Absence of p53 mutations in benign and pre-malignant male genital lesions with over-expressed p53 protein

Mutations of the tumor-suppressor gene p53 are common in epithelial tumors. Clonal mutations of p53 have been found in cervical and vulvar carcinomas negative for human papillomavirus (HPV), though at least in cervical cancer HPV infection and p53 mutations are not mutually exclusive. We have previously shown that about 40% of male genital warts and bowenoid papulosis lesions exhibit immunohistochemically detectable aberrant p53 protein, irrespective of the presence of HPV DNA. We studied p53 mutations in exons 4–8 with SSCP and sequencing in 13 male patients with 1 to 3 therapy-resistant genital warts or intra-epithelial neoplasias each and in 4 patients with penile squamous cell carcinoma. Thus, 13 genital warts, 6 bowenoid papulosis, 1 Queyrat's erythroplasia and 1 carcinoma in situ were studied. p53 protein was detected immunohistochemically, and HPV status was analyzed with DNA in situ hybridization and amplification of HPV-specific DNA. There was no correlation between p53 protein expression and HPV status. No mutations in exons 5–8 of the p53 gene were found in any of the lesions, and furthermore, no exon 4 mutations were found in lesions positive in p53 immunohistochemistry. In conclusion, over-expression of p53 does not indicate a p53 mutation in male genital warts, pre-malignant lesions or malignant squamous cell carcinomas. Our study thus suggests that p53 mutations are not important, or at least not early, events in male genital carcinogenesis. Int. J. Cancer 77:674–678, 1998. © 1998 Wiley-Liss, Inc.     

抗癌灵治疗原发性肝癌的实验研究

 目的　观察抗癌灵水煎剂对小鼠移植性肝癌 (H2 2 )的治疗作用。方法　以瘤株 (H2 2 )接种小鼠创作模型 ,设正常对照组、荷瘤模型组及抗癌灵治疗组 ,共 3组 ,观察各组小鼠瘤重和抑瘤率、肿瘤坏死因子(TNF)的体内活性表达、各组H2 2 小鼠生命延长率以及对肝癌细胞增殖周期的影响等指标。结果　用抗癌灵治疗后的H2 2 小鼠其抑瘤率达 61 .2 0 % ,明显高于模型组 (P <0 .0 1 ) ,并能抑制荷瘤肌体TNF水平的异常表达 ,提高H2 2 荷瘤小鼠的生命延长率 ,而且该药还能阻滞肿瘤细胞周期G1期向S期进展 ,从而阻断DNA的合成和复制。结论　抗癌灵对小鼠肝癌 (H2 2 )具有明显的杀伤作用     

In primary human breast carcinomas mutations in exons 5 and 6 of the p53 gene are associated with a high s-phase index

A series of 121 human breast tumors was screened for point mutations in exons 5 through 8 of the p53 gene, by SSCP analysis. On the same tumor samples, the S-phase index (SPI) was determined by the incorporation of BUdR in fresh tissue. p53 mutations were observed in 29% of the cases. The frequency of point mutations for the individual exons was: exon 5, 10.0%; exon 6, 9.9%; exon 7, 7.1% and exon 8, 5.5%. Two mutations detected by SSCP were confirmed by sequencing the p53 cDNA. The presence of a p53 mutation, irrespective of its location, correlates (p = 0.003) with a high SPI. This association appears to primarily reflect mutations in exon 5 (p = 0.0002) and exon 6 (p = 0.05), since mutations in exons 7 and 8 failed to show any association. These results indicate that mutations in the p53 gene identify highly proliferating tumors, and that the position of the p53 mutation may have different effects upon the proliferative activity of tumor cells in vivo.     

Human papilloma viruses and p53 mutations in normal,pre-malignant and malignant oral epithelia

HPV infections have been previously observed in oral cancers, and inactivation of the p53 gene has been shown to be one of the most common genetic alterations in human tumors. We examined 179 oral specimens from 70 individuals with histologic findings of either normal mucosa (n = 6) or oral disease that ranged from mild dysplasia to invasive squamous-cell carcinoma (n = 64) to determine the occurrence of both HPV infection and p53 mutations and their relationship with several clinical factors. HPV infection was detected by PCR amplification of viral DNA, and the presence of p53 mutations was assayed using the single-strand conformation polymorphism (SSCP)-PCR technique. HPV infection was found in 31% of individuals with oral disease and was not seen in healthy individuals. Mutations in exons 5, 6, 7 or 8 of the p53 gene were detected in 37.5% of patients with oral lesions and in a biopsy from 1 healthy individual who was a heavy smoker. Approximately one-third of lesions classified as pre-malignant (dysplasia and carcinoma in situ) and 42% of invasive carcinomas contained p53 mutations. The majority of these mutations were G:T transversions located within exons 7 and 8. Tumor tissues from 6 patients with oral lesions were found both to be HPV-16-positive and to contain p53 mutations; of these, 4 were poorly differentiated carcinomas that were diagnosed as late-stage disease. In this study, p53 mutations were detected in the early stages of cancer development. © 1996 Wiley-Liss, Inc.     

p53 and human papillomavirus dna in renal pelvic and ureteral carcinoma including dysplastic lesions

Ninety-eight cases of transitional-cell carcinoma (TCC) of the renal pelvis and ureter, including dysplastic lesions, were studied for tumor incorporation of human papillomavirus (HPV) type-16 and type-18 DNA by in situ hybridization (ISH) with DNA probes for each HPV viral type. Immunohistochemical analysis of p53 expression was also performed. Fresh tumor tissues from 26 patients were also studied for p53 mutations in exons 4 through 9 by direct sequencing and for HPV infection by polymerase chain reaction (PCR). Thirty-two tumors were positive for HPV DNAs, including 6 double-positive cases. Among these tumors, adjacent dysplastic lesions in 21 cases (66%) also revealed identical reactivity. Overexpressed p53 was detected in 26 cases. Expression of p53 was also detected in dysplastic lesions in 19 out of these 26 cases (73%). Three cases were positive for both HPV DNA and p53 antibody. p53 point mutation was detected in 7 of 26 cases, 6 of which were also positive for p53. HPV type-16 DNA was detected in 6 cases by PCR, 4 of which were also ISH-positive. Overexpressed p53 was frequently detected in invasive and non-papillary tumors (p < 0.01) and in high-grade tumors (p < 0.05). HPV infection was more common in non-invasive and papillary tumors (p < 0.05). These findings suggest that HPV infection or overex-pression (mutation) of p53 may be an early event and be related to phenotypes of tumor-cell growth patterns and progression. © 1995 Wiley-Liss, Inc.     

Detection of Human Papillomavirus DNA and State of p53 Gene in Japanese Penile Cancer

The frequency of integration with human papillomavirus (HPV)and its genotypes in Japanese penile cancer was examined withrelation to p53 gene mutations using polymerase chain reactionamplification. Tissues were obtained from 13 patients (eightfrom freshly frozen and five from paraffin-embedded samples).HPV DNA was detected in seven out of the 13 (54%), and theirgenotypes were type 16 in four, type 31 in one and type 33 intwo cases. Neither HPV-detected nor -undetected tissues showedmutated alterations in exons 4-9 of p53 genes. The results suggestHPV to be, at least to some extent, involved in the oncogenesisof penile cancer, and that p53 gene mutations may not correlatewith the development of penile cancer.     

Pathogenetic significance of p53 and c-Ki-ras gene mutations and human papillomavirus dna integration in adenocarcinoma of the uterine cervix and uterine isthmus

The pathogenetic significance of p53 and c-Ki-ras gene mutations and genomic integration of human papillomavirus (HPV) DNA was examined in surgically resected specimens of adenocarcinomas of the uterine cervix and isthmus using polymerase chain reaction (PCR), single-strand-conformation polymorphism and Southern blotting analysis. Among 25 cervical adeno-carcinomas, p53 gene mutations between exons 5 and 8 were detected in 32%, and the incidence of these mutations was higher in cases at advanced clinical stages and with high grades of nuclear and structural atypia both in endocervical and in endometrioid types. HPV DNA type 16 or 18 in cervical adenocarcinomas was detected in 35% of cases by PCR and in 29% by Southern blotting, and, in contrast to the p53 mutations, the majority of cases with the HPV DNA were at a relatively early clinical stage with low-grade histological atypia. c-Ki-ras gene mutation was detected in only 4% of cervical adenocarcinomas. Among 8 isthmus adenocarcinomas, the incidence of p53 and c-Ki-ras gene mutations, and the presence and integration of HPV DNA type 16 or 18 were 38%, 50%, 57% and 25% respectively. The pattern of p53 mutations differed between isthmus and cervical adenocarcinomas: all of the mutations in the former were one-base substitutions of the transition type, whereas in the latter nearly half of the mutations were of the transversion type. Among cervical adenocarcinomas, p53 mutations between exons 5 and 8 were indicated as being mostly involved in the pathogenesis and development of biologically aggressive tumors, whereas HPV type 16 or 18 infection appeared to be involved in less aggressive cases. In isthmus adenocarcinoma, c-Ki-ras gene mutation, apart from p53 mutation and HPV-type-16 or ?18 infection, appeared to be involved frequently in cancer development.     

The carcinogenic role of oncogenic HPV and p53 gene mutation in cervical adenocarcinomas

Thirty tumors were collected from our archive of cervical adenocarcinomas. They were examined with respect to the content of oncogenic HPV and presence of mutations in the p53 gene exons 5 through 8. Furthermore, available clinical information on the cases was reviewed. For the detection of p53 gene and presence of oncogenic HPV, PCR followed by direct sequence analysis of the amplified DNA was employed. Seventeen tumors were identified as HPV-positive, comprising both HPV types 18 and 16. Six cases showed a p53 gene mutation, of which five were of the missence and one of the silent type. No statistical correlation between the occurrence of oncogenic HPV and presence of p53 gene mutation (p=0.67) was recorded. Among the tumors with p53 gene mutation, three were HPV-positive and three were HPV-negative. The determination of p53 gene mutations was not related to clinical findings such as the stage of the tumor or presence of metastases of the lymph nodes. However, p53 gene mutations were somewhat more prevalent in low differentiated tumors (p<0.02). The results indicate that oncogenic HPV and p53 gene mutations have independent carcinogenic roles in cervical adenocarcinomas.     

ras,p53 and hpv status in benign and malignant prostate tumors

To study the role of ras, p53 genes and HPV virus (16 and 18) in the development of prostate cancer, we analyzed tissue sections from 27 patients affected with carcinomas (stages A to D) and from 24 patients with adenomas. Mutations of H, K and N-ras and p53 (exons 2-9) were studied by SSCP and DNA sequencing. Accumulation of p53 protein was studied by immu-nohistochemistry on tissue sections. Tumors were also analyzed for the presence of HPV 16 and -18 sequences by PCR and DNA hybridization with sequence-specific oligonucleotides. No mutation was found in the three ras genes studied, either in carcinomas or adenomas. By SSCP analysis we identified p53 mutations in only 2 of 19 carcinomas studied, both in exon 7. Immunohistochemical results strongly correlate with the SSCP results: p53 protein was positive in tumors with p53 mutation but not in others; 32% of studied adenomas had detectable HPV16 DNA, while 53% of carcinomas were HPV16+. Among these I presented a p53 mutation. No HPV 18 E6 sequence could be detected. Our data show that in prostate tumors from France, mutations of p53 and ras are rare events but that these tumors display detectable HPV 16 DNA at a high frequency. The low incidence of p53 mutation, associated to a significant proportion of tumors showing HPV16 DNA, could suggest that in prostate cancer HPV 16 infection could participate in p53 inactivation by E6. © 1995 Wiley-Liss, Inc.     

Mutations of the p53 tumor suppressor gene in neoplasms of the human nervous system

A variety of neoplasms of the human nervous system were analyzed for the presence of mutations in the p53 tumor suppressor gene. DNA was extracted from frozen or formalin-fixed, paraffin-embedded material. Single-strand conformation polymorphism (SSCP) analysis for exons 5–8 was followed by direct DNA sequencing. Mutations leading to an amino acid change were found in three of 11 (27%) low-grade (World Health Organization (WHO) Grade II) astrocytomas. They were located in codon 183 (TCA → TGA) of exon 5, codon 237 (ATG → ATA) of exon 7, and codon 273 (CGT → CAT) of exon 8. In one of these cases, the sequence indicated loss of the wild-type allele. Of 12 juvenile pilocytic astrocytomas (WHO Grade I), none contained a p53 mutation, suggesting a different molecular basis for this childhood neoplasm. Except for a mutation in one of seven (14%) meningeal hemangiopericytomas (codon 238; TGT → TTT, Cys → Phe), no mutations were observed in exons 5–8 of the p53 gene in any of the following tumors of the nervous system and its coverings: 13 schwannomas, 12 central neurocytomas, 22 meningiomas, 10 choroid plexus papillomas and carcinomas, and 30 neuroblastomas of the sympathetic nervous system. These and published data support the view that p53 mutations are frequently involved both in low-grade and progressive (anaplastic) astrocytomas, including glioblastomas multiforme. Oligodendrogliomas, medulloblastomas, meningiomas, and hemangiopericytomas rarely (<15%) show p53 mutations in exons 5–8, whereas none of the remaining nervous system neoplasms revealed evidence of an involvement of the p53 gene in their development.     

Screening for p53 mutations in c3h/he mouse liver tumors derived spontaneously or induced with diethylnitrosamine or phenobarbitone

Distinct differences have been described in the development of C3H/He mouse liver tumors induced by the genotoxic carcinogen diethylnitrosamine (DEN) and by the nongenotoxic agent phenobarbitone (PB) in terms of pathology and the frequency of mutation at codon 61 of the Ha-ras oncogene. To further define the mechanisms involved, we screened the tumor suppressor gene p53 for mutations in exons 5, 7, and 8 using polymerase chain reaction (PCR)–single-strand conformation polymorphism (SSCP) analysis. Nearly all the mutations so far described have been found within these three exons. In this study a total of six spontaneous tumors, eight tumors induced by PB, 14 tumors induced by DEN, and five samples of normal liver tissue were screened, and no mutations were found in any of the tumors examined. The positive control, the plasmid LTRp53cG (val), had a point mutation in exon 5 that was detected by PCR-SSCP. Since many of the tumors were late-stage hepatocellular carcinomas, we concluded that mutations in exons 5, 7, and 8 of the p53 gene do not play an important role in the development of chemically induced liver tumors in the C3H/He mouse. © 1994 Wiley-Liss, Inc.     

Diagnosis of bladder cancer by analysis of the allelic loss of the p53 gene in urine samples using blunt-end single-strand conformation polymorphism

The novel approach of blunt-end single-strand conformation polymorphism (SSCP) has been applied in the analysis of urine samples from bladder-cancer patients for detecting loss of heterozygosity (LOH) of 3 polymorphic markers in the p53 gene. Of the 28 urine samples examined by SSCP analysis of blunt-ended DNA fragments using a fluorescence-based automated sequencer, 16 were informative in more than 1 of the 3 polymorphic markers at the p53 locus and 8 (50.0%) showed allelic loss of the p53 gene. In analysis of resected tumor tissues, LOH of the p53 gene was detected in 8 of 8 informative samples (100%) with T1 and higher stages and/or Grade 2 and Grade 3 tumors, while it was detected in 6 (75.0%) urine samples obtained from these 8 patients. This new diagnostic modality enables sensitive detection of tumor cells in urine samples and would be applicable for diagnostic bladder cancer with invasive character. Int. J. Cancer 74:403–406, 1997. © 1997 Wiley-Liss, Inc.     

Enhanced TGFalpha-EGFR expression and P53 gene alterations contributes to gastric tumors aggressiveness

We determined whether alterations in the expression of p53, p16(INK4) and p21(WAF1/CIP1) influence the invasiveness of a subset of gastric adenocarcinomas co-expressing TGFalpha and EGFR. Immunopositivity for TGFalpha-EGFR (26%) was observed in both early and advanced adenocarcinomas, and 88% of these showed immunoreactivity for p53. SSCP analysis revealed that in 81% of these tumors the p53 gene was mutated in exons 5-8. The intensity of p53 immunoreactivity was significantly higher (P < 0.013) in deeply invasive tumors. p16(INK4) and p21(WAF1/CIP1) immunoreactivity was detected in 93 and 76% of the samples co-expressing TGFalpha-EGFR but the levels were not correlated with those of p53 and other clinico-pathological parameters. We conclude that gastric adenocarcinomas potentially dependent upon the TGFalpha-EGFR autocrine loop for growing exhibit increased aggressiveness in the presence of aberrant p53.     

人乳头瘤病毒感染和p53基因突变与宫颈癌的关系

目的：探讨人乳头瘤病毒１６ 和１８ 型及抑癌基因ｐ５３ 突变对宫颈的致癌作用以及 Ｈ Ｐ Ｖ 感染与ｐ５３ 基因突变的相关性。方法：采用聚合酶链反应（ Ｐ Ｃ Ｒ） 技术和限制性酶切片段多态性分析（ Ｒ Ｆ Ｌ Ｐ） 技术对３４ 例原发性宫颈癌组织及３０ 例正常宫颈组织 Ｈ Ｐ Ｖ１６ ,１８ 型 Ｄ Ｎ Ａ 及抑癌基因ｐ５３ 的突变进行了检测。结果： Ｈ Ｐ Ｖ１６ ,１８ Ｄ Ｎ Ａ 在宫颈癌的总阳性率为６４７ ％ （２２／３４） ,正常宫颈组织只有６７ ％ 阳性,８例宫颈癌组织出现ｐ５３ 基因第６ 外显子突变,其中２ 例为 Ｈ Ｐ Ｖ１６ Ｄ Ｎ Ａ 阳性、１ 例 Ｈ Ｐ Ｖ１８ Ｄ Ｎ Ａ 阳性。结论：宫颈癌的发病与 Ｈ Ｐ Ｖ 感染及ｐ５３ 基因突变有关,宫颈癌组织中ｐ５３ 基因突变与 Ｈ Ｐ Ｖ 感染无关。     

Mutation analysis and loss of heterozygosity of PEDF in central nervous system primitive neuroectodermal tumors

Deletion of 17p is the most frequent abnormality observed in central nervous system (CNS) primitive neuroectodermal tumors (PNETs), implicating the presence of a tumor suppressor gene which maps to 17p. The gene for pigment epithelium-derived factor (PEDF) has been cloned and mapped to 17p13. PEDF belongs to the serine protease inhibitor (SERPIN) gene family. The PEDF protein has neurotrophic and neuronal-survival activities and is expressed in the CNS. Twenty tumor and matched normal DNA samples from patients with PNETs were screened by single-strand conformation polymorphism (SSCP) analysis to determine loss of heterozygosity (LOH) and to identify potential mutations within the 8 exons of the PEDF gene. Ten of the 20 tumors demonstrated LOH, consistent with the deletion status of 17p determined by cytogenetic or fluorescence in situ hybridization studies. SSCP analysis of the genomic DNA from the 10 cases with LOH demonstrated several polymorphisms in exons 4 and 7, but no mutations. Our results are consistent with a loss of alleles on 17p in 50% of CNS PNETs, but do not suggest that PEDF is a candidate for the PNET suppressor gene in 17p13. Int. J. Cancer 72:277–282, 1997. © 1997 Wiley-Liss, Inc.     

Prognostic significance of p53 gene alterations in node-negative breast cancer

Summary Mutations in the p53 gene can play a role in the transformation of normal to malignant cells. Because these mutations are more frequently reported later in the course of transformation, their presence could reflect a greater malignant potential of the tumor and, thus, an increased probability of metastasis and recurrence after local therapy. In a pilot study using single-stranded conformation polymorphism analysis (SSCP), 200 node-negative breast tumors were examined for mutations in the region encompassing exons 5 through 9 of the p53 gene. Exons 5 through 9 were tested because they contain 80–90% of known p53 gene mutations. The tumors ranged in size from 1 to 3 cm. 28 tumors were found to have an abnormal band pattern on both initial and repeat analysis. 4 of these tumors were sequenced; 3 contained a p53 mutation and the 4th had a rare neutral polymorphism. Disease-free survival (DFS) at 5 years for women with tumors having an abnormal SSCP analysis was 57% (± 10%), compared to a 79% (± 3%) DFS for the group with a normal pattern. By the log rank test, this difference was highly significant, p 0.01. The relative risk of recurrence for the group with an abnormal SSCP pattern was 2.2. In a multivariate analysis including ER, PgR, ploidy, S-phase, age, and tumor size, an abnormal p53 by SSCP analysis and patient age were the only factors that independently predicted DFS at 5 years. Conclusion: Women with node-negative breast cancer who have tumors with alterations in the p53 gene, as indicated by SSCP analysis, have a significantly poorer prognosis and a higher rate of relapse at 5 years. The prognostic significance is maintained in a multivariate analysis including many established prognostic factors.We regret to report that Dr. McGuire died March 25, 1992, while this work was in progress.     

p53 mutations in primary human lung tumors and their metastases

In a total of 26 primary human lung tumors and 60 metastases derived from them, exons 5–8 of the p53 tumor suppressor gene were analyzed by single-strand conformation polymorphism and subsequent direct DNA sequencing of amplified DNA. Mutational inactivation of the p53 gene was identified in four of five squamous cell carcinomas, three of nine adenocarcinomas, and two of nine small-cell carcinomas, the overall incidence being 35%. Point mutations occurred at a similar incidence in exons 5–8, with a preference for G←T transversions. In seven of nine cases (78%), mutations were identical in the primary tumor and all of its metastases, indicating that in lung tumors, p53 mutations usually precede metastasis and that hematogenic and lymphogenic dissemination of tumor cells to other tissues is not associated with a selection against p53 inactivation. In one case, a kidney metastasis had the same mutation as the primary squamous cell carcinoma, whereas a liver metastasis had no mutation, indicating heterogeneity of the primary lung neoplasm and selective metastasis of mutated and nonmutated tumor cells to kidney and liver, respectively. Only in one liver metastasis was a mutation identified that was neither present in the primary lung tumor nor in a kidney metastasis, suggesting that occasionally p53 mutations occur after metastatic spread. © 1994 Wiley-Liss, Inc.     

Mutations in the p53 tumor suppressor gene in rat esophageal papillomas induced by N-nitrosomethylbenzylamine

Mutations in thep53 tumor suppressor gene have been implicatedin the pathogenesis of a wide variety of human neoplasms. Thelocation and type of p53 gene mutation can reflect exposureof humans to certain types of carcinogenic agents. Much lessis known about the role of p53 mutational inactivation in rodenttumors. Using both ‘Hot’ (radioactive) and ‘Cold’(non-radioactive) single strand conformation polymorphism (SSCP)analyses, the present study analyzed exons 5–8 and theexon-intron junction of the p53 gene from rat esophageal papillomasinduced by N-nitrosomethylamine (NMBA) for mutations. Nine of30 (30%) esophageal papillomas contained SSCP mobility shifts,principally within exons 5 and 7. These positive SSCP findingswere further validated by direct DNA sequencing analysis. Eightof the nine mutations were G: C     

A subset of head and neck squamous cell carcinomas exhibits integration of HPV 16/18 DNA and overexpression of p16INK4A and p53 in the absence of mutations in p53 exons 5-8

Besides well-known risk factors such as tobacco use and alcohol consumption, oncogenic human papillomavirus (HPV) infection also has recently been suggested to promote head and neck tumorigenesis. HPV is known to cause cancer by inactivation of cell cycle regulators p53 and pRb via expression of viral oncoproteins E6 and E7. This indicates that p53 mutations are not a prerequisite in HPV-induced tumor development. However, discrepancy exists with respect to the frequency of head and neck squamous cell carcinomas (HNSCC) harboring DNA of oncogenic HPV and the fraction of these tumors showing p53 mutations. In our study, we examined the frequency of HNSCC demonstrating HPV 16/18 integration as identified by fluorescence in situ hybridization (FISH) and investigated their p53 (mutation) status by immunohistochemistry and single-strand conformation polymorphism (SSCP) analysis of exons 5-8. Paraffin-embedded, archival biopsy material from 27 premalignant mucosal lesions and 47 cases of HNSCC were analyzed. Ten of the 47 (21%) HNSCC unequivocally exhibited HPV 16 integration, including 8 of 12 (67%) tonsillar carcinomas. This is supported by the immunohistochemical detection of p16(INK4A) overexpression in all 10 HPV-positive tumors. Although FISH is considered to be less sensitive than PCR-based methods for HPV detection, our data clearly demonstrate clonal association of HPV with these tumors, as illustrated by the presence of integrated HPV 16 in both the primary tumor and their metastases in 2 patients. In contrast, HPV 16/18 DNA could not be detected in the premalignant lesions. In 30 of 47 (64%), HNSCC accumulation of p53 was observed, including 8 of the 10 HPV-positive carcinomas. However, in none of the latter cases could mutations in exons 5-8 be identified, except for a polymorphism in codon 213 of exon 6 in one patient. Evaluation of clinical data revealed a significant inverse relation between tobacco use with or without alcohol consumption, and HPV positivity of the tumors.     

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.