Assuntos
Separação Celular/métodos , Células Epiteliais/citologia , Biologia Molecular/métodos , Ácidos Nucleicos/isolamento & purificação , Neoplasias do Colo , DNA de Neoplasias/genética , Genes p53 , Globinas/genética , Humanos , Queratinas/genética , Glomérulos Renais , Perda de Heterozigosidade , Masculino , PróstataRESUMO
BACKGROUND: Carcinogenesis is a multistep process, which may begin as a consequence of chromosomal changes. Deletions in the short arm of chromosome 9 (9p) have been observed in lung carcinomas. In addition, morphologically recognizable preneoplastic lesions, frequently multiple in number, precede onset of invasive carcinomas. PURPOSE: We tested for deletions and loss of heterozygosity (LOH) at 9p loci in preneoplastic and neoplastic foci in lungs of patients with non-small-cell lung carcinomas (NSCLCs). METHODS: Seven archival, paraffin-embedded, surgically resected NSCLC specimens were selected. They were predominantly from patients with adenocarcinomas and contained multiple preneoplastic lesions, including hyperplasia, metaplasia, dysplasia, and carcinoma in situ (CIS). Fifty-three histologically identified preneoplastic and malignant lesions present in bronchi, bronchioles, and alveoli were precisely microdissected from stained tissue sections with a micromanipulator. Stromal lymphocytes were used to determine constitutional heterozygosity. The specimens were analyzed for LOH using polymerase chain reaction-based assays for polymorphism in dinucleotide repeats (microsatellite markers) in interferon alfa (IFNA) and D9S171 loci on 9p. RESULTS: All seven cases were constitutionally heterozygous for one or both microsatellite markers. Five of seven cases had LOH at one or both 9p loci in the invasive primary cancers (doubly informative cases). Four of these five cases also revealed LOH in preneoplastic foci. In the doubly informative cases, LOH was detected in five (38%) of 13 foci of hyperplasia, four (80%) of five foci of dysplasia, and three (100%) of three CIS lesions. LOH was detected in preneoplastic lesions from all regions of the respiratory tract, including bronchi, bronchioles, and alveoli, and involved five different cell types. The identical allele was lost from both the preneoplastic lesions and the corresponding tumors (12 of 12 lesions, 17 of 17 comparisons), a phenomenon we have referred to as "allele-specific mutation." Statistical analyses employing a cumulative binomial test demonstrated that the probabilities of such findings occurring by chance are 2.4 x 10(-4) and 7.6 x 10(-6), respectively. From comparisons with the previously published data on other chromosomal abnormalities in the same tissue specimens, it appears that LOH at 3p and 9p loci occurred early in the hyperplasia stage, but the ras gene point mutations were relatively late, at the CIS stage. CONCLUSIONS: LOH at 9p loci occurs at the earliest stage in the pathogenesis of lung cancer and involves all regions of the respiratory tract. LOH in NSCLC is not random but targets a specific allele in individuals. Studying preneoplastic lesions may help identify intermediate markers for risk assessment and chemoprevention.
Assuntos
Alelos , Carcinoma Pulmonar de Células não Pequenas/genética , Deleção Cromossômica , Cromossomos Humanos Par 8/genética , Neoplasias Pulmonares/genética , Síndromes Paraneoplásicas/genética , Adenocarcinoma/genética , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/complicações , Feminino , Genes ras/genética , Heterozigoto , Humanos , Neoplasias Pulmonares/complicações , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Síndromes Paraneoplásicas/complicaçõesRESUMO
BACKGROUND: Several molecular changes, including loss of heterozygosity (i.e., deletion of one copy of allelic DNA sequences) and alterations in microsatellite DNA, have been detected early in the pathogenesis of lung cancer, even in histologically normal epithelium. In the bronchial epithelium of patients with lung cancer, we have determined the frequency, size, and patterns of molecularly abnormal clonal patches. METHODS: We studied formalin-fixed, paraffin-embedded samples from 16 surgically resected lung carcinomas (five squamous cell carcinomas, four small-cell carcinomas, six adenocarcinomas, and one large-cell carcinoma). From each carcinoma, we microdissected foci (each containing about 200 cells) of tumor tissue and equivalent samples of histologically normal and abnormal epithelium. Furthermore, multiple discontinuous foci of bronchial epithelium were analyzed from methanol-fixed samples from three additional patients with lung cancer (two with squamous cell carcinoma and one with adenocarcinoma). We used two-step polymerase chain reaction-based assays involving 12 microsatellite markers at seven chromosomal regions frequently deleted in lung cancer. RESULTS: Two hundred eighteen foci of nonmalignant bronchial epithelium (195 of histologically normal or slightly abnormal epithelium and 23 of dysplastic epithelium) were studied from the 19 surgically resected lobectomy specimens. Thirteen (68%) of the 19 specimens had at least one focus of bronchial epithelium with molecular changes. At least one molecular abnormality was detected in 32% of the 195 histologically normal or slightly abnormal foci and in 52% of the 23 dysplastic foci. Extrapolating from our two-dimensional analyses, we estimate that most clonal patches contain approximately 90 000 cells. Although, in a given individual, tumors appeared homogeneous with respect to molecular changes, the clonally altered patches of mildly abnormal epithelium were heterogeneous. CONCLUSIONS: Our findings indicate that multiple small clonal or subclonal patches containing molecular abnormalities are present in normal or slightly abnormal bronchial epithelium of patients with lung cancer.
Assuntos
Brônquios/patologia , Carcinoma/genética , Carcinoma/patologia , Células Clonais/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/patologia , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Epitélio/patologia , Humanos , Perda de Heterozigosidade , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Most lung cancers are attributed to smoking. These cancers have been associated with multiple genetic alterations and with the presence of preneoplastic bronchial lesions. In view of such associations, we evaluated the status of specific chromosomal loci in histologically normal and abnormal bronchial biopsy specimens from current and former smokers and specimens from nonsmokers. METHODS: Multiple biopsy specimens were obtained from 18 current smokers, 24 former smokers, and 21 nonsmokers. Polymerase chain reaction-based assays involving 15 polymorphic microsatellite DNA markers were used to examine eight chromosomal regions for genetic changes (loss of heterozygosity [LOH] and microsatellite alterations). RESULTS: LOH and microsatellite alterations were observed in biopsy specimens from both current and former smokers, but no statistically significant differences were observed between the two groups. Among individuals with a history of smoking, 86% demonstrated LOH in one or more biopsy specimens, and 24% showed LOH in all biopsy specimens. About half of the histologically normal specimens from smokers showed LOH, but the frequency of LOH and the severity of histologic change did not correspond until the carcinoma in situ stage. A subset of biopsy specimens from smokers that exhibited either normal or preneoplastic histology showed LOH at multiple chromosomal sites, a phenomenon frequently observed in carcinoma in situ and invasive cancer. LOH on chromosomes 3p and 9p was more frequent than LOH on chromosomes 5q, 17p (17p13; TP53 gene), and 13q (13q14; retinoblastoma gene). Microsatellite alterations were detected in 64% of the smokers. No genetic alterations were detected in nonsmokers. CONCLUSIONS: Genetic changes similar to those found in lung cancers can be detected in the nonmalignant bronchial epithelium of current and former smokers and may persist for many years after smoking cessation.
Assuntos
Brônquios/metabolismo , Deleção Cromossômica , Dano ao DNA , Fumar/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Autorradiografia , Brônquios/patologia , Epitélio/metabolismo , Epitélio/patologia , Feminino , Heterozigoto , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Genético , Abandono do Hábito de Fumar , Fatores de TempoRESUMO
BACKGROUND: Retinoic acid plays an important role in lung development and differentiation, acting primarily via nuclear receptors encoded by the retinoic acid receptor-beta (RARbeta) gene. Because receptor isoforms RARbeta2 and RARbeta4 are repressed in human lung cancers, we investigated whether methylation of their promoter, P2, might lead to silencing of the RARbeta gene in human lung tumors and cell lines. METHODS: Methylation of the P2 promoter from small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cell lines and tumor samples was analyzed by the methylation-specific polymerase chain reaction (PCR). Expression of RARbeta2 and RARbeta4 was analyzed by reverse transcription-PCR. Loss of heterozygosity (LOH) was analyzed by PCR amplification followed by electrophoretic separation of PCR products. Statistical differences were analyzed by Fisher's exact test with continuity correction. RESULTS: The P2 promoter was methylated in 72% (63 of 87) of SCLC and in 41% (52 of 127) of NSCLC tumors and cell lines, and the difference was statistically significant (two-sided P:<.001). By contrast, in 57 of 58 control samples, we observed only the unmethylated form of the gene. Four tumor cell lines with unmethylated promoter regions expressed both RARbeta2 and RARbeta4. Four tumor lines with methylated promoter regions lacked expression of these isoforms, but demethylation by exposure to 5-aza-2'-deoxycytidine restored their expression. LOH at chromosome 3p24 was observed in 100% (13 of 13) of SCLC lines and 67% (12 of 18) of NSCLC cell lines, and the difference was statistically significant (two-sided P: =.028). CONCLUSIONS: Methylation of the RARbeta P2 promoter is one mechanism that silences RARbeta2 and RARbeta4 expression in many lung cancers, particularly SCLC. Chemical demethylation is a potential approach to lung cancer therapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Pequenas/metabolismo , Perda de Heterozigosidade , Neoplasias Pulmonares/metabolismo , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Pequenas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Metilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
To identify the major tumor suppressor gene (TSG) loci involved in the pathogenesis of lung cancer, we have conducted a high-resolution (10 cM), genome-wide search of loss of heterozygosity (LOH). Thirty-six lung cancer cell lines [14 small cell lung cancers (SCLCs) and 22 non-SCLCs (NSCLCs)] and their matched control DNAs were analyzed using 399 fluorescent microsatellite markers from the ABI Prism linkage mapping set v.2 on an ABI 377 sequencer/genotyper. Overall, 22 different regions with more than 60% LOH were identified: (a) 13 regions with a preference for SCLC; (b) 7 regions with a preference for NSCLC; and (c) 2 regions affecting both SCLC and NSCLC. The chromosomal arms with the most frequent LOH were 1p, 3p, 4p, 4q, 5q, 8p, 9p (p16), 9q, 10p, 10q, 13q (Rb), 15q, 17p (p53), 18q, 19p, Xp, Xq. In addition, new homozygous deletions were found at 2p23, 8q24, 18q11, and Xq22. On average, 34% (SCLC) to 36% (NSCLC) of markers showed allele loss in individual tumors, with an average size of subchromosomal region of loss of five to six markers (50-60 cM). Whereas SCLC and NSCLC had different regions of frequent LOH (hot spots), and NSCLC had more of these regions (n = 22) than SCLC (n = 17), in all other parameters (fractional allelic loss, number of breakpoints, and number of microsatellite alterations), SCLC and NSCLC were not significantly different. Clustering analysis revealed correlations between LOH on different chromosomes that suggest previously unknown genetic interactions for lung cancer development. We conclude that (a) in lung cancer cell lines, at least 17-22 chromosomal regions with frequent allele loss are involved, suggesting that the same number of putative TSGs are inactivated; (b) SCLC and NSCLC frequently undergo different specific genetic alterations; and (c) clusters of TSGs are likely to be inactivated together. Overall, these data provide global estimates of the extent of genetic changes leading to lung cancer and will be useful for the positional cloning of new TSGs and for the identification of multiple new biomarkers for translational research.
Assuntos
Alelos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Pequenas/genética , Genoma Humano , Neoplasias Pulmonares/genética , Análise por Conglomerados , DNA de Neoplasias/genética , Feminino , Genes Supressores de Tumor/genética , Marcadores Genéticos , Homozigoto , Humanos , Perda de Heterozigosidade/genética , Masculino , Família Multigênica/genética , Células Tumorais CultivadasRESUMO
Aberrant methylation of CpG islands acquired in tumor cells in promoter regions is one method for loss of gene function. We determined the frequency of aberrant promoter methylation (referred to as methylation) of the genes retinoic acid receptor beta-2 (RARbeta), tissue inhibitor of metalloproteinase 3 (TIMP-3), p16INK4a, O6-methylguanine-DNA-methyltransferase (MGMT), death-associated protein kinase (DAPK), E-cadherin (ECAD), p14ARF, and glutathione S-transferase P1 (GSTP1) in 107 resected primary non-small cell lung cancers (NSCLCs) and in 104 corresponding nonmalignant lung tissues by methylation-specific PCR. Methylation in the tumor samples was detected in 40% for RARbeta, 26% for TIMP-3, 25% for p16INK4a, 21% for MGMT, 19% for DAPK, 18% for ECAD, 8% for p14ARF, and 7% for GSTP1, whereas it was not seen in the vast majority of the corresponding nonmalignant tissues. Moreover, p16INK4a methylation was correlated with loss of p16INK4a expression by immunohistochemistry. A total of 82% of the NSCLCs had methylation of at least one of these genes; 37% of the NSCLCs had one gene methylated, 22% of the NSCLCs had two genes methylated, 13% of the NSCLCs had three genes methylated, 8% of the NSCLCs had four genes methylated, and 2% of the NSCLCs had five genes methylated. Methylation of these genes was correlated with some clinicopathological characteristics of the patients. In comparing the methylation patterns of tumors and nonmalignant lung tissues from the same patients, there were many discordancies where the genes methylated in nonmalignant tissues were not methylated in the corresponding tumors. This suggests that the methylation was occurring as a preneoplastic change. We conclude that these findings confirm in a large sample that methylation is a frequent event in NSCLC, can also occur in smoking-damaged nonmalignant lung tissues, and may be the most common mechanism to inactivate cancer-related genes in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Neoplasias Pulmonares/genética , Regiões Promotoras Genéticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose , Caderinas/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Proteínas Quinases Associadas com Morte Celular , Feminino , Genes p16/genética , Glutationa S-Transferase pi , Glutationa Transferase/genética , Humanos , Imuno-Histoquímica , Isoenzimas/genética , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , O(6)-Metilguanina-DNA Metiltransferase/genética , Reação em Cadeia da Polimerase/métodos , Proteínas/genética , Receptores do Ácido Retinoico/genética , Fatores de Risco , Taxa de Sobrevida , Inibidor Tecidual de Metaloproteinase-3/genética , Proteína Supressora de Tumor p14ARFRESUMO
We investigated preneoplastic lesions associated with lung cancer to determine at what stage in lung carcinogenesis K-ras mutations appear. We selected six archival lung cancer resection cases that had ras mutations. We precisely microdissected 74 relevant areas from paraffin-embedded sections. K-ras mutations at codons 12, 13, and 61 were determined by the designed restriction fragment length polymorphism method using mismatched nested primers and confirmed by direct sequencing. All samples of invasive and metastatic cancers had K-ras mutations, as did four of five lesions of noninvasive cancer. Mutations were detected in only 1 of 12 dysplastic lesions and were absent from hyperplastic and normal-appearing cells. In all cases, the specific point mutations and the mutational pattern in the tumors, metastases, and the corresponding noninvasive lesions were identical. These results indicate that K-ras mutations arise relatively late in the pathogenesis of lung cancer and may be associated with the appearance of the malignant phenotype.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Genes ras/genética , Neoplasias Pulmonares/genética , Mutação Puntual/genética , Lesões Pré-Cancerosas/genética , Códon/genética , Humanos , Polimorfismo de Fragmento de RestriçãoRESUMO
Allelic losses on the short arm of chromosome 8 (8p) have been reported as frequent events in several cancers, including lung. However, no comprehensive mapping analysis of chromosome 8p in lung cancer tumors has been performed, and no data are available about the stage at which these abnormalities occur during the multistage development of lung cancer. Using 26 microsatellite markers, we mapped the chromosome 8 regions frequently deleted in lung cancer in 13 small cell carcinoma and 17 non-small cell lung carcinoma cell lines and in 68 microdissected archival primary lung tumors (22 small cell lung carcinomas, 25 squamous cell carcinomas, and 21 adenocarcinomas). We also studied the role of 8p deletions in lung cancer pathogenesis by examining 95 microdissected normal epithelium and preneoplastic samples from 11 surgically resected squamous cell lung carcinomas and from 58 bronchoscopy biopsy samples obtained from 31 current and former smokers. High frequencies of deletions at 8p21-23 regions were detected in lung cancer cell lines and in primary lung tumors. Deletions commenced early during the multistage development of lung cancer at the hyperplasia/metaplasia stage in cancer patients and in smokers without cancer. Allelic deletions persisted for up to 48 years after smoking cessation. There was a progressive increase of the overall 8p21-23 loss of heterozygosity frequency and in the size of the deleted region with increasing severity of histopathological preneoplastic changes. In epithelial samples from resected squamous cell lung carcinomas, we compared the presence of loss of heterozygosity at 8p21-23 with deletions at chromosomes 3p and 9p. Of interest, the pattern of deletions was not random, and 8p21-23 allelic losses always followed 3p deletions and usually followed 9p deletions. We conclude that 8p21-23 deletions are frequent and early events in the pathogenesis of lung carcinomas.
Assuntos
Cromossomos Humanos Par 8 , Perda de Heterozigosidade , Neoplasias Pulmonares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Brônquios/fisiologia , Deleção Cromossômica , Mapeamento Cromossômico , Epitélio/fisiologia , Feminino , Genes Supressores de Tumor , Humanos , Masculino , Pessoa de Meia-Idade , Fumar/genéticaRESUMO
Although gallbladder carcinoma is one of the most frequent neoplasms in Chile, there is limited information about the molecular changes involved in its pathogenesis. We investigated the incidence of ras gene mutations and loss of heterozygosity (LOH) at the following genes/loci: p53, DCC, rb, 5q 3p, 8p, and 9p. We precisely microdissected 194 relevant areas from paraffin-embedded microslides from 25 gallbladder carcinomas and their accompanying nonneoplastic lesions (which were present in 15 cases) from patients in Chile. The specimens were analyzed by PCR-based assays for LOH, and we designed a RFLP method for ras mutations and immunohistochemistry for p53 protein overexpression. We determined that LOH at p53 (91%), 9p (50%), 8p (44%) and DCC (31%) are frequent events and that LOH at p53, 9p, and DCC are early events, while ras mutations and LOH at 3p, rb, and 5q occurred occasionally. LOH at p53 occurred more frequently and earlier than protein overexpression. The mean number of mutations present in invasive carcinomas was 2.1, and in six cases, LOH at the p53 gene was the sole mutation detected. The same allele was lost in 61 (93%) of 71 nonneoplastic foci as in the corresponding invasive carcinomas for all four mutations studied. The odds of this occurring by chance are approximately 4 x 10(-15). Although clonality cannot be excluded, allelic loss appears to be highly directed, but the mechanism for allele-specific mutations remains to be determined.
Assuntos
Alelos , Deleção Cromossômica , Cromossomos Humanos , Neoplasias da Vesícula Biliar/genética , Mutação , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Sequência de Bases , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Carcinoma de Células Pequenas/cirurgia , Chile/epidemiologia , Epitélio/patologia , Vesícula Biliar/patologia , Neoplasias da Vesícula Biliar/epidemiologia , Neoplasias da Vesícula Biliar/patologia , Neoplasias da Vesícula Biliar/cirurgia , Genes do Retinoblastoma , Genes p53 , Genes ras , Marcadores Genéticos , Humanos , Metaplasia , Invasividade Neoplásica , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Sequências Repetitivas de Ácido NucleicoRESUMO
Expression of some members of the cadherin family is reduced in several human tumors, and CDH13 (H-cadherin), located on chromosome 16q24.2-3, may function as a tumor suppressor gene. In human tumors, loss of expression of many tumor suppressor genes occurs by aberrant promoter region methylation. We examined the methylation status of the CDH13 promoter in breast and lung cancers and correlated it with mRNA expression using methylation-specific PCR and reverse transcription-PCR. Methylation was frequent in primary breast tumors (18 of 55, 33%) and cell lines (7 of 20, 35%). In lung cancers, methylation was present more frequently in non-small cell lung cancer tumors (18 of 42, 43%) and cell lines (15 of 30, 50%) than in small cell lung cancer cell lines (6 of 30, 20%; P = 0.03). Only the methylated or unmethylated forms of the gene were present in most (73 of 80, 91%) tumor cell lines. CDH13 expression was present in 24 of 30 (80%) of nonmethylated tumor lines. All 18 methylated lines tested lacked expression irrespective of whether the unmethylated form was present, confirming biallelic inactivation in methylated lines. Gene expression was restored in all five methylated cell lines tested after treatment with the demethylating agent 5'-aza-2-deoxycytidine. Our results demonstrate frequent aberrant methylation of CDH13 in breast and lung cancers accompanied by loss of gene expression, although expression may occasionally be lost by other mechanisms.
Assuntos
Neoplasias da Mama/genética , Caderinas/genética , Metilação de DNA , Neoplasias Pulmonares/genética , Neoplasias da Mama/metabolismo , Caderinas/biossíntese , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/metabolismo , DNA de Neoplasias/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Humanos , Perda de Heterozigosidade , Neoplasias Pulmonares/metabolismo , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
Allele loss and loss of expression of fragile histidine triad (FHIT), a putative tumor suppressor gene located in chromosome region 3p14.2, are frequent in several types of cancers. Tumor-acquired methylation of promoter region CpG islands is one method for silencing tumor suppressor genes. We investigated 5' CpG island methylation of the FHIT gene in 107 primary non-small cell lung cancer (NSCLC) samples and corresponding nonmalignant lung tissues, 39 primary breast carcinomas, as well as in 49 lung and 22 breast cancer cell lines by a methylation-specific PCR assay. In addition, we analyzed brushes from the bronchial epithelium of 35 heavy smokers without cancer. FHIT methylation was detected in 37% of primary NSCLCs, 31% of primary breast cancers, and 65% of lung and 86% of breast cancer cell lines. The frequency of methylation in small cell and NSCLC cell lines were identical. Methylation was found in 9% of the corresponding nonmalignant lung tissues and in 17% of bronchial brushes from heavy cigarette smokers. FHIT methylation was significantly correlated with loss of FHIT mRNA expression by Northern blot analysis in lung cancer cell lines and with loss of Fhit expression in NSCLC and breast tumors by immunostaining. We conclude that methylation of FHIT is a frequent event in NSCLC and breast cancers and is an important mechanism for loss of expression of this gene. Methylation of FHIT commences during lung cancer pathogenesis and may represent a marker for risk assessment.
Assuntos
Hidrolases Anidrido Ácido , Azacitidina/análogos & derivados , Neoplasias da Mama/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Ilhas de CpG , Metilação de DNA , Inativação Gênica , Neoplasias Pulmonares/genética , Proteínas de Neoplasias , Proteínas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Biomarcadores Tumorais/genética , Northern Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Ilhas de CpG/genética , Decitabina , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Perda de Heterozigosidade , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Allelotyping studies suggest that allelic losses at one or both arms of chromosome 4 are frequent in several tumor types, but information about breast cancer is scant. A recent comparative genomic hybridization analysis revealed frequent losses of chromosome 4 in breast carcinomas. In an effort to more precisely locate the putative tumor suppressor gene(s) on chromosome 4 involved in the pathogenesis of breast carcinomas, we performed loss of heterozygosity studies using 19 polymorphic microsatellite markers. After precise microdissection of archival surgical cases, we analyzed DNA obtained from 44 breast carcinomas for loss of heterozygosity. In addition, DNA from tumor cell lines derived from 14 of these 44 breast carcinomas were also analyzed. We observed deletions of chromosome 4 at multiple sites in both tumor cell lines and breast carcinomas. The deletions in cell lines and their corresponding tumors were extensive in nature, whereas they were more localized in noncultured breast carcinomas. The localized deletions in the noncultured breast carcinomas clearly defined four nonoverlapping regions of frequent deletions: 4q33-34 (76%); 4q25-26 (63%); 4p15.1-15.3 (57%); and 4p16.3 (50%). Our results suggest that there may be multiple putative tumor suppressor genes, located on both arms of chromosome 4, whose inactivation is important in the pathogenesis of breast cancer.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Cromossomos Humanos Par 4/genética , Deleção de Genes , Alelos , Neoplasias da Mama/patologia , Carcinoma/patologia , Mapeamento Cromossômico , DNA de Neoplasias/genética , Feminino , Genes Supressores de Tumor , Humanos , Perda de Heterozigosidade , Repetições de Microssatélites , Células Tumorais CultivadasRESUMO
We investigated the aberrant promoter methylation profile of bladder cancers and correlated the data with clinicopathological findings. The methylation status of 10 genes was determined in 98 surgically resected bladder cancers, and we calculated the median methylation index (MI), a reflection of the methylated fraction of the genes tested. Methylation frequencies of the genes tested in bladder cancers were 36% for CDH1, 35% for RASSF1A and APC, 29% for CDH13, 16% for FHIT, 15% for RAR beta, 11% for GSTP1, 7% for p16(INK4A), 4% for DAPK, and 2% for MGMT. Methylation of four of the individual genes (CDH1, RASSF1A, APC, and CDH13) and the MI were significantly correlated with several parameters of poor prognosis (tumor grade, growth pattern, muscle invasion, tumor stage, and ploidy pattern). Methylation of CDH1, FHIT, and a high MI were associated with shortened survival. CDH1 methylation positive status was independently associated with poor survival in multivariate analyses. Our results suggest that the methylation profile may be a potential new biomarker of risk prediction in bladder cancer.
Assuntos
Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/patologia , Metilação de DNA , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Carcinoma de Células de Transição/cirurgia , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Regiões Promotoras Genéticas , Fatores de Risco , Neoplasias da Bexiga Urinária/cirurgiaRESUMO
To study the molecular abnormalities involved in the multistage development of cervical carcinoma (CC), we investigated the presence of oncogenic human papillomavirus (HPV) sequences, loss of heterozygosity (LOH), and microsatellite alterations at several genes/loci at 3p (3p14.2 at the FHIT gene, 3p14.3-21.1, 3p21, and 3p22-24.2), 9p21, RB and P53, and P53 gene point mutations in precisely microdissected archival tissues from 20 CCs and their accompanying precursor lesions (cervical intraepithelial neoplasia, CIN; n = 40) and normal epithelia (n = 20). In all HPV-positive cases (90% of CCs), HPV sequences were detected as the earliest appearing molecular change or simultaneously with other changes. LOH at any 3p region was found in 70% of CCs, and 3p14.2 (FHIT gene/FRA3B fragile site) (56%) and 3p21 (57%) were the most frequent 3p sites of loss. LOH at some 3p region was in the CIN I stage, and the 3p deletions in precursor CIN lesions were smaller than the 3p losses found in the associated invasive CC. LOH at the other regions studied and P53 gene mutations were less frequent and later events. Microsatellite alterations were detected in 35% of CCs, and identical abnormalities were detected in the associated precursor lesions. Although infection with oncogenic HPV strains is the earliest and most frequent molecular event, progressive deletions at one or more 3p regions (particularly at 3p14.2, and 3p21) are also frequent events occurring early in the pathogenesis of CC.
Assuntos
Hidrolases Anidrido Ácido , Carcinoma de Células Escamosas/genética , Deleção Cromossômica , Cromossomos Humanos Par 3 , Proteínas de Neoplasias , Neoplasias do Colo do Útero/genética , Carcinoma de Células Escamosas/virologia , DNA Viral/análise , Feminino , Genes do Retinoblastoma/genética , Genes p53/genética , Heterozigoto , Humanos , Repetições de Microssatélites/genética , Mutação , Papillomaviridae/genética , Proteínas/genética , Estudos Retrospectivos , Neoplasias do Colo do Útero/virologia , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/virologiaRESUMO
Allele loss involving chromosome arm 3p is one of the most frequent and earliest known genetic events in lung cancer pathogenesis and may affect several potential tumor suppressor gene regions. To further study the role of chromosome 3p allele loss in the pathogenesis of lung cancer, we performed high resolution loss of heterozygosity (LOH) studies on 97 lung cancer and 54 preneoplastic/preinvasive microdissected respiratory epithelial samples using a panel of 28 3p markers. Allelic losses of 3p were detected in 96% of the lung cancers and in 78% of the preneoplastic/preinvasive lesions. The allele losses were often multiple and discontinuous, with areas of LOH interspersed with areas of retention of heterozygosity. Most small cell lung carcinomas (91%) and squamous cell carcinomas (95%) demonstrated larger 3p segments of allele loss, whereas most (71%) of the adenocarcinomas and preneoplastic/preinvasive lesions had smaller chromosome areas of 3p allele loss. There was a progressive increase in the frequency and size of 3p allele loss regions with increasing severity of histopathological preneoplastic/preinvasive changes. In analyses of the specific parental allele lost comparing 42 preneoplastic/preinvasive foci with those lost in the lung cancer in the same patient (n = 10), the same parental allele was lost in 88% of 244 comparisons for 28 3p markers (P = 1.2 x 10(-36) for this occurring by chance). This indicates the occurrence of allele-specific loss in these foci similar to that seen in the tumor by a currently unknown mechanism. Analysis of all of the data indicated multiple regions of localized 3p allele loss including telomere-D3S1597, D3S1111-D3S2432, D3S2432-D3S1537, D3S1537, D3S1537-D3S1612, D3S4604/Luca19.1-D3S4622/Luca4.1, D3S4624/Luca2.1, D3S4624/Luca2.1-D3S1582, D3S1766, D3S1234-D3S1300 (FHIT/FRA3B region centered on D3S1300), D3S1284-D3S1577 (U2020/DUTT1 region centered on D3S1274), and D3S1511-centromere. A panel of six markers in the 600-kb 3p21.3 deletion region showed loss in 77% of the lung cancers, 70% of normal or preneoplastic/preinvasive lesions associated with lung cancer, and 49% of 47 normal, mildly abnormal, or preneoplastic/preinvasive lesions found in smokers without lung cancer; however, loss was seen in 0% of 18 epithelial samples from seven never smokers. The 600-kb 3p21.3 region and the 3p14.2 (FHIT/FRA3B) and 3p12 (U2020/DUTT1) regions were common, independent sites of breakpoints (retention of heterozygosity by some markers and LOH by other markers in the immediate region). We conclude that 3p allele loss is nearly universal in lung cancer pathogenesis; involves multiple, discrete, 3p LOH sites that often show a "discontinuous LOH" pattern in individual tumors; occurs in preneoplastic/preinvasive lesions in smokers with and without lung cancer (multiple lesions often lose the same parental allele); frequently involves breakpoints in at least three very small defined genomic regions; and appears to have allele loss and breakpoints first occurring in the 600-kb 3p21.3 region. These findings are consistent with previously reported LOH studies in a variety of tumors showing allele loss occurring by mitotic recombination and induced by oxidative damage.
Assuntos
Brônquios/patologia , Quebra Cromossômica , Cromossomos Humanos Par 3 , Neoplasias Pulmonares/genética , Lesões Pré-Cancerosas/genética , Mucosa Respiratória/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Deleção Cromossômica , Mapeamento Cromossômico , Feminino , Marcadores Genéticos , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Lesões Pré-Cancerosas/patologia , Células Tumorais CultivadasRESUMO
A tumor cell line, HCC1937, was established from a primary breast carcinoma from a 24-year-old patient with a germ-line BRCA1 mutation. A corresponding B-lymphoblastoid cell line was established from the patient's peripheral blood lymphocytes. BRCA1 analysis revealed that the tumor cell line is homozygous for the BRCA1 5382insC mutation, whereas the patient's lymphocyte DNA is heterozygous for the same mutation, as are at least two other family members' lymphocyte DNA. The tumor cell line is marked by multiple additional genetic changes including a high degree of aneuploidy, an acquired mutation of TP53 with wild-type allele loss, an acquired homozygous deletion of the PTEN gene, and loss of heterozygosity at multiple loci known to be involved in the pathogenesis of breast cancer. Comparison of the primary tumor with the cell line revealed the same BRCA1 mutation and an identical pattern of allele loss at multiple loci, indicating that the cell line had maintained many of the properties of the original tumor. This breast tumor-derived cell line may provide a useful model system for the study of familial breast cancer pathogenesis and for elucidating BRCA1 function and localization.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Genes BRCA1 , Mutação em Linhagem Germinativa , Heterozigoto , Células Tumorais Cultivadas , Adulto , Alelos , DNA de Neoplasias/genética , Éxons , Feminino , Humanos , Cariotipagem , Linhagem , Polimorfismo Conformacional de Fita SimplesRESUMO
We studied the pattern of aberrant methylation during the multistage pathogenesis of cervical cancers. We analyzed a total of 73 patient samples and 10 cervical cancer cell lines. In addition, tissue samples [peripheral blood lymphocytes (n = 10) and buccal epithelial cells (n = 12)] were obtained from 22 healthy volunteers. On the basis of the results of preliminary analysis, the cervical samples were grouped into three categories: (a) nondysplasia/low-grade cervical intraepithelial neoplasia (CIN; n = 37); (b) high-grade CIN (n = 17); and (c) invasive cancer (n = 19). The methylation status of six genes was determined (p16, RARbeta, FHIT, GSTP1, MGMT, and hMLH1). Our main findings are as follows: (a) methylation was completely absent in control tissues; (b) the frequencies of methylation for all of the genes except hMLH1 were >20% in cervical cancers; (c) aberrant methylation commenced early during multistage pathogenesis and methylation of at least one gene was noted in 30% of the nondysplasia/low-grade CIN group; (d) an increasing trend for methylation was seen with increasing pathological change; (e) methylation of RARbeta and GSTP1 were early events, p16 and MGMT methylation were intermediate events, and FHIT methylation was a late, tumor-associated event; and (f) methylation occurred independently of other risk factors including papillomavirus infection, smoking history, or hormone use. Although our findings need to be extended to a larger series, they suggest that the pattern of aberrant methylation in women with or without dysplasia may help identify subgroups at increased risk for histological progression or cancer development.
Assuntos
Hidrolases Anidrido Ácido , Metilação de DNA , Proteínas de Neoplasias , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Colo do Útero/metabolismo , Feminino , Genes p16/genética , Glutationa S-Transferase pi , Glutationa Transferase/metabolismo , Humanos , Isoenzimas/metabolismo , Metilação , Proteínas/metabolismo , Receptores do Ácido Retinoico/genética , Células Tumorais CultivadasRESUMO
Although human lung tumor-derived cell lines play an important role in the investigation of lung cancer biology and genetics, there is no comprehensive study comparing the genotypic and phenotypic properties of lung cancer cell lines with those of the individual tumors from which they were derived. We compared a variety of properties of 12 human non-small cell lung carcinoma (NSCLC) cell lines (cultured for a median period of 39 months; range, 12-69) and their corresponding archival tumor tissues. There was, in general, an excellent concordance between the lung tumor cell lines and their corresponding tumor tissues for morphology (100%), the presence of aneuploidy (100%), immunohistochemical expression of HER2/neu (100%) and p53 proteins (100%), loss of heterozygosity at 13 chromosomal regions analyzed (97%) using 37 microsatellite markers, microsatellite alterations (MAs, 75%), TP53 (67%), and K-ras (100%) gene mutations. In addition, there was 100% concordance for the parental allele lost in all 115 comparisons of allelic losses. Some discrepancies were found; more aneuploid subpopulations of cells were detected in the cell lines as well as higher incidences of TP53 mutations (4 of 10 mutations not found in the tumors) and microsatellite alterations (two cell lines with MAs not detected in the tumors). Similar loss of heterozygosity frequencies by chromosomal regions and mean fractional allelic loss index were detected between successfully cultured and 40 uncultured lung tumors (0.45 and 0.49, respectively), indicating that both groups were similar. Our findings indicate that the NSCLC cell lines in the large majority of instances retain the properties of their parental tumors for lengthy culture periods. NSCLC cell lines appear very representative of the lung cancer tumor from which they were derived and thus provide suitable model systems for biomedical studies of this important neoplasm.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Células Tumorais Cultivadas/patologia , Adulto , Idoso , Alelos , Aneuploidia , Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/genética , Técnicas de Cultura de Células/métodos , Análise Mutacional de DNA , DNA de Neoplasias/genética , Feminino , Genes p53 , Genes ras , Humanos , Perda de Heterozigosidade , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Receptor ErbB-2/análise , Fatores de Tempo , Células Tumorais Cultivadas/química , Proteína Supressora de Tumor p53/análiseRESUMO
Accumulation of wild-type or mutant p53 protein occurs in approximately 50% of human malignancies. This overexpression may generate antigenic epitopes recognized by CTLs. Because normal cells have undetectable levels of p53, these CTLs are likely to be tumor specific. Here, for the first time, we test the hypothesis that full-length wild-type p53 protein can be used for generation of an immune response against tumor cells with p53 overexpression. T cells obtained from nine HLA-A2-positive cancer patients and three HLA-A2-positive healthy individuals were stimulated twice with dendritic cells (DCs) transduced with an adenovirus wild-type p53 (Ad-p53) construct. Significant cytotoxicity was detected against HLA-A2-positive tumor cells with accumulation of mutant or wild-type p53 but not against HLA-A2-positive tumor cells with normal (undetectable) levels of p53 or against HLA-A2-negative tumor cells. This response was specific and mediated by CD8+ CTLs. These CTLs recognized HLA-A2-positive tumor cells expressing normal levels of p53 protein after their transduction with Ad-p53 but not with control adenovirus. Stimulation of T cells with Ad-p53-transduced DCs resulted in generation of CTLs specific for p53-derived peptide. These data demonstrate that DCs transduced with the wild-type p53 gene were able to induce a specific antitumor immune response. This offers a new promising approach to immunotherapy of cancer.