient. Okadaic acid and phorbol 12-myristate 13-acetate significantly decreased heme-mediated induction of heme oxygenase-1 mRNA in both Huh-7 and HepG2 cells. Wortmannin diminished the heme-mediated induction of heme oxygenase-1 mRNA in HepG2 cells, but not Huh-7 cells. These results report a novel property of okadaic acid to affect heme metabolism in human cell lines.     

Treatment of myeloid leukemic cells with the phosphatase inhibitor okadaic acid induces cell cycle arrest at either G1/S or G2/M depending on dose.

The phosphatase inhibitor okadaic acid was found to induce cell cycle arrest of human myeloid leukemic cell lines HL-60 and U937 in a concentration- and time-dependent manner. Exposure to low concentrations of okadaic acid (2-8nM) for 24-48 hr caused greater than 70% of cells to arrest at G2/M, with up to 40% of the cells arrested in early mitosis. Cell viability decreased rapidly after 48 hr of treatment, and morphological and DNA structure analysis indicated that this was primarily due to the induction of apoptosis. The cells arrested in mitosis by 8 nM okadaic acid could be highly enriched by density gradient centrifugation and underwent apoptosis when further cultured either with or without okadaic acid, indicating that the effects of okadaic acid were irreversible. In contrast to the effects of low concentrations of okadaic acid, high concentrations (500 nM), inhibited proliferation in less than 3 hr. Remarkably, the majority of cells also entered a mitosis-like state characterized by dissolution of the nuclear membrane and condensation and partial separation of chromosomes. However, these cells had a diploid content of DNA, indicating that the cell cycle arrest occurred at G1/S with premature chromosome condensation (PCC), rather than at G2/M. If cells were first blocked at G1/S with hydroxyurea and then treated with okadaic acid, greater than 90% developed PCC in less than 3 hr without replicating their DNA. Caffeine was not able to induce PCC in these cells, either with or without prior inhibition of DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative study of toxicological and cell cycle effects of okadaic acid and dinophysistoxin-2 in primary rat hepatocytes

AimsTo determine the relative toxicity and effects on the cell cycle of okadaic acid and dinophysistoxin-2 in primary hepatocyte cultures.Main methodsCytotoxicity was determined by the MTT method, caspase-3 activity and lactate dehydrogenase release to the medium. The cell cycle analysis was performed by imaging flow cytometry and the effect of the toxins on cell proliferation was studied by quantitative PCR and confocal microscopy.Key findingsWe show that dinophysistoxin-2 is less toxic than okadaic acid for primary hepatocytes with a similar difference in potency as that observed in vivo in mice after intraperitoneal injection. Both toxins induced apoptosis with caspase-3 increase. They also inhibited the hepatocytes cell cycle in G1 affecting diploid cells and diploid bi-nucleated cells. In proliferating hepatocytes exposed to the toxins, a decrease of p53 gene expression as well as a lower protein level was detected. Studies of the tubulin cytoskeleton in toxin treated cells, showed nuclear localization of this molecule and a granulated tubulin pattern in the cytoplasm.SignificanceThe results presented in this work show that the difference in toxicity between dinophysistoxin-2 and okadaic acid in cultured primary hepatocytes is the same as that observed in vivo after intraperitoneal injection. Okadaic acid and dinophysistoxin-2 arrest the cell cycle of hepatocytes at G1 even in diploid bi-nucleated cells. p53 and tubulin could be involved in the cell cycle inhibitory effect.     

Okadaic acid co-induces vimentin expression and cell cycle arrest in MPC-11 mouse plasmacytoma cells

The effect of the tumor promoter okadaic acid on cell cycle progression and on vimentin expression in MPC-11 mouse plasmacytoma cells was compared with that of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Cell cycle progression of asynchronously grown MPC-11 cells was inhibited by both agents, but, in contrast to the G1 phase arrest caused by TPA, okadaic acid gave rise to G2/M phase and S phase arrest. This effect of okadaic acid was delayed significantly compared to the TPA-caused arrest. Furthermore, okadaic acid was able to induce vimentin expression to an extent comparable to the TPA response. However, vimentin expression was markedly delayed in okadaic acid-treated relative to TPA-treated cells. Another protein phosphatase inhibitor, calyculin A, also induced cell cycle changes and vimentin expression at concentrations at or above 1 × 10^?9M. Based on these observations, we suggest an involvement of protein phosphatase 1 (possibly also phosphatase 2A and/or other phosphatases) in both the G2/M cell cycle block and the induction of vimentin expression in MPC-11 cells by okadaic acid. © 1995 Wiley-Liss, Inc.     

The tumour suppressor gene product APC blocks cell cycle progression from G0/G1 to S phase.

The APC gene is mutated in familial adenomatous polyposis (FAP) as well as in sporadic colorectal tumours. The product of the APC gene is a 300 kDa cytoplasmic protein associated with the adherence junction protein catenin. Here we show that overexpression of APC blocks serum-induced cell cycle progression from G0/G1 to the S phase. Mutant APCs identified in FAP and/or colorectal tumours were less inhibitory and partially obstructed the activity of the normal APC. The cell-cycle blocking activity of APC was alleviated by the overexpression of cyclin E/CDK2 or cyclin D1/CDK4. Consistent with this result, kinase activity of CDK2 was significantly down-regulated in cells overexpressing APC although its synthesis remained unchanged, while CDK4 activity was barely affected. These results suggest that APC may play a role in the regulation of the cell cycle by negatively modulating the activity of cyclin-CDK complexes.     

Involvement of p27Kip1 in the G1- and S/G2-phase lengthening mediated by glucocorticoids in normal human lymphocytes.

Glucocorticoids inhibit cell proliferation by inducing cell cycle lengthening. In this report, we have analyzed, in normal peripheral blood lymphocytes, the involvement of p27Kip1 in this slowing of proliferation. Following dexamethasone (DXM) treatment, p27Kip1 expression and regulation varied differently with the level of lymphocyte stimulation. In quiescent cells, DXM inhibited p27Kip1 protein expression by decreasing its rate of synthesis, whereas its half-life and mRNA steady state remained constant. In contrast, in stimulated lymphocytes, DXM increased p27Kip1 expression by enhancing its mRNA steady state. This increase is not only a consequence of the DXM-induced interleukin 2 inhibition: we also found an increase in p27Kip1 mRNA stability that was not observed in quiescent lymphocytes. Cyclin/cyclin-dependent kinase (CDK) complexes immunoprecipitated with p27Kip1 are differentially modified by DXM addition: (a) G1 kinasic complexes (cyclin D/CDK4 or CDK6) associated with p27Kip1 are strongly decreased by DXM, (b) S-phase complexes (CDK2/cyclin E and A) remained stable or increased, and (c) the association of p27Kip1 with the phosphorylated forms of CDK1 is increased by DXM. In addition, CDK2 kinase activity was decreased in DXM-treated cells: we suggest that p27Kip1 might participate in inhibiting its catalytic activity. These results indicated that, in normal lymphoid cells, p27Kip1 may be involved in DXM antiproliferative effects. The increase of p27Kip1 expression and a decrease in G1 mitogenic factors, together with the redistribution of p27Kip1 to S/G2-M regulatory complexes, may explain the lengthening of G1 and S/G2 after DXM treatment in lymphocytes.     

Cyclin E and SV40 small T antigen cooperate to bypass quiescence and contribute to transformation by activating CDK2 in human fibroblasts

Cyclin E overexpression is observed in multiple human tumors and linked to poor prognosis. We have previously shown that ectopic expression of cyclin E is sufficient to induce mitogen-independent cell cycle entry in a variety of tumor/immortal cell lines. Here we have investigated the rate-limiting step leading to cell cycle entry in quiescent normal human fibroblasts (NHF) ectopically expressing cyclin E. We found that in serum-starved NHF, cyclin E forms inactive complexes with CDK2 and fails to induce DNA synthesis. Coexpression of SV40 small t antigen (st), but not other tested oncogenes, efficiently induces mitogen-independent CDK2 phosphorylation on Thr-160, CDK2 activation, and DNA synthesis. Additionally, in contact-inhibited NHF ectopically expressing cyclin E, st induces cell cycle entry, continued proliferation, and foci formation. Coexpression of cyclin E and st also bypasses G(0)/G(1) arrests induced by CDK inhibitors. Although CDK2 is dispensable for G(0)/G(1) cell cycle entry and normal proliferation in mammals, CDK2 activity is an essential rate-limiting step in NHF with deregulated cyclin E expression and altered PP2A activity, which endows primary cells with transformed features. Consequently, CDK2 could be targeted therapeutically in tumors that involve these alterations. These data also suggest that alterations prior to cyclin E deregulation facilitate proliferation of tumor cells by bypassing mitogenic requirements and negative regulation by adjacent cells.     

Regulation of the cell cycle in response to inhibition of mitochondrial generated energy

Cell cycle control is regulated through the temporal action of both cyclin-dependent kinases and cyclin binding partners. Previously, we have demonstrated that low doses of oligomycin result in a cell cycle arrest of HL-60 cells in G(1) [S. Sweet, G. Singh, Accumulation of human promyelocytic leukemic (HL-60) cells at two energetic cell cycle checkpoints, Cancer Res. 55 (1995) 5164-5167]. In this study, we provide the molecular mechanisms for the observed G(1) arrest following mitochondrial ATPase inhibition. Protein expression of cyclin E and CDK2, the kinase activity of complexed cyclin E/CDK2, and protein expression of p16, p21, and p27 were all unaffected by oligomycin administration. While CDK4 levels were unchanged following oligomycin treatment, a dramatic reduction in cyclin D(1) was observed. Moreover, increased amounts of hypo-phosphorylated retinoblastoma protein (Rbp) and Rbp bound E2F were observed following mitochondrial ATP synthase inhibition. These data provide further evidence that surveillance of available energy occurs during G(1) and ATP deprivation results in cell cycle arrest via a reduction in cyclin D.     

Coiled body heterogeneity induced by G(1) arrest with amiloride + bumetanide

Ki67 is a nuclear protein expressed in proliferating cells, but not in quiescent or G(0)-arrested cells. Similar to the proliferating cell nuclear antigen and several other well-characterized molecules, Ki67 exhibits a repeating pattern of regulated expression and redistribution during the cell cycle, making it a useful marker for cell cycle phase. In addition to other structures labeled, concentrated foci may be observed in the nucleus and sometimes the cytoplasm. We observed that these Ki67 foci can be found at any stage of the endothelial cell cycle. They are not coincident with coiled bodies (CB), as determined in double-label immunofluorescence experiments with anti-Ki67 and antibodies to the CB marker protein pigpen. However, arrest of BPA47 endothelial cells in G(1) with amiloride + bumetanide induces colocalization of pigpen and Ki67 in 40% of cells exhibiting Ki67 foci. We conducted a series of experiments to examine the possibilities that pigpen was exported from CB and into unique, Ki67-containing foci or that Ki67 was imported into pigpen-containing CB. Our results showed us that although CB typically contain both coilin and pigpen, amiloride + bumetanide-induced G(1) arrest reconfigured the CB compartment into three populations of foci: one containing pigpen without coilin, the second containing coilin without pigpen, and a third containing both pigpen and coilin together. Furthermore, G(1) arrest resulted in Ki67 redistribution into both coilin- and pigpen-containing foci. The results suggest that under certain conditions, "resident" CB proteins can be differentially redistributed, and proteins not previously recognized as resident in CB can be driven into that compartment. Our observations underscore the fluid nature of CB. They demonstrate that previously reported heterogeneity in the CB compartment can be amplified by a specific experimental manipulation. This may be useful in future analyses of protein trafficking within the CB compartment and between CB and other cellular compartments. Finally, the redistribution of Ki67 into CB represents a new finding for a widely expressed but poorly understood molecule, one that may be useful in elucidating function.     

CDK1 inhibition sensitizes normal cells to DNA damage in a cell cycle dependent manner

Cyclin-dependent kinase 1 (CDK1) orchestrates the transition from the G2 phase into mitosis and as cancer cells often display enhanced CDK1 activity, it has been proposed as a tumor specific anti-cancer target. Here we show that the effects of CDK1 inhibition are not restricted to tumor cells but can also reduce viability in non-cancer cells and sensitize them to radiation in a cell cycle dependent manner.

Radiosensitization by the specific CDK1 inhibitor, RO-3306, was determined by colony formation assays in three tumor lines (HeLa, T24, SQ20B) and three non-cancer lines (HFL1, MRC-5, RPE). Initial results showed that CDK1 inhibition radiosensitized tumor cells, but did not sensitize normal fibroblasts and epithelial cells in colony formation assays despite effective inhibition of CDK1 signaling. Further investigation showed that normal cells were less sensitive to CDK1 inhibition because they remained predominantly in G1 for a prolonged period when plated in colony formation assays. In contrast, inhibiting CDK1 a day after plating, when the cells were going through G2/M phase, reduced their clonogenic survival both with and without radiation. Our finding that inhibition of CDK1 can damage normal cells in a cell cycle dependent manner indicates that targeting CDK1 in cancer patients may lead to toxicity in normal proliferating cells. Furthermore, our finding that cell cycle progression becomes easily stalled in non-cancer cells under normal culture conditions has general implications for testing anti-cancer agents in these cells.     

Regulation of cyclin-dependent kinase 2 activity by ceramide

Cyclin-dependent kinases have been implicated in the inactivation of retinoblastoma (Rb) protein and cell cycle progression. Recent studies have demonstrated that the lipid molecule ceramide is able to induce Rb hypophosphorylation leading to growth arrest and cellular senescence. In this study, we examined the underlying mechanisms of Rb hypophosphorylation and cell cycle progression utilizing the antiproliferative molecule ceramide. C6-Ceramide induced a G0/G1 arrest of the cell cycle in WI38 human diploid fibroblasts. Employing immunoprecipitation kinase assays, we found that ceramide specifically inhibited cyclin-dependent kinase CDK2, with a mild effect on CDC2 and significantly less effect on CDK4. The effect of ceramide was specific such that C6-dihydroceramide was not effective. Ceramide did not directly inhibit CDK2 in vitro but caused activation of p21, a major class of CDK-inhibitory proteins, and led to a greater association of p21 to CDK2. Using purified protein phosphatases, we showed that ceramide activated both protein phosphatase 1 and protein phosphatase 2A activities specific for CDK2 in vitro. Further, calyculin A and okadaic acid, both potent protein phosphatase inhibitors, together almost completely reversed the effects of ceramide on CDK2 inhibition. Taken together, these results demonstrate a dual mechanism by which ceramide inhibits the cell cycle. Ceramide causes an increase in p21 association with CDK2 and through activation of protein phosphatases selectively regulates CDK2. These events may lead to activation of Rb protein and subsequent cell cycle arrest.     

Dynamic changes in subnuclear NP95 location during the cell cycle and its spatial relationship with DNA replication foci

We determined the expression and subcellular localization of nuclear protein NP95 during the cell cycle in mouse 3T3 cells. The levels of NP95 mRNA and protein were extremely low in quiescent cells; however, stimulation with 10% serum increased their expressions in a time course similar to that of the late growth-regulated gene proliferating cell nuclear antigen (PCNA). Subnuclear location of NP95 dynamically changed during the cell cycle. Double immunostaining for NP95 and chromatin-bound PCNA, a marker of DNA replication sites, revealed that NP95 was almost exclusively colocalized with chromatin-bound PCNA throughout the nucleus in early S phase and partly in mid-S phase. Distinct localization of the two proteins, however, became evident in mid-S phase, and thereafter, many chromatin-bound PCNA foci not carrying NP95 foci could be detected. In G2 phase, nodular NP95 foci were still identified without any chromatin-bound PCNA foci. Chromatin-bound PCNA was observed as a pre-DNA replication complex at the G1/S boundary synchronized by hydroxyurea treatment, while NP95 was detected in nucleolar regions as unique large foci. There was no significant redistribution of NP95 foci shortly after DNA damage by gamma-irradiation. Nodular NP95 foci characteristically seen in G2 phase were also detected in G2-arrested cells following gamma-irradiation. Taken together, our results indicate that NP95 is assigned to a late growth-regulated gene and suggest that NP95 does not take a direct part in DNA replication as part of the DNA synthesizing machinery, like PCNA, but is presumably involved in other DNA replication-linked nuclear events.