Infrequent p53 gene mutations in medulloblastomas

Cytogenetic and molecular studies of medulloblastomas have demonstrated frequent loss of sequences from the short arm of chromosome 17, possibly implicating loss or inactivation of the p53 tumor suppressor gene. We amplified exons 5 through 8 of the p53 gene by the polymerase chain reaction technique. These segments, which encompass the regions usually mutated in human tumors, were sequenced to search for p53 mutations in 12 medulloblastoma tumors, 8 xenografts, and 3 permanent cell lines. Mutation of the p53 gene was found in only 1 of 3 cell lines tested and in none of the xenografts or primary tumors studied. Our results suggest that p53 is mutated in an unusual way or that a second tumor suppressor gene on the short arm of chromosome 17 is involved in the pathogenesis of medulloblastoma.     

Mutational analysis of p73 and p53 in human cancer cell lines.

p73 is a candidate tumor suppressor gene with substantial DNA and protein homology to the p53 tumor suppressor gene. We have investigated two hypotheses: (a) p73 is mutated in diverse types of human cancer, and (b) p73 is functionally redundant with p53 in carcinogenesis so that mutations would be exclusive in these two genes. The entire coding region and intronic splice junctions of p73 were examined in 54 cancer cell lines. Three lung cancer cell lines contained mutations that affected the amino acid sequence. One amino acid substitution was in a region with homology to the specific DNA binding region of p53 and two microdeletions were outside the region of homology. Two of the cell lines with p73 mutations also carried p53 mutations. Although our results are inconsistent with the two hypotheses tested, p73 mutations may contribute infrequently to the molecular pathogenesis of human lung cancer.     

p53 mutations in non-small cell lung cancer in Japan: association between mutations and smoking.

The p53 gene has been implicated as a tumor suppressor gene involved in the pathogenesis of lung cancer. Our previous study revealed that the p53 gene is frequently mutated with a distinct nucleotide substitution pattern in small cell lung cancer specimens in Japanese patients. In this study, we examined 30 primary, resected non-small cell lung cancer samples in Japanese patients using complementary DNA-polymerase chain reaction and sequencing. Mutations changing the p53 coding sequence were found in 14 of 30 tumor samples (47%), while G:C to T:A transversions which are uncommon in other cancers such as colon cancer were the most frequently observed mutations, in agreement with an earlier report on non-small cell lung cancer in American patients. Furthermore, the present study shows for the first time that in univariate and multivariate analyses, the presence of p53 mutations is closely associated with lifetime cigarette consumption.     

p53 gene mutations in primary lung tumors are conserved in brain metastases

The p53 product is frequently mutated in human tumors. Both acquired and inherited mutations have been described. These mutations transform p53 from a growth suppressor gene to a transforming oncogene. We examined tissue from 6 patients with primary lung carcinoma and the corresponding brain metastases for the presence of p53 mutations by immunohistochemistry. We then confirmed and characterized the mutations by single strand conformation analysis and by direct sequence analysis. All 6 patients had primary and metastatic tumor expressing a mutant p53. The mutations were all G-T transversions and mapped to exons 5, 6, 7, and 8. The mutations in the primary tumors were precisely conserved in the brain metastases.     

p53 and RAS gene mutations in multiple myeloma.

We analysed genomic DNA from 30 patients with multiple myeloma (MM), searching for alterations in the p53 and RAS genes by a combination of polymerase chain reaction and single-strand conformation polymorphism techniques. Mutations in the p53 gene were observed in 20% (6 out of 30) of the patients, and were located in conserved sequence blocks within exons 5 and 7. These were single-nucleotide substitutions and consisted predominantly (4/6) of G:C to A:T transitions. Of the six patients with a mutated p53 gene, four were in the terminal phase of the disease. RAS gene mutations were found more frequently since they occurred in 47% (14 out of 30) of the patients. Mutations consisted of single-nucleotide substitutions, located in codons 12, 13 and 61 of either K- or N-RAS, to the exclusion of H-RAS. Moreover, one patient bore two simultaneous mutations, affecting simultaneously the K- and the N-RAS genes. RAS gene mutations were more frequently observed in patients with fulminating disease (10/15, 67%) than in patients with less aggressive forms of the disease (4/15, 26%). We also analysed genomic DNAs from 10 human myeloma cell lines, of which two bore mutations affecting codon 12 of the K-RAS gene, and one codon 12 of the N-RAS gene. The first two cell lines were obtained from freshly explanted tumor cells in which we observed identical mutations. Results presented here show that activating mutations in the RAS genes are, in MM, more frequent than those affecting the p53 gene and suggest that both events are related to terminal phases of the disease.     

p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features.

We screened 77 non-small-cell lung cancer (NSCLC) cell lines for mutations of the p53 gene using a single-strand conformation polymorphism (SSCP) assay. We found that 57 cell lines (74%) had mutations of the p53 gene. Three cell lines had a deletion of the p53 gene. Of the remaining 54 cell lines, 49 cell lines were sequenced and 52 mutations were confirmed. In contrast to previously published p53 mutations in other human tumors, the p53 gene mutations in NSCLC were diverse with regard to the location and nature of the mutations. The region corresponding to codons 144-166, which is outside the evolutionarily conserved regions, was a frequent site of p53 gene mutations in NSCLC. The presence of a p53 gene mutation was not associated with age, sex, histological types, culture site, treatment intent, presence of prior cytotoxic treatment, neuroendocrine differentiation, median culture time or patient survival. The prevalence of p53 mutations in cell lines with ras mutations did not differ from that in cell lines without ras mutations. However, p53 gene mutations in NSCLC cell lines with ras mutations tended to cluster in exon 8, suggesting the presence of a functional domain of the p53 gene relating to interaction with the ras gene. We conclude that p53 and ras mutations are frequent and apparently independent genetic alterations which play different roles in the pathogenesis, progression and prognosis of NSCLC.     

Infrequency of p53 gene mutations in ependymomas

Summary Ependymomas, which comprise 5% of central nervous system tumors, have not been extensively characterized genetically. The p53 tumor suppressor gene is frequently mutated in human cancer, and is important in the pathogenesis of other central nervous system (CNS) tumors. Chromosomal DNA corresponding to the p53 tumor suppressor gene was amplified by the polymerase chain reaction (PCR) from 31 archival ependymoma specimens. DNA was screened for the presence of p53 mutations by single strand conformational polymorphism (SSCP) analysis; samples with altered mobility were further tested for the presence of mutation by direct DNA sequence analysis. Of the 31 ependymomas tested, one contained a detectable DNA sequence change in the p53 gene. Sequencing revealed a silent mutation in exon 6, at codon 213, which represents a known p53 sequence polymorphism. These findings suggest that in contrast to many other human cancers, p53 mutation is not important in the pathogenesis or progression of ependymomas.     

Wild-type but not mutant p53 suppresses the growth of human lung cancer cells bearing multiple genetic lesions.

Accumulating evidence indicates that lung cancer arises due to multiple genetic changes in both dominant oncogenes, such as ras, and tumor suppressor genes, such as p53. In this report we examined whether the wild-type p53 gene is able to suppress in vitro and/or in vivo cellular growth of lung cancer cell lines which carry multiple genetic abnormalities. Introduction of a wild-type p53 complementary DNA expression vector into lung cancer cell lines carrying either a homozygous deletion (NCI-H358) or a missense mutation (NCI-H23) in the p53 gene greatly suppressed tumor cell growth. In contrast, p53 expression vectors bearing lung cancer derived mutations affecting single amino acids had lost this growth suppressing ability.     

TP53 mutations and molecular epidemiology

Tumor suppressor p53 protein is activated by a variety of cellular stresses through several pathways and transactivates its downstream genes, including regulators of cell cycle, apoptosis and DNA repair. The loss of p53 function by TP53 gene mutations therefore fails to activate these genes and is thought to be a critical cause of carcinogenesis and/or tumor progression. TP53 is one of the most frequently mutated genes in human cancer. TP53 mutations are found in about 50% of human cancers, although the frequency of TP53 mutations differs among tumor types. However, the degree of functional disorder of mutant p53 varies according to the type of TP53 mutation. And the effects of p53 on cancer formation and/or progression are influenced by the degree of p53 dysfunction. So it is important to analyze the effects of TP53 mutations carefully according to the oncogenicity of each mutation from the molecular epidemiological point of view. Here, together with some cautions needed for analyzing and interpreting the significance of TP53 gene mutations, we present some examples of the identified specific mutation spectrum and the correlation between the prognosis and TP53 mutation in some cancers.     

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.     

WAF1 expression is independent of p53 gene alterations and protein expression in human lung cancer cells

Accumulation of wild-type p53 protein arrests cells primarily at G(1)-S. This arrest is characterized by accumulation of the cyclin-dependent kinase inhibitor p21 (WAF1/CIP1) protein. Since the WAF1 gene is itself a target of p53, we investigated p53 gene expression to understand the mechanism 3 of p21 regulation in cellular transformation in human non-small cell lung cancer lines and tumor tissues. Northern and Western blot analyses in cell lines carrying wild-type or mutated p53 showed that WAFI mRNA and protein expression varied among different cell lines. WAFI expression was not directly correlated with the p53 status of the cells. WAFI was also expressed at high levels in the absence of p53 expression in a p53-deleted cell line (H358). Abrogation of p53 protein in H226b (wild-type p53) and H322 (mutated p53) cell lines by antisense RNA reduced WAFI expression in both lines, indicating that mutations in the p53 gene may not necessarily abrogate p53-mediated regulation of WAFI expression. Fourteen primary lung tumors were analyzed for p53 status by SSCP analysis and DNA sequencing. Eight of 14 lung tumor tissues examined contained p53 mutations. Six of 14 contained wildtype p53. Enhanced p21 expression was detected in two lung tumors containing p53 mutations by immunohistochemistry. Together, these data indicate that WAFI expression is independent of p53 gene alterations and protein expression in non-small cell lung tumor cells.     

A Comparative Study of Glioma Cell Lines for p16, p15, p53 and p21 Gene Alterations

A total of 10 glioma cell lines were examined for alterations of the p16, p15, p53 and p21 genes, which are tumor suppressor genes or candidates with direct or indirect CDK-inhibitory functions. Genetic alterations (deletions or mutations) were frequently seen in the p16, p15 and p53 genes in these cell lines, but not in the p21 gene. When the states of the p16, p15 and p53 genes were compared among cell lines, all the cell lines showed abnormalities in at least 1 gene, often in 2 or 3 genes coincidentally, suggesting that dysfunction of these genes is closely related to glioma cell growth. Although alteration of all 3 genes was most frequent, there were cell lines having either p16/p15 or p53 or p16 and p53 gene alterations, suggesting that the time order of these genetic alterations was variable depending on the cell line. Among cell lines examined, one with homozygous p53 gene deletion seemed of particular practical value, since such a cell line might be useful in various studies, including investigation of the functions of various mutant p53 genes in the absence of heteromeric protein formation. On examination of the primary tumor tissues, the same alterations of the p16/p15 and p53 genes as detected in the cell lines were demonstrated in all 6 cases examined: p16/p15 gene deletion in 1, p16 gene mutation in 1 and p53 gene mutations in 5 cases. This suggested that the p16/p15 and the p53 gene alterations and their combinations in at least some glioma cell lines reflected those in the primary glioma tissues.     

p53 mutations in human lung tumors.

Mutation of one p53 allele and loss of the normal p53 allele [loss of heterozygosity (LOH)] occur in many tumors including lung cancers. These alterations apparently contribute to development of cancer by interfering with the tumor suppressor activity of p53. We directly sequenced amplified DNA in the mutational hot spots (exons 4-8) of p53 in DNA samples from 40 lung cancers. Most (31 of 40) samples were preselected for LOH in the region of p53. We detected 23 p53 mutations within these exons in 22 lung cancers; no p53 mutations were found in normal tissue of the patients. One-half of the mutations were G to T transversions on the nontranscribed strand, consistent with mutagenesis by tobacco smoke. Mutations of C to A on the nontranscribed strand, which would result from G to T mutations on the transcribed strand, were detected only in one sample. Three of 23 mutations were nonsense mutations; to date, nonsense mutations of p53 have not been reported in lung cancer. Mutation of this p53-coding region was detected in 20 of 27 small cell lung cancer samples, representing a 70% occurrence. Mutation of the p53 gene is apparently very frequent in small cell lung cancers. When LOH in the p53 region could be determined, complete concordance occurred between a sample having both a p53 mutation and LOH in the region of p53 (18 of 18 samples). Twelve samples of lung cancer had LOH in the region of p53, but the samples had no detectable p53 mutations, suggesting either alterations outside the known mutational hot spots of p53 or alterations of another unidentified tumor suppressor gene in the region of p53.     

Frequent p53 mutations in head and neck cancer.

Squamous cell carcinomas of the head and neck (SCCHN) are associated strongly with the use of tobacco and alcohol, but little is known about the molecular pathogenesis of these tumors. In the present study, we analyzed SCCHN for mutations in the tumor suppressor gene p53 by immunocytochemistry and complementary DNA sequencing. Overexpression of p53 protein was detected in 13 (100%) of 13 SCCHN cell lines and in tumor cells cultured directly from 10 (77%) of 13 patients with SCCHN. Direct evidence for p53 mutations was obtained by sequencing p53 complementary DNA from eight SCCHN cell lines and two tumor xenografts. The genetic alterations included seven missense mutations resulting in single amino acid substitutions, a mutation encoding a stop codon, one 10-base pair deletion, and one 2-base pair addition. All seven missense mutations were G to T transversions, five of which were clustered at codons 245 and 248. A similar high frequency of G to T transversions predominates in lung cancer, another tobacco-related disease. Mutation of the p53 gene is the most common genetic alteration detected in SCCHN and implicates this gene locus as a critical site of specific damage by mutagenic carcinogens in tobacco, one of the important risk factors in the etiology of this disease.     

Absence of ras Mutations and Low Incidence of p53 Mutations in Renal Cell Carcinomas Induced by Ferric Nitrilotriacetate

Renal cell carcinomas induced in male Wistar rats by iron chelate of nitrilotriacetate (Fe-NTA) were examined for mutations in ras oncogenes and p53 tumor suppressor gene. Fourteen primary tumors and two metastatic tumors from 11 animals were evaluated. Exons 1 and 2 of the H-, K-, and N- ras genes were amplified by polymerase chain reaction (PCR), and the presence of mutations was examined by direct sequencing. Exon 5 through exon 7 of p53 gene, including the 3'half of the conserved region II and the entire conserved region III through V, were surveyed for point mutations by PCR-single stranded conformation polymorphism (SSCP) analysis. Direct sequencing of the ras genes showed no mutations in codon 12, 13, or 61 among the tumors evaluated. SSCP analysis of p53 gene exon 6 indicated conformational changes in two primary tumors. One tumor had a CCG-to-CTG transition at codon 199, and the other had an ATC-to-ATT transition at codon 229 and two nonsense C-to-T transitions. These results suggest that neither ras genes nor p53 gene play a major role in the development of renal cell carcinomas induced by Fe-NTA.     

Ras and p53 expression in non-small-cell lung-cancer patients - p53 over-expression correlates with a poor prognosis

Expression of the tumor suppressor gene p53 and the ras oncogene were examined in 46 tumor and nodal specimens of non-small cell lung cancer (NSCLC) using the antibodies p53 pAb 240 and ras Y13-259 respectively. p53 expression was elevated in 46% and ras p21 was over-expressed in 85% of the tumor specimens analyzed. Fifteen cases of benign lessions were also assessed for both ras p21 and p53 expression; all were found to have negative staining. p53 over-expression was found to correlate with a poor prognosis in both the tumor specimens (p<0.05) and in the nodal tissues (p<0.005). Ras p21 over-expression was found to be associated with survival (p<0.1) in both the tumor and the nodal specimens. Stage of the disease correlated with survival; similarly both p53 and ras p21 over-expression correlated with stage. No correlations were found with the pathological grade of the tumors nor with a history of smoking or duration of smoking. No K-ras mutations at codon 12 were observed in a further 15 NSCLC specimens analyzed. These results indicate that the p53 gene in particular plays a role in the stages of NSCLC.     

Analysis of p53 status in human cell lines using a functional assay in yeast: detection of new non-sense p53 mutation in codon 124

p53 tumor suppressor is a sequence-specific DNA-binding protein that controls the expression of many genes in response to diverse stress stimuli. p53 gene is often mutated in human cancer and in cancer cell lines. Several methods are available for identification of p53 mutations, including functional analysis of separated alleles in yeast (FASAY). FASAY distinguishes yeast colonies expressing functional p53 protein from colonies producing a dysfunctional p53 protein simply on the basis of color. We analyzed the p53 status of 26 human cell lines of different tissue origin using the functional assay in yeast. Wild-type p53 was found in six cell lines and various p53 aberrations in the remaining twenty. FASAY detected temperature-sensitive p53 mutations in breast cancer cell line, BT474, and leiomyosarcoma cell line, SK-LMS-1, and two independent p53 point mutations in SK-UT-1 and SK-LMS-1 leiomyosarcoma cell lines. In addition, the assay revealed that a recombination occurred between the two mutated p53 alleles producing a stable ratio of p53 wild-type alleles (11.7 or 2.7% respectively). In the case of acute myeloid leukemia cell line, ML-1, we detected both wild-type and heterozygous p53 status, depending on the source of the cell line. In Hs913T, HL60 and Saos-2 cell lines, FASAY failed to assess p53 status due to a large deletion/rearrangement of the p53 gene. In acute lymphoid leukemia HPB cell line, we disclosed unknown non-sense mutation in codon 124 of the p53 gene.