p53 protein accumulation and gene mutations in human glioma cell lines

Mutations in, and aberrant expression of, the p53 tumor suppressor gene were assessed in 17 cell lines derived from human malignant brain tumors (glioblastoma multiforme). Ex-ons 5 through 8 were screened by single strand conformational polymorphism analysis (SSCP), followed by direct DNA sequencing. Mutations were found in 6 of 17 glioma cell lines, i.e., at a frequency similar to that found in primary malignant gliomas. Loss of the wild type allele was observed in 4 of the mutated cell lines. Two cell lines had the same mutation (CGG → TGG; Arg → Trp) in codon 248. Five of 6 mutations were transitions, 4 of which occurred at CpG dinucleotides. In one cell line a 10-bp deletion at the intron 4/exon 5 junction was found. Five of 6 glioma cell lines contained a mutation identical to that in the respective primary tumor despite prolonged in vitro culture (140-221 passages). Thus, the acquisition of p53 mutations during culture appears to be infrequent. Two cell lines derived from heterozygous tumors maintained the wild type p53 allele during long term culture. p53 protein levels were assessed by immunofluorescence cytochemistry and immunoprecipitation followed by Western blot analysis and revealed elevated levels of the p53 protein, although to a variable extent, in all cell lines with p53 mutations. A marked p53 protein accumulation was also observed in two cell lines lacking p53 mutations in exons 5 through 8, indicating that a prolonged half life of the gene product is not solely dependent on an aberrant coding sequence. The remaining cell lines had either low levels or no detectable p53 protein; one of the latter contained a gross rearrangement of the p53 gene. Our results suggest that with respect to p53 gene status, glioma cell lines usually resemble the original tumors and may, therefore, be suitable for studying the biological changes associated with p53 mutations in glial tumors.     

p53-dependent radioresistance in ovarian carcinoma cell lines

In the present study, we investigated the radiosensitivity profiles of three established human ovarian carcinoma cell lines, PA-1, Caov-3, and SK-OV-3, using the adenosine triphosphate-cell viability assay (ATP-CVA). We have correlated radioresponsiveness with the p53 status and the p53 accumulation after irradiation as well as with the Bcl-2 expression and the growth rate of these cell lines. The p53 status was examined by immunocytochemistry and a functional assay (functional analysis of separated alleles in yeast, FASAY); the p53 accumulation was determined by immunocyotochemistry and flow cytometry. Futhermore, the Bcl-2 expression before and after irradiation was examined by immunocytochemistry. PA-1, expressing wild-type p53, showed an unequivocal accumulation of p53 protein following exposure to irradiation. This cell line was found to be strongly sensitive to irradiation. The two p53 mutant cell lines Caov-3 and SK-OV-3 showed radioresistance at different degrees and irradiation did not result in p53 accumulation. None of the cell lines examined expressed Bcl-2 protein and no change was seen after irradiation. Furthermore, the most sensitive cell line to irradiation, PA-1, showed the highest proliferative activity, while Caov-3 and SK-OV-3, the more resistant cell lines, exhibited lower growth rates. Our findings indicate that the presence of p53 protein is a possible determinant for the cytotoxicity induced by irradiation in the investigated ovarian carcinoma cell lines. Bcl-2 expression does not seem to determine the response to irradiation in these cell lines. Additionally, an association between radioresponsiveness and the growth rate is suggested in PA-1, Caov-3, and SK-OV-3.     

p53 protein accumulation and p53 gene mutation in colorectal cancer

Comparison of immunohistochemical methods for detection of protein p53 accumulation and molecular techniques for analysis p53 gene mutation in colorectal cancer is presented. Thirty eight patients were included: all underwent surgery without preoperative treatment. Sex of patients, tumor localisation, macro and microscopic type of cancer and staging according to Astler-Coller and Jass classifications were evaluated. Protein p53 accumulation was detected by the streptavidin-biotin method using DO-7 (Dako) antibody. The number of cells stained were classified semiquanititatively according to a scoring system: (-)no positive cells, (+) : 10-30% positive cells, (++) : 40-70% positive cells, (+++) : >70% positive cells. For all cancer samples, exons 5 to 9 of p53 gene were amplified from isolated genomic DNA. PCR products were subjected to single standed conformational polymorphism analysis. All product were also directly sequenced on ABI PRISM 377 apparatus using fluorescent dideoxyterminators chemistry. The protein p53 accumulation was detected in 53% (20/38), whereas p53 gene mutation was seen in 55% (21/38). Among them, 15 patients (39%) with overexpression showed mutation in exon 5-8 gene p53. Discrepancies between results were noted in 29%. In conclusion, the necessity of both methods immunohistochemical and molecular is indicated for the objective evaluation of functional and structural status of p53 gene and protein.     

Effect of glutathione on DNA repair in cisplatin-resistant human ovarian cancer cell lines

We have studied the effect of glutathione reduction by buthionine sulfoximine (BSO), a specific inhibitor of gamma -glutamyl cysteine synthetase, on DNA repair after cisplatin damage in an ovarian cancer cell line with in vitro induced resistance to cisplatin. In addition, we have examined the effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, in combination with BSO on cisplatin-associated DNA repair. BSO treatment was found to partially inhibit DNA repair, and the addition of aphidicolin caused nearly a 100% inhibition in DNA repair activity. Treatment of cells with glutathione ester after BSO resulted in complete recovery of DNA repair activity or partial recovery if aphidicolin was present. The significance of these results to the chemosensitizing effects of BSO medicated glutathione reduction is discussed.     

DDP-induced cytotoxicity is not influenced by p53 in nine human ovarian cancer cell lines with different p53 status.

Nine human ovarian cancer cell lines that express wild-type (wt) or mutated (mut) p53 were used to evaluate the cytotoxicity induced by cisplatin (DDP). The concentrations inhibiting the growth by 50% (IC50) were calculated for each cell line, and no differences were found between cells expressing wt p53 and mut p53. Using, for each cell line, the DDP IC50, we found that these concentrations were able to induce an increase in p53 levels in all four wt-p53-expressing cell lines and in one out of five mut-p53-expressing cell lines. WAF1 and GADD45 mRNAs were also increased by DDP treatment, independently of the presence of a wt p53. Bax levels were only marginally affected by DDP, and this was observed in both wt-p53- and mut-p53-expressing cells. DDP-induced apoptosis was evident 72 h after treatment, and the percentage of cells undergoing apoptosis was slightly higher for wt-p53-expressing cells. However, at doses near the IC50, the percentage of apoptotic cells was less than 20% in all the cell lines investigated. We conclude that the presence of wt p53 is not a determinant for the cytotoxicity induced by DDP in human ovarian cancer cell lines.     

拓扑替康促人卵巢癌顺铂耐药细胞株凋亡机制的初步研究

目的探讨拓扑替康(TPT)对人卵巢癌顺铂耐药细胞株A2780/DDP和COC1/DDP的杀伤和诱导凋亡活性及其作用机制.方法用MTT比色法测定TPT对人卵巢癌顺铂耐药细胞株A2780/DDP和COC1/DDP的杀伤效应;透射电镜和DNA凝胶电泳研究TPT对靶细胞的凋亡诱导活性;Western blot检测凋亡相关蛋白bcl-2和bax的表达.结果TPT对A2780/DDP和COC1/DDP细胞均有较强的体外杀伤作用,其IC50值分别为102.91 ng/ml和111.75 ng/ml;TPT能诱导A2780/DDP和COC1/DDP细胞产生凋亡,DNA凝胶电泳呈现典型的凋亡梯度,透射电镜观察到细胞核染色质固缩、边集,胞质浓缩,出现空泡等典型的凋亡超微结构改变;TPT不影响bcl-2蛋白表达,却能上调bax蛋白表达,增大bax/bcl-2比值,且呈剂量依赖性.结论TPT对人卵巢癌顺铂耐药细胞株A2780/DDP和COC1/DDP均有明显的杀伤和促凋亡作用,其机制可能与bax基因表达上调而提高bax/bcl-2比值有关.     

Expression of p53 in human leukemic cell lines

The cell-encoded p53 antigen seems to be tightly associated with various human malignancies. We have analyzed biochemical properties of p53 in two different cell lines derived from patients with ALL or ANLL. p53 was found in elevated levels in both leukemic cell lines compared to unstimulated or stimulated normal lymphocytes. High levels of p53 in these cell lines are due to an extended stability of p53 protein rather than to different rates of synthesis. p53 from both cell lines formed low- and high-molecular weight oligomers which revealed that p53 exists in a heterogenous population in these tumor cells. The presence of immunologically different subsets of p53 was demonstrated by sequential immunoprecipitation experiments with different p53 specific monoclonal antibodies. Our results showed structural and immunological variabilities of p53 in cell lines derived from human tumors and may thus provide an insight into the role p53 may play in human malignancies.     

p53 Mutations in human immortalized epithelial cell lines

Although rodent cells have been immortalized following transfectionwith a mutant p53 gene, the role of p53 in the immortalizationof human cells is unknown. Therefore, human epithelial celllines were examined for p53 mutations in exons 4–9 whichinclude the evolutionarily conserved regions. A spontaneouslyimmortalized skin keratinocyte cell line, HaCat, and three ras-transfectedclones, have a p53 mutational spectrum that is typical of ultravioletlight induced mutations. A normal finite lifespan cell strain(184) and two benzo[a]pyrene immortalized mammary epithelialcell lines derived from 184 (184A1 and 184B5) contain wild typep53 sequences in exons 4–9, although elevated levels ofnuclear p53 indicate an alteration in the stability of the normallytransient protein. Wild type p53 was found in human bronchial,esophageal and hepatic epithelial cells immortalized by SV40T antigen gene and human renal epithelial cells immortalizedby adenovirus 5. BEAS-2B, an SV40 T antigen immortalized bronchialepithelial cell line and two subclones, have a germline polymorphismat codon 47. Inactivation of p53 by mechanisms such as mutationor complexing with proteins of DNA tumor viruses appears tobe important in the immortalization of human epithelial cells.     

p53 in chronic myeloid leukemia cell lines.

Four human chronic myeloid leukemia (CML) cell lines, BV173, K562, KCL-22, and KYO-1, were studied for inactivation of human tumor suppressor gene p53. Southern blotting showed allele deletion in KCL-22 and cytogenetic studies showed a chromosome 17 deletion in KYO-1 but no gross structural abnormalities in the other two lines. Northern blotting showed increased amounts of normal size p53 mRNA in BV-173 and KYO-1, trace amounts in KCL-22, and none in K562. Direct sequencing of p53 cDNA revealed a missense point mutation in KYO-1 and a single base pair deletion consistent with a coding frame shift in KCL-22. Both abnormalities in these myeloid cell lines were located in the highly conserved region of p53. Studies with two monoclonal antibodies showed that the three cell lines with p53 mRNA had readily detectable p53 proteins. In KYO-1 and BV173 cells the p53 protein was located mainly in the nuclei but KCL-22 cells had weak staining in the cytoplasm. Our data support the assumption that inactivation of p53 tumor suppressor function in myeloid blast transformation of CML may result from point mutations or deletions that produce mutant proteins.     

鼻咽癌中p53蛋白积聚对瘤细胞有丝分裂和凋亡的影响

目的：观察疗前鼻咽癌组织中Ｐ５３蛋白的积聚及其对瘤细胞有丝分裂和凋亡的影响。方法随机收集１９９７年疗前鼻咽癌活检标本４３例,采用免疫组化ＬＳＡＢ法ＤＯ－７一抗检测Ｐ５３蛋白的表达。在Ｈ＆Ｅ染色切片上位细胞死亡试剂盒检测瘤细胞凋亡,平均每个高倍视野下的凋亡瘤细胞数为凋亡指数（ＴＵＮＥＬ ｉｎｄｅｘ,Ｔ１）。比较高于位细胞死亡试剂盒检测瘤细胞凋亡,平均每个高倍视野下的细胞瘤细胞数为凋亡指数（ＴＵＮＥＬ     

p53 protein accumulation in multiple oesophageal squamous cell carcinoma: relationship to risk factors

To clarify mechanisms involved in the carcinogenesis of multiple oesophageal squamous cell carcinoma, the expression of p53 protein in 46 lesions surgically excised from 13 Japanese patients was investigated immunohistochemically and the relation of p53 protein accumulation to the patient's history of alcohol consumption and cigarette smoking was analyzed. p53 protein accumulation was observed in 13 main lesions, that is in 6 (85.7%) of 7 subjects with a history of heavy drinking and smoking, but only in 1 (16.7%) of 6 with no such history (Fisher's exact test, p = 0.025). As regards the 46 lesions, p53 protein accumulation was evident in 22 (88.0%) of 25 lesions of the high-risk patients, but in 7 (33.3%) of 21 lesions of the other subjects (Fisher's exact test, p < 0.001). p53 protein accumulation was similarly recognized in all oesophageal lesions in 5 of 7 high-risk patients. Thus, use of both alcohol and cigarettes is clearly associated with a high frequency of p53 protein accumulation in multiple oesophageal squamous cell carcinoma present at the same time. These findings are considered to support the concept of field carcinogenesis of the oesophagus.     

p53 mutations, ras mutations, and p53-heat shock 70 protein complexes in human lung carcinoma cell lines.

The p53 tumor suppressor gene is frequently mutated and the K-ras oncogene is occasionally mutated in primary specimens of human lung carcinomas. These mutated genes also cooperate in the immortalization and neoplastic transformation of rodent cells. To determine whether these mutations are necessary for maintenance of the immortalized and/or neoplastically transformed states of human bronchial epithelial cells, the p53 gene and regions of the ras (K-, H-, and N-) genes were sequenced in nine human lung carcinoma cell lines. Detection of p53 mutations by polymerase chain amplification and direct DNA sequencing was corroborated by p53 immunocytochemistry and coimmunoprecipitation of p53 with heat shock protein 70. p53 and ras genes were frequently, but not always, mutated in the carcinoma cell lines. These data are consistent with the hypothesis that multiple genetic changes involving both protooncogenes and tumor suppressor genes occur during lung carcinogenesis.     

High incidence of p53 gene mutation in human ovarian cancer and its association with nuclear accumulation of p53 protein and tumor DNA aneuploidy.

Using the polymerase chain reaction and single-strand conformation polymorphism analysis, p53 gene mutations were examined in 24 cases of ovarian tumor including 14 ovarian carcinomas and 2 borderline cases of common epithelial type, 7 germ cell tumors, and one stromal tumor. Abnormal bands indicating mutations were detected in 12 (50%) of the cases examined, being present most frequently in common "epithelial" ovarian carcinoma (71%, 10/14). One case each of squamous cell carcinoma originating in a dermoid cyst and anaplastic dysgerminoma were positive for mutation. Direct sequencing confirmed 12 mutations and revealed G-->A and G-->C nucleotide changes in 5 and 3 cases (42% and 25%), respectively. The mutation was localized at the CpG site of the gene in 3 cases. Immunohistochemical examination of p53 protein in 21 cases and DNA flow-cytometrical analysis in 17 cases were also performed. Nuclear accumulation of the p53 protein and DNA aneuploidy pattern were detected in 11 (52%) and 9 (53%) cases, respectively. These were significantly correlated with p53 gene mutation (P < 0.01 and P < 0.05, respectively; Fisher's exact test). Neither mutation of the p53 gene, nuclear accumulation of p53 protein nor DNA aneuploidy was detected in borderline cases of common "epithelial" type, typical dysgerminoma and immature teratoma. These results suggest that p53 gene mutation, nuclear accumulation of the protein and the DNA aneuploidy pattern are events occurring almost simultaneously in the progression of ovarian tumors, and that p53 abnormalities seem to be correlated with a high grade of malignancy.     

细胞色素c 介导的半胱天冬氨酸蛋白酶-3活性改变与人卵巢癌顺铂耐药的关系

目的：探讨人卵巢癌顺铂耐药细胞株A2780／DDP、COC1／DDP中抗凋亡基因bcl-XL、细胞色素c的表达和半胱天冬氨酰蛋白酶－3（caspase-3)活性对人卵巢癌顺铂耐药的影响。方法：采用逆转录聚合酶链反应（RT－PCR）和Western blot检测人卵巢癌顺铂敏感细胞株A2780、COC1和顺铂耐药株A2780／DDP、COC1／DDP中bcl-XL的表达,以及顺铂作用后细胞色素c的含量和caspase-3活性的变化,并应用流式细胞仪测定顺铂作用后A2780、COC1、A2780／DDP、COC1／DDP细胞的凋亡率。结果：bcl-XL在A2780/DDP、COC1／DDP细胞中的表达明显高于A2780、COC1细胞;顺铂作用后,细胞色素c在A2780／DDP、COC1／DDP细胞中的表达明显减少,caspase-3活性和凋亡率也较A2780和COC1细胞明显降低（P＜0．05）。结论：人卵巢部细胞对顺铂产生耐药可能与细胞内bcl-XL过度表达、细胞色素c释放受抑制和caspase-3活性下降有关。     

Induction of p53 protein by carbon-ion and proton beam irradiation in two close human lymphoblastoid cell lines with different p53 status

In this study, we compared the kinetics of the postirradiation p53 protein expression for carbon ion beam (290 MeV/n, LET 75 keV/mu m) and proton beam (65 MeV) with that of (13)7Cs-gamma ray. We used two human lymphoblastoid cell lines derived from the same donor with different p53 status. Wild-type p53 protein increased after irradiation and it was dose-dependent. Meanwhile, the mutated p53 protein level did not show any increase with irradiation. With the three forms of radiation, there was no significant difference as regards the p53 protein kinetics.     

Synergistic growth inhibition by combination of adenovirus mediated p53 transfer and cisplatin in ovarian cancer cell lines

Objective

This study was to investigate the synergistic growth inhibitory effect by combination of adenovirus mediated p53 gene transfer and cisplatin in ovarian cancer cell lines with different p53 gene mutation patterns.

Methods

Three ovarian cancer cell lines, p53 deleted SKOV3, p53 mutated OVCAR-3, and PA-1 with wild-type p53 were transduced with human adenovirus vectors carrying p53 gene (Ad-p53) and treated with a sublethal concentration of cisplatin before and after Ad-p53. The cell number was counted daily for 5 days after Ad-p53 transduction. Western blotting was used to identify p53 and p21 protein expressions, and flow cytometric analysis was performed to investigate any change of DNA ploidy after Ad-p53 transfer.

Results

Ad-p53 transduced cells successfully expressed p53 and p21 proteins after 48 hours of Ad-p53 transduction. Synergistic growth inhibition by combination of Ad-p53 and cisplatin was detected only in SKOV3 and OVCAR-3 cells, but not in PA-1 cells. In p53 deleted SKOV3 cells, cisplatin treatment after Ad-p53 showed higher growth inhibition than the treatment before Ad-p53 transduction, and reverse relationship was observed in p53 mutated OVCAR-3 cells. In SKOV3 cells, the fraction of cells at G2/M phase increased after cisplatin treatment, however, it decreased dramatically with Ad-p53 transduction.

Conclusion

The synergistic growth inhibition by combination of Ad-p53 and cisplatin may depend on the p53 status and the temporal sequence of cisplatin treatment, suggesting judicious selective application of this strategy in clinical trials.