Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome). We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53. We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele. Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.     

Loss of p53 protein during radiation transformation of primary human mammary epithelial cells.

The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated gamma-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletion within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G1 arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells.     

Human breast cancer: frequent p53 allele loss and protein overexpression

A sample of 114 primary breast tumors and corresponding constitutional DNA were tested for loss of heterozygosity (LOH) of the YNZ22 and p53 genes, both located in the 17p13 region. Loss of the p53 allele was found in 28 of 44 primary breast carcinomas (64%). In contrast LOH in only 26 of 61 tumors (43%) was detected with the variable number of tandem repeats (VNTR) probe YNZ22 mapping at 17p13.3 close to the p53 locus at 17p13.1. Among 19 tumors informative for both probes allele loss at 17p13.3 never occurred without p53 involvement. These data suggest, that p53 is the target of 17p13 allelic deletions in human breast cancer. Immunohistochemistry showed overexpression of the p53 protein in 25 of 50 cases (50%) presumably reflecting activating point mutations. Overexpression was not correlated with allele loss but seemed to be closely related to the presence of point mutations in this study. No homozygous deletions or rearrangements of the p53 gene were detected. This would argue for an important role of heterozygous p53 mutations in human breast cancer.     

Allelic and haplotype frequencies of the p53 polymorphisms in brain tumor patients

The polymorphisms of the tumor suppressor gene p53 in exon 4 (p53 BstUI) and in intron 6 (p53 MspI) have been suggested to be associated with the genetically determined susceptibility in diverse types of human cancer. In our hospital-based case-control study, we examined the allele and genotype incidence of these polymorphisms as well as their haplotype combinations in 60 brain tumor patients (27 males and 33 females) and 183 controls without malignancies. The genotype characteristics were determined by the PCR-based RFLP method using DNA extracted from peripheral blood. In this study we show that the p53 BstUI and the p53 MspI polymorphisms are not associated with increased risk of brain tumors. Thus, we conclude that the p53 BstUI and the p53 MspI polymorphic sites within the tumor suppressor gene p53 do not represent genetic determinants of susceptibility to brain tumors.     

Multiple mutations of the p53 gene in human mammary carcinoma

Alteration of the p53 tumor suppressor gene is the most common genetic abnormality in human cancer. In breast cancer, depending on the stage of disease and method of detection, mutation rates of 25-60% have been observed. Multiple mutations of p53 gene in the same tumor however, are rarely reported. In this study we explored the frequency of multiple mutations of p53 gene in mammary carcinoma in a cohort of south Florida patients. Three hundred eighty-four cases of primary breast cancer diagnosed between 1984 and 1986 at the University of Miami, Jackson Medical Center were subjects of this study. Sequence analysis of exons 5 through 8 of p53 was performed on cloned PCR-amplified DNA of formalin-fixed, paraffin-embedded tumors. Two hundred thirty-four of 384 breast cancers (61%) had p53 mutation. Of those, 36 tumors showed more than one mutation; 31 tumors had two mutations, three showed three, one tumor had five mutations, and one case carried six mutations. The majority of mutations were missense (43) followed by silent (35); and most occurred within a single exon. Our study suggests that multiple mutations of p53 suppressor gene in breast cancer are more common than currently believed.     

Absence of p53 germ-line mutations in bilateral breast cancer patients

Summary The cause of Li-Fraumeni syndrome, a rare group syndrome of familial cancers, has recently been identified. Patients with this inherited condition are highly susceptible to specific neoplasms, including early-onset breast cancers. The available evidence links Li-Fraumeni syndrome to inherited mutations of the tumor suppressor gene p53. Moreover, somatically acquired p53 mutations and gene deletions are common feature in breast cancer of sporadic origin. These findings suggest that germline p53 mutations are important in familial and, possibly sporadic, breast tumors. We have therefore screened lymphocyte DNA from 19 unrelated bilateral cancer patients for germline p53 mutations in exons 5, 6, 7 and 8. We have however detected no germline mutations by means of the single-strand confirmation polymorphism technique in any of the lymphocyte DNAs examined and conclude that p53 mutations are not generally involved in bilateral breast cancer.     

Structure of the rat p53 tumor suppressor gene.

Aberration within the p53 tumor suppressor gene is the most frequently identified genetic damage in human cancer. Regulatory functions proposed for the p53 protein include modulation of the cell cycle, cellular differentiation, signal transduction, and gene expression. Additionally, the p53 gene product may guard the genome against incorporation of damaged DNA. To facilitate study of its role in carcinogenesis using a common animal model, we determined the structure of the rat p53 gene. We identified 18 splice sites and defined 25 bases of the intervening sequences adjacent to these sites. We also discovered an allelic polymorphism that occurs within intron 5 of the gene. The rat gene approximates the mouse ortholog. It is 12 kb in length with the non-coding exon 1 separated from exon 2 by 6.2 kb of intervening sequence. The location and size of all rat gene introns approximate those of the mouse. Whereas the mouse and human genes each contain 11 exons, the rat p53 gene is composed of only 10. No intervening sequence occurs between the region of the rat gene corresponding to exons 6 and 7 of the mouse and human p53 genes. This implies intron 6 may be functionally insignificant for species in which it is retained. To extrapolate to p53 involvement in human tumorigenesis, we suggest that mutational events within intron 6 may not be of pathological significance unless splicing is hindered.     

Reconstitution of p53 expression in a nonproducer Ab-MuLV-transformed cell line by transfection of a functional p53 gene

L12 are Ab-MuLV-transformed cells that express the abl p120 oncogene product but lack the cellularly encoded p53. The functional p53 gene in these cells has been inactivated by the insertion of Moloney virus-like sequences into the first p53 intron. Transfection of L12 cells with a functional p53 gene, contained in a 16 kb Eco RI genomic cloned fragment gave rise to L12-derived cell lines with novel p53 sequences of various sizes and copy number. A high percentage of L12-derived clones efficiently transcribed p53 mRNA and synthesized the p53 protein. Whereas injection of L12 parental cells into syngeneic mice caused the development of local tumors that later regressed, L12-derived clones that expressed p53 caused lethal tumors in syngeneic mice, thus behaving similarly to other Ab-MuLV-transformed p53-producer cell lines. These results suggest that the expression of p53 is essential for tumor cells to exhibit a fully transformed phenotype, manifested in lethal tumors in syngeneic mice.     

Association of TP53 mutation, p53 overexpression, and p53 codon 72 polymorphism with susceptibility to apoptosis in adult patients with diffuse astrocytomas

Clarification of TP53 alterations is important to understand the mechanisms underlying the development of diffuse astrocytomas. It has been suggested that the alleles of TP53 at codon 72 differ in their ability to induce apoptosis in human cancers. The aim of this study was to analyze the possible association of TP53 mutation, p53 overexpression, and p53 codon 72 polymorphism with susceptibility to apoptosis in adult Brazilian patients with diffuse astrocytomas. We analyzed 56 surgical specimens of diffuse astrocytomas for alterations of TP53, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) direct sequencing. p53 and cleaved caspase 3 protein expression were assessed by immunohistochemistry. We found TP53 mutations in 19.6% (11 out of 56) of tumors tested, with the lowest mutation rate found in the cases of glioblastomas (8.8%) (p = 0.03). Only 16.1% of tumors tested showed cleaved caspase 3-positive staining, demonstrating that apoptosis is very inhibited in these tumors. All tumors having TP53 mutation and p53 accumulation had no expression of cleaved caspase 3. Additionally, no association was observed in tumors having proline and arginine alleles and expression of cleaved caspase 3. We concluded that clarification of the TP53 alterations allows a better understanding of the mechanisms involved in the progression of diffuse astrocytomas, and the allele status at codon 72 was not associated with apoptosis in these tumors.     

Allelic losses on chromosomes 1p, 3p, 11p, 11q in non small cell lung cancers

Recent studies have shown that lung cancer patients frequently suffer inactivation of antioncogenes such as Rb gene (13q14) and p53 gene (17p13). In a study of 48 cases of non-small cell lung cancer (28 squamous-cell carcinomas, 11 adenocarcinomas, 4 large-cell carcinomas, and 5 other types) using restriction fragment length polymorphism analysis, we found DNA sequence deletions from chromosomes 1p32-36, 3p21, 11p15.5, and 11q13. The frequencies of allele loss on chromosome 1p, 3p, 11p and 11q are 31, 57, 20 and 49% of informative cases in this patient group, respectively. Of them, 19 tumors show one allele loss and 10 patients suffer two or more allele losses from different chromosomes.     

A variation in the structure of the protein-coding region of the human p53 gene

An extensive analysis of genomic DNA preparations from a number of normal and malignant tissues revealed BglII site polymorphism of the human p53 gene. Approximately 10% of p53 gene alleles were found to contain an additional BglII site localized in a region of intron I. This allelic form of p53 gene was also responsible for p53 protein having altered electrophoretic mobility. Molecular cloning and sequencing of both the alleles of p53 gene revealed a base-pair change in codon 72 causing arginine → proline substitution in the allele with the additional BglII site. Both variants of the p53 gene may occur in homozygous state and are therefore functional.     

Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53

Galectin-7 was initially described as a marker of epithelial differentiation expressed in the stratified epithelium of various tissues. Like other members of the galectin family, its expression level is often significantly altered in cancer cells. In breast cancer, its expression is significantly augmented in aggressive molecular subtypes, most notably in estrogen receptor-negative tumors and in cell lines with a basal-like phenotype. Studies using experimental mouse models have further shown high expression of galectin-7 was sufficient to increase the metastatic behavior of poorly metastatic breast cancer cells, rendering them more resistant to apoptosis. This expression pattern in breast cancer cells is unexpected because galectin-7 was originally identified as a p53-induced gene. To address this paradox, we have examined the molecular mechanisms regulating galectin-7 in breast cancer cells. Our results showed that transfection of breast cancer cells with expression vectors encoding mutant p53 was sufficient to induce galectin-7 at both mRNA and protein levels. Doxorubicin treatment of breast cancer cells harboring a mutant p53 also induced galectin-7. This induction was specific since knockdown of endogenous mutant p53 inhibited doxorubicin-induced galectin-7 expression. The p53-induced galectin-7 expression in breast cancer cells correlated with increased NF-κB activity and was inhibited by NF-κB inhibitors, indicating that the ability of mutant p53 to induce galectin-7 was dependent on NF-κB activity. The implication of NF-κB was further supported by data showing that NF-κB bound to the endogenous galectin-7 promoter and that TNFα-induced galectin-7 expression was abolished by NF-κB inhibitors. Taken together, our data provide an explanation to the observed high galectin-7 expression levels in cancer cells and suggest that galectin-7 could be part of a common pathway used by mutant p53 to promote cancer progression.     

Mutation analysis of p53 in ovarian tumors by DHPLC

Up to now, ovarian carcinomas represent a major health problem among female cancers because they are the leading cause of death from gynecological malignancy. A high proportion of these tumors selects for mutations in the p53 gene. There is evidence that inactivation of the p53 protein could indicate poor prognosis and chemoresistance of patients. To set up a fast and sensitive test for p53 defects in tumor tissues, we analyzed ovarian cancer cells by denaturing high-performance liquid chromatography (DHPLC). A primer set spanning the whole coding region of p53 with seven fragments was designed and appropriate heteroduplex detection in DHPLC analysis was elaborated. The analysis of 45 ovarian tumor specimens yielded 17 genetic alterations (38%) occurring exclusively in the malignant tissue of the patients. In addition, frequent polymorphisms present in normal compared to tumor tissue could serve as a tool for the rapid identification of loss of heterozygosity (LOH) in the tumor. We observed that LOH in intron 2 or 3 correlated well with a lack of one allele in mutated fragments. In conclusion, DHPLC screening appears to be a sensitive and effective test for genetic alterations in tumors with p53 involvement. Since p53 mutations point to a poor prognosis state in several cancers, a fast screening of tumor material for genetic variations may have implications for further individual treatment of patients.     

P53 mutations, polymorphisms, and haplotypes in Pakistani ethnic groups and breast cancer patients

Inactivation of the p53 gene has been found to be associated with the pathogenesis of several neoplasias. Three biallelic polymorphisms in the p53 gene have been linked to predisposition to the development of various malignancies. These include a 16-bp duplication in intron 3 and BstU I and Msp I restriction fragment length polymorphisms (RFLPs) in exon 4 and intron 6, respectively. The prevalence of these polymorphisms was studied in breast cancer patients and nine major ethnic groups of Pakistan. Differences in allele frequencies for all three polymorphisms were observed among the various ethnic groups and breast cancer patients. The absence of the 16-bp duplication was common among the northern ethnic groups, being highest in the Hazara (0.90). The Msp I A1 allele frequency in the southern Makrani population was significantly higher in comparison with the other ethnic groups. In the cancer patients, the absence of the 16-bp duplication in combination with the BstU I Pro and absence of Msp I restriction site were the most frequent. In these patients, ten substitution mutations were found in the p53 gene, seven of which have been reported previously for breast cancer. The remaining three mutations have been found in other malignancies, but not in carcinoma of the breast.