Increased expression of CHK2 in human gastric carcinomas harboring p53 mutations

Human Chk1 and Chk2 are DNA damage-activated protein kinases that function as downstream mediators of ataxia-telangiectasia mutated (ATM), which is involved in G(2)/M cell cycle arrest. To clarify the relation between the expression of Chk1/Chk2 and p53 gene status in human gastric carcinomas, we examined expression of Chk1, Chk2 and p53 proteins in 87 gastric carcinomas by Western blotting and immunohistochemistry. We found a significant correlation between the expression levels of Chk1 and p53 proteins in gastric carcinomas (p = 0.016). Significant statistical association was also observed between levels of Chk2 and p53 proteins (p = 0.00024). To clarify the genetic alterations of p53 in gastric carcinomas, we performed PCR-SSCP analysis on 47 gastric carcinomas. Although we found that 5 of 7 (71%) gastric cancers expressed elevated levels of Chk1 had p53 mutation, there was not a statistically significant correlation between expression of Chk1 and genetic status of p53. We also found that 7 of 11 (78%) gastric carcinomas expressed elevated levels of Chk2 had p53 mutation, and this correlation was significant (p = 0.0157). We used a highly quantitative 5' nuclease fluorogenic RT-PCR method (TaqMan) to analyze the expression of Chk2 mRNA in 22 gastric carcinomas. Chk2 mRNA expression was higher in gastric carcinomas with p53 mutations compared to those harboring wild-type p53. A significant association was recognized between the expression of Chk2 mRNA and p53 mutational status (p = 0.031). Our findings support the hypothesis that expression of Chk2 protein is increased in gastric carcinomas with mutant p53. Chk1 and Chk2 may play important roles in the checkpoint function in human gastric carcinomas harboring p53 mutation when their functions are preserved to prevent cell cycle progression.     

Effect of melphalan and hyperthermia on p34cdc2 kinase activity in human melanoma cells.

We previously reported that combined treatment with melphalan and mild hyperthermia (1 h at 42 degrees C) caused a synergistic cytotoxic effect in JR8 melanoma cells, paralleled by a stabilisation of a melphalan-induced G2-phase cell block. In this study, we investigated the effect of melphalan and hyperthermia on proteins that regulate G2-M transition. Neither hyperthermia nor melphalan at a concentration of 2.5 micrograms ml-1, which had no antiproliferative effect at 37 degrees C, interfered with cyclin B1 expression or p34cdc2 kinase activity. At a concentration of 8.5 micrograms ml-1, which reduced cell growth by 50% at 37 degrees C, melphalan inhibited p34cdc2 kinase activity as a consequence of an increased tyrosine phosphorylation of the protein. A similar inhibitory effect on p34cdc2 kinase was obtained when the lowest melphalan concentration (2.5 micrograms ml-1) was used under hyperthermic conditions. Our results indicate that thermal enhancement of melphalan cytotoxicity could be mediated at least in part by an inhibition of p34cdc2 kinase activity, which prevents cell progression into mitosis.     

CIS-diamminedichloroplatinum(II) inhibits p34cdc2 protein kinase in human lung-cancer cells

cis-Diamminedichloroplatinum(II) (CDDP) induced G₂-phase arrest in PC-9 human cancer cells. To elucidate how CDDP acts on cell-cycle regulation, we analyzed the effect of CDDP on cell-cycle regulators such as p34^cdc2 protein kinase. p34^cdc2 protein kinase activity was maximum in G₂ phase and decreased after G₂/M transition in synchronized PC-9 human lung cancer cells. Evidence for a phosphorylated p34^cdc2 protein kinase complexed with cyclin B was obtained from cells in G₂ phase and the p34^cdc2 protein kinase appeared to be dephosphorylated at M phase. After exposure to CDDP in G, phase, PC-9 cells were arrested in G₂ phase. The activation of p34^cdc2 protein kinase was inhibited by CDDP. Cyclin A and wee-1 kinase were not affected by the exposure to CDDP. Cyclin B was degraded in M phase in PC-9 cells. Exposure to CDDP did not affect the degradation of cyclin B. Our data suggest that the effect of CDDP on cell-cycle phase might be regulated by the dephosphorylation of p34^cdc2 protein kinase. To determine whether the p34cdc2 protein kinase is a primary target for CDDP, we examined the direct effect of CDDP on tyrosine dephosphorylation of p34^cdc2 protein kinase in cellular extracts. Cell lysates from synchronized PC-9 in G₂ phase were immunoprecipitated with p 13-Sepharose beads. In vitro dephosphorylation of phosphotyrosine of p34^cdc2 protein kinase was observed after exposure to okadaic acid in a concentration-dependent manner. The dephosphorylation of p34^cdc2 protein kinase by okadaic acid was inhibited by CDDP. We hypothesize that inhibition of p34^cdc2 dephorphorylation by CDDP is important for its growth-inhibiting properties.     

卵巢上皮源性肿瘤Polo样激酶1的表达及其与p34cdc2的相关性

目的:探讨Polo样激酶1(plk1)、p34cdc2在卵巢上皮源性肿瘤组织中的表达及与卵巢癌各临床病理特征的关系.方法:免疫组化SP法对正常卵巢及卵巢上皮良性肿瘤、卵巢癌组织中的plk4、p34cdc2进行检测.结果:卵巢癌组织中的plk1、p34cdc2阳性表达率分别为80.8%(38/47)和63.8%(30/47).plk1与p34cdc2的表达在正常卵巢、卵巢上皮良性肿瘤及卵巢癌组织中呈明显递增趋势,且两者在卵巢癌组织中的表达显著高于其余2组,P均<0.01.plk1的表达与卵巢癌的组织学分级、临床分期及病理学类型无关;p34cdc2的阳性表达与卵巢癌的组织学分级、临床分期呈明显相关.在47倒卵巢癌组织中,plk1的表达与p34cdc2的表达呈正相关,r=0.388,P=0.004.结论:plkl和p340de2的高表达可能与卵巢癌的发生、发展密切相关,可望成为卵巢癌治疗的理想靶点.     

Diallyl disulfide inhibits p34(cdc2) kinase activity through changes in complex formation and phosphorylation

Previous studies from our laboratory demonstrated that diallyl disulfide (DADS), an oil-soluble allyl sulfur compound found in processed garlic, markedly suppressed p34(cdc2) kinase activity and induced a G(2)/M phase arrest in cultured human colon tumor (HCT-15) cells. The present studies reveal that suppression of p34(cdc2) kinase activity by DADS does not result from direct interactions with the protein, but through changes in factors influencing the formation and conversion of the enzyme to its active form. Flow cytometric analyses showed that the increased proportion of cells in the G(2)/M phase following DADS treatment was accompanied by an increase in cyclin B(1) protein expression. A temporal and dose-dependent response in cyclin B(1) expression was observed in cells treated with DADS. Western blot analysis revealed that 50 microM DADS did not influence the quantity of p34(cdc2) protein expressed, but did decrease the amount associated with cyclin B(1) by 26% (P < 0.05). Exposure of unsynchronized cells to 25 or 50 microM DADS caused a trend towards increased p34(cdc2) hyperphosphorylation (17 and 22%, respectively). Exposure of synchronized cells to 100 microM DADS increased p34(cdc2) hyperphosphorylation by 15% (P < 0.05). Consistent with its ability to slightly increase the quantity of hyperphosphorylated p34(cdc2), DADS, 25 or 50 microM, decreased cdc25C protein expression by 23 and 46%, respectively (P < 0.05). The present studies suggest that the ability of DADS to inhibit p34(cdc2) kinase activation occurs because of decreased p34(cdc2)/cyclin B(1) complex formation and modest p34(cdc2) hyperphosphorylation.     

Increased expression and phosphorylation of sh-ptp2 (syp) in human gastric carcinomas

The expression of SH-PTP2 (SYP), a novel protein tyrosine phosphatase with src homology 2, was examined in human gastric carcinoma cell lines and gastric carcinoma tissues as well as corresponding non-neoplastic mucosas by Northern and Western blotting. The expression of SH-PTP2 mRNA was detected in all 8 gastric carcinoma cell lines at various levels. Most of the cell lines expressed SH-PTP2 protein of 70 kd which corresponds to tyrosine-phosphorylated form, whereas only one cell line expressed unphosphorylated form of SH-PTP2; Fourteen (66.6%) of 21 gastric carcinomas expressed SH-PTP2 mRNA at higher level than non-neoplastic mucosas. Furthermore, most of the gastric carcinomas displayed higher levels of tyrosine-phosphorylated form of SH-PTP2, while non-neoplastic mucosas tended to express unphosphorylated SH-PTP2 protein. These results suggest that the increased expression and tyrosine-phosphorylation of SH-PTP2 may participate in stomach carcinogenesis.     

Inhibition of p34cdc2 kinase activation, p34cdc2 tyrosine dephosphorylation, and mitotic progression in Chinese hamster ovary cells exposed to etoposide.

p34cdc2 kinase, an enzyme essential for mitosis in mammalian cells, may play a role in etoposide-induced G2 phase arrest of Chinese hamster ovary cells. In this study, etoposide is shown to cause inhibition of a specific p34cdc2 kinase activation pathway, that of tyrosine dephosphorylation. Exposure of asynchronously dividing cells to etoposide caused a simultaneous rapid decline of both mitotic index and p34cdc2 kinase activity, suggesting that the kinase was not activated and that the arrest point was in late G2 phase. Using synchronized cells, p34cdc2 kinase exhibited maximal activity at the G2/M transition. Activation of the kinase and the onset of mitosis were accompanied by increased electrophoretic mobility and tyrosine dephosphorylation of the p34cdc2 protein. A 1-h exposure to etoposide during early G2 phase inhibited p34cdc2 kinase activation, its shift in electrophoretic mobility, and its tyrosine dephosphorylation, all of which correlated with a delay in mitotic progression. The interaction between the p34cdc2 and cyclin B proteins appeared unaffected under etoposide exposure conditions which resulted in greater than 70% inhibition of p34cdc2 kinase activity and almost complete cessation of transition into mitosis. These data suggest that mammalian cells express a DNA damage-responsive mechanism which controls mitotic progression at the level of p34cdc2 tyrosine dephosphorylation.     

Altered protein kinase C activity in biopsies of human colonic adenomas and carcinomas

Protein kinase C (PK-C) seems to be involved in the regulation of growth and differentiation of normal epithelial cells. Colonic adenomas and carcinomas show increased proliferation and decreased differentiation. We investigated the activity and subcellular distribution of PK-C in biopsies of normal, neoplastic, and malignant colonic epithelium to evaluate alterations in enzyme activity. In the control group (n = 7), the activity of PK-C was highest in the distal ileum (597 pmol/min/mg protein) and declined to the lowest amounts in rectal mucosa (225 pmol/min/mg protein). In patients with colonic adenomas (n = 16), total PK-C activity was significantly reduced as compared to adjacent mucosa (146 versus 336 pmol/min/mg protein, P less than 0.05) and to values determined in the control group (372 pmol/min/mg protein, P less than 0.01). The reduction of total PK-C activity in the adenoma group was even more evident in intraindividual comparison to paired adjacent mucosa (41.8% of adjacent mucosa, P less than 0.001). Specific activity of membrane-associated PK-C was equally decreased in colonic adenomas (36.3 pmol/min/mg protein) when compared to adjacent mucosa (102 pmol/min/mg protein, P less than 0.05) or to the control group (107 pmol/min/mg protein). In patients with colonic carcinomas (n = 10), the amount of total PK-C activity was also decreased (198 pmol/min/mg protein) when compared to adjacent mucosa or to the control group (P less than 0.05). In addition, the amount of membrane-associated PK-C activity (89.1 pmol/min/mg protein) was significantly reduced in carcinoma when compared to adjacent mucosa (P less than 0.05). The ratio of membrane-associated/total PK-C was not altered in adenomas, while in patients bearing carcinomas the relative fraction of membrane-associated PK-C activity was increased in samples from carcinomas and equally from adjacent colonic mucosa (45.0 and 44.6 versus 28.9%, P less than 0.05) when compared to controls. These results indicate that alterations within the protein kinase C pathway occur as early events in the adenoma-carcinoma sequence of intestinal mucosa, suggesting an important role of PK-C in epithelial differentiation and growth.     

Prognostic significance of p34cdc2 expression in tongue squamous cell carcinoma

We evaluated the prognostic significance of p34cdc2 expression in 50 tongue squamous cell carcinomas (SCC) using immunohistochemical methods. The p34cdc2 protein was expressed in 33 cases of 50 tumor tissues (66.0%), compared with 15 cases of 42 controlled epithelia (35.7%). The expression of p34cdc2 was significantly correlated with the histological grade of tongue carcinoma (P<0.01). In addition, on evaluation of prognosis of tumor, the p34cdc2 protein was overexpressed in recurrent tumors or in those with lymph node metastasis. Statistics showed a significant reduction in the 5-year accumulative survival rate of p34cdc2 positive cases compared with p34cdc2 negative cases (P<0.01). Namely, the p34cdc2 positive patients had worse prognosis. The results suggested that the expression of p34cdc2 suited to the histological grade might reflect the malignant degree of tongue carcinoma biologically. Therefore, the evaluation of p34cdc2 expression was of benefit in elucidating the nature of tumor malignancy and the prognostic prediction of tongue SCC.     

pp60c-src protein kinase activity in human gastric carcinomas

We examined pp60c-src protein kinase activity in human gastric carcinoma cell lines and gastric carcinoma tissues as well as normal mucosa. pp60c-src kinase activity was detected in all 5 carcinoma cell lines at various levels. Of 16 gastric carcinoma tissues, 8 showed higher pp60c-src kinase activity in tumor tissues than in corresponding normal mucosa. However, the levels of expression of pp60c-src detected by Western blotting were not always consistent with the activities of pp60c-src protein kinase. These findings suggest that the increase in pp60c-src protein kinase activity might be brought about by post-translational changes.     

The p53 nuclear localisation signal is structurally linked to a p34cdc2 kinase motif

We have identified a region of human p53 protein with striking homology to a sequence motif on Simian Virus 40 T antigen which includes the nuclear localisation signal. Mutation of basic amino acid residues in this region of p53 (residues 312 to 323; SSSPQPKKKP) compromises transport of p53 protein to the nucleus. The sequence functions efficiently as a nuclear localisation signal when fused to E. coli beta galactosidase. Serine 315 within this p53 structural motif is phosphorylated in vitro by the cell cycle kinase p34cdc2. Thus in both T antigen and p53, nuclear localisation signal and p34cdc2 kinase acceptor residue map to a contiguous region of primary amino acid sequence.     

Increased expression of the retinoblastoma gene in human colorectal carcinomas relative to normal colonic mucosa

We report the first evidence of increased levels of the retinoblastoma (Rb) message in a majority of colorectal cancers when compared with normal mucosa. Southern blot analysis showed an increase in Rb gene copy number in at least 28% of colorectal carcinomas relative to normal mucosa. These results plus previous reports of nonrandom chromosome 13 gains in approximately 50% of colorectal cancers suggest that an increase in Rb gene copy number occurs frequently in these tumors. Possible mechanisms pertaining to overexpression of the Rb gene are discussed in relation to its role as a recessive cancer gene.     

Possible role for p34cdc2 kinase in etoposide-induced cell death of Chinese hamster ovary cells

In an effort to shed light upon the processes of antitumor drug-induced cell death, we have carried out a systemic study of the effects of the anti-topoisomerase II agent, etoposide, on Chinese hamster ovary cells. Treatment of Chinese hamster ovary cells for 1 h with a 2-log cell-killing concentration of etoposide induces a high incidence of DNA single-strand breaks which are rapidly repaired upon drug removal. p34cdc2 kinase activity is inhibited within 1 h of addition of etoposide. Following removal of drug, cells accumulate transiently in G2. Upon recovery of p34cdc2 kinase activity (between 12 and 24 h posttreatment), approximately 50% of cells progress through mitosis which results in micronucleation. Examination of mitotic figures at various posttreatment incubation times indicates that micronucleation of daughter cells could be attributed to abnormal segregation of chromosomes during mitosis. Unexpectedly, p34cdc2 kinase activity remains elevated relative to untreated controls until 36 h post-etoposide treatment, a point where no further cell division takes place. This activity decreases by 48 h posttreatment, concomitant with a decrease in cell viability as estimated by the ability to exclude trypan blue. These results indicate that etoposide may induce cytotoxicity via gross chromosomal fragmentation, and that p34cdc2 kinase may be involved in this process.     

Inhibition of p34cdc2 kinase activity by etoposide or irradiation as a mechanism of G2 arrest in Chinese hamster ovary cells

The mammalian homologue of the yeast cdc2 gene product, p34cdc2, is a cell cycle-regulated protein essential for mitosis. We have used polyclonal antisera raised against a peptide corresponding to the carboxyl terminus of the sequence of human cdc2 to study p34cdc2 in Chinese hamster ovary (CHO) cells. Major bands are immunoprecipitated at a molecular weight of 34,000, although not in the presence of competing antigenic peptide. p34cdc2 is coimmunoprecipitated with proteins of molecular weights of 52,000 and 57,000. Immunoprecipitates express histone H1 kinase activity which varies throughout the cell cycle, maximal activity being observed in G2-M. The activity of the p34cdc2 kinase varies according to its association with the Mr 52,000 and 57,000 proteins and according to their phosphorylation state. Treatment of either asynchronous CHO cells or an enriched G2 population with the antitumor agent, etoposide, results in rapid inhibition of immunoprecipitated p34cdc2 kinase activity, which is not due to a direct effect of drug upon the enzyme. p34cdc2 kinase activity recovers as cells arrest in G2 and a second etoposide treatment further inhibits p34cdc2 kinase activity and prolongs G2 arrest. Exposure of asynchronous CHO cells to gamma-irradiation also inhibits p34cdc2 kinase activity within 1 h. Again this activity recovers as cells accumulate in G2. These results suggest that DNA damage in CHO cells elicits a response which results in inhibition of p34cdc2 kinase activity and, consequently, G2 arrest.     

p34~(cdc2)和cyclin B1在宫颈癌中的表达及临床意义

目的:研究细胞周期蛋白p34cdc2和cyclin B1在官颈癌组织中的表达水平,并探讨它们的表达与宫颈癌患者临床病理资料之间的相关性.方法:采用实时荧光定量PCR(real-time fluorogentic quantitative PCR,RFQ-PCR)法和Western印迹法,对62例原发性宫颈癌和15例对照宫颈组织进行检测,定量分析p34cdc2和cyclin B1的mRNA和蛋白质的表达水平.结果:在宫颈癌组织中p34cdc2和cyelin B1的mRNA和蛋白质表达水平明显高于对照宫颈组织(P=0.004,P=0.013和P=0.016,P=0.029),而且主要表现为过表达.p34cdc2和cyclin B1之间存在显著性正相关,mRNA和蛋白质表达的相关系数分别为0.527(P=0.001)和0.432(P=0.022).p34cdc2和cyclin B1的mRNA表达与官颈癌淋巴结转移之间存在密切关系(P=0.02,P=0.001),但与患者的年龄、临床分期、病理类型和分化程度无关(P＞0.05).结论:p34cdc2和cyelin B1是宫颈癌中的重要调控分子,可促发癌细胞克服细胞周期G2/M期控节制点进入M期,促进肿瘤的发生与发展.p34cdc2和cyclin B1的高表达可能成为研究宫颈癌发生机制和淋巴转移的新的分子标志.     

A p34(cdc2) survival checkpoint in cancer

A checkpoint surveying the entry into mitosis responds to defects in spindle microtubule assembly/stability. This has been used to trigger apoptosis in cancer cells, but how the spindle checkpoint couples to the cell survival machinery has remained elusive. Here, we report that microtubule stabilization engenders a survival pathway that depends on elevated activity of p34(cdc2) kinase and increased expression of the apoptosis inhibitor and mitotic regulator, survivin. Pharmacologic, genetic, or molecular ablation of p34(cdc2) kinase after microtubule stabilization resulted in massive apoptosis independent of p53, suppression of tumor growth, and indefinite survival without toxicity in mice. By ablating this survival checkpoint, inhibitors of p34(cdc2) kinase could safely improve the efficacy of microtubule-stabilizing agents used to treat common cancers.     

Expression of epidermal growth factor receptor in human gastric and colonic carcinomas

The expression of epidermal growth factor (EGF) receptor was examined immunohistochemically in a total of 122 gastric and 61 colonic carcinomas, out of which 16 gastric and 8 colonic carcinomas were also examined by 125I-labeled EGF binding analysis and Western blotting. The values of EGF binding were 12.68 +/- 1.98 (SE; n = 16) fmol/mg protein in gastric carcinomas and 5.72 +/- 2.15 (n = 8) fmol/mg protein in nonneoplastic gastric mucosa, the difference being significant (P less than 0.01). In the colonic tissue, the binding capacities in carcinomas and nonneoplastic mucosa were 13.29 +/- 4.17 (n = 8) and 10.68 +/- 0.41 (n = 3) fmol/mg protein, respectively. Scatchard analysis of 125I-labeled EGF binding indicated a single class of receptors in gastric and colonic carcinomas with an apparent Kd value of from 111 to 277 (n = 4) and from 87.4 to 341 fM (n = 5), respectively, except for one gastric carcinoma having two classes of receptors (Kd = 15.9 and 896 fM). In Western blotting using monoclonal anti-EGF receptor antibody, various levels of EGF receptor expression were detected in 12 (85.7%) of the 14 gastric carcinomas and in 7 (87.5%) of the 8 colonic carcinomas. Immunohistochemically, EGF receptor immunoreactivity was detected in one (3.8%) of the 26 early gastric carcinomas, while it was observed in 33 (34.4%) of the 96 advanced gastric carcinomas, the incidence between the two being significantly different (P less than 0.01). In the colonic carcinomas, 47 (77.1%) of the 61 cases showed positive immunoreactivity to EGF receptor, which did not differ by histological type.