Activation of protein kinase C relays distinct signaling pathways in the same cell type: differentiation and caspase-mediated apoptosis

Activation of PKC with 5 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h in human U937 myeloid leukemia cells is associated with induction of adherence, followed by monocytic differentiation and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these effects about 25% of U937 cells accumulated in an apoptotic subG1 phase after TPA treatment. The appearance of these apoptotic suspension cells was detectable throughout the time course of the culture and was independent of TPA concentrations between 0.5 and 500 nM. Experiments with cells synchronized by centrifugal elutriation revealed dominant susceptibility of G1-phase cells to TPA-mediated apoptosis. While adherent cells expressed differentiation markers including the integrin CD11c, this effect was less pronounced in the TPA-treated suspension fraction. Moreover, previous work has demonstrated cell cycle arrest in differentiating U937 cells. Accordingly, PKC activation by TPA treatment was associated with a significant expression of the cdk/cyclin inhibitor p21WAF/CIP/sdi-1 in the adherent population and subsequent G0/G1 cell cycle arrest. In contrast, suspension cells failed to induce significant levels of p21WAF/CIP/sdi-1 after TPA stimulation. Immunoblotting experiments demonstrated no difference in the expression of the pro-apoptotic factors Bax, Bad, and Bak in either control U937 and TPA-treated adherent or suspension cells, respectively. However, anti-apoptotic factors including Bcl-2, Bcl-xL, and Mcl-1 were significantly induced in the adherent population whereas no induction was detectable in the suspension cells. In this context, incubation with the caspase-3/caspase-7 specific tetrapeptide inhibitor DEVD prior to TPA treatment prevented an accumulation of cells in subG1, respectively, demonstrating an involvement of these caspases. Taken together, these data suggest that PKC activation can relay distinct signaling pathways such as induction of adherence coupled with monocytic differentiation and growth arrest, or induction of caspase-mediated apoptosis coupled with the failure to adhere and to differentiate.     

Down-modulation of poly(ADP-ribose) polymerase-1 (PARP-1) in human TUR leukemia cells restores transcriptional responsiveness for differentiation and cell cycle arrest

Aggressive tumor developing human TUR myeloid leukemia cells continued cell cycle progression in the presence of the differentiation-inducing phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Similar results were obtained after stable transfection of TUR cells with the pTracer control vector (pTracer TUR cells). In contrast, TUR transfectants containing a constitutively active poly(ADP-ribose) polymerase-1 (PARP-1) gene fragment in antisense orientation within the pTracer vector (asPARP TUR cells) demonstrated increasing cell attachment and differentiation after TPA treatment. Moreover, asPARP TUR cells ceased to divide upon TPA stimulation. Cell cycle analysis revealed a predominant G0/G1 arrest and a partial G2/M arrest in TPA-treated asPARP TUR cells, whereas little if any population was detectable in S phase. Microarray gene expression analysis exhibited a significant down-regulation of cell cycle genes in phorbol ester-stimulated asPARP TUR and markedly elevated levels of differentiation-associated factors in contrast to TPA-incubated wild-type TUR cells. Whereas PARP-1 can associate with the 20S proteasome in leukemia cells, a significant reduction of this proteolytic activity was observed in asPARP TUR cells. Conversely, protein levels of manganese superoxide dismutase and the matrix metalloproteinases MMP-1 and MMP-9 were progressively increased in TPA-treated asPARP TUR cells, respectively. These findings underscore an important function of PARP-1 in human leukemia cells to connect cell cycle progression and control of differentiation.     

Regulation of Cdc2p and Cdc13p is required for cell cycle arrest induced by defective RNA splicing in fission yeast

Screening of cdc mutants of fission yeast for those whose cell cycle arrest is independent of the DNA damage checkpoint identified the RNA splicing-deficient cdc28 mutant. A search for mutants of cdc28 cells that enter mitosis with unspliced RNA resulted in the identification of an orb5 point mutant. The orb5+ gene, which encodes a catalytic subunit of casein kinase II, was found to be required for cell cycle arrest in other mutants with defective RNA metabolism but not for operation of the DNA replication or DNA damage checkpoints. Loss of function of wee1+ or rad24+ also suppressed the arrest of several splicing mutants. Overexpression of the major B-type cyclin Cdc13p induced cdc28 cells to enter mitosis. The abundance of Cdc13p was reduced, and the phosphorylation of Cdc2p on tyrosine 15 was maintained in splicing-defective cells. These results suggest that regulation of Cdc13p and Cdc2p is required for G2 arrest in splicing mutants.     

1,25-dihydroxyvitamin D(3)-induced retardation of the G(2)/M traverse is associated with decreased levels of p34(cdc2) in HL60 cells.

Cellular differentiation of neoplastic cells after exposure to 1, 25-dihydroxyvitamin D(3) (1,25 D(3)) is accompanied by altered cell cycle regulation. In previous studies, blocks in both G(1)/S and G(2)/M checkpoints have been observed in 1,25D(3)-treated HL60 cells, but the mechanism of the 1,25D(3)-induced G(2)/M block has not been previously reported. In this study, we show by cell cycle analysis, using bromodeoxyuridine pulse-chase labeling, that the G(2)/M block in 1,25D(3)-treated HL60 cells is incomplete. We also demonstrate that although the 1,25D(3)-treated cells exhibit elevated levels of cyclin B1, Cdc25C, and Cdk7, which are positive regulators of the G(2)/M traverse, these cells have decreased protein levels of p34(cdc2) and decreased p34(cdc2) kinase activity. This provides potential mechanisms for the observed accumulation of cells in the G(2) cell cycle compartment and occasional polyploidization following treatment of HL60 cells with 1,25D(3). The data also suggest that the ability of some cells to traverse this block may be the result of cellular compensatory mechanisms responding to decreased p34(cdc2) activity by increasing the levels of other regulators of the G(2) traverse, such as cyclin B1, Cdc25C, and Cdk7.     

c-Fos/activator protein-1 transactivates wee1 kinase at G(1)/S to inhibit premature mitosis in antigen-specific Th1 cells

M-phase promoting factor is a complex of cdc2 and cyclin B that is regulated positively by cdc25 phosphatase and negatively by wee1 kinase. We isolated the wee1 gene promoter and found that it contains one AP-1 binding motif and is directly activated by the immediate early gene product c-Fos at cellular G(1)/S phase. In antigen-specific Th1 cells stimulated by antigen, transactivation of the c-fos and wee1 kinase genes occurred sequentially at G(1)/S, and the substrate of wee1 kinase, cdc2-Tyr15, was subsequently phosphorylated at late G(1)/S. Under prolonged expression of the c-fos gene, however, the amount of wee1 kinase was increased and its target cdc2 molecule was constitutively phosphorylated on its tyrosine residue, where Th1 cells went into aberrant mitosis. Thus, an immediate early gene product, c-Fos/AP-1, directly transactivates the wee1 kinase gene at G(1)/S. The transient increase in c-fos and wee1 kinase genes is likely to be responsible for preventing premature mitosis while the cells remain in the G(1)/S phase of the cell cycle.     

Modulation of cell-cycle regulatory signaling network by 2-methoxyestradiol in prostate cancer cells is mediated through multiple signal transduction pathways

2-Methoxyestradiol (2-ME(2)), a promising anticancer drug, induces growth arrest and apoptosis in various androgen-dependent (LNCaP) and -independent (DU145 and PC-3) prostate cancer cell lines. Moreover, flow cytometric analysis indicated a novel dual impact of 2-ME(2) on the cell division cycle of prostate cancer cells. Chronic exposure of high doses of 2-ME(2) enhance the accumulation of cells in S and G2/M phases, while cell numbers in the G1 phase were reduced significantly by this treatment. Because cyclin B1 overexpression, induction of cdc2 phosphorylation, and its regulatory proteins wee1 and phospho-cdc25C (interphase and mitotic forms) by 2-ME(2) treatment correlated with the induction of apoptosis, growth arrest at the G2/M phase, and accumulation of the S phase, we reasoned that cyclin B1 and cdc2 phosphorylation and its upstream regulatory molecular networks may be associated with the ultimate impacts of 2-ME(2). Because phosphorylation of cdc2 and upregulation of wee1 by 2-ME(2) can be abolished by both extracellular receptor kinase (ERK) inhibitor (U0126) and c-Jun N-terminal kinase (JNK) inhibitor (SP600125), our studies indicate that the 2-ME(2)-induced upregulation of wee1 and subsequent cdc2 phosphorylation are mediated through mitogen-activated protein kinase (MAPK)-ERK-JNK signaling pathways.     

Cell cycle-dependent vimentin expression in elutriator-synchronized, TPA-treated MPC-11 mouse plasmacytoma cells.

We correlated cell cycle progression and vimentin expression at the single cell level by multiparameter flow cytometry in populations of MPC-11 cells enriched in different cell cycle phases by centrifugal elutriation and subsequently treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Synchronized, untreated cultures showed a uniform, synchronous progression through the cell cycle during further cultivation. A 6-h TPA treatment of G1-phase-enriched cultures induced both a partial G1-phase arrest in the same cycle and a moderate fraction of cells to become vimentin positive. However, nearly all cells of the cultures enriched in S- or in G2/M-phase cells could be arrested by TPA treatment at the earliest in the G1 phase of the second cell cycle and displayed higher fractions of positive cells as well as higher average levels of vimentin. After 20 h of treatment, the G1-phase arrest was almost complete. In terms of fractions of vimentin-positive cells as well as of average cellular vimentin content, the differences between the cultures resembled, albeit on a higher level, those between the respective cultures treated with TPA for 6 h. These observations might explain the striking bimodal distribution of individual cellular vimentin content detectable in G1-phase fractions of asynchronous, TPA-treated cultures. The pattern of vimentin mRNA accumulation in synchronized cultures after short-term TPA treatment strongly suggests that the cell cycle-dependent pattern of vimentin expression is caused, at least in part, by different levels of vimentin mRNA accumulated in the cells. Since proteinaceous mediator(s) are obviously involved in TPA-induced vimentin expression in MPC-11 cells, cell cycle-dependent vimentin expression in these cells may be dependent on cell cycle-dependent regulation of the activity and/or concentration of such mediator(s).     

12-O-tetradecanoylphorbol-13-acetate induces transient cell cycle arrest in G1 and G2 in metastatic melanoma cells: inhibition of phosphorylation of p34cdc2.

The growth of Demel human metastatic melanoma cells was inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA) and other nonphorbol tumor promoters including palytoxin and okadaic acid. Using flow cytometry, we have demonstrated that the cells arrested growth in G1 and G2 phases of the cell cycle. Detailed analysis of the kinetics of the growth arrest in unsynchronized cells showed that (a) the growth arrest was transient and peaked 16-20 h following addition of TPA; (b) effects of TPA on cell growth began within 1-2 h after the addition; and (c) cells completed S phase and arrested in G2. In addition, TPA induced a pronounced morphological change, which peaked by 1 h and gradually subsided over 24 h. In populations of cells synchronized in G1 using lovastatin, (a) addition of TPA blocked the onset of DNA synthesis up to the end of G1; (b) the lag between addition of the drug and onset of DNA synthesis was less than 30 min; and (c) addition of TPA at the end of G1 prevented the increased phosphorylation of p34cdc2, as determined by immunoprecipitation. The experiments reported here show that TPA transiently blocked the proliferation of Demel melanoma cells at the G1-S border and in G2, thus preventing cells from progressing through the cell cycle. These experiments suggest that pathways involving protein kinase C interact with and rapidly alter the molecular pathways involving p34cdc2 which regulate the onset of DNA synthesis and the G2-M transition.     

TGFbeta regulates the expression and activities of G2 checkpoint kinases in human myeloid leukemia cells

Transforming Growth Factor-beta (TGFbeta) is known to be a negative regulator of G1 cyclin/cdk activity. It is not clear whether TGFbeta has any effect on G2 checkpoint kinases. We have found that TGFbeta downregulated the expression of several G2 checkpoint kinases including cdc2, cyclin B1, and cdc25c without causing cell accumulation in G2/M phases in two human leukemia cell lines. The inhibition was time-dependent with a maximal inhibition being observed by 24h for cyclin B1 and cdc2 and by 48h for cdc25c. The inhibition was not a result of G1 arrest but a direct effect of TGFbeta which downregulates their expression at mRNA level. In proliferating cells, there was a significant formation of cdc2-pRb complexes, which was decreased to 30% of control levels by 48h after initiating TGFbeta treatment. Cdc2 showed a marked kinase activity on GST-Rb protein in proliferating cells detected by in vitro kinase assay, which was downregulated in response to TGFbeta. In addition, TGFbeta caused a rapid and transient dephosphorylation of cdc2 (Tyr15) and cdc25c (Ser216) for about 2-3h before a dramatic decrease of both molecules by 48h. Taken together, our data suggest that TGFbeta has a direct inhibitory effect on G2 checkpoint kinases, which is regulated at mRNA level. The transient activation of cdc2 and cdc25c and subsequent inhibition of cdc2, cyclin B1, and cdc25c could amplify TGFbeta-induced G1 arrest and growth inhibition.     

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.     

TNFalpha-mediated cell death is independent of cdc25A

Tumor necrosis factor (TNFs) have been shown to be synthesized by ovarian carcinomas, and may therefore affect tumor cells in an autocrine manner. Therefore, we investigated the effects of recombinant TNFs on ovarian carcinoma cells N.1 and examined expression of the proto-oncogenes c-myc and cdc25A which are known to play a prominent role in apoptosis. TNFalpha elicited apoptosis in N.1 cells within 72 h which was shown by typical morphological changes, DNA fragmentation and signature type cleavage of poly(ADP-ribose) polymerase into a 89 kDa proteolytic peptide. TNFalpha-induced apoptosis was accompanied by constitutive c-Myc expression, although the mRNA level of phosphatase cdc25A was suppressed within 24 h of TNFalpha treatment and the protein level decreased after 48 h. Cdc25A tyrosine phosphatase is an activator of the cdk2-cyclin E complex which allows for cell cycle progression. As expected, we found TNFalpha-mediated Cdc25A down-regulation to inhibit Cdk2 activity. Cdc25A suppression was related to TNFalpha-induced apoptosis but not to a TNFalpha-induced G0 arrest because cyclin D1 expression was unaffected and the gene gas6 (growth arrest specific 6) was not induced. Arresting cells by treatment with genistein prevented TNFalpha-triggered apoptosis and inhibited c-myc expression. TNFalpha-induced apoptosis is not accompanied by cell cycle arrest which may be due to constitutive c-Myc expression, although Cdc25A and Cdk2 activity is also down-regulated. High c-Myc and low Cdc25A activity might present conflicting signals to the cell cycle machinery which are incompatible with cell survival.     

TIS21/BTG2/PC3 is expressed through PKC-delta pathway and inhibits binding of cyclin B1-Cdc2 and its activity, independent of p53 expression

Signal transduction pathway and a new function of TIS21/BTG2/PC3 were investigated in p53 null U937 cells; Expression of TIS21 by 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulation was mediated by PKC-delta activation, however, was strongly inhibited by cPKC isozymes. When U937 cells were treated with TPA+Go6976, but not TPA+Go6850, the level of TIS21 mRNA was maintained over that of TPA alone. When analyzed by FACS, TPA-induced G2/M arrest was significantly inhibited by Go6850, but not by Go6976, suggesting the involvement of TIS21 and nPKC isozymes. Indeed, PKC-delta was found to be a regulator of the G2/M arrest and TIS21 expression, confirmed by employing rottlerin and dnPKC-delta experiments. In vivo accumulation of TIS21 protein significantly induced cell death through caspase 3 activation, which was supported further by degradations of procaspase 3, full-length PKC-delta, pRB, and p21(WAF1) in TIS21DeltaC expresser. When the cells were synchronized by nocodazole, TIS21 overexpressers inhibited degradations of cyclin A and cyclin B1 in 3 h after release from the synchronization. Furthermore, TIS21 inhibited cyclin B1-Cdc2 binding and its kinase activity in vivo. In summary, TPA-induced TIS21 mRNA expression is mediated by PKC-delta, and TIS21 induces G2/M arrest and cell death by inhibiting cyclin B1-Cdc2 binding and the kinase activity through its binding to Cdc2.     

Regulation of acid phosphatase activity in human promyelocytic leukemic cells induced to differentiate in culture

Induction of differentiation of a human promyelocytic leukemic cell line (HL60) in culture is accompanied by changes in acid phosphatase (Acpase) activity. The increase in activity is less than twofold when the leukemic cells are stimulated by dimethylsulfoxide (DMSO) to differentiate into metamyelocytes and granulocytes but is eightfold when the cells are stimulated by the tumor-promoting agent 12-0- tetradecanoylphorbol 13-acetate (TPA) to differentiate into macrophage- like cells. Five different isozymes of Acpase were separated by acrylamide gel electrophoresis. Isozyme 1, the most anodal isozyme, was found to be present in undifferentiated, DMSO-treated and TPA-treated cells; isozyme 2 was a very faint band observed both in DMSO- and TPA- treated cells, the isoenzymes 3a and 3b were present only in TPA- induced cells; and isozyme 4, the most cathodal isozyme, was present both in TPA- and DMSO-induced cells. A time sequence study on the appearance of the various forms after TPA treatment indicated that the expression of the isozymes is regulated in an uncoordinated fashion. Acpase activity has been shown by ultrastructural cytochemistry to be localized in the entire rough endoplasmic reticulum (RER) and in areas of the smooth endoplasmic reticulum (SER) located near the Golgi complex in differentiating cells but to be extremely weak, if at all detectable, in undifferentiated promyelocytes.     

Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity.

The Vpr accessory gene product of human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus is believed to play a role in permitting entry of the viral core into the nucleus of nondividing cells. A second role for Vpr was recently suggested by Rogel et al. (M. E. Rogel, L. I. Wu, and M. Emerman, J. Virol. 69:882-888, 1995), who showed that Vpr prevents the establishment in vitro of chronically infected HIV producer cell lines, apparently by causing infected cells to arrest in the G2/M phase of the cell cycle. In cycling cells, progression from G2 to M phase is driven by activation of the p34cdc2/cyclin B complex, an event caused, in part, by dephosphorylation of two regulatory amino acids of p34cdc2 (Thr-14 and Tyr-15). We show here that Vpr arrests the cell cycle in G2 by preventing the activation of the p34cdc2/cyclin B complex. Vpr expression in cells caused p34cdc2 to remain in the phosphorylated, inactive state, p34cdc2/cyclin B complexes immunoprecipitated from cells expressing Vpr were almost completely inactive in a histone H1 kinase assay. Coexpression of a constitutively active mutant p34cdc2 molecule with Vpr relieved the G2 arrest. These findings strongly suggest that Vpr arrests cells in G2 by preventing the activation of the p34cdc2/cyclin B complex that is required for entry into M phase. In vivo, Vpr might, by preventing p34cdc2 activation, delay or prevent apoptosis of infected cells. This would increase the amount of virus each infected cell produced.     

Relationships between cdc2 kinase, DNA cross-linking, and cell cycle perturbations induced by nitrogen mustard.

M phase-promoting factor (MPF) consists of a p34cdc2 (cdc2) kinase and cyclin B complex which in its active form promotes G2 to M transition. The role of MPF in G2 arrest following DNA damage, however, has remained largely uncharacterized. We have investigated whether nitrogen mustard (HN2) interfered with either the formation of MPF or its activation. For this purpose, we measured cdc2 kinase activity relative to cdc2 and cyclin B protein turnover and the phosphorylation status of cdc2. Studies were performed in two exceptional human lymphoma cell lines, which differed in HN2 sensitivity by 5-fold (CA46, 50% growth-inhibitory dose = 1.0 microM; JLP119, 50% growth-inhibitory dose = 0.2 microM) but exhibited virtually identical DNA interstrand and DNA-protein cross-link exposure. Following HN2 treatment, CA46 cells ceased to enter mitosis and exhibited a marked delay in G2 phase. Failure to enter mitosis paralleled inhibition of cdc2 kinase. Inhibition was not due to decreased levels of cdc2 or cyclin B protein; rather, G2 arrest correlated with the accumulation of both tyrosine-phosphorylated cdc2 and cyclin B. These findings implied that G2 arrest resulted from a down-regulation of the processes that activate MPF. We also found that JLP119 cells, within a few hours of mitosis at the time of drug treatment, evaded checkpoint control and continued cell division unabated by DNA damage. Furthermore, despite similar DNA cross-link exposure, JLP119 cells within the window of checkpoint control were more susceptible to S phase delay than CA46 cells. Altered cell cycle responses correlated with the greater susceptibility of JLP119 cells to the cytotoxic effects of HN2.