RESUMO
OBJECTIVES: Physical activity is recommended for disability prevention in the older adult population; however, the level of physical activity required for older adults with chronic kidney disease (CKD) remains unknown. This study aimed to examine the associations between daily physical activity and disability incidence in older adults with and without CKD to determine relevant daily physical activity levels. DESIGN: Prospective observational study. SETTING AND PARTICIPANTS: 3,786 community-dwelling older adults aged ≥65 years. MEASUREMENTS: Mean daily times spent in light- (LPA) and moderate-to-vigorous physical activity (MVPA) were measured using triaxial accelerometers. CKD was defined by a creatinine estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2. Disability incidence was identified as long-term care insurance certification during a 60-month follow-up period. Associations between physical activity and disability incidence were examined using Cox proportional hazard models stratified by the CKD status. Non-linear and linear associations were tested using the restricted cubic spline. RESULTS: A total of 1,054 individuals were identified to have CKD. Disability incidence was higher in the CKD group than in the non-CKD group. The adjusted cox proportional hazard models indicated that a 10-minute increase in MVPA time was associated with lower disability incidence in the non-CKD group (hazard ratio [HR], 0.838; 95% confidence interval [CI]: 0.764-0.918) and the CKD group (HR, 0.859; 95% CI: 0.766-0.960). Linear associations were observed in MVPA for the non-CKD and CKD groups. CONCLUSION: Increasing MVPA was associated with lower disability incidence in older adults with and without CKD. These findings can help devise disability prevention strategies for older CKD patients.
Assuntos
Pessoas com Deficiência , Insuficiência Renal Crônica , Idoso , Exercício Físico , Taxa de Filtração Glomerular , Humanos , Vida IndependenteRESUMO
OBJECTIVE: To analyse the expression of individual forms of cytochrome P450 (CYP) at the mRNA level in five homogenized oral buccal tissue samples from four individuals with or without oral malignancy. METHOD: Individual forms of CYPs were studied by reverse transcriptase-polymerase chain reaction (RT-PCR), using specific primers for CYPs 2B6, 2C, 2D6, 2E1, 3A3/4 and 3A5, and oral CYP expressions were compared with CYP expression in liver tissue. RESULTS: Consistent expression of CYPs 2C, 2E1 and 3A5 was observed in oral buccal tissue at mRNA level. CONCLUSIONS: These particular CYPs have possible roles in the protection of the body against orally ingested xenobiotics as well as influence the bioavailability of therapeutic compounds.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Mucosa Bucal/enzimologia , Idoso , Sistema Enzimático do Citocromo P-450/genética , Perfilação da Expressão Gênica , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Fígado/enzimologia , Pessoa de Meia-Idade , Especificidade de Órgãos , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We found a significant correlation between lung cancer in smokers and the expression of a human gene, D40, predominantly expressed in testis and cancers. In an attempt to clone a novel human gene, we screened a cDNA library derived from a human B cell line and obtained a cDNA clone that we refer to as D40. A search for public databases for sequence homologies showed that the D40 gene is identical to AF15q14. D40 mRNA is predominantly expressed in normal testis tissue. However, this gene is also expressed in various human tumour cell lines and primary tumours derived from various organs and tissues, such as lung cancer. We examined the relationship between D40 expression and clinico-pathological characteristics of tumours in primary lung cancer. D40 expression did not significantly correlate with either histological type or pathological tumour stage. However, D40 expression was observed more frequently in poorly differentiated tumours than in well or moderately differentiated ones. Furthermore, the incidence of D40 expression was significantly higher in tumours from patients who smoke than in those from non-smokers. D40/AF15q14 is the first gene in the cancer/testis family for which expression is related to the smoking habits of cancer patients.
Assuntos
Cromossomos Humanos Par 15 , Neoplasias Pulmonares/genética , Fumar/genética , Testículo , Idoso , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Primers do DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The development of second primary tumors (SPTs) in patients with head and neck squamous cell carcinoma (HNSCC) has become an increasingly important factor in clinical treatment decisions. Currently, clinical and histologic parameters are used to determine whether or not SPT is present. Recent studies suggest that many SPTs in the upper aerodigestive tract have a common clonal origin, challenging the longstanding multiclonal origin concept. To determine genetic relationships among multiple oral cancerous and precancerous lesions (MOCP), we analysed 100 lesions from 26 Japanese patients. Lesion development was synchronous and metachronous. We looked for patterns of microsatellite alterations (MA) using seven markers at chromosomes 3p14, 9p21, and 17p13, where MA occurs early in oral carcinogenesis. Loss of heterozygosity (LOH) was found in 52.6% (41/78), 62.5% (60/96), and 59.3% (32/54) of informative MOCP at 3p14, 9p21, and 17p13, respectively. Microsatellite instability (MI) was observed in 11, 26 and 13% of the samples at 3p14, 9p21, and 17p13 markers, respectively. Patterns of MA were concordant in only nine (14%) of 63 lesions from four (18%) of 22 patients who initially presented with noninvasive lesions. However, two of four patients with invasive cancer as indexed lesion showed 16 (43%) clonally related MOCP among 37 lesions (P=0.003). The results suggest that the majority of MOCP arise from clonally independent cells affected by field cancerization. However, the probability of mucosal spread of clonal malignant or premalignant cells may increase along with malignant progression.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Bucais/genética , Neoplasias Primárias Múltiplas/genética , Segunda Neoplasia Primária/genética , Carcinoma de Células Escamosas/patologia , Estudos de Coortes , Humanos , Perda de Heterozigosidade , Repetições de Microssatélites/genética , Neoplasias Bucais/patologia , Neoplasias Primárias Múltiplas/patologia , Segunda Neoplasia Primária/patologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologiaRESUMO
Oral squamous-cell carcinoma (SCC) is the most common neoplasm in Sri Lanka, accounting for approximately 30% of all cancers in males. Epidemiologic evidence indicates that there is an unequivocal relationship between betel chewing and oral carcinogenesis, suggesting that there may be specific genetic targets of betel-quid ingredients. The p53 gene has been indicated to be a tumor-suppressor gene that is found in mutated form in common human cancers; however, there are few reports about "carcinogen-specific" p53 mutation. Because of this background, primary resected specimens from 23 oral SCCs, 7 leukoplakias and 2 oral submucous fibrosis were collected from oral SCC patients in Sri Lanka and were used for p53 mutation analysis. Exons 5 through 8 of the p53 gene were examined by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing. Mutations in the p53 gene were frequent (10/23) in oral SCC specimens from Sri Lanka. Moreover, the mutations clustered significantly in exon 5 (7/10) of the p53 gene, and small deletions and inclusions other than point mutations were observed. These results indicate that 1) betel-quid chewing may cause specific genetic changes, including mutation in the p53 gene; 2) mutations in the p53 gene are not rare events in SCC patients who are betel-quid chewers, which contrasts with other reports; 3) exon 5 of the p53 gene could be one of the specific targets for some betel-quid ingredients; and 4) betel-quid chewing may be a critical environmental factor in the development of oral SCC.
Assuntos
Areca/efeitos adversos , Carcinoma de Células Escamosas/genética , DNA de Neoplasias/genética , Genes p53/genética , Neoplasias Bucais/genética , Mutação , Plantas Medicinais , Adulto , Idoso , Carcinoma de Células Escamosas/induzido quimicamente , Primers do DNA , Éxons , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/induzido quimicamente , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Sri LankaRESUMO
To determine the timing and actual incidence of p53 mutations in oral epithelial lesions, we examined 33 primary squamous cell carcinomas (SCCs), 14 dysplasias and six hyperplasias from Japanese patients by a combination of yeast functional assay and DNA sequencing. The assay detects mutations of p53 mRNA between codons 67 and 347 on the basis of the DNA-binding activity of the protein. Twenty-six SCCs (79%) and five dysplasias (36%) were positive for p53 mutation, while all six hyperplasias were negative for the mutation. Human papillomavirus type 16 E6 mRNA was detected in one of seven p53 mutation-negative SCCs by reverse transcription polymerase chain reaction (RT-PCR). We further examined p53 mutations in 17 Sri Lankan oral SCCs using the yeast functional assay and the single-strand conformation polymorphism analysis of PCR-amplified DNA fragments (PCR-SSCP) of exon 5-8. The mutations were confirmed by DNA sequencing and the detection sensitivity was compared between the two methods. Six samples (35%) were positive for p53 mutation in PCR-SSCP analysis, while nine samples (53%) were positive in yeast functional assay. This suggests that the incidence of p53 mutations has been considerably underestimated in the conventional SSCP analysis. The present data indicate that p53 mutations are extremely frequent in oral cancers in the Japanese, and suggest that the timing and significance of p53 mutation in oral tumor progression vary in different ethnic populations and areas.
Assuntos
Carcinoma de Células Escamosas/genética , Técnicas Genéticas , Neoplasias Bucais/genética , Mutação , Proteínas Repressoras , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA Viral/análise , Epitélio/patologia , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo Conformacional de Fita Simples , Lesões Pré-Cancerosas , Sensibilidade e Especificidade , Sri Lanka , Leveduras/genéticaRESUMO
Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression was caused by immunological mechanisms, because the tumor cells were renogenized. In this study, we have tried to find out whether tumor-associated antigen (TAA) expression in these xenogenized tumor cells can be modulated by xenogenization. FV-KMT-17 cells (1 x 10(7)), which were subcutaneously transplanted into ten rats, spontaneously regressed after temporary growth. All rats which rejected FV-KMT-17 cells showed strong resistance to rechallenge with KMT-17 (1 x 10(6)) cells. To reveal the chronological modulation of TAA and virus-associated antigen (VAA), a single-cell suspension was obtained from the subcutaneous tumors and expression of these antigens was chronologically measured. TAA, termed CE7 antigen, was examined by anti-CE7 monoclonal antibody (MoAb) and VAA was examined by anti-FK1 MoAb which recognizes the FV env gene product (gp 70). Expression of VAA was not modulated through either the progression or the regression phase, but expression of TAA was strongly enhanced in the regression phase. These results show that enhancement of TAA expression occurs during the regression phase of FV-KMT-17 growth in vivo and that TAA-expressing cells may stimulate anti-tumor immunity, resulting in acquisition of resistance against parental KMT-17 cells.
Assuntos
Antígenos de Neoplasias/biossíntese , Antígenos Virais/biossíntese , Fibrossarcoma/patologia , Vírus da Leucemia Murina de Friend/genética , Animais , Anticorpos Monoclonais , Feminino , Fibrossarcoma/imunologia , Mutagênese , Transplante de Neoplasias , Ratos , Ratos WistarRESUMO
Pulmonary metastasis formation after local radiotherapy against a rat fibrosarcoma was investigated. KMT-17 fibrosarcoma cells were transplanted into the hind leg in syngeneic WKA rats and two different doses (30Gy, 60Gy) of irradiation from a 60Co source were applied 5 days after transplantation. Pulmonary metastasis was inhibited by 30Gy irradiation rather than 60Gy irradiation, which was enough to almost completely cure the local tumors. This inhibitory effect of 30Gy irradiation was induced by the continued presence of irradiated tumors. As for pulmonary metastasis, the different effects of irradiation doses were not recognized when the tumor was removed surgically 1 day after irradiation, but when it was removed 4 days after 30Gy irradiation significantly inhibited metastasis. Expression of tumor-associated antigen (TAA), termed CE7 antigen, on the cell surface was enhanced effectively and continuously by 30Gy irradiation rather than by 60Gy. With this increase in CE7-expressing cells, the enhancement of anti-tumor immunity of spleen cells was observed in an in vitro 125I-IudR release assay and an in vivo tumor-neutralizing assay (Winn assay). The above results suggest that an appropriate dose of irradiation such as 30Gy, to a local tumor can efficiently enhance the TAA expression and that TAA-expressing cells may stimulate anti-tumor immunity, resulting in inhibition of pulmonary metastasis. This phenomenon may offer the possibility of resistance to micrometastasis through the induction of antitumor effector cells.
Assuntos
Antígenos de Neoplasias/análise , Fibrossarcoma/radioterapia , Neoplasias Pulmonares/secundário , Animais , Feminino , Fibrossarcoma/imunologia , Fibrossarcoma/patologia , RatosRESUMO
The p53 gene has been indicated to be a tumour suppressor gene that is found in mutated form in common human cancers. Human papillomavirus (HPV) has oncogenic activity in cervical and oral squamous cell carcinomas (SCCs). The E6 protein of HPV is known to bind with p53 protein and inactive the tumor suppressor activity by promoting p53 degradation. Because of this background, we examined 38 primary, resected specimens of oral SCCs for detection of p53 mutations and HPV DNAs. Exons 5 through 8 of the p53 Mutations were observed in nine cases (24%). HPV-DNA detection and typing were performed using PCR with ¿high risk group' HPV-specified primers. HPV DNA sequences were detected in eight cases (21%). The AvaII digestion pattern of PCR-amplified HPV DNA showed that HPV-16 was present in all eight cases. Seven cases were p53 mutation-positive/HPV-negative, six cases were p53 mutation-negative/HPV-positive, and two intraosseus SCC cases were p53 mutation-positive/ HPV-positive. Thus, 15/38 (40%) cases had inactivation of the p53 protein. Interestingly, p53 mutation-negative/ HPV-negative cases had a poorer prognosis than p53 mutation positive or HPV-positive cases (P < 0.01). We conclude that (1) mutation in the p53 gene and/or HPV infection are frequent (40%) in oral SCC; (2) inactivation of p53 function by mutation and HPV infection are important genetic events in the development of 40% integral of oral SCCs; (3) p53 mutation and HPV infection are not mutually exclusive events and (4) other oncogenes or tumor suppressor genes may be crucial in the development of oral SCC if the prognosis is poor.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virologia , Genes p53/genética , Neoplasias Bucais/genética , Mutação , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Neoplasias Bucais/virologia , Estadiamento de Neoplasias , Prognóstico , Distribuição Tecidual , Infecções Tumorais por Vírus/genéticaRESUMO
We have previously reported that rat fibrosarcoma KMT-17 cells and their in vitro counterparts, cloned A3 cells, shed a tumor-associated antigen (TAA), termed CE7, from the cell surface on vesicular membranes, under growth-enhancing conditions. This study shows that irradiation (1 approximately Gy) from a 60Co source, inhibited A3 cell growth dose-dependently and correspondingly increased CE7 expression by A3 cells as determined by anti-CE7 monoclonal antibody using flow cytometry. CE7 expression gradually increased with increasing doses of irradiation and reached a peak level at 30Gy. After 30Gy irradiation, CE7 expressing A3 cells were fixed with 1% paraformaldehyde and were used to intradermally immunize syngenic rats. Immunized rats developed transplantation resistance to the parent KMT-17 cells as compared to rats immunized with unirradiated A3 cells. Rat MHC class 1 antigen expression was slightly decreased by irradiation and therefore, resistance to tumor transplantation appeared to arise solely due to the enhancing effects of irradiation on TAA expression which increases the antigenicity of the tumor cells coverting them to an effective stimulator of antitumor effector cells. This phenomenon may offer a possibility of the resistance to the re-emergence and metastasis of the tumor like a KMT-17 through the induction of antitumor memory cells.
Assuntos
Antígenos de Neoplasias/biossíntese , Fibrossarcoma/imunologia , Fibrossarcoma/radioterapia , Animais , Anticorpos Monoclonais , Divisão Celular/efeitos da radiação , Feminino , Fibrossarcoma/metabolismo , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/efeitos da radiação , Imunoterapia Ativa , Ratos , Ratos WistarRESUMO
BACKGROUND: The etiology of oral squamous cell carcinoma (SCC) is still obscure. Since human papillomavirus (HPV) DNAs are associated with carcinoma of the uterine cervix, carcinomas of the oral cavity were investigated to ascertain if these viruses are present in squamous carcinomas of this anatomic site. METHODS: Seventy-seven oral mucosal SCCs were examined for the presence of HPV DNAs by polymerase chain reaction and dot blot hybridization. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and p53 was performed and single strand conformation polymorphism analysis for p53 was undertaken. In situ hybridization detection of HPV-16 DNA also was performed. RESULTS: Human papillomavirus-16 DNA was detected in 23 cases of oral SCC and both HPV-16 and HPV-18 DNA were detected in one case of tongue SCC. Human papillomavirus DNAs were detected of 11 of 33 tongue, 4 of 15 gingival, 2 of 4 palate, 2 of 5 buccal mucosa, 3 of 7 maxillary sinus, and 2 of 11 the floor of the mouth SCCs. None were detected in SCCs of the retromolar region (0/2). Immunohistochemical examination for p53 was performed in 26 cases of oral SCC and the accumulation of p53 protein was observed in 6 cases (i.e., in 4 of 17 HPV DNA-negative cases and in 2 of 9 HPV DNA-positive cases). Single strand conformation polymorphism analysis confirmed gene mutations in all 6 cases. Human papillomavirus-16 DNA was predominantly identified in cancer cells that showed a morphologic resemblance to basal cells and its hybridized signal in keratinized cells was reduced by in situ hybridization detection. Immunohistochemical detection of PCNA revealed its cooccurrence with HPV-16 DNA in cancer cells. CONCLUSIONS: These results suggest that HPV-16 DNA sequences may have the capability to maintain the proliferative state of epithelial cells, and may contribute to the production of malignant phenotypes.
Assuntos
Carcinoma de Células Escamosas/virologia , DNA Viral/análise , Neoplasias Bucais/virologia , Papillomaviridae/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/metabolismo , Feminino , Genes Supressores de Tumor/genética , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Neoplasias Bucais/genética , Neoplasias Bucais/imunologia , Neoplasias Bucais/metabolismo , Mutação , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Proteína Supressora de Tumor p53/genéticaRESUMO
We investigated the correlation of p53 abnormalities with survival in 85 patients with non-small cell lung cancer (NSCLC) who had undergone resection with curative intent as part of Lung Cancer Study Group (LCSG) 871. Our previous studies showed that only a subset of p53 mutations in lung cancers result in overexpression. In addition, protein overexpression has been described in the absence of mutation. Therefore, we determined both p53 protein overexpression (by immunostaining) and p53 and ras gene mutations (by single-strand conformation polymorphism and DNA sequencing) in this set of resected tumor specimens. Clinical follow-up data were available for 75 cases. Of the studied patients, 64% showed p53 overexpression and 51% had mutant p53 sequences; however, the concordance rate was only 67%. There was a negative survival correlation with positive p53 immunostaining (p = 0.05), but not with the presence of gene mutations (p = 0.62) in this group of patients. Overexpression of p53 protein determined by immunostaining may contribute to adverse outcome due to the ability of p53 to act as a dominant oncogene, or alternatively, overexpression may reflect ongoing DNA damage in the tumor as a marker for a more aggressive behavior. When adjusted for stage, age, and gender by multivariate analysis, however, there was no independent impact of p53 overexpression on survival.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Expressão Gênica , Genes p53/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Mutação , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Análise Mutacional de DNA , Feminino , Genes ras , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Polimorfismo Conformacional de Fita Simples , Prognóstico , Taxa de SobrevidaRESUMO
CE7 antigen is shed from A3 cell surfaces by cells grown in medium containing a sufficient (10%) amount of fetal calf serum (FCS), but shedding of the antigen decreases with a decrease in FCS content in the culture medium. However, the cells contain similar amounts of antigen as evidenced by Western blotting, indicating that low FCS levels interfere with antigen shedding but not antigen synthesis. Antigen expression by A3 cells treated with mitomycin C gradually shifted from negative to a strong positive with time, and on day 2, two peaks corresponding to negative and positive cells within the population can be observed. In contrast, A3 cells treated with bleomycin and cyclophosphamide shifted as a whole from negative to weakly positive. When A3 cells in media containing 10% FCS were incubated at 4 degrees C, although the cells did not proliferate, antigen expression could not be detected by flow cytometry.
Assuntos
Antígenos de Neoplasias/biossíntese , Antineoplásicos/farmacologia , Fibrossarcoma/imunologia , Animais , Antígenos de Neoplasias/imunologia , Western Blotting , Temperatura Baixa , Meios de Cultura , Citometria de Fluxo , Ratos , Ratos Endogâmicos , Células Tumorais CultivadasRESUMO
The p53 gene was examined in primary lymphoblasts of 25 pediatric patients with acute lymphoblastic leukemia by the RNase protection assay and by single strand conformation polymorphism analysis in 23 of 25 cases. p53 mutations were found to occur, but at a low frequency (4 of 25). While all four mutations were identified by single strand conformation polymorphism, the comparative sensitivity of RNase protection was 50% (2 of 4). Heterozygosity was retained at mutated codons in 3 of 4 cases. One pedigree was consistent with the Li-Fraumeni syndrome, and bone marrow from both diagnosis and remission indicated a germline G to T transversion at codon 272 (valine to leucine). Although members of another family were affected with leukemia, a 2-bp deletion in exon 6 was nonhereditary. The other two nonhereditary p53 mutations included a T to G transversion at codon 270 (phenylalanine to cysteine) and a G to C transversion at codon 248 (arginine to proline). These data support the role of both hereditary and acquired p53 mutations in the pathogenesis and/or progression of some cases of childhood acute lymphoblastic leukemia.
Assuntos
Genes p53 , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Adulto , Sequência de Bases , Linfoma de Burkitt/genética , Criança , Pré-Escolar , Deleção Cromossômica , Humanos , Lactente , Recém-Nascido , Síndrome de Li-Fraumeni/genética , Dados de Sequência Molecular , Polimorfismo GenéticoRESUMO
We have compared the effects of specific point mutations on the tertiary and quaternary structure of the human p53 protein. Eight mutants, each derived from primary resected tissues of lung carcinomas, were expressed in vitro under strictly defined conditions, such that the only known variant was the point mutation present in each p53 mRNA. All the mutations were located in highly conserved domains. The tertiary structure of each mutant protein was investigated by reactivity with anti-p53 monoclonal antibodies directed against conformation-dependent epitopes. Quaternary structure was examined by gel filtration. Although all the mutant proteins exhibited abnormal tertiary structures, their quaternary structures appeared similar to wild type, the one exception being p53-tyr135, which contains tyrosine in place of cysteine at residue 135. The conformational phenotype of mutant human p53 was found to be dependent upon (i) the locus of the mutation and (ii) the nature of the amino acid substitution: two different substitutions at residue 273 yielded two mutants with differing structural properties. We have discovered three mutants of human p53 that are temperature sensitive for conformation; one is mutated at codon 273, a 'hotspot' for p53 mutation in human cancer.
Assuntos
Carcinoma/química , Genes p53/genética , Neoplasias Pulmonares/química , Mutação/genética , Proteína Supressora de Tumor p53/química , Humanos , Fenótipo , Biossíntese de Proteínas , Conformação Proteica , Temperatura , Proteína Supressora de Tumor p53/análiseRESUMO
Twenty-six primary breast tumors were examined for mutations in the p53 tumor suppressor gene by an RNase protection assay and nucleotide sequence analysis of PCR-amplified p53 complementary DNAs. Each method detected p53 mutations in the same three tumors (12%). One tumor contained two mutations in the same allele. Single strand conformation polymorphism analysis of genomic DNA and complementary DNA proved more sensitive in the detection of mutations. Combining this technique with the other two a total of 12 mutations in the p53 gene were demonstrated in 11 tumors (46%), and a polymorphism at codon 213 was detected in another tumor. Loss of heterozygosity on chromosome 17p was detected by Southern blot analysis in 30% of the tumor DNAs. Not all of the tumors containing a point mutation in p53 also had loss of heterozygosity of the remaining allele, suggesting that loss of heterozygosity may represent a later event.
Assuntos
Neoplasias da Mama/genética , Genes p53 , Mutação , Sequência de Bases , Mapeamento Cromossômico , DNA de Neoplasias/análise , Feminino , Heterozigoto , Humanos , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , RNA Neoplásico/análiseRESUMO
This report describes a rare polymorphism at codon 213 (silent alteration of CGA to CGG) within the coding region of the p53 gene. The rare polymorphic allele was present in six cases out of 189 lung and breast cancer DNAs analyzed (3.2%) and resulted in the loss of a TaqI site. This allele could be mistaken for a mutation when screening methods of mutation analysis are used without comparison with normal tissue DNA.
Assuntos
Códon/genética , Genes p53 , Polimorfismo Genético , Sequência de Bases , Neoplasias da Mama/genética , DNA de Neoplasias/genética , Feminino , Humanos , Neoplasias Pulmonares/genéticaRESUMO
We explored the state of the p53 gene in gastric cancer. Using one or more methods, we examined 15 specimens from primary carcinomas (14 tumors, one cell line), five cell lines derived from metastases, and seven paired samples of nonmalignant gastric mucosa. Sequence analyses of complementary DNA containing the entire p53 gene open reading frame demonstrated abnormalities in one of five samples from primary tumors and in all five samples from metastases. The single cell line derived from a primary carcinoma had no abnormality of the gene. The six abnormalities included four point mutations, one base-pair deletion resulting in a frame shift, and a 24 base-pair deletion caused by an intronic point mutation (as determined by sequence analysis of genomic DNA). Four of the six mutations mapped to regions highly conserved among species or involved in simian virus 40 T-antigen binding. Restriction fragment length polymorphism studies confirmed that chromosome 17p allelic deletions occur only in a minority of primary tumors, but that they may occur more frequently in metastases. Northern blotting and ribonuclease protection assays detected only a fraction of the p53 gene abnormalities detected by sequencing. Our findings indicate that mutations of the p53 gene are relatively rare in primary gastric tumors but appear to be relatively frequent in cell lines derived from metastatic lesions. Our results may help in understanding the molecular events associated with progression and metastasis in gastric carcinoma.
Assuntos
Genes p53/genética , Neoplasias Gástricas/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , DNA de Neoplasias/genética , Expressão Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/genética , Neoplasias Gástricas/patologia , Células Tumorais CultivadasRESUMO
Point mutations in genes can be etiologic of pulmonary diseases, as in the case of the inherited disorders alpha-1-antitrypsin deficiency and cystic fibrosis or in the context of dominant and recessive oncogenes in lung cancer. Various methodologies have been developed to screen for single-base mutations. These techniques include direct DNA sequencing, RNase protection, denaturing gradient gel electrophoresis, and chemical mismatch cleavage. The latter method offers the advantages of rapid and efficient analysis of genomic or cDNA and is thus ideally suited to screening applications. Furthermore, all possible single-base changes can theoretically be detected. In the present work, chemical mismatch cleavage was utilized to detect mutations in the p53 gene in small cell and non-small cell lung cancer. This technique was modified by using a two-step, hemi-nested PCR procedure for preparation of target genomic DNAs permitting an expanded target size for analysis. Evaluation by chemical mismatch cleavage of eight p53 cDNAs derived from lung tumors shown to have different mutations by DNA sequencing correctly detected the presence of a point mutation in all instances. Analysis of six additional tumor genomic DNAs with defined mutations in the corresponding p53 cDNAs accurately confirmed the mutation at the level of the genome. The technique also identified codon 72 and intron 6 polymorphisms. Using the intron 6 polymorphism, loss of heterozygosity at the p53 locus in tumor DNA was readily detected by chemical mismatch cleavage. Finally, utilizing this technique for scanning analysis of the p53 gene of uncharacterized lung tumor DNAs, additional mutations were identified in a prospective manner which were confirmed by sequence analysis.(ABSTRACT TRUNCATED AT 250 WORDS)