Splicing mutations in TP53 in human squamous cell carcinoma lines influence immunohistochemical detection.

The mutational status of the tumor suppressor gene TP53 is often examined by immunohistochemistry. We compared the incidence of TP53 mutations in 12 permanent squamous cell carcinoma lines of the head and neck with the immunohistochemical staining obtained with two different antibodies. The mutational status of the TP53 gene was assessed by sequencing the complete coding frame of the TP53 mRNA. All 12 tumor cell lines had TP53 mutations. Six of them showed missense mutations and five had premature stop codons caused either by splicing mutations or nonsense mutations or by exon skipping. One tumor cell line was heterozygous, with a truncating splicing mutation and an additional missense mutation located on different alleles. In one case, an in-frame insertion of 23 extra codons was found. All missense mutations were positive in immunhistochemistry and Western blotting. The truncated p53 was not immunohistochemically detected in three cases with the DO-7 antibody and in five cases with the G59-12 antibody, giving false-negative results in 25% or 40%, respectively, of all tumor cell lines examined. We conclude that splicing mutations are common in squamous cell carcinoma lines and that the incidence of p53 inactiviation by erroneous splicing is higher than yet reported. Sequencing of only the exons of TP53 may miss intronic mutations leading to missplicing and may therefore systematically underestimate the TP53 mutation frequency.     

Ineffectiveness of the presence of H-ras/p53 combination of mutations in squamous cell carcinoma cells to induce a conversion of a nontumorigenic to a tumorigenic phenotype

Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells ability to exhibit a malignant potential in nude mice.     

Alterations of the p53 gene in Epstein-Barr virus-associated immunodeficiency-related lymphomas.

Mutations of the p53 tumor suppressor gene are among the most common genetic alterations found in many different human malignancies, including those of the colon, lung, and breast. Alterations in wild-type p53 lead to loss of the suppressor function and thus contribute to tumorigenesis. The potential role of p53 mutations in a sampling of B-cell lymphomas, the majority of which were associated with Epstein-Barr virus (EBV), was investigated. Twenty-six biopsy specimens from immunocompromised patients, including allograft recipients and patients with AIDS, Wiscott-Aldrich syndrome, and human T-cell leukemia virus type 1 infection, in comparison with three Burkitt lymphomas and four Burkitt lymphoma cell lines were analyzed. Mutation in p53 was detected in all four Burkitt lymphoma cell lines as well as the three Burkitt lymphoma biopsy specimens. In patients with AIDS, 5 of 10 lymphomas were EBV positive, and 1 had a mutation in p53. Mutation in p53 was not detected in 14 EBV-positive lymphomas which arose in transplant recipients. These data indicate that with the exception of Burkitt lymphomas, p53 mutations are not involved in the majority EBV-positive B-cell lymphomas which develop in immunocompromised patients.     

p53 mutations in tumors derived from irradiated human thyroid epithelial cells

A non-tumorigenic human thyroid epithelial cell line (HTori-3) has been transformed into tumorigenic cells by exposure in vitro to alpha particles or gamma-radiation. These transformants were tumorigenic in athymic nude mice and tumors were transplantable into other nude mice. To further characterize processes involved in neoplastic progression, the tumor cell lines derived from these radiation-induced primary tumors were screened for mutations in the p53 tumor suppressor gene. p53 mutation was detected by single-strand conformation polymorphism (SSCP) analysis of exons 5 to 8 inclusive. Mutations detected by SSCP analysis were confirmed by sequencing. Mutations were detected in all four exons analysed, although there was no correlation between dose, LET or mutation position or frequency. Mutations in p53 exons 6 and 7 have been reported in the childhood papillary thyroid carcinomas in Belarus presumably as a result of radioiodine fall-out. Similarly here, p53 mutations are induced experimentally during the development of human thyroid tumors generated by irradiation of a human thyroid epithelial cell line in vitro.     

The oncogenic roles of p53 mutants in mouse models

Tumors associated with p53 usually contain missense mutations in the p53 tumor suppressor gene rather than deletions of p53, suggesting a growth advantage for cells with missense mutations. The oncogenic roles of p53 mutants have been examined extensively in cell lines. Mouse models that inherit p53 mutations expressed at physiological levels have now been generated to examine the activities of mutant p53 upon tumorigenesis in vivo. Mice with p53 mutations develop tumor spectrums and metastatic phenotypes different from those of mice with a p53-null allele. Embryo fibroblasts with mutant p53 also show increased proliferative and transformation properties. One mechanism for this gain-of-function potential is the inhibition of function of the p53 family members p63 and p73.     

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.     

Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome

The p53 tumor suppressor gene is commonly altered in human tumors, predominantly through missense mutations that result in accumulation of mutant p53 protein. These mutations may confer dominant-negative or gain-of-function properties to p53. To ascertain the physiological effects of p53 point mutation, the structural mutant p53R172H and the contact mutant p53R270H (codons 175 and 273 in humans) were engineered into the endogenous p53 locus in mice. p53R270H/+ and p53R172H/+ mice are models of Li-Fraumeni Syndrome; they developed allele-specific tumor spectra distinct from p53+/- mice. In addition, p53R270H/- and p53R172H/- mice developed novel tumors compared to p53-/- mice, including a variety of carcinomas and more frequent endothelial tumors. Dominant effects that varied by allele and function were observed in primary cells derived from p53R270H/+ and p53R172H/+ mice. These results demonstrate that point mutant p53 alleles expressed under physiological control have enhanced oncogenic potential beyond the simple loss of p53 function.     

Establishment and characterization of four human pancreatic carcinoma cell lines. Genetic alterations in the TGFBR2 gene but not in the MADH4 gene

We characterized four pancreatic carcinoma cell lines (designated SNU-213, SNU-324, SNU-410, and SNU-494) established from histopathologically varied primary or liver metastatic tumor samples of Korean patients. Three cell lines grew as adherent monolayers and one as adherent and floating cell clumps. All lines had: (1) relatively high viability; (2) an absence of mycoplasma or bacterial contamination; (3) genetic heterogeneity as assessed by DNA-fingerprinting analysis; (4) an absence of MADH4 mutation. Among the lines, three lines had mutations in codon 12 in K- ras, two lines harbored p53 mutations within the DNA-binding domain; two lines had homozygous deletions in both p16 and p15 genes; and one line had a missense mutation. Two lines (SNU-324 and SNU-410) had genetic alterations in the TGFBR2 gene: the SNU-324 line had a -1-bp or +1-bp mutation in 10-bp polydeoxyadenine repeat tracts; the SNU-410 line had a genomic deletion in this gene. Mutation analysis of mismatch repair genes demonstrated that SNU-324 has two heterozygous missense mutations in different exons of the hMLH1 gene. In addition, this line showed microsatellite instability and harbored frameshift mutations in simple repeated sequences of the coding regions of the TGFBR2, BAX, and hMSH3 genes. These defects of microsatellite instability and mismatch repair genes suggest the possibility of a new mutator phenotype for pancreatic carcinogenesis. These cell lines should be very useful for studying the biology of pancreatic carcinoma, particularly those related to mutator phenotype and genetic alterations in the TGFBR2 gene.     

Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells.

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.     

p53 tumor suppressor gene mutations in fibroblast-like synoviocytes from erosion synovium and non-erosion synovium in rheumatoid arthritis

Abnormalities in the p53 tumor suppressor gene have been detected in rheumatoid arthritis (RA) and could contribute to the pathogenesis of chronic disease. To determine whether synoviocytes from invasive synovium in RA have an increased number of mutations compared with non-erosion synoviocytes, p53 cDNA subclones from fibroblast-like synoviocytes (FLS) derived from erosion and non-erosion sites of the same synovium were examined in patients requiring total joint replacement. Ten erosion FLS lines and nine non-erosion FLS lines were established from nine patients with RA. Exons 5-10 from 209 p53 subclones were sequenced (114 from erosion FLS, 95 from non-erosion FLS). Sixty percent of RA FLS cell lines and 8.6% of the p53 subclones isolated from FLS contained p53 mutations. No significant differences were observed between the erosion and non-erosion FLS with regard to the frequency or type of p53 mutation. The majority of the mutations were missense transition mutations, which are characteristic of oxidative damage. In addition, paired intact RA synovium and cultured FLS from the same joints were evaluated for p53 mutations. Matched synovium and cultured synoviocytes contained p53 mutations, although there was no overlap in the specific mutations identified in the paired samples. Clusters of p53 mutations in subclones were detected in some FLS, including one in codon 249, which is a well-recognized 'hot spot' associated with cancer. Our data are consistent with the hypothesis that p53 mutations are randomly induced by genotoxic exposure in small numbers of RA synoviocytes localized to erosion and non-erosion regions of RA synovium. The determining factor for invasiveness might be proximity to bone or cartilage rather than the presence of a p53 mutation.     

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。     

Transforming activity of mutant human p53 alleles

Mutant forms of the p53 gene have been shown to cooperate with an activated ras gene in transforming primary cells in culture. The aberrant proteins encoded by p53 mutants are thought to act in a dominant negative manner in these assays. In vivo data, however, reveal that where p53 has undergone genetic change in tumors, both alleles have been affected. We previously identified a case of human acute myelogenous leukemia (AML) in which both alleles of the p53 gene had undergone independent missense mutations (at codons 135 cys to ser and 246 met to val). In these blasts, p53 mutations appear to be acting recessively. We have assayed the transforming potential of these p53 mutations, as well as that of another mutation at codon 273, also identified in a human neoplasm. Both mutations from the AML blasts (codon 135 and codon 246) confer transforming ability on the mutant protein. While transformation assays may define functionally different subsets of p53 mutations, the overexpression phenotype of mutants in this assay may not accurately reflect the pathological effects of p53 mutations in vivo.     

Frequent retention of heterozygosity for point mutations in p53 and Ikaros in N-ethyl-N-nitrosourea-induced mouse thymic lymphomas

In agreement with Knudson's two-hit theory, recent findings indicate that the inactivation of tumor suppressor genes is not only mediated by the loss of function but also by the dominant-negative or gain-of-function activity. The former generally accompanies loss of a wild-type allele whereas in the latter a wild-type allele is retained. N-Ethyl-N-nitrosourea (ENU), which efficiently induces point mutations, reportedly leads to the development of tumors by activating ras oncogenes. Little is known about how ENU affects tumor suppressor genes and, therefore, we examined ENU-induced mutations of p53 and Ikaros in thymic lymphomas and compared these with mutations of Kras. In addition, loss of heterozygosity was examined for chromosome 11 to which both p53 and Ikaros were mapped. The frequency of point mutations in p53 and Ikaros was 30% (8/27) and 19% (5/27), respectively, comparable to that observed in Kras (33%: 9/27). In total, 14 of the 27 thymic lymphomas examined (52%) harbored mutations in at least one of these genes. One Ikaros mutation was located at the splice donor site, generating a novel splice isoform lacking zinc finger 3, Ik (F3del). Interestingly, 90% (10/11) of the tumors with point mutations retained wild-type alleles of p53 and Ikaros. Sequence analysis revealed that the most common nucleic acid substitutions were T>A (4/8) in p53, T>C (4/5) in Ikaros and G>A/T (8/9) in Kras, suggesting that the spectrum of mutations was gene dependent. These results suggest that point mutations in tumor suppressor genes without loss of the wild-type allele play an important role in ENU-induced lymphomagenesis.     

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.     

Functional evidence for a second tumor suppressor gene on human chromosome 17.

The development and progression of human tumors often involves inactivation of tumor suppressor gene function. Observations that specific chromosome deletions correlate with distinct groups of cancer suggest that some types of tumors may share common defective tumor suppressor genes. In support of this notion, our initial studies showed that four human carcinoma cell lines belong to the same complementation group for tumorigenic potential. In this investigation, we have extended these studies to six human soft tissue sarcoma cell lines. Our data showed that hybrid cells between a peripheral neuroepithelioma (PNET) cell line and normal human fibroblasts or HeLa cells were nontumorigenic. However, hybrid cells between the PNET cell line and five other soft tissue sarcoma cell lines remained highly tumorigenic, suggesting at least one common genetic defect in the control of tumorigenic potential in these cells. To determine the location of this common tumor suppressor gene, we examined biochemical and molecular polymorphic markers in matched pairs of tumorigenic and nontumorigenic hybrid cells between the PNET cell line and a normal human fibroblast. The data showed that loss of the fibroblast-derived chromosome 17 correlated with the conversion from nontumorigenic to tumorigenic cells. Transfer of two different chromosome 17s containing a mutant form of the p53 gene into the PNET cell line caused suppression of tumorigenic potential, implying the presence of a second tumor suppressor gene on chromosome 17.     

Analysis of p53 inactivation in a human T-cell leukemia virus type 1 Tax transgenic mouse model

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). The HTLV-1 Tax protein has been strongly linked to oncogenesis and is considered to be the transforming protein of this virus. A Tax transgenic mouse model was utilized to study the contribution of p53 inactivation to Tax-mediated tumorigenesis. These mice develop primary, peripheral tumors consisting of large granular lymphocytic (LGL) cells, which also infiltrate the lymph nodes, bone marrow, spleen, liver, and lungs. Primary Tax-induced tumors and tumor-derived cell lines exhibited functional inactivation of the p53 apoptotic pathway; such tumors and tumor cell lines were resistant to an apoptosis-inducing stimulus. In contrast, p53 mutations in tumors were found to be associated with secondary organ infiltration. Three of four identified mutations inhibited transactivation and apoptosis induction activities in vitro. Furthermore, experiments which involved mating Tax transgenic mice with p53-deficient mice demonstrated minimal acceleration in initial tumor formation, but significantly accelerated disease progression and death in mice heterozygous for p53. These studies suggest that functional inactivation of p53 by HTLV-1 Tax, whether by mutation or another mechanism, is not critical for initial tumor formation, but contributes to late-stage tumor progression.     

p53 gene mutations in neoplastic transformation of C3H 10T1/2 and severe combined immunodeficiency fibroblasts.

The relevance of p53 mutations to the neoplastic malignant transformation of rodent fibroblasts by genotoxic physical and chemical agents is not clear. In the present study, we investigated p53 mutations (in exons 5-8) in non-transformed and neoplastically transformed C3H 10T1/2 and severe combined immunodeficiency (SCID) cells. No p53 mutations were detected in 15 neoplastically transformed (two spontaneous, one 3-methylcholanthrene-induced, seven gamma-ray-induced and five 'hot particle'-induced) and two non-transformed 10T1/2 cells. Wild-type p53 gene was also detected in all non-transformed (immortalized) SCID cell lines analyzed (four lines) whereas all three neoplastically transformed (two spontaneous, one gamma-ray-induced) cell lines displayed missense mutations in the p53 gene. These mutations were all transitions: A > G in codon 123, G > A in codon 152, and C > T in codon 238. We conclude that mutation in the p53 gene appears to be an infrequent event in 10T1/2 cells regardless of the transforming agent, but a frequent event in the neoplastic transformation of immortalized SCID cells. Non-transformed SCID cells are deficient in repair of DNA double-strand breaks, and neoplastically transformed cells are assumed to be deficient as well.