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1.
We present a classification analysis of the mutation spectra of the p53 gene and construct maps of hotspots for the germline (Li-Fraumein syndrome), different types of tumors and their derived cell lines. While spectra from solid tumors share common hotspots with the germline spectrum, they also contain unique sets of somatic hotspots that are not observed in the germline. All these hotspots correspond to amino acid replacements in the DNA-binding interface of p53. The mutation spectra of lymphomas and cell lines derived from lymphomas and lung cancers contained few hotspots compared to solid tumors. Thus, the distribution of hotspots in the p53 gene appears to depend on the tumor type and cell growth conditions; this specificity is missed by the bulk hotspot analysis. A negative correlation was detected between the amino acid replacement propensity in tumors and evolutionary variability: the hotspots are located in the positions that are highly conserved in p53 and its paralogs, p63 and p73. In all the mutation spectra, substitutions leading to amino acid replacements strongly dominate over silent substitutions, indicating that functional sites evolving under strong purifying selection are subject to intensive positive selection in p53-dependent tumors. These results are compatible with the gain-of-function concept of the role of p53 in tumorigenesis. 相似文献
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Mutational hot spots in the human p53 gene are well established in tumors in the human population and are frequently negative prognosticators of the clinical outcome. We previously developed a mouse model of skin cancer with mutations in the xeroderma pigmentosum group C gene (Xpc). UVB radiation-induced skin cancer is significantly enhanced in these mice when they also carry a mutation in one copy of the Trp53 gene (Xpc-/-Trp53+/-). Skin tumors in these mice often contain inactivating mutations in the remaining Trp53 allele and we have previously reported a novel mutational hot spot at a non-dipyrimidine site (ACG) in codon 122 of the Trp53 gene in the tumors. Here we show that this mutation is not a hot spot in Xpa or Csa mutant mice. Furthermore, the mutation in codon T122 can be identified in mouse skin DNA from (Xpc-/-Trp53+/-) mice as early as 2 weeks after exposure to UVB radiation, well before histological evidence of dysplastic or neoplastic changes. Since this mutational hot spot is not at a dipyrimidine site and is apparently Xpc-specific, we suggest that some form of non-dipyrimidine base damage is normally repaired in a manner that is distinct from conventional nucleotide excision repair, but that requires XPC protein. 相似文献
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H. Lee D. Li T. Prior B.C. Casto C.M. Weghorst C.F. Shuler G.E. Milo 《Cell biology and toxicology》1997,13(6):419-434
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. 相似文献
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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. 相似文献
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T Frebourg N Barbier Y X Yan J E Garber M Dreyfus J Fraumeni Jr F P Li S H Friend 《American journal of human genetics》1995,56(3):608-615
Germ-line mutations of the tumor-suppressor gene p53 have been observed in some families with the Li-Fraumeni syndrome (LFS), a familial cancer syndrome in which affected relatives develop a diverse set of early-onset malignancies including breast carcinoma, sarcomas, and brain tumors. The analysis of the p53 gene in LFS families has been limited, in most studies to date, to the region between exon 5 and exon 9. In order to determine the frequency and distribution of germ-line p53 mutations in LFS, we sequenced the 10 coding exons of the p53 gene in lymphocytes and fibroblast cell lines derived from 15 families with the syndrome. Germ-line mutations were observed in eight families. Six mutations were missense mutations located between exons 5 and 8. One mutation was a nonsense mutation in exon 6, and one mutation was a splicing mutation in intron 4, generating aberrant shorter p53 RNA(s). In three families, a mutation of the p53 gene was observed in the fibroblast cell line derived from the proband. However, the mutation was not found in affected relatives in two families and in the blood from the one individual, indicating that the mutation probably occurred during cell culture in vitro. In four families, no mutation was observed. This study indicates that germ-line p53 mutations in LFS are mostly located between exons 5 and 8 and that approximately 50% of patients with LFS have no germ-line mutations in the coding region of the p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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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. 相似文献
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In vitro antibody evolution targeting germline hot spots to increase activity of an anti-CD22 immunotoxin 总被引:4,自引:0,他引:4
Recombinant immunotoxin BL22, containing the Fv portion of an anti-CD22 antibody, produced complete remissions in most patients with drug-resistant hairy cell leukemia but had less activity in leukemias with low CD22 expression. Complementarity-determining region (CDR) mutagenesis is used to increase antibody affinity but can be difficult to perform successfully. We previously showed that antibodies with increased affinity and immunotoxins with increased activity could be obtained by directing mutations at specific DNA residues called hot spots. Because hot spots can arise either by somatic mutation or be present in the germline, we examined which type of hot spot is preferred for increasing antibody affinity. Initially, a second generation antibody phage-display library targeting a germline hot spot (Ser(30)-Asn(31)) within CDR1 of the antibody light chain was mutated. Substitution of serine 30 or asparagine 31 with arginine produced mutant immunotoxins with an affinity (0.8 nM) increased 7-fold over BL22 (5.8 nM) and 3-fold over the first generation mutant HA22 (2.3 nM). More importantly, a 10-fold increase in activity over BL22 and a 2-3-fold increase over HA22 were observed in various B lymphoma cell lines including WSU-CLL that contains only 5500 CD22 sites per cell. For comparison, two phage-display libraries targeting non-germline hot spots in heavy chain CDR1 and CDR3 were generated but did not produce Fv with increased affinity. Our results demonstrate that germline hot spots but not non-germline hot spots are effective for in vitro antibody affinity maturation. 相似文献
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Wolfgang Eicheler Daniel Zips Annegret D?rfler Reidar Grénman Michael Baumann 《The journal of histochemistry and cytochemistry》2002,50(2):197-204
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. 相似文献
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Microarray fabrication with covalent attachment of DNA using bubble jet technology 总被引:22,自引:0,他引:22
We have developed a method for fabricating DNA microarrays that uses a Bubble Jet ink jet device to eject 5'-terminal-thiolated oligonucleotides to a glass surface. The oligonucleotides are covalently attached to the glass surface by heterobifunctional crosslinkers that react with the amino group on the substrate and a thiol group on the oligonucleotide probe. Using this method, we fabricated DNA microarrays that carried 64 groups of 18-mer oligonucleotides encoding all possible three-base mutations in the mutational "hot spot" of the p53 tumor-suppressor gene. These were screened with a fluorescently labeled synthetic 18-mer oligonucleotide derived from the p53 gene, or segments of the p53 gene that had been PCR amplified from genomic DNA of two cell lines of human oral squamous cell carcinoma (SCC). This allowed us to discriminate between matched hybrids and 1 bp-mismatched hybrids. 相似文献
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We have developed a fluorescence-based single strand conformation polymorphism (SSCP) method that offers fast and sensitive screening for mutations in exons 5-8 of the human p53 gene. The method uses an ABI 377 DNA sequencer for unique color detection of each strand, plus accurate alignment of lanes for better detection of mobility shifts. To validate the method, 21 cell lines with reported mutations in p53 exons 5-8 were analyzed by SSCP using various gel conditions. The sensitivity for mutation detection was 95% for all cell lines studied, and no false positives were seen in 10 normal DNA samples for all four exons. Experiments mixing known amounts of tumor and normal DNA showed that mutations were detected even when tumor DNA was mixed with 80% normal DNA. Fluorescent SSCP analysis using the ABI sequencer is a useful tool in cancer research, where screening large numbers of samples for p53 mutations is desired. 相似文献
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N-hydroxy-4-aminobiphenyl-DNA binding in human p53 gene: sequence preference and the effect of C5 cytosine methylation 总被引:4,自引:0,他引:4
4-Aminobiphenyl (4-ABP) is a major etiological agent for human bladder cancer. Metabolically activated 4-ABP is able to interact with DNA to form adducts that may induce mutations and initiate carcinogenesis. Thirty to sixty percent of bladder cancer has a mutation in the tumor suppressor p53 gene, and the mutational spectrum bears unique features. To date the DNA binding spectrum of 4-ABP in the p53 gene is not known due to the lack of methodology to detect 4-ABP-DNA adducts at nucleotide sequence level. We have found that UvrABC nuclease, a nucleotide excision repair complex isolated from Escherichia coli, is able to incise specifically and quantitatively DNA fragments modified with N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), an activated intermediate of 4-ABP. Using the UvrABC nuclease incision method, we mapped the binding spectrum of N-OH-4-ABP in DNA fragments containing exons 5, 7, and 8 of the human p53 gene and also determined the effect of C5 cytosine methylation on N-OH-4-ABP-DNA binding. We found that codon 285, a mutational hotspot at a non-CpG site in bladder cancer, is the preferential binding site for N-OH-4-ABP. We also found that C5 cytosine methylation greatly enhanced N-OH-4-ABP binding at CpG sites, and that two mutational hotspots at CpG sites, codons 175 and 248, became preferential binding sites for N-OH-4-ABP only after being methylated. These results suggest that both the unique DNA binding specificity of 4-ABP and cytosine methylation contribute to the mutational spectrum of the p53 gene in human bladder cancer. 相似文献
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Hot-spot p53 mutants interact specifically with two cellular proteins during progression of the cell cycle. 总被引:1,自引:0,他引:1
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Inactivation of both alleles of the p53 gene is commonly found in human cancers. In contrast to mutations of the retinoblastoma gene, certain altered forms of p53 gain growth-promoting functions. To explore the mechanisms underlying this gain of function, we have identified two nuclear proteins, with molecular masses of 42 and 38 kDa, respectively, that are specifically associated with p53 mutated within the simian virus 40 T-antigen-binding domain, "hot spots" found in many human tumors. These mutants transactivate the multiple-drug resistance gene promoter and cause cells to grow to higher density. Both the mutated p53 complex with p42 and p38 increase when cells enter S phase of the cell cycle but decrease in G1 and M phases, suggesting that they may have a role in promoting cell growth. 相似文献
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Chavali S Mahajan A Ghosh S Mondal B Bharadwaj D 《Biochemical and biophysical research communications》2011,(4):716-722
Molecular epidemiology studies have used the counts of different mutational types like transitions, transversions, etc. to identify putative mutagens, with little reference to gene organization and structure–function of the translated product. Moreover, geographical variation in the mutational spectrum is not limited to the mutational types at the nucleotide level but also have a bearing at the functional level. Here, we developed a novel measure to estimate the rate of spontaneous detrimental mutations called “mutation index” for comparing the mutational spectra consisting of all single base, missense, and non-missense changes. We have analyzed 1609 mutations occurring in 38 exons in 24 populations in three diseases viz. hemophilia B (F9 gene – 420 mutations in 9 populations across 8 exons), hemophilia A (F8 gene – 650, 8 and 26, respectively) and ovarian carcinoma (TP53 gene – 539, 7 and 4, respectively). We considered exons as units of evolution instead of the entire gene and observed feeble differences among populations implying lack of a mutagen-specific effect and the possibility of mutation causing endogenous factors. In all the three genes we observed elevated rates of detrimental mutations in exons encoding regions of significance for the molecular function of the protein. We propose that this can be extended to the entire exome with implications in exon-shuffling and complex human diseases. 相似文献
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Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome 总被引:13,自引:0,他引:13
Lang GA Iwakuma T Suh YA Liu G Rao VA Parant JM Valentin-Vega YA Terzian T Caldwell LC Strong LC El-Naggar AK Lozano G 《Cell》2004,119(6):861-872
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. 相似文献
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Although extensive data indicate that the tumor suppressor TP53 modifies the radiation responses of human and rodent cells, the exact relationship between TP53 and radiation responsiveness remains controversial. To elucidate the relevance of endogenous TP53 genomic status to radiosensitivity in a cell-type-independent manner, different cells of 10 human tumor cell lines with different tissues of origin were examined for TP53 status. The TP53 status was compared with radiation-related cell survival parameters (D(q), D(0), SF2) and with the mode of cell death. Different modes of cell death were examined by measuring radiation-induced micronucleation, apoptosis and abnormal cells. Alterations of the TP53 gene were detected in eight cell lines. No splicing mutation was found. Five cell lines showed codon 68 polymorphism. Codon 72 alterations were found in four cell lines. "Hot spot" alterations were detected in only two of 10 cell lines. Although the cells differed widely in survival parameters (D(q), D(0), SF2) and modes of cell death (micronucleation/apoptosis/abnormal cells) after irradiation, significant cell-type-independent correlations were obtained between the multiple cell death parameter micronucleation/apoptosis/abnormal cells and SF2 (P < 0.001) and D(q) (P = 0.003). Moreover, cells with a wild-type TP53 gene were more resistant to X rays than cells with a mutated TP53 gene or cells that were TP53-deficient. The alterations within exons 5-10 of the TP53 correlated with a enhanced radiosensitivity. For the first time, we demonstrated a correlation between endogenous genetic alterations within exons 5-10 of TP53 and radiation-related cell survival and cell death. This indicates a new molecular relevance of TP53 status to intrinsic cellular radiosensitivity. 相似文献