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Biomedical subjects

A F Gazdar

Publications and source records attributed to A F Gazdar.

At least 19 recordsLinked to original sources
PubMed2001

Aberrant promoter methylation profile of bladder cancer and its relationship to clinicopathological features.

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.

Adult↗
PubMed2001

Inhibition of lung cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene SEMA3B.

Semaphorins SEMA3B and its homologue SEMA3F are 3p21.3 candidate tumor suppressor genes (TSGs), the expression of which is frequently lost in lung cancers. To test the TSG candidacy of SEMA3B and SEMA3F, we transfected them into lung cancer NCI-H1299 cells, which do not express either gene. Colony formation of H1299 cells was reduced 90% after transfection with wild-type SEMA3B compared with the control vector. By contrast, only 30-40% reduction in colony formation was seen after the transfection of SEMA3F or SEMA3B variants carrying lung cancer-associated single amino acid missense mutations. H1299 cells transfected with wild-type but not mutant SEMA3B underwent apoptosis. We found that lung cancers (n = 34) always express the neuropilin-1 receptor for secreted semaphorins, whereas 82% expressed the neuropilin-2 receptor. Because SEMA3B and SEMA3F are secreted proteins, we tested conditioned medium from COS-7 cells transfected with SEMA3B and SEMA3F and found that medium from wild-type SEMA3B transfectants reduced the growth of several lung cancer lines 30-90%, whereas SEMA3B mutants or SEMA3F had little effect in the same assay. Sequencing of sodium bisulfite-treated DNA showed dense methylation of CpG sites in the SEMA3B 5' region of lung cancers not expressing SEMA3B but no methylation in SEMA3B-expressing tumors. These results are consistent with SEMA3B functioning as a TSG, the expression of which is inactivated frequently in lung cancers by allele loss and promoter region methylation.

Animals↗
PubMed2001

hTR repressor-related gene on human chromosome 10p15.1.

Somatic cells express genes that suppress telomerase activity and these genes may be inactivated in tumour cells. We postulated that cancer cells acquire immortality by activation of telomerase by the loss of such a gene. We have reported recently that a telomerase repressor gene may be located on 10p15.1 by deletion mapping using microcell-mediated chromosome transfer (MMCT), radiated microcell fusion (RMF), fluorescent in situ hybridization (FISH) and STS analysis. To independently confirm this result, we correlated expression of RNA component of telomerase (hTR) as a marker of telomerase expression by in situ hybridization with allelic loss in pulmonary carcinoid tumours. Unlike most malignant tumours, pulmonary carcinoids (which are low-grade malignant tumours) are heterogeneous for telomerase expression. Loss of 5 closely spaced polymorphic markers on 10p15.1, especially D10S1728, were highly correlated with hTR expression. In an additional experiment, 10p15.1 showed higher and more significant correlation than any region of 3p where it has been predicted as another chromosomal location of telomerase repressor with allelic loss of the region. Our findings strongly suggest that 10p15.1 harbours a gene involved in repression of telomerase RNA component in human somatic cells and each putative repressor (on 3p and 10p) may act independently.

Carcinoid Tumor↗
PubMed2001

Inactivation of human SRBC, located within the 11p15.5-p15.4 tumor suppressor region, in breast and lung cancers.

A cDNA clone encoding human SRBC [serum deprivation response factor (sdr)-related gene product that binds to c-kinase] was isolated in a yeast two-hybrid screening, with amino acids 1-304 of BRCA1 as the probe. The human SRBC gene (hSRBC) was mapped to chromosome region 11p15.5-p15.4, close to marker D11S1323, at which frequent loss of heterozygosity (LOH) has been observed in sporadic breast, lung, ovarian, and other types of adult cancers as well as childhood tumors. hSRBC-coding region mutations including frame shift and truncation mutations were detected in a few ovarian and lung cancer cell lines. More significantly, the expression of hSRBC protein was down-regulated in a large fraction [30 (70%) of 43] of breast, lung, and ovarian cancer cell lines, whereas strong expression of hSRBC protein was detected in normal mammary and lung epithelial cells. The down-regulation of hSRBC expression in cancer cells was associated with hypermethylation of CpG dinucleotides in its promoter region, and 3 (60%) of 5 primary breast tumors and 11 (79%) of 14 primary lung tumors were also found to be hypermethylated. Treatment of breast cancer MCF7 cells with 5'azacytidine and Trichostatin A resulted in expression of hSRBC, confirming DNA methylation as the mode of inactivation. Our results suggest that epigenetic or mutational inactivation of hSRBC may contribute to the pathogenesis of several types of human cancers, marking hSRBC as a candidate tumor suppressor gene.

Amino Acid Sequence↗
PubMed2001

Aberrant methylation and simian virus 40 tag sequences in malignant mesothelioma.

Aberrant promoter methylation and resultant silencing of several genes plays an important role in the pathogenesis of many tumor types. We compared the methylation profile of 66 malignant mesotheliomas (MMs) and 40 lung adenocarcinomas using methylation-specific PCR for seven genes frequently methylated in lung cancer. We also compared the methylation frequencies of these genes as well as the methylation index, a reflection of all of the gene frequencies, with the presence of SV40 large T-antigen (Tag) sequences, histological subtype, and patient survival. Our major findings are: (a) with the exception of the RASSF1A promoter of the RASSF1 gene, frequencies of aberrant methylation were significantly lower in MMs than in adenocarcinomas; (b) the frequency of RASSF1A aberrant methylation and the value of the methylation index were significantly higher in SV40 sequence positive MM than in negative MM; and (c) the methylation index was higher in epithelial MM than in sarcomatous/mixed MM. Our results demonstrate a relationship between SV40 and aberrant methylation in MMs.

Adenocarcinoma↗
PubMed2001

Genetic alteration of the beta-catenin gene (CTNNB1) in human lung cancer and malignant mesothelioma and identification of a new 3p21.3 homozygous deletion.

The beta-catenin gene (CTNNB1) has been shown to be genetically mutated in various human malignancies. To determine whether the beta-catenin gene is responsible for oncogenesis in thoracic malignancies, we searched for the mutation in 166 lung cancers (90 primary tumors and 76 cell lines), one blastoma and 10 malignant mesotheliomas (two primary tumors and eight cell lines). Among the lung cancers, including 43 small cell lung cancers (SCLCs) and 123 non-small cell lung cancers (NSCLCs), we identified four alterations in exon 3, which is the target region of mutation for stabilizing beta-catenin. One primary adenocarcinoma had a somatic mutation from C to G, leading to an amino acid substitution from Ser to Cys at codon 37. Among the cell lines, SCLC NCI-H1092 had a mutation from A to G, leading to an Asp to Gly substitution at codon 6, NSCLC HCC15 had a mutation from C to T, leading to a Ser to Phe substitution at codon 45, and NSCLC NCI-H358 had a mutation from A to G, leading to a Thr to Ala substitution at codon 75. One blastoma also had a somatic mutation from C to G, leading to a Ser to Cys substitution at codon 37. Among the 10 malignant mesotheliomas, we identified a homozygous deletion in the NCI-H28 cell line. Cloning of the rearranged fragment from NCI-H28 indicated that all the exons except exon 1 of the beta-catenin gene are deleted and that the deletion junction is 13 kb downstream from exon 1. Furthermore, Northern blot analysis of 26 lung cancer and eight mesothelioma cell line RNAs detected ubiquitous expression of the beta-catenin messages except NCI-H28, although Western blot analysis showed that relatively less amounts of protein products were expressed in some of lung cancer cell lines. Our findings suggest that the beta-catenin gene is infrequently mutated in lung cancer and that the NCI-H28 homozygous deletion of the beta-catenin gene might indicate the possibility of a new tumor suppressor gene residing in this region at 3p21.3, where various types of human cancers show frequent allelic loss.

Base Sequence↗
PubMed2001

Loss of expression and aberrant methylation of the CDH13 (H-cadherin) gene in breast and lung carcinomas.

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.

Breast Neoplasms↗
PubMed2001

Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression.

BACKGROUND: The recently identified RASSF1 locus is located within a 120-kilobase region of chromosome 3p21.3 that frequently undergoes allele loss in lung and breast cancers. We explored the hypothesis that RASSF1 encodes a tumor suppressor gene for lung and breast cancers. METHODS: We assessed expression of two RASSF1 gene products, RASSF1A and RASSF1C, and the methylation status of their respective promoters in 27 non-small-cell lung cancer (NSCLC) cell lines, in 107 resected NSCLCs, in 47 small-cell lung cancer (SCLC) cell lines, in 22 breast cancer cell lines, in 39 resected breast cancers, in 104 nonmalignant lung samples, and in three breast and lung epithelial cultures. We also transfected a lung cancer cell line that lacks RASSF1A expression with vectors containing RASSF1A complementary DNA to determine whether exogenous expression of RASSF1A would affect in vitro growth and in vivo tumorigenicity of this cell line. All statistical tests were two-sided. RESULTS: RASSF1A messenger RNA was expressed in nonmalignant epithelial cultures but not in 100% of the SCLC, in 65% of the NSCLC, or in 60% of the breast cancer lines. By contrast, RASSF1C was expressed in all nonmalignant cell cultures and in nearly all cancer cell lines. RASSF1A promoter hypermethylation was detected in 100% of SCLC, in 63% of NSCLC, in 64% of breast cancer lines, in 30% of primary NSCLCs, and in 49% of primary breast tumors but in none of the nonmalignant lung tissues. RASSF1A promoter hypermethylation in resected NSCLCs was associated with impaired patient survival (P =.046). Exogenous expression of RASSF1A in a cell line lacking expression decreased in vitro colony formation and in vivo tumorigenicity. CONCLUSION: RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.

Adult↗
PubMed2001

5' CpG island methylation of the FHIT gene is correlated with loss of gene expression in lung and breast cancer.

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.

Acid Anhydride Hydrolases↗
PubMed2001

Genome-wide allelotyping analysis reveals multiple sites of allelic loss in gallbladder carcinoma.

Although gallbladder carcinoma (GBC) is a highly malignant neoplasm, there is very limited information about the molecular changes involved in its pathogenesis. To identify the chromosomal locations of putative tumor suppressor gene loci involved in the pathogenesis of GBC, we conducted a genome-wide allelotyping or loss of heterozygosity (LOH) analysis of GBCS: Microdissected tissue from 24 archival GBCs and their matched control DNAs were analyzed for PCR-based LOH using 169 microsatellite markers spanning all nonacrocentric autosomal arms and the X chromosome. The chromosomal arms with the greatest frequencies of LOH (> or = 60%) were 3p, 6q, 7q, 8p, 9p, 9q, 11q, 12q, 17p, 18q, 19p, 22q, and XQ: The average fractional allele loss index in GBC cases was high (0.43) and frequent breakpoints were detected in gallbladder tumors. Of interest, 21 different regions of frequent LOH (hot spots) defined as > or = 50% for individual GBC samples were detected in this neoplasm, nearly half of them confined to one microsatellite marker. We conclude that in GBC at least 21 chromosomal regions with frequent allele losses are involved, suggesting that several putative tumor suppressor genes are inactivated in its pathogenesis. Overall, these data provide global estimates of the extent of genetic changes leading to GBC and will be useful for the identification of new tumor suppressor genes and for multiple new markers for translational research.

Adenocarcinoma↗
PubMed2001

Expression of the RNA component of human telemorase (hTR) in ThinPrep preparations from bladder washings.

BACKGROUND: The enzyme telomerase is associated with cellular immortality and is expressed in the vast majority of human neoplasms. The expression of the RNA component of human telomerase (hTR) shows excellent concordance with enzyme activity. METHODS: In this study, hTR expression was analyzed in a series of 18 perioperative bladder washings and compared with histologic diagnoses from material obtained in the same setting. The hTR expression analysis used an 35S-based in-situ hybridization assay. ThinPrep preparations fixed in PreservCyt solution (Cytyc Corporation, Boxborough, MA) were hybridized with sense and antisense hTR probes. A 1-4+ grading scheme was used, with appropriate positive and negative controls. RESULTS: Five of six (83%) lesions with benign histology had hTR expression that was 2+ or less in the exfoliated urothelial cells. In contrast, 11 of 12 (93%) lesions with malignant histology had an hTR expression that was focally 3+ or more, with 7 of 12 (58%) lesions having 4+ hTR expression in at least some urothelial clusters. Although increased hTR expression was present in smears with malignant urothelial cells, a similar trend was not seen with muscularis propria invasion or higher grades of TCC on subsequent histology. CONCLUSIONS: The use of in situ hybridization technique bypasses the need for stringent specimen processing and allows identification of the specific cell type that expresses telomerase. Cancer (Cancer Cytopathol)

Carcinoma, Transitional Cell↗
PubMed2001

Searching for microsatellite mutations in coding regions in lung, breast, ovarian and colorectal cancers.

RepX represents a new informatics approach to probe the UniGene database for potentially polymorphic repeat sequences in the open reading frame (ORF) of genes, 56% of which were found to be actually polymorphic. We now have performed mutational analysis of 17 such sites in genes not found to be polymorphic (<0.03 frequency) in a large panel of human cancer genomic DNAs derived from 31 lung, 21 breast, seven ovarian, 21 (13 microsatellite instability (MSI)+ and eight MSI-) colorectal cancer cell lines. In the lung, breast and ovarian tumor DNAs we found no mutations (<0.03-0.04 rate of tumor associated open reading frame mutations) in these sequences. By contrast, 18 MSI+ colorectal cancers (13 cancer cell lines and five primary tumors) with mismatch repair defects exhibited six mutations in three of the 17 genes (SREBP-2, TAN-1, GR6) (P<0.000003 compared to all other cancers tested). We conclude that coding region microsatellite alterations are rare in lung, breast, ovarian carcinomas and MSI (-) colorectal cancers, but are relatively frequent in MSI (+) colorectal cancers with mismatch repair deficits.

Base Pair Mismatch↗
PubMed2001

Functional evidence for a telomerase repressor gene on human chromosome 10p15.1.

Based on the sites of frequent allelic loss in hepatocellular carcinoma, five normal human chromosomes (2, 4, 5, 10 and 16) were transferred individually into a telomerase-positive human hepatocellular carcinoma cell line, Li7HM, by microcell-mediated chromosome transfer (MMCT). Chromosome 10, but not the others, repressed telomerase activity immediately and stopped cell growth after 50 population doublings (PDs). Loss of the transferred 10p loci resulted in the emergence of revertant cells that continued to proliferate and expressed telomerase activity, suggesting the presence of a telomerase repressor gene on this chromosomal arm. Transfer of a series of defined fragments from chromosome 10p successfully narrowed down the responsible region: a 28.9-cM region on 10p15 (between WI-4752 and D10S249), but not a 26.2-cM region (between D10S1728 and D10S249), caused repression of telomerase activity and progressive telomere shortening. A strong correlation between the expression level of telomerase catalytic subunit gene (hTERT) and telomerase activity was observed. These findings suggest that a novel telomerase repressor gene which controls the expression of hTERT is located on the 2.7-cM region (between WI-4752 and D10S1728) on chromosome 10p15.1.

Carcinoma, Hepatocellular↗
PubMed2001

Aberrant promoter methylation of multiple genes in non-small cell lung cancers.

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.

Adult↗
PubMed2001

Association of SV40 with human tumours.

SV40 was discovered as a contaminant of poliovirus vaccines that were inadvertently administered to millions of people in Europe and the United States between 1955 and 1963. Shortly afterwards, SV40 was proven to be oncogenic in rodents and capable of transforming human and animal cells in vitro. The possibility that SV40 might cause tumours in humans thus became a subject of scientific and public interest and scrutiny. However, largely due to a lack of significant epidemiological evidence, interest in assessing SV40's potential carcinogenic role in humans diminished. Recently, many laboratories have reported the presence of SV40-like DNA in a high proportion of human mesotheliomas, ependymomas and osteosarcoma (the three main types of tumours caused by virus in hamsters), renewing the question whether SV40 might be a human tumour virus. Molecular data from these studies are reviewed to re-evaluate the potential role of SV40 as a human carcinogen.

Animals↗