Reduction to homozygosity involving p53 in esophageal cancers demonstrated by the polymerase chain reaction.

Loss of heterozygosity affecting chromosome 17p has been detected at high frequencies in a variety of human tumors, including cancers of the colon, breast, lung, and brain. One presumed target of these losses is p53, a tumor suppressor gene located on 17p. To our knowledge, loss of heterozygosity has not yet been reported at any locus, including p53, in human esophageal cancer. Moreover, current methods of detecting loss of heterozygosity depend on the availability of large amounts of high molecular weight DNA, making the study of small biopsy specimens or paraffin-embedded tissues problematic. We examined 52 primary human esophageal neoplasms for loss of heterozygosity affecting the p53 gene by using the polymerase chain reaction. Loss of one allele was detected in 52% of informative cases and was more common in squamous carcinomas than in adenocarcinomas. Southern blot analysis was used to confirm polymerase chain reaction-derived data. The identification of allelic loss in approximately half of the tumors analyzed supports the hypothesis that inactivation of p53 is involved in the pathogenesis of esophageal cancer.     

Benzo[a]pyrene-induced murine skin tumors exhibit frequent and characteristic G to T mutations in the p53 gene.

Human tobacco-related cancers exhibit a high frequency of G to T transversions in the mutation hot spot region of the p53 tumor suppressor gene, possibly the result of specific mutagens in tobacco smoke, most notably benzo[a]pyrene (B[a]P). No in vivo animal model of B[a]P-induced tumorigenesis has been used, however, to substantiate these molecular epidemiological data experimentally. Direct DNA sequence analysis of the hot spot region (exons 5-8 inclusive) of murine p53 was performed in 20 skin tumors induced by a complete carcinogenesis protocol with B[a]P. Sequence analyses revealed numerous heterozygous missense mutations in carcinomas, specifically in exons 7 and 8 of the p53 gene, and targeting exclusively guanine residues. Moreover, 70% (5/7) of the mutations characterized were G to T transversions. In contrast, direct DNA sequence analysis of 36 skin tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) in either a complete carcinogenesis protocol or in a two-stage carcinogenesis protocol revealed a 30% frequency of heterozygous p53 mutations, with the majority of mutations found in carcinomas, but only a single G to T transversion (1/8). Thus, while mutation frequencies are similar, the pattern and type of p53 mutations in B[a]P-induced skin tumors differs significantly from the mutation spectra in DMBA-induced squamous neoplasias. These in vivo findings in B[a]P-induced tumors lend support to in vitro and molecular epidemiological evidence, suggesting that the p53 tumor suppressor gene may be a selective target of metabolically activated B[a]P species etiologically associated with human tobacco-related cancers.     

The clinicopathological significance of p21 and p53 expression in esophageal squamous cell carcinoma: an analysis of 153 patients

OBJECTIVE: The p21 gene is thought to play a central role in tumor suppression. The aim of this study was to examine the clinicopathological role of p21 and p53 in esophageal squamous cell carcinomas. METHODS: The expression of p21 and p53 proteins in 153 Chinese patients (131 men, 22 women) with resected esophageal squamous cell carcinomas was investigated by the immunohistochemical method. Correlation between p21 and p53 expression and clinicopathological features was examined. RESULTS: The expression of p21 and p53 was detected in 70% and 64% of the tumors, respectively. The staining of p21 and p53 was also found in squamous carcinoma in situ, dysplasia, and nontumor epithelium. p21 expression was often weak in the suprabasal cells and found in better differentiated tumors. There was no significant correlation between the expression of p21 and the abnormal accumulation of p53. The prognosis of the patients depended on the size, stage, and p21 expression of the lesion. In stage III lesions with tumor diameter < or = 7.5 cm (n = 93), patients with loss of p21 expression had better survival. The survival rates of patients were worse if they had expression of both p21 and p53. CONCLUSIONS: Thus, p21 and p53 had prognostic value for esophageal squamous cell carcinomas. Loss of p21 expression was shown without p53 alternations, indicating that other mechanisms are also involved in turning off the gene. The pattern of p21 and p53 expression predicts an aggressive clinical course of esophageal squamous cell carcinomas.     

A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.

Sunlight is a carcinogen to which everyone is exposed. Its UV component is the major epidemiologic risk factor for squamous cell carcinoma of the skin. Of the multiple steps in tumor progression, those that are sunlight-related would be revealed if they contained mutations specific to UV. In a series of New England and Swedish patients, we find that 14/24 (58%) of invasive squamous cell carcinomas of the skin contain mutations in the p53 tumor suppressor gene, each altering the amino acid sequence. Involvement of UV light in these p53 mutations is indicated by the presence in three of the tumors of a CC----TT double-base change, which is only known to be induced by UV. UV is also implicated by a UV-like occurrence of mutations exclusively at dipyrimidine sites, including a high frequency of C----T substitutions. p53 mutations in internal malignancies do not show these UV-specific mutations. The dipyrimidine specificity also implicates dipyrimidine photoproducts containing cytosine as oncogenic photoproducts. We believe these results identify a carcinogen-related step in a gene involved in the subsequent human cancer.     

Mutations of p53 gene exons 4-8 in human esophageal cancer

AIM: To characterize the tumor suppressor gene p53 mutations in exon 4, esophageal cancer and adjacent non-cancerous tissues. METHODS: We performed p53 (exons 4-8) gene mutation analysis on 24 surgically resected human esophageal cancer specimens by PCR, single-strand conformation polymorphism, and DNA sequencing. RESULTS: p53 gene mutations were detected in 9 of 22 (40.9%) esophageal cancer specimens and 10 of 17 (58.8%) adjacent non-cancerous tissues. Eight of sixteen (50.0%) point mutations detected were G-A transitions and 9 of 18 (50.0%) p53 gene mutations occurred in exon 4 in esophageal cancer specimens. Only 1 of 11 mutations detected was G-A transition and 4 of 11 (36.4%) p53 gene mutations occurred in exon 4 in adjacent non-cancerous tissues. CONCLUSION: Mutation of p53 gene in exon 4 may play an important role in development of esophageal cancer. The observation of p53 gene mutation in adjacent non-cancerous tissues suggests that p53 gene mutation may be an early event in esophageal carcinogenesis. Some clinical factors, including age, sex, pre-operation therapy and location of tumors, do not influence p53 gene mutation rates.     

Nonhereditary p53 mutations in T-cell acute lymphoblastic leukemia are associated with the relapse phase

We have previously reported that greater than 60% of human leukemic T- cell lines possess mutations in the p53 tumor suppressor gene. To determine whether T-cell acute lymphoblastic leukemia (T-ALL) patient samples possess p53 mutations, we screened peripheral blood-and bone marrow-derived leukemia samples, taken at diagnosis and at relapse, for p53 mutations. Exons 4 through 9 and selected intron regions of the p53 gene were analyzed using polymerase chain reaction-single-strand conformation polymorphism and direct sequencing. p53 mutations were found in 0 of 15 T-ALL diagnosis samples, as compared with 10 of 36 (28%) T-ALL relapse samples. To determine whether p53 mutations play a role in the recurrence (relapse) of T-ALL, two special groups of T-ALL patients were studied: (1) a group of 8 relapse patients whose disease was refractory to chemotherapeutic treatment, and (2) a group of 6 "paired" T-ALL cell samples from patients for whom we possess both diagnosis and relapse samples. Three of 8 relapsed patients (37.5%) whose disease was refractory to the reinduction of remission by chemotherapy possessed missense mutations of the p53 gene. All 3 cases had mutations in exon 5. Among the paired samples, 3 of 6 patients harbored p53 mutations at disease recurrence, but possessed only wild- type p53 alleles at diagnosis. One case had mutation on exon 4, 1 case in exon 5, and 1 case in exon 8 with loss of heterozygosity. These data clearly indicate that recurrence of T-ALL is associated with missense mutations in p53. Our results indicate that (1) mutations of p53 do occur in T-ALL in vivo, and such mutations are associated with the relapse phase of the disease; and (2) p53 mutation is involved in the progression of T-ALL. This conclusion is supported by our observation that the introduction of T-ALL-derived mutant p53 expression constructs into T-ALL cell lines further increases their growth rate in culture, enhances cell cloning in methylcellulose, and increases tumor formation in nude mice.     

Somatic mutation analysis of p53 and ST7 tumor suppressor genes in gastric carcinoma by DHPLC

AIM: To verify the effectiveness of denaturing high-performance liquid chromatography (DHPLC) in detecting somatic mutation of p53 gene in gastric carcinoma tissues. The superiority of this method has been proved in the detection of germline mutations, but it was not very affirmative with respect to somatic mutations in tumor specimens. ST7 gene, a candidate tumor suppressor gene identified recently at human chromosome 7q31.1, was also detected because LOH at this site has also been widely reported in stomach cancer. METHODS: DNA was extracted from 39 cases of surgical gastric carcinoma specimen and their correspondent normal mucosa. Seven fragments spanning the 11 exons were used to detect the mutation of p53 gene and the four exons reported to have mutations in ST7 gene were amplified by PCR and directly analyzed by DHPLC without mixing with wild-type allele. RESULTS: In the analysis of p53 gene mutation, 9 aberrant DHPLC chromatographies were found in tumor tissues, while their normal-adjacent counterparts running in parallel showed a normal shape. Subsequent sequencing revealed nine sequence variations, 1 polymorphism and 8 mutations including 3 mutations not reported before. The mutation rate of p53 gene (21%) was consistent with that previously reported. Furthermore, no additional aberrant chromatography was found when wild-type DNA was added into the DNA of other 30 tumor samples that showed normal shapes previously. The positivity of p53 mutations was significantly higher in intestinal-type carcinomas (40%) than that in diffuse-type (8.33%) carcinomas of the stomach. No mutation of ST7 gene was found. CONCLUSION: DHPLC is a very convenient method for the detection of somatic mutations in gastric carcinoma. The amount of wild type alleles supplied by the non-tumorous cells in gastric tumor specimens is enough to form heteroduplex with mutant alleles for DHPLC detection. ST7 gene may not be the target gene of inactivation at 7q31 site in gastric carcinoma.     

FASAY: a simple functional assay in yeast for identification of p53 mutation in tumors.

Alteration of the p53 tumor suppressor gene is the most common genetic defect known to occur in human tumors. Germ-line p53 mutations significantly increase the risk of developing diverse malignancies. FASAY is a simple functional assay for germ-line and somatic mutations in the p53 gene altering the transactivation capability of the p53 protein. The method was successfully used for mutation analysis of p53 in various cell lines, somatic tumor cells and blood cells. In addition, FASAY was also found effective as a tool for basic research of binding of mutant p53 proteins to promoters of different p53 target genes.     

Alterations of the p53 tumor suppressor gene in diffuse large cell lymphomas with translocations of the c-MYC and BCL-2 proto-oncogenes

Diffuse large cell lymphomas are aggressive tumors of B-cell origin. In some cases they arise from low-grade follicular lymphomas carrying the t(14;18) translocation, an event that leads to the overexpression of the BCL-2 gene product. More frequently, however, they lack the t(14;18) translocation. Rearrangements of the c-MYC proto-oncogene and mutations of the p53 tumor suppressor gene have also been documented in these lymphomas. This study examines the extent to which alterations in the BCL-2, c-MYC, and p53 genes co-exist within individual lymphomas. Eight diffuse large cell lymphoma cell lines and 11 diffuse large cell lymphoma tumors were assessed for genetic alterations in these three genes. Our results indicate that there is a heterogeneity in the oncogene/suppressor gene profile among diffuse large cell lymphomas. Two cell lines and one tumor carried alterations in all three genes, one cell line carried alterations of c-MYC and p53, and one primary tumor and one cell line carried p53 mutations and the t(14;18). Single alterations of BCL-2 and p53 were also observed. Another cell line had no alterations in any of these genes. The heterogeneity indicates that varied mechanisms may be involved in the generation of diffuse large cell lymphomas.     

Rat p53 gene mutations in primary Zymbal gland tumors induced by 2-amino-3-methylimidazo[4,5-f]quinoline, a food mutagen.

There are reports of p53 gene mutations in various human cancers but not in rat tumor cell lines or rat primary tumor tissue. We found a p53 gene mutation in a cell line of a spontaneous squamous cell carcinoma of the rat Zymbal gland, SCC131, at codon 171 by direct sequencing of cDNA fragments amplified by PCR. We tested for p53 gene mutations in 15 primary Zymbal gland tumors induced by 2-amino-3-methylimidazo[4,5-f]quinoline by single-strand conformation polymorphism analysis of the PCR-amplified cDNA products. Samples of four tumors showed mobility shifts. Direct sequencing revealed that all these tumors had mutations in conserved regions or in scattered conserved residues. Single-strand conformation polymorphism analysis of cDNA suggested that mRNA from the wild-type allele of the p53 gene was not present in tumor cells of three of four positive cases, although genomic DNA analysis indicated that the wild-type allele was retained in all the cases. All mutations were found at a guanine base: three mutations were guanine----pyrimidine transversions and one was a deletion of a guanine base within a G+C-rich sequence. These findings indicate that 2-amino-3-methylimidazo[4,5-f]quinoline may be directly involved in induction of these mutations by forming DNA adducts at various sites in the p53 gene.     

Clinical implication of hot spot BRAF mutation,V599E,in papillary thyroid cancers

Activating mutations in the BRAF kinase gene have recently been reported in human cancers. The aim of the present study was to determine the frequency of BRAF mutations in thyroid cancer and their correlation with clinicopathological parameters. We analyzed exons 11 and 15 of BRAF gene in six human thyroid cancer cell lines and 207 paraffin-embedded thyroid tumor tissues. A missense mutation was found at T1796A (V599E) in exon 15 in four of the six cell lines and 51 of 207 thyroid tumors (24.6%; 0 of 20 follicular adenoma, 0 of 11 follicular carcinoma, 49 of 170 papillary carcinomas, and 2 of 6 undifferentiated carcinomas). Activation of MAPK kinase-MAPK pathway was observed in cell lines harboring BRAF mutation. BRAF mutation-associated enhanced cell growth was suppressed by MAPK kinase inhibitor, U0126. Examination of 126 patients with papillary thyroid cancer showed that BRAF mutation correlated significantly with distant metastasis (P = 0.033) and clinical stage (P = 0.049). Our results indicate that activating mutation of BRAF gene could be a potentially useful marker of prognosis of patients with advanced thyroid cancers.     

Mutations in p53 as potential molecular markers for human breast cancer.

Based on the high incidence of loss of heterozygosity for loci on chromosome 17p in the vicinity of the p53 locus in human breast tumors, we investigated the frequency and effects of mutations in the p53 tumor suppressor gene in mammary neoplasia. We examined the p53 gene in 20 breast cancer cell lines and 59 primary breast tumors. Northern blot analysis, immunoprecipitation, and nucleotide sequencing analysis revealed aberrant mRNA expression, over-expression of protein, and point mutations in the p53 gene in 50% of the cell lines tested. A multiplex PCR assay was developed to search for deletions in the p53 genomic locus. Multiplex PCR of genomic DNA showed that up to 36% of primary tumors contained aberrations in the p53 locus. Mutations in exons 5-9 of the p53 gene were found in 10 out of 59 (17%) of the primary tumors studies by single-stranded conformation polymorphism analysis. We conclude that, compared to amplification of HER2/NEU, MYC, or INT2 oncogene loci, p53 gene mutations and deletions are the most frequently observed genetic change in breast cancer related to a single gene. Correlated to disease status, p53 gene mutations could prove to be a valuable marker for diagnosis and/or prognosis of breast neoplasia.     

Mutation hotspots due to sunlight in the p53 gene of nonmelanoma skin cancers.

To identify the sites in the p53 tumor suppressor gene most susceptible to carcinogenic mutation by sunlight, the entire coding region of 27 basal cell carcinomas (BCCs) of the skin was sequenced. Fifty-six percent of tumors contained mutations, and these were UV-like: primarily CC-->TT or C-->T changes at dipyrimidine sites. Such mutations can alter more than half of the 393 amino acids in p53, but two-thirds occurred at nine sites at which mutations were seen more than once in BCC or in 27 previously studied squamous cell carcinomas of the skin. Seven of these mutation hotspots were specific to skin cancers. Internal-cancer hotspots not located at dipyrimidine sites were not mutated in skin cancers; moreover, UV photoproducts were absent at these nucleotides. The existence of hotspots altered the process of inactivating p53 in BCC compared to other cancers: allelic loss was rare, but 45% of the point mutations were accompanied by a second point mutation on the other allele. At least one of each pair was located at a hotspot. Sunlight, acting at mutation hotspots, appears to cause mutations so frequently that it is often responsible for two genetic events in BCC development.     

tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors

TP53 is the most frequently mutated tumor suppressor gene in human cancer, with nearly 50% of all tumors exhibiting a loss-of-function mutation. To further elucidate the genetic pathways involving TP53 and cancer, we have exploited the zebrafish, a powerful vertebrate model system that is amenable to whole-genome forward-genetic analysis and synthetic-lethal screens. Zebrafish lines harboring missense mutations in the tp53 DNA-binding domain were identified by using a target-selected mutagenesis strategy. Homozygous mutant fish from two of these lines were viable and exhibited mutations similar to those found in human cancers (tp53(N168K) and tp53(M214K)). Although homozygous tp53(N168K) mutants were temperature-sensitive and suppressed radiation-induced apoptosis only at 37 degrees C, cells in the tp53(M214K) embryos failed to undergo apoptosis in response to gamma radiation at both 28 and 37 degrees C. Unlike wild-type control embryos, irradiated tp53(M214K) embryos also failed to up-regulate p21 and did not arrest at the G(1)/S checkpoint. Beginning at 8.5 months of age, 28% of tp53(M214K) mutant fish developed malignant peripheral nerve sheath tumors. In addition to providing a model for studying the molecular pathogenic pathways of malignant peripheral nerve sheath tumors, these mutant zebrafish lines provide a unique platform for modifier screens to identify genetic mutations or small molecules that affect tp53-related pathways, including apoptosis, cell-cycle delay, and tumor suppression.     

p53 gene mutations and protein accumulation in human ovarian cancer.

Mutations of the p53 gene on chromosome 17p are a common genetic change in the malignant progression of many cancers. We have analyzed 38 malignant tumors of ovarian or peritoneal müllerian type for evidence of p53 variations at either the DNA or protein levels. Genetic studies were based on single-strand conformation polymorphism analysis and DNA sequencing of exons 2 through 11 of the p53 gene; mutations were detected in 79% of the tumors. These data show a statistically significant association between mutations at C.G pairs and a history of estrogen therapy. Two of 20 patients whose normal tissue could be studied carried germ-line mutations of p53. Immunohistochemical analysis of the p53 protein was carried out using monoclonal antibody PAb1801. Ninety-six percent of the missense mutations were associated with abnormal accumulation of p53 protein, but nonsense mutations, a splicing mutation, and most deletions did not result in p53 protein accumulation. A statistically significant association between p53 protein accumulation in poorly differentiated stage III serous carcinomas and small primary tumor size at diagnosis was found, perhaps suggesting that p53 protein accumulation accelerates the metastatic spread from a primary tumor. Overall, our findings indicate that alterations of p53 play a major role in ovarian cancer, including predisposition to the disease in some patients, and suggest a possible mechanism for somatic mutations leading to this cancer.     

Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations.

At present, mutation of the p53 gene appears to be the most common genetic alteration found in human cancers. These mutations can occur within many different regions of the gene. We have developed a modification of denaturing gradient gel electrophoresis termed "constant denaturant gel electrophoresis" (CDGE), which provides a rapid and sensitive method to screen the four conserved regions within the p53 gene where the majority of p53 mutations have been reported. The sensitivity of CDGE was first tested with known p53 mutations in all four conserved regions. The CDGE technique was then used to screen 32 breast carcinomas that had been analyzed by immunohistochemical methods for altered p53 protein levels and whose DNA had already been shown to have loss of heterozygosity for a chromosome 17p marker. By immunostaining techniques, only 6 of the 32 tumors had elevated p53 expression. However, CDGE detected p53 mutations in 11 of the 32 tumors. DNA sequence analysis was performed to determine the nucleotide positions of these mutations in all 11 samples. Loss of heterozygosity for the pYNZ22 or p144D6 markers did not associate with either the loss of heterozygosity at the p53 locus or the mutations detected by CDGE. We conclude that CDGE is a rapid and effective technique to screen for p53 mutations.     

Kinase requirements in human cells: V. Synthetic lethal interactions between p53 and the protein kinases SGK2 and PAK3

Cervical carcinomas are initiated through a series of well-defined stages that rely on the expression of human papillomavirus (HPV) oncogenes. A panel of 100 small hairpin RNAs that target essential kinases in many tumor types was used to study the stepwise appearance of kinase requirements during cervical tumor development. Twenty-six kinases were commonly required in three cell lines derived from frank carcinomas, and each kinase requirement was traced to the specific stage in which the requirement emerged. Six kinases became required following HPV-induced immortalization, and the requirement for two kinases, SGK2 and PAK3, was mapped to the inactivation of p53 in primary human epithelial cells. Loss of the p53 tumor suppressor in other primary epithelial cells also induced dependence on SGK2 and PAK3. Hence, SGK2 and PAK3 provide important cellular functions following p53 inactivation, fulfilling the classical definition of synthetic lethality; loss of p53, SGK2, or PAK3 alone has little effect on cell viability, whereas loss of p53 together with either SGK2 or PAK3 loss leads to cell death. Whereas tumor suppressor gene mutations are not directly druggable, other proteins or pathways that become obligatory to cell viability following tumor suppressor loss provide theoretical targets for tumor suppressor-specific drug discovery efforts. The kinases SGK2 and PAK3 may thus represent such targets for p53-specific drug development.