Levels Of P34cdc2 Research Articles

Indirect semiquantitative determination of p34cdc2 levels in G1 and G2 cells of the carbohydrate-starved root meristems in Vicia faba var. minor

In eukaryotes, the 34kDa kinase (p34) encoded by the <em>cdc2</em> gene is a key regulator of both the onset of DNA synthesis (G<sub>1</sub> to S phase transition) and the onset of mitosis (G<sub>1</sub> to M phase transition). Using mouse anti-human PSTAIRE and FITClabelled goat antibodies, indirect semiquantitative determination of p34<sup><em>cdc2</em></sup> levels was performed in meristematic cells from the control (intact) and excised, carbohydrate-starved main roots of Vicia faba var. minor. No evident differences in the intensity of fluorescence was found either between the G<sub>1</sub> and G<sub>2</sub> cells or between the control cells and the cells arrested at both Principal Control Points by carbohydrate starvation. It seems thus, that the cell cycle block induced in meristematic cells of <em>V. faba</em> var. <em>minor</em> is not correlated with the absolute level of the key cell cycle enzyme responsible for phosphory-lution of cellular proteins, but primarily with the altered activity of p34<sup><em>cdc2</em></sup>.

Induction of autophagy during in vitro maturation improves the nuclear and cytoplasmic maturation of porcine oocytes

While a critical role of autophagy in mammalian early embryogenesis has been demonstrated, few studies have been conducted regarding the role of autophagy in in vitro maturation (IVM) of immature oocytes. In the present study we investigated the effect of rapamycin, a chemical autophagy inducer, on the nuclear and cytoplasmic maturation of porcine oocytes. Rapamycin treatment led to increased expression of LC3-II, an autophagy marker. Compared with the control group, as well as the 5 and 10nM rapamycin treatment groups, the rate of MII oocyte production was higher in the 1nM rapamycin treatment group, indicating improvement in nuclear maturation. In the analyses of cytoplasmic maturation, we found that the level of p34(cdc2), a cytoplasmic maturation marker, and the monospermic fertilisation rate were higher in the 1nM rapamycin treatment group than in the other groups. Moreover, the beneficial effect of 1nM rapamycin on cytoplasmic maturation of MII oocytes was further evidenced by increases in blastocyst formation rate, total cell number and cell survival. In the blastocyst embryos, anti-apoptotic Bcl-xL transcript levels were elevated in the 1nM rapamycin-treated group, whereas pro-apoptotic Bax transcript levels were decreased. Collectively, these results suggest that induction of autophagy during IVM contributes to enhancement of the nuclear and cytoplasmic maturation of porcine oocytes.

Silibinin Triggers Apoptosis and Cell‐Cycle Arrest of SGC7901 Cells

Silibinin, a flavonoid compound, has shown to be of chemopreventive potential against many cancers. However, its efficacy against gastric cancer has not been well elucidated. Here, we assessed the activity of Silibinin on apoptosis and cell-cycle arrest in human gastric cells culture system using SGC-7901 as the model. Silibinin treatment could inhibit the cell growth and cause a prominent G2 phase arrest and apoptosis in dose- and time-dependent manner. In mechanistic studies, Silibinin decreased the protein level of p34cdc2, which might be the possible molecular mechanism of Silibinin efficacy on the growth inhibition in SGC-7901 cells. In addition, Silibinin caused an increase in p53 and p21 protein level as well as mRNA levels. Interestingly, Silibinin-induced apoptosis in SGC-7901 cells was independent of caspases activation. These results indicated that Silibinin is a cell-cycle regulator and apoptosis inducer in human gastric carcinoma SGC-7901 cells and might be used as a candidate chemopreventive agent for gastric carcinoma prevention and intervention.

Influence of the G2 cell cycle block abrogator pentoxifylline on the expression and subcellular location of cyclin B1 and p34cdc2 in HeLa cervical carcinoma cells.

The progression of cells from G2 into mitosis is mainly controlled by formation of the cyclin B1/p34cdc2 complex. The behaviour of this complex in the irradiation-induced G2 cell cycle delay is still unclear. A prior study demonstrated that the expression of the cyclin B1 protein is reduced by irradiation, and restored to control levels by the methylxanthine drug pentoxifylline, which is a potent G2 block abrogator. The present study shows that irradiation, and 2 mM pentoxifylline affect the expression of the cyclin-dependent kinase p34cdc2 in HeLa cells. Irradiation induces p34cdc2 levels to increase and cyclin B1 levels to decrease. Addition of pentoxifylline at the G2 maximum reverses these trends. This is also evident from the cyclin B1/p34cdc2 ratios which decline after irradiation and are rapidly restored to control levels upon addition of pentoxifylline. It is concluded that cyclin BI and p34cdc2 protein expression are important events and act in concert to control the irradiation induced G2 block. Analysis of cyclin B1 expression in whole cells and in isolated nuclei furthermore show that cyclin B1 is translocated from the nucleus into the cytoplasm when the G2 block is abrogated by pentoxifylline.

1,25-dihydroxyvitamin D3-induced retardation of the G2/M traverse is associated with decreased levels of p34cdc2 in HL60 cells

Cellular differentiation of neoplastic cells after exposure to 1,25-dihydroxyvitamin D3 (1,25 D3) is accompanied by altered cell cycle regulation. In previous studies, blocks in both G1/S and G2/M checkpoints have been observed in 1,25D3-treated HL60 cells, but the mechanism of the 1,25D3-induced G2/M block has not been previously reported. In this study, we show by cell cycle analysis, using bromodeoxyuridine pulse-chase labeling, that the G2/M block in 1,25D3-treated HL60 cells is incomplete. We also demonstrate that although the 1,25D3-treated cells exhibit elevated levels of cyclin B1, Cdc25C, and Cdk7, which are positive regulators of the G2/M traverse, these cells have decreased protein levels of p34cdc2 and decreased p34cdc2 kinase activity. This provides potential mechanisms for the observed accumulation of cells in the G2 cell cycle compartment and occasional polyploidization following treatment of HL60 cells with 1,25D3. 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 p34cdc2 activity by increasing the levels of other regulators of the G2 traverse, such as cyclin B1, Cdc25C, and Cdk7. J. Cell. Biochem. 75:226–234, 1999. © 1999 Wiley-Liss, Inc.

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.

Deregulation of cdc2 gene expression correlates with overexpression of a 110 kDa CCAAT box binding factor in transformed cells.

Eukaryotic cell cycle progression is regulated by an orderly and sequential activation of several cyclin-dependent kinases, which phosphorylate key substrates during this process. p34cdc2, the catalytic subunit of cdc2 kinase, is expressed at the late G1/S boundary and is required for the G2-->M phase transition. Transactivation of the human cdc2 promoter by the DNA tumor virus-encoded oncogenic protein SV40 large T antigen is mediated by induction of a novel 110 kDa CCAAT box binding factor (CBF/cdc2). To investigate whether induction of CBF/cdc2 is an intrinsic property of the viral oncoprotein or is a common event during transformation of normal cells, expression of CBF/cdc2 was analyzed in many human tumor cell lines and in rodent cells spontaneously transformed or stably expressing various oncogenes. Our results showed that CBF/cdc2 was overexpressed in all transformed cells examined, including human 293, MCF-7, HeLa and HepG2 cells. Moreover, expression of CBF/cdc2 was elevated in spontaneously transformed rat liver epithelial cells (C4T), but not detectable in the non-tumorigenic parental (RLE) cells. The elevated levels of CBF/cdc2 expression in C4T cells correlated well with increased cdc2 mRNA and p34cdc2 levels. CBF/cdc2 was also overexpressed in a rat liver epithelial cell line (WB) stably transfected with various oncogenes, v-myc, v-Ha-ras and mutated rat neu and v-src. Using an electrophoretic mobility shift assay, specific binding of CBF/cdc2 to the CCAAT box motifs of the human cdc2, cycA and cdc25C promoters was detected, suggesting that transcription of these cell cycle regulatory genes are coordinately activated by CBF/cdc2.

Contribution of the cyclin‐dependent kinase inhibitor p27KIP1 to the confluence‐dependent resistance of HT29 human colon carcinoma cells

We have previously shown that growth of HT29 human colorectal cancer cells at confluence increased their resistance to the cytotoxic agent cisplatin. This study further explores the mechanisms of this resistance phenotype. DNA platination induced by cisplatin exposure is slightly reduced by confluence. However, at an equivalent DNA platination level, non-confluent cells accumulate in the G2/M phase of the cell cycle, demonstrate aberrant mitotic figures and die by apoptosis, while confluent cells progress slowly through the cell cycle, do not reach mitosis and are more resistant to drug-induced cell death. At a molecular level, cisplatin enhances cyclin B and p34cdc2 levels and histone H1 kinase activity in non-confluent, but not in confluent, cells. Furthermore, when HT29 cells reach confluence, expression of the cyclin-dependent kinase inhibitor p27Kip1 increases and cells accumulate in the G0/G1 phase of the cell cycle. Transfection-mediated over-expression of p27Kip1 in non-confluent HT29 cells decreases the cytotoxic activity of cisplatin as well as its ability to trigger apoptosis. Non-confluent HT29 cells over-expressing p27Kip1 are also more resistant to doxorubicin, etoposide and 5-fluorouracil. Our results suggest that p27Kip1 contributes to the confluence-dependent resistance phenotype. Int. J. Cancer 77:796–802, 1998. © 1998 Wiley-Liss, Inc.

Contribution of the cyclin-dependent kinase inhibitor p27KIP1 to the confluence-dependent resistance of HT29 human colon carcinoma cells.

We have previously shown that growth of HT29 human colorectal cancer cells at confluence increased their resistance to the cytotoxic agent cisplatin. This study further explores the mechanisms of this resistance phenotype. DNA platination induced by cisplatin exposure is slightly reduced by confluence. However, at an equivalent DNA platination level, non-confluent cells accumulate in the G2/M phase of the cell cycle, demonstrate aberrant mitotic figures and die by apoptosis, while confluent cells progress slowly through the cell cycle, do not reach mitosis and are more resistant to drug-induced cell death. At a molecular level, cisplatin enhances cyclin B and p34cdc2 levels and histone H1 kinase activity in non-confluent, but not in confluent, cells. Furthermore, when HT29 cells reach confluence, expression of the cyclin-dependent kinase inhibitor p27Kip1 increases and cells accumulate in the G0/G1 phase of the cell cycle. Transfection-mediated over-expression of p27Kip1 in non-confluent HT29 cells decreases the cytotoxic activity of cisplatin as well as its ability to trigger apoptosis. Non-confluent HT29 cells over-expressing p27Kip1 are also more resistant to doxorubicin, etoposide and 5-fluorouracil. Our results suggest that p27Kip1 contributes to the confluence-dependent resistance phenotype.

P34cdc2 expression and meiotic competence in growing goat oocytes.

The expression of the catalytic subunit of the maturation promoting factor (MPF), p34cdc2, was analyzed during meiosis and final growth of goat oocytes. Western blot analysis revealed the presence of p34cdc2 in fully grown oocytes (follicles > 3 mm in diameter) prior to and during meiotic maturation. p34cdc2 was present in partially competent oocytes at the germinal vesicle stage (follicles 0.5 to 0.8 mm and 1 to 1.8 mm in diameter). In contrast, p34cdc2 was not expressed in meiotically incompetent oocytes from small antral follicles (follicles < 0.5 mm in diameter). The amount of p34cdc2 increased with oocyte growth and acquistion of meiotic competence. Moreover, p34cdc2 accumulated in partially competent and incompetent oocytes within 27 hr of culture, but the level of p34cdc2 in incompetent oocytes remained very low and was not sufficient to allow spontaneous resumption of meiosis. The expression of the cdc2 gene was analyzed by polymerase-chain-reaction (PCR) on reverse transcribed mRNA. The presence of the cdc2 transcript was evidenced in both competent and incompetent oocytes at the germinal vesicle stage. These data indicate that a deficiency in the expression of p34cdc2 that could be regulated at the translational level, may be a limiting factor for meiotic competence acquistion in goat oocytes. We further investigated the mechanisms of MPF activation in competent and incompetent oocytes by using okadaic acid, a protein phosphatase inhibitor. Okadaic acid induced the premature resumption of meiosis associated with MPF activation in competent oocytes. In partially competent oocytes, okadaic acid induced premature meiosis reinitiation and MPF activation, but only after pre-culture for 10 hr. Acquisition of sensitivity to okadaic acid treatment was dependent on protein synthesis since it failed to occur when cycloheximide was added during the pre-culture period. Incompetent oocytes responded to okadaic acid treatment only after 27 hr of culture, when they had accumulated small amounts of p34cdc2. These data suggest that okadaic acid may bypass the subthreshold level of p34cdc2, provided the oocytes have synthesized some additional factors that remain to be identified.

P34cdc2 expression and meiotic competence in growing goat oocytes

The expression of the catalytic subunit of the maturation promoting factor (MPF), p34cdc2, was analyzed during meiosis and final growth of goat oocytes. Western blot analysis revealed the presence of p34cdc2 in fully grown oocytes (follicles >3 mm in diameter) prior to and during meiotic maturation. p34cdc2 was present in partially competent oocytes at the germinal vesicle stage (follicles 0.5 to 0.8 mm and 1 to 1.8 mm in diameter). In contrast, p34cdc2 was not expressed in meiotically incompetent oocytes from small antral follicles (follicles <0.5 mm in diameter). The amount of p34cdc2 increased with oocyte growth and acquisition of meiotic competence. Moreover, p34cdc2 accumulated in partially competent and incompetent oocytes within 27 hr of culture, but the level of p34cdc2 in incompetent oocytes remained very low and was not sufficient to allow spontaneous resumption of meiosis. The expression of the cdc2 gene was analyzed by polymerase-chain-reaction (PCR) on reverse transcribed mRNA. The presence of the cdc2 transcript was evidenced in both competent and incompetent oocytes at the germinal vesicle stage. These data indicate that a deficiency in the expression of p34cdc2 that could be regulated at the translational level, may be a limiting factor for meiotic competence acquisition in goat oocytes. We further investigated the mechanisms of MPF activation in competent and incompetent oocytes by using okadaic acid, a protein phosphatase inhibitor. Okadaic acid induced the premature resumption of meiosis associated with MPF activation in competent oocytes. In partially competent oocytes, okadaic acid induced premature meiosis reinitiation and MPF activation, but only after pre-culture for 10 hr. Acquisition of sensitivity to okadaic acid treatment was dependent on protein synthesis since it failed to occur when cycloheximide was added during the pre-culture period. Incompetent oocytes responded to okadaic acid treatment only after 27 hr of culture, when they had accumulated small amounts of p34cdc2. These data suggest that okadaic acid may bypass the subthreshold level of p34cdc2, provided the oocytes have synthesized some additional factors that remain to be identified. Mol. Reprod. Dev. 50:251–262, 1998. © 1998 Wiley-Liss, Inc.

A measure of the mitotic index: studies of the abundance and half-life of p34cdc2 in cultured cells and normal and neoplastic tissues.

The cdc2 gene encodes a protein kinase, p34cdc2, that is essential for mitosis, and is present at high levels in dividing cells. Classical studies of the levels of this protein in dividing and resting cells used antibodies that cross-react with other members of the CDK family, in particular with CDK2. We have therefore re-examined the abundance of p34cdc2 in a variety of tissues and cell lines, using a highly specific, epitope-mapped monoclonal antibody that does not react with CDK2. We observed high levels of p34cdc2 in proliferating cells, especially those in neoplastic tissues. Cells that have withdrawn from the cell cycle have low or undetectable levels. At the end of mitosis, the level of p34cdc2 declines, with simple first-order kinetics, with a half-life which is never less than 6h and is more typically about 18h. The persistence of p34cdc2 after the last cell division is comparable to that of PCNA, a commonly used marker of proliferation. The immunochemical detection of p34cdc2 provides an accurate, reliable and meaningful measure of the proliferative activity of cells in tissues. We suggest that p34cdc2 should be considered as the most authentic molecular marker of the mitotic index.

Different mechanisms for inhibition of cell proliferation via cell cycle proteins in PC12 cells by nerve growth factor and staurosporine

PC12 cells have previously been shown to cease cell division during nerve growth-factor (NGF)-induced differentiation by affecting specific cell cycle proteins. Staurosporine, a protein kinase inhibitor, also causes PC12 cell differentiation, independently of neurotrophins or plasma membrane receptors. We have investigated the relationship of the tumor suppressor protein, p53, and other cell cycle proteins to the antiproliferative effects of NGF and staurosporine in PC12 cells. NGF treatment of PC12 cells stimulated an increase of p53 protein in the nucleus and, more slowly, an increase in total cellular p53 protein. Levels of the cyclin-kinase inhibitor p21/WAF1, cyclin D1, and cyclin G, all downstream transcriptional targets of p53, increased after short times of NGF treatment. Cessation of replication and differentiation occurred more rapidly in defined medium (2 days) than in serum medium (6 days), in correspondence with the more rapid changes in both p53 and p21/WAF1 levels in defined medium (1 hour) than in serum (1 day). Levels of p34cdc2 and p33cdk2 kinase dropped after 6 to 10 days treatment with NGF in serum, close to the time of terminal differentiation. Staurosporine, on the other hand, inhibited DNA replication of PC12 cells in a time- and dose-dependent fashion by affecting cyclin-dependent kinases. Staurosporine had no effect on the protein levels of p53, p21/WAF1, or cyclin G. The kinase activity of both p34cdc2 and p33cdk2 were inhibited in vitro with IC50 values of 20 nM and 75 nM, respectively. In vivo p34cdc2 kinase activity was inhibited within 1 day, before the decrease in the levels of p34cdc2 protein at days 2 to 3. In contrast, in vivo p33cdk2 kinase activity only decreased in concert with protein levels. Although both NGF and staurosporine inhibit DNA replication concomitant with induction of differentiation by affecting the activity of p34cdc2 and p33cdk2, the mechanism of the two agents is quite different. NGF achieves inhibition of activity of these cyclin-dependent kinases by signalling through the TrkA receptor to the tumor suppressor protein p53 and then to p21/WAF1. In contrast, staurosporine directly inhibits the activity of p34cdc2 and p33cdk2 by binding to them and also indirectly by alteration of their phosphorylation through other regulatory kinases.

Different mechanisms for inhibition of cell proliferation via cell cycle proteins in PC12 cells by nerve growth factor and staurosporine

PC12 cells have previously been shown to cease cell division during nerve growth-factor (NGF)-induced differentiation by affecting specific cell cycle proteins. Staurosporine, a protein kinase inhibitor, also causes PC12 cell differentiation, independently of neurotrophins or plasma membrane receptors. We have investigated the relationship of the tumor suppressor protein, p53, and other cell cycle proteins to the antiproliferative effects of NGF and staurosporine in PC12 cells. NGF treatment of PC12 cells stimulated an increase of p53 protein in the nucleus and, more slowly, an increase in total cellular p53 protein. Levels of the cyclin-kinase inhibitor p21/WAF1, cyclin D1, and cyclin G, all downstream transcriptional targets of p53, increased after short times of NGF treatment. Cessation of replication and differentiation occurred more rapidly in defined medium (2 days) than in serum medium (6 days), in correspondence with the more rapid changes in both p53 and p21/WAF1 levels in defined medium (1 hour) than in serum (1 day). Levels of p34cdc2 and p33cdk2 kinase dropped after 6 to 10 days treatment with NGF in serum, close to the time of terminal differentiation. Staurosporine, on the other hand, inhibited DNA replication of PC12 cells in a time- and dose-dependent fashion by affecting cyclin-dependent kinases. Staurosporine had no effect on the protein levels of p53, p21/WAF1, or cyclin G. The kinase activity of both p34cdc2 and p33cdk2 were inhibited in vitro with IC50 values of 20 nM and 75 nM, respectively. In vivo p34cdc2 kinase activity was inhibited within 1 day, before the decrease in the levels of p34cdc2 protein at days 2 to 3. In contrast, in vivo p33cdk2 kinase activity only decreased in concert with protein levels. Although both NGF and staurosporine inhibit DNA replication concomitant with induction of differentiation by affecting the activity of p34cdc2 and p33cdk2, the mechanism of the two agents is quite different. NGF achieves inhibition of activity of these cyclin-dependent kinases by signalling through the TrkA receptor to the tumor supressor protein p53 and then to p21/WAF1. In contrast, staurosporine directly inhibits the activity of p34cdc2 and p33cdk2 by binding to them and also indirectly by alteration of their phosphorylation through other regulatory kinases. J. Neurosci. Res. 49:461–474, 1997. © 1997 Wiley-Liss, Inc.

Identification and preliminary characterization of p31, a new PSTAIRE-related protein in fission yeast.

One of the defining characteristics of the catalytic subunit of the cyclin-dependent protein kinases (cdks) is the so-called PSTAIRE motif. Western blots of fission yeast cytosolic extracts using a monoclonal antibody against the PSTAIRE peptide revealed two bands at 34 kDa (p34cdc2) and 31 kDa (p31). Polyclonal antibodies to the C-terminus of p34cdc2 or to the full-length protein recognized the 34 kDa band but not p31. Overexpression of the cdc2+ gene resulted in the increase of the 34 kDa band but not p31. Like p34cdc2, the level of p31 revealed no obvious cell cycle regulation but the protein was present in spores where p34cdc2 was barely detectable. p31 expression was unaffected by removal of either phosphate or ammonium from the growth medium, although the level of p34cdc2 was reduced in the absence of phosphate. p31 was not associated with cyclin B, nor was it adsorbed to p13suc1 Sepharose beads, two characteristics of p34cdc2. p31 did, however, interact with p15, the starfish homologue of p13suc1. p31 was present in cells in which cdc2+ was replaced by its budding yeast homologue CDC28. When fission yeast cytosolic extracts were subjected to gel filtration chromatography, p31 eluted in two peaks, one at approximately 100 kDa, the other at approximately 30 kDa. We conclude that p31 is a novel fission yeast PSTAIRE protein and therefore, potentially, a new cdk.

Association between p34cdc2 levels and meiotic arrest in pig oocytes during early growth.

The molecules involved in determining meiotic competence were determined in porcine oocytes isolated from preantral and antral follicles of different sizes. Oocytes isolated from preantral follicles had a mean diameter of 78 microns, contained diffuse filamentous chromatin in the germinal vesicle and were incapable of progressing from the G2 to the M phase of the cycle even after 72 h in culture. Oocytes from early antral follicles had a mean diameter of 105 microns, showed a filamentous chromatin configuration and about half resumed meiosis but arrested at metaphase I (MI) when cultured. Oocytes from mid-antral (3-4 mm) and large antral follicles (5-6 mm) had mean oocyte diameters of 115 and 119 microns respectively, contained condensed chromatin around the nucleolus and progressed to metaphase II (MII) in 48% and 93% of instances respectively. Analysis of p34cdc2, the catalytic subunit of maturation promoting factor (MPF), by immunoblotting indicates that the inability of small (78 microns) oocytes to resume meiosis is due, at least in part, to inadequate levels of the catalytic subunit of MPF. On the other hand, the inability of intermediate-sized (105 microns) oocytes from antral follicles to complete the first meiotic division by progressing beyond MI appears not to be limited by levels of p34cdc2, which are maximal by this stage. We postulate that an inadequacy of molecules other than p34cdc2 limits progression of MI to MII; the acquisition of these molecules during the final stages of growth may be correlated with the formation of the perinucleolar chromatin rim in the germinal vesicle.

Activation-Induced T-Cell Death Is Cell Cycle Dependent and Regulated by Cyclin B

Developing thymocytes and some T-cell hybridomas undergo activation-dependent programmed cell death. Although recent studies have identified some critical regulators in programmed cell death, the role of cell cycle regulation in activation-induced cell death in T cells has not been addressed. We demonstrate that synchronized T-cell hybridomas, irrespective of the point in the cell cycle at which they are activated, stop cycling shortly after they reach G2/M. These cells exhibit the diagnostic characteristics of apoptotic cell death. Although p34cdc2 levels are not perturbed after activation of synchronously cycling T cells, cyclin B- and p34cdc2-associated histone H1 kinase activity is persistently elevated. This activation-dependent induction of H1 kinase activity in T cells is associated with a decrease in the phosphotyrosine content of p34cdc2. We also demonstrate that transient inappropriate coexpression of cyclin B with p34cdc2 induces DNA fragmentation in a heterologous cell type. Finally, in T cells, cyclin B-specific antisense oligonucleotides suppress activation-induced cell death but not cell death induced by exposure to dexamethasone. We therefore conclude that a persistent elevation of the level of cyclin B kinase is required for activation-induced programmed T-cell death.

Smooth muscle cell proliferation. Expression and kinase activities of p34cdc2 and mitogen-activated protein kinase homologues.

Rat vascular smooth muscle cells were synchronized to the quiescent state (G0) by serum deprivation and then stimulated to enter the cell cycle by serum refeeding. At various times of the cell cycle, cells were analyzed for the expression of p34cdc2 and mitogen-activated protein kinase homologues by immunoblotting and for kinase activity toward histone H1, myelin basic protein, and caldesmon. A small amount of p34cdc2 was expressed in the G0/G1 phase (0 to 8 hours). At the G1/S transition (12 hours), the level of p34cdc2 started to accumulate and increased by 60-fold at G2/M (18 hours), accompanied by a more slowly migrating band. Histone H1 kinase activity was undetectable in anti-p34cdc2 immunoprecipitates in the G0/G1 cells but appeared around the G1/S boundary and peaked at G2/M (18 hours). The caldesmon kinase activity exhibited two distinct phases: the first appeared at G0/G1 (0 to 8 hours), and the second appeared at G1/S and continued through G2/M. Two mitogen-activated protein kinase isoforms were expressed throughout the cell cycle. Anti-mitogen-activated protein kinase immunoprecipitates possessed kinase activities toward myelin basic protein and caldesmon, which were activated within 15 minutes after serum stimulation and declined within a few hours. These findings suggest that p34cdc2 and mitogen-activated protein kinase homologues may play significant roles in regulating the progression of the cell cycle of smooth muscle cells, the former at the G2/M transition and the latter at the G0/G1 transition.

Differential expression and activity of p34cdc2 in cultured aortic adventitial fibroblasts derived from spontaneously hypertensive and Wistar-Kyoto rats.

The present investigation was undertaken to determine whether p34cdc2, a cell-cycle regulatory kinase, is involved in the manifestation of the altered proliferation evident in fibroblasts isolated from spontaneously hypertensive rats (SHR). Experiments were performed on quiescent aortic adventitial fibroblasts stimulated to re-enter the cell cycle in order to examine the timing of cell cycle-related events. The cell-cycle phase was determined by flow cytometry and was related to the cellular content and kinase activity of p34cdc2. SHR fibroblasts displayed a heightened basal level of p34cdc2 at quiescence relative to Wistar-Kyoto (WKY) rat cells. Both SHR and WKY fibroblasts showed a cell cycle-dependent increase in p34cdc2 content, beginning in S phase. However, the SHR adventitial fibroblasts exited G0-G1 several hours earlier than the WKY fibroblasts as indicated by the time of initiation of DNA synthesis and increase in activity of p34cdc2. SHR aortic adventitial fibroblasts appear to have a heightened proliferative capacity relative to WKY fibroblasts, which is evident in a quicker exit from G0 and faster transition to DNA synthesis, followed by the earlier activation of p34cdc2.

Inactivation of cdc2 kinase during mitosis requires regulated and constitutive proteins in a cell-free system.

Inactivation of the cyclin-p34cdc2 protein kinase complex is a major requirement for anaphase onset and exit from mitosis. To facilitate identification of specific molecules that regulate this event in mammalian cells, I have developed a cell-free assay in which cdc2 kinase associated with a chromosomal fraction from metaphase tissue culture cells is inactivated by a cell-cycle-regulated cytosolic system. In vitro kinase inactivation requires ATP, Mg2+ and the dephosphorylation of one or more sites in the chromosomal fraction by protein phosphatase 1 and/or 2A. Cyclin B is destroyed during inactivation, while the level of p34cdc2 remains constant. Ammonium sulfate fractionation resolves the cytosolic inactivating system into at least two distinct protein components that are both required for inactivation and are differentially regulated during mitosis.