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.     

Single base pair germ-line deletion in the p53 gene in a cancer predisposed family

A family with an aggregation of adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, and early onset breast cancer was referred to our laboratory. Because this aggregation was reminiscent of Li-Fraumeni syndrome, germ-line mutation of the p53 tumor suppressor gene was sought in the DNA of two affected members. The highly conserved regions spanning exons 5 to 8 of the p53 gene were screened by a previously validated denaturing gradient gel electrophoresis method. A single base pair deletion at codon 215 was detected in constitutional DNA of the two patients, and in the DNA extracted from an adrenocortical carcinoma tumor specimen of the propositus. This deletion is predicted to lead to the formation of a truncated p53 protein, a relatively rare event in Li-Fraumeni families. The spectrum of tumors observed in this family does not differ markedly from the spectrum observed in families with missense p53 mutations.     

Li-Fraumeni Syndrome: a p53 Family Affair

The p53 alterations frequently found in human tumors are missense mutations in the DNA binding domain. These p53 mutations have been shown to have gain-of-function or dominant negative properties in multiple experiments. The consequences of these p53 mutations at physiological levels on the development of a tumor were unclear. Using mouse models, three recent papers have shed light on the mechanisms of mutant p53 and its family members, p63 and p73, in tumorigenesis. Interestingly, the p53 point mutant mice had a similar phenotype to p53 family compound mutant mice suggesting that there is an interplay between the p53 family members in tumorigenesis and Li-Fraumeni syndrome.     

Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles.

Loss of cell cycle control and acquisition of chromosomal rearrangements such as gene amplification often occur during tumor progression, suggesting that they may be correlated. We show here that the wild-type p53 allele is lost when fibroblasts from patients with the Li-Fraumeni syndrome (LFS) are passaged in vitro. Normal and LFS cells containing wild-type p53 arrested in G1 when challenged with the uridine biosynthesis inhibitor PALA and did not undergo PALA-selected gene amplification. The converse occurred in cells lacking wild-type p53 expression. Expression of wild-type p53 in transformants of immortal and tumor cells containing mutant p53 alleles restored G1 control and reduced the frequency of gene amplification to undetectable levels. These studies reveal that p53 contributes to a metabolically regulated G1 check-point, and they provide a model for understanding how abnormal cell cycle progression leads to the genetic rearrangements involved in tumor progression.     

A database of germline p53 mutations in cancer-prone families.

We created a comprehensive database covering all published cases of germline p53 mutations. The current version lists 580 tumours in 448 individuals belonging to 122 independent pedigrees. The database describes each p53 mutation (type of the mutation, exon and codon affected by the mutation, nucleotide and amino acid change), each family (family history of cancer, diagnosis of Li-Fraumeni syndrome), each affected individual (sex, generation, p53 status, from which parent the mutation was inherited) and each tumour (type, age of onset, p53 status-loss of heterozygosity, immunostaining). Each entry contains the original reference(s). The database is freely available and can be obtained from http://www.lf2.cuni.cz     

p53 and disease: when the guardian angel fails

The p53 tumor suppressor gene (TP53) is mutated more often in human cancers than any other gene yet reported. Of importance, it is mutated frequently in the common human malignancies of the breast and colorectum and also, but less frequently, in other significant human cancers such as glioblastomas. There is also one inherited cancer predisposing syndrome called Li-Fraumeni that is caused by TP53 mutations. In this review, we discuss the significance of p53 mutations in some of the above tumors with a view to outlining how p53 contributes to malignant progression. We also discuss the usefulness of TP53 status as a prognostic marker and its role as a predictor of response to therapy. Finally, we outline some evidence that abnormalities in p53 function contribute to the etiology of other non-neoplastic diseases.     

Spontaneous in vitro immortalization of breast epithelial cells from a patient with Li-Fraumeni syndrome.

Individuals with germ line mutations in the p53 gene, such as Li-Fraumeni syndrome (LFS), have an increased occurrence of many types of cancer, including an unusually high incidence of breast cancer. This report documents that normal breast epithelial cells obtained from a patient with LFS (with a mutation at codon 133 of the p53 gene) spontaneously immortalized in cell culture while the breast stromal fibroblasts from this same patient did not. Spontaneous immortalization of human cells in vitro is an extremely rare event. This is the first documented case of the spontaneous immortalization of breast epithelial cells from a patient with LFS in culture. LFS patient breast stromal fibroblasts infected with a retroviral vector containing human papillomavirus type 16 E7 alone were able to immortalize, whereas stromal cells obtained from patients with wild-type p53, similarly infected with human papillomavirus type 16 E7, did not. The present results indicate a protective role of normal pRb-like functions in breast stromal fibroblasts but not in breast epithelial cells and reinforces an important role of wild-type p53 in the regulation of the normal growth and development of breast epithelial tissue.     

Aberrant regulation and function of wild-type p53 in radioresistant melanoma cells.

Sporadic human tumors and the hereditary cancer predisposition syndrome Li-Fraumeni are frequently associated with mutations in the p53 tumor suppressor gene that compromise its ability to function as a DNA damage checkpoint. A subset of Li-Fraumeni patients with wild-type p53 alleles have mutations in chk2/hcds1, one of the genes signaling the presence of DNA damage to the p53 protein. This suggests that p53 may be kept inactive in human cancer by mutations targeting DNA damage signaling pathways. Melanoma cells are highly radioresistant, yet they express wild-type p53 protein, raising the possibility of defects in the pathways that activate p53 in response to DNA damage. We have described a chk2/hcds1-independent DNA damage signaling pathway that targets Ser-376 within the COOH terminus of p53 for dephosphorylation and leads to increased p53 functional activity. We now report that in several human melanoma cell lines that express wild-type p53, the phosphorylation state of Ser-376 was not regulated by DNA damage. In these cell lines, neither the endogenous wild-type p53 protein nor high levels of ectopic wild-type p53 led to cell cycle arrest or apoptosis. Thus, defective activation of p53 in response to DNA damage may underlie the radioresistance of human melanoma cells.     

Delayed chromosome changes in gamma-irradiated normal and Li-Fraumeni fibroblasts

Knockout mice with only one Trp53 allele (+/- genotype) are highly susceptible to radiation-induced cancers, possibly through numerical chromosome changes. Patients with the Li-Fraumeni syndrome, having heterozygous TP53 germline mutations (+/mut genotype), are also susceptible to spontaneous and radiogenic cancers. We have investigated the susceptibility of six Li-Fraumeni syndrome +/mut and six normal fibroblast strains to induced numerical and unstable structural aberrations at six population doublings after exposure to 3 or 6 Gy gamma rays. Four of the irradiated Li-Fraumeni syndrome strains showed small increases in both aberration types, similar to those seen in the normal strains. In two irradiated Li-Fraumeni syndrome strains, there were high levels of induced structural changes, and one of these showed a modest increase in hyperploidy. We suggest that enhanced sensitivity to delayed radiation-induced chromosome changes in Li-Fraumeni syndrome cells requires other genetic alterations in addition to TP53 heterozygosity, apparently in contrast to the situation in Trp53 heterozygous null mice. If such additional alterations occur in vivo in Li-Fraumeni syndrome patients, they may predispose them to radiogenic cancers, mainly through enhanced structural rather than numerical chromosome changes. Our findings raise questions about the validity of quantitative extrapolation of cytogenetic data from Trp53-defective mice to radiogenic cancer risk in humans.     

突变体p53研究进展

抑癌基因突变是癌症发生过程中一个极为关键的事件。p53作为体内最重要的抑癌基因之一, 在人类癌症中发生突变的频率高达50%。同时, p53突变也是人类遗传病Li-Fraumeni综合征的主要病因。p53最常见的突变形式是错义突变, 所形成的突变体p53不但失去了野生型p53的抑癌功能, 而且还获得了一系列类似于癌基因的功能, 促进了肿瘤的进程。文章拟对突变体p53的结构功能改变, 获得癌基因活性的分子机制, 以及近年来对封闭突变体p53活性所进行的探索等研究方向所取得的进展做一综述。     

Functional interaction of p53 and BLM DNA helicase in apoptosis

The Bloom syndrome (BS) protein, BLM, is a member of the RecQ DNA helicase family that also includes the Werner syndrome protein, WRN. Inherited mutations in these proteins are associated with cancer predisposition of these patients. We recently discovered that cells from Werner syndrome patients displayed a deficiency in p53-mediated apoptosis and WRN binds to p53. Here, we report that analogous to WRN, BLM also binds to p53 in vivo and in vitro, and the C-terminal domain of p53 is responsible for the interaction. p53-mediated apoptosis is defective in BS fibroblasts and can be rescued by expression of the normal BLM gene. Moreover, lymphoblastoid cell lines (LCLs) derived from BS donors are resistant to both gamma-radiation and doxorubicin-induced cell killing, and sensitivity can be restored by the stable expression of normal BLM. In contrast, BS cells have a normal Fas-mediated apoptosis, and in response to DNA damage normal accumulation of p53, normal induction of p53 responsive genes, and normal G(1)-S and G(2)-M cell cycle arrest. BLM localizes to nuclear foci referred to as PML nuclear bodies (NBs). Cells from Li-Fraumeni syndrome patients carrying p53 germline mutations and LCLs lacking a functional p53 have a decreased accumulation of BLM in NBs, whereas isogenic lines with functional p53 exhibit normal accumulation. Certain BLM mutants (C1055S or Delta133-237) that have a reduced ability to localize to the NBs when expressed in normal cells can impair the localization of wild type BLM to NBs and block p53-mediated apoptosis, suggesting a dominant-negative effect. Taken together, our results indicate both a novel mechanism of p53 function by which p53 mediates nuclear trafficking of BLM to NBs and the cooperation of p53 and BLM to induce apoptosis.     

The Li-Fraumeni syndrome

Li-Fraumeni syndrome (LFS) has been the most common terminology used for the syndrome. It is a rare familial dominantly inherited cancer syndrome characterized by a wide spectrum of neoplasms occurring in children and young adults. The canonical definition of LFS includes a proband diagnosed with sarcoma before 45 years of age, a first-degree relative with cancer before this same age and another first- or second-degree relative in the lineage with any cancer before this age or sarcoma at any age. Multiple studies have reported p53 germline mutations in LFS families in various parts of the world. As in sporadic tumors, loss of heterozygosity leading to the inactivation of the wild-type allele by deletion or mutation is observed in LFS tumors. Cancer-risk in mutation carriers has been estimated to be 73% in males and nearly 100% in females, the difference almost entirely explained by breast cancer. The identification of germline p53 mutations in rare cancer-prone families has given rise to the medical, counseling, psychological and ethical problems.     

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.     

Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome

Individuals with Li-Fraumeni syndrome carry inherited mutations in the p53 tumor suppressor gene and are predisposed to tumor development. To examine the mechanistic nature of these p53 missense mutations, we generated mice harboring a G-to-A substitution at nucleotide 515 of p53 (p53+/515A) corresponding to the p53R175H hot spot mutation in human cancers. Although p53+/515A mice display a similar tumor spectrum and survival curve as p53+/- mice, tumors from p53+/515A mice metastasized with high frequency. Correspondingly, the embryonic fibroblasts from the p53515A/515A mutant mice displayed enhanced cell proliferation, DNA synthesis, and transformation potential. The disruption of p63 and p73 in p53-/- cells increased transformation capacity and reinitiated DNA synthesis to levels observed in p53515A/515A cells. Additionally, p63 and p73 were functionally inactivated in p53515A cells. These results provide in vivo validation for the gain-of-function properties of certain p53 missense mutations and suggest a mechanistic basis for these phenotypes.     

Decreased DNA repair efficiency by loss or disruption of p53 function preferentially affects removal of cyclobutane pyrimidine dimers from non-transcribed strand and slow repair sites in transcribed strand

The tumor suppressor protein p53 plays a central role in modulating the cellular responses to DNA damage. Several recent studies, undertaken with the whole genomic DNA or full-length gene segments, have shown that p53 is involved in nucleotide excision repair and it selectively influences the adduct removal from the non-transcribed strand in the genome. In this study, we have analyzed the damage induction at nucleotide resolution by ligase-mediated polymerase chain reaction and compared the repair of ultraviolet radiation-induced cyclobutane pyrimidine dimers within exon 8 of p53 gene in normal and Li-Fraumeni syndrome fibroblasts as well as in normal and human papillomavirus 16 E6 and E7 protein-expressing human mammary epithelial cells. The results demonstrate that (i) loss or disruption of p53 function decreases efficiency of DNA repair, by preferentially affecting the repair of non-transcribed strand and of intrinsically slow repair sites in transcribed strand; (ii) mutant p53 protein affects DNA repair, at least of non-transcribed strand, in a dominant negative manner; and (iii) pRb does not have an effect on the repair of DNA damage within transcribed or non-transcribed strand. The overall data suggest that p53 could regulate excision repair or related events through direct protein-protein interaction.