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Biomedical subjects

C C Harris

Publications and source records attributed to C C Harris.

At least 145 records · Page 8Linked to original sources
PubMed1994

Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3.

Chronic active hepatitis caused by infection with hepatitis B virus, a DNA virus, is a major risk factor for human hepatocellular carcinoma. Since the oncogenicity of several DNA viruses is dependent on the interaction of their viral oncoproteins with cellular tumor-suppressor gene products, we investigated the interaction between hepatitis B virus X protein (HBX) and human wild-type p53 protein. HBX complexes with the wild-type p53 protein and inhibits its sequence-specific DNA binding in vitro. HBX expression also inhibits p53-mediated transcriptional activation in vivo and the in vitro association of p53 and ERCC3, a general transcription factor involved in nucleotide excision repair. Therefore, HBX may affect a wide range of p53 functions and contribute to the molecular pathogenesis of human hepatocellular carcinoma.

Cell Line↗
PubMed1994

Clonal variation of tumorigenic potential in v-Ha-ras-transformed human bronchial epithelial cells: relationship to ras oncogene expression and CAD gene amplification.

Infection of an SV40 large-T antigen-"immortalized" human bronchial epithelial cell line with a Zip-v-Ha-ras retroviral vector resulted in a mass culture that was tumorigenic in athymic nude mice. A tumor cell line derived from passage of the mass culture in vivo, however, exhibited increased tumorigenicity and v-Ha-ras expression. To examine and compare the molecular events involving the ras oncogene during cell transformation in vitro and subsequent tumor formation in vivo, clonal cell populations were isolated from the v-Ha-ras-transformed mass culture. While the clonal cell lines exhibited diverse tumorigenic profiles, these differences did not correlate with v-Ha-ras expression. However, the expression of the activated ras gene, while not necessary for growth in vitro, did appear to be associated with a selective growth advantage in vivo. In addition, the modulation of gene amplification ability in these cells was not associated with the induction of tumorigenicity or v-Ha-ras expression.

Animals↗
PubMed1994

Activation of promutagens in a human bronchial epithelial cell line stably expressing human cytochrome P450 1A2.

Normal human bronchial epithelial (NHBE) cells are the putative progenitor cells of all types of lung cancer. NHBE cells immortalized by SV40 T-antigen retain many characteristics of the primary cells and are a useful model for investigating the role of oncogenes, tumor suppressor genes, and certain chemical carcinogens in the molecular pathogenesis of lung cancer. In this study, SV40 T-antigen-positive cells (BEAS-2B) were characterized for their metabolic functions and were shown to continue to express epoxide hydrolase, glutathione S-transferase pi, glutathione peroxidase, and catalase. To increase their metabolic activity towards human procarcinogens, human cytochrome P450 1A2 (CYP1A2) was stably expressed by introducing CYP1A2 cDNA into BEAS-2B cells either by infection with a high-titer recombinant retrovirus (pXT-1A2) or by transfection with a CYP1A2 expression vector (pCMV1A2), which produced the cell lines B-1A2 and B-CMV1A2, respectively. Cell lines established with either expression system expressed enzymatically active CYP1A2 protein and were 50- to 400-fold more sensitive to the cytotoxic effect of the carcinogen aflatoxin B1 (AFB1) than the corresponding control cell lines. The cytotoxic effects of AFB1 were paralleled by increased metabolism of AFB1 and enhanced formation of the AFB1-N7 guanine adduct in B-CMV1A2 cells. Cytotoxicity and adduct formation correlated with a significantly higher protein expression of CYP1A2 by the cytomegalovirus promoter-driven plasmid. Since this human epithelial cell line is the precursor cell type of lung cancer, has normal phase II enzymes, and exhibits highly reproducible expression of phase I enzymes, this in vitro model should aid in the evaluation of putative human carcinogens and anticarcinogens.

Aflatoxin B1↗
PubMed1994

p53 mutations at A:T base pairs in angiosarcomas of vinyl chloride-exposed factory workers.

Mutations in the p53 tumor suppressor gene are commonly found in the major human cancers and the mutational spectrum in some cancer types is consistent with the genotoxic effects of the associated environmental risk factors. Thus far there is little information on p53 mutations in cancers of factory workers with a history of carcinogen exposure in the workplace. Occupational exposure to vinyl chloride causes liver angiosarcomas (ASL) and also increases the risk of several other cancers. Loss of p53 function in osteo- and fibrosarcomas can occur by two different mechanisms, p53 mutation and amplification of the MDM2 gene. We examined tumors from five vinyl chloride-exposed patients, four with ASL and one with hepatocellular carcinoma (HCC), for evidence of MDM2 proto-oncogene amplification or p53 mutation in exons 5-8. Amplification of MDM2 was not found, but in two of the angiosarcomas an A:T to T:A missense mutation was detected. p53 sequence analysis of vinyl chloride associated cancers may provide valuable information on the relationship between carcinogen exposure and DNA damage in cancer-related genes.

Adenine↗
PubMed1994

Determination of the allelic frequencies of an L-myc and a p53 polymorphism in human lung cancer.

The L-myc and p53 genes have been implicated in lung cancer. Both of these genes have restriction fragment length polymorphisms (RFLPs) that could account for differential expression or activity of variant forms. An EcoRI restriction site in the L-myc gene was previously reported to be a predictor of poor prognosis in Japanese lung cancer patients. There are several RFLPs in the p53 gene. In exon 4 there is a polymorphism that codes for either an arginine or proline residue at codon 72. We previously reported the frequency of DNA-RFLPs at these gene loci revealed by EcoRI and AccII respectively. Here we report results from a study comparing lung cancer cases (n = 31) with chronic obstructive pulmonary disease controls (n = 49). No association was found between these RFLPs and disease status. Previous observations that the frequencies of these RFLPs varied by race were confirmed. The p53 arginine allele was found to be more common in Caucasians (0.71) than African-Americans (0.50). The EcoRI restriction site present allele in L-myc was more frequent in African-Americans (0.71) than Caucasians (0.49). Thus, the allelic frequency for L-myc was similar in African-Americans to that reported for Japanese, and the allelic frequency for p53 was similar in Caucasians to that reported for Japanese.

Base Sequence↗
PubMed1994

Pathobiological effects of acetaldehyde in cultured human epithelial cells and fibroblasts.

The ability of acetaldehyde, a respiratory carcinogen present in tobacco smoke and automotive emissions, to affect cell viability, thiol status and intracellular Ca2+ levels and to cause DNA damage and mutations has been studied using cultured human cells. Within a concentration range of 3-100 mM, a 1 h exposure to acetaldehyde decreases colony survival and inhibits uptake of the vital dye neutral red in bronchial epithelial cells. Acetaldehyde also causes both DNA interstrand cross-links and DNA protein cross-links whereas no DNA single strand breaks are detected. The cellular content of glutathione is also decreased by acetaldehyde, albeit, without concomitant changes in the glutathione redox status or in the content of protein thiols. Transient or sustained increases in cytosolic Ca2+ occur within minutes following exposure of cells to acetaldehyde. Moreover, acetaldehyde significantly decreases the activity of the DNA repair enzyme O6-methylguanine-DNA methyltransferase. Finally, a 5 h exposure to acetaldehyde causes significant levels of 6-thioguanine resistance mutations in an established mutagenesis model involving skin fibroblasts. The results indicate that mM concentrations of acetaldehyde cause a wide range of cytopathic effects associated with multistep carcinogenesis. The fact that acetaldehyde, in relation to its cytotoxicity, causes comparatively higher genotoxicity and inhibits DNA repair more readily than other major aldehydes in tobacco smoke and automotive emissions is discussed.

Acetaldehyde↗
PubMed1994

Constitutive expression of inducible nitric oxide synthase in human bronchial epithelial cells induces c-fos and stimulates the cGMP pathway.

Two major roles have been defined for nitric oxide (NO): cell-cell communication mediated by the stimulation of cyclic guanosine 3',5'-monophosphate (cGMP) synthesis and cytotoxicity by direct or indirect interaction of the free radical NO with cellular targets. Thus, pathologic states might result from an alteration of NO pathways, e.g., by deregulated activity of NO synthase. To investigate this hypothesis, we introduced the murine-inducible NO synthase (iNOS) sequence into immortalized human bronchial epithelial cells (BEAS-2B). iNOS activity, measured by conversion of [14C]arginine to [14C]citrulline in the presence of 1 mM EGTA, was higher than 100 pmol/min/mg protein in early passages of iNOS-transfected cells but decreased with cell subculturing. No iNOS activity could be detected in control vector-transfected cells. NO stimulated cGMP production in iNOS-transfected cells, and this effect was inhibited by the iNOS inhibitor NG-monomethyl-L-arginine. In addition, NO production induced c-fos expression and did not interfere with clonal cell growth. These results suggest that BEAS-2B cells constitute a suitable model to study the consequences of iNOS activity on signal transduction pathways in bronchial epithelium.

Amino Acid Oxidoreductases↗
PubMed1994

Induction of anchorage independent growth and serum resistance in immortalized human bronchial epithelial cells by alteration of the cytoskeleton.

Malignant transformation is frequently accompanied by obvious changes in cytoarchitecture, but the importance of these changes has been difficult to assess in view of the large number of other cellular changes that also occur. In this study, we transfected the SV40-immortalized human bronchial epithelial cell line, BEAS-2B, with human wild-type beta or gamma actin gene expression plasmids to induce cytoskeletal changes and to determine whether this was associated with altered cellular growth properties. Cells expressing the exogenous full-length actin genes underwent a fibroblastoid change in morphology which was reflected in changes in their pattern of actin cable organization, and acquired both the ability to grow under anchorage-independent conditions and resistance to the normal growth inhibitory effects of fetal bovine serum. These phenotypic changes correlated with changes in actin mRNA levels, but not with changes in actin protein levels. The phenotypically altered cells were not tumorigenic when injected subcutaneously in athymic nude mice, and they retained the ability to suppress the tumorigenic potential of a lung carcinoma cell line, HuT-292. Therefore, alteration of the cytoskeleton of immortalized human bronchial epithelial cells resulted in the acquisition of some properties commonly found in malignant cells, but did not result in tumorigenicity.

Actins↗
PubMed1994

Database of p53 gene somatic mutations in human tumors and cell lines.

A data base is described in which over 2,500 mutations in the p53 gene of human tumors and tumor cell lines are compiled from a systematic search of reports published before 1 January 1994. Data from 1994 are being added intermittently, with a systematic search and update scheduled for December, 1994. The compilation has been deposited with the EMBL Data Library and is available in electronic form free of charge. This report contains a rationale for the compilation, a brief summary of the major findings and a description of the data base.

DNA Mutational Analysis↗
PubMed1994

Analysis of cytochrome P450 2E1 genetic polymorphisms in relation to human lung cancer.

Human cancer risk assessment using molecular genetic techniques is a rapidly emerging field. Many studies suggest that both inherited and acquired genetic predispositions play an important role in carcinogenesis. Cytochrome P450 (CYP) 2E1 is involved in the metabolic activation of N-nitrosamines and other low molecular weight compounds. A recently described genetic polymorphism of CYP2E1 [DraI restriction fragment length polymorphism (RFLP)] has been associated with an increased risk of lung cancer in Japanese. We have assessed the allelic frequency of three RFLPs (PstI, RsaI, and DraI) in African-Americans (n = 109), Caucasian Americans (n = 153), and octogenarian Japanese (n = 42), and also in a United States case-control study of lung cancer (histologically confirmed lung cancer, n = 58; controls, n = 56; total, n = 114). The relationship of the CYP2E1 DraI polymorphism to other CYP2E1 polymorphisms (PstI and RsaI RFLP) was examined. The allelic frequency of the DraI C minor allele for all subjects was 0.09 in Caucasians, 0.09 in African-Americans, and 0.31 in Japanese. In the case-control study of lung cancer, no association of the CYP2E1 DraI genotype with lung cancer was found (odds ratio, 1.57; 95% confidence interval, 0.59-4.18). Comparison after discordant CYP2E1 genotypes suggests the presence of different haplotypes in Americans and Japanese. These results indicate that the CYP2E1 DraI RFLP is probably not a cancer risk factor in United States Caucasian or African-Americans, although statistical power is limited given the low frequency of the CYP2E1 DraI C minor alleles.

Aged↗
PubMed1993

DNA strand bias in the repair of the p53 gene in normal human and xeroderma pigmentosum group C fibroblasts.

We have measured the gene-specific and strand-specific DNA repair of UV-induced cyclobutane pyrimidine dimers in the p53 tumor suppressor gene in a normal, repair-proficient human fibroblast strain and in fibroblasts from a patient with the repair deficient disorder xeroderma pigmentosum, complementation xeroderma pigmentosum group C (XP-C). In both cell strains, repair was measured in the p53 gene and in its individual DNA strands. For comparison, the repair also was measured in other genomic regions in these human fibroblast strains, including the housekeeping gene dihydrofolate reductase, and two inactive genomic regions, the delta globin gene, and the 754 locus of the X chromosome. In both cell strains, we find that the p53 gene is repaired faster than the dihydrofolate reductase gene and much more efficiently than the inactive genomic regions. Selective repair of the transcribed DNA strand of p53 is observed in both human cell strains; the strand bias of repair is particularly distinct in XP-C. Mutations specific to the nontranscribed strand may occur due to replication errors at the sites of unrepaired DNA damage. Therefore, our results predict that the majority of mutations in skin cancers, especially those from patients with XP-C, would occur on the nontranscribed strand of the p53 gene. Indeed, Dumasz et al. (Proc. Natl. Acad. Sci. USA, in press, 1993) report such a strand bias of p53 mutation in skin cancers from XP-C patients.

DNA↗
PubMed1993

Altered expression of the cyclin D1 and retinoblastoma genes in human esophageal cancer.

We have examined DNA from four human esophageal carcinoma cell lines and 50 primary esophageal carcinomas obtained from China, Italy, and France for amplification of the cyclin D1 gene. We also examined 36 of these 50 carcinomas for expression of the cyclin D1 and retinoblastoma (RB) proteins by immunohistochemistry. We found a 3- to 10-fold amplification of the cyclin D1 gene in 16 of the 50 (32%) tumors and in two of the four cell lines. Cyclin D1 protein was overexpressed in 12 of 13 tumors and the two cell lines that showed gene amplification when compared to normal controls. Studies on RB protein expression indicated that 6 of the 36 (17%) tumor samples examined and one cell line did not show detectable expression of this protein. The tumors and cell lines that had cyclin D1 gene amplification and overexpression exhibited normal levels of expression of RB protein. By contrast, the tumors and cell line that did not appear to express the RB protein did not show amplification of the cyclin D1 gene and expressed only low levels of the cyclin D1 protein (P = 0.03). These results suggest that the inhibitory effect of RB on cell cycle progression can be abrogated during tumor development either by loss of expression of the RB gene or by increased expression of the cyclin D1 gene.

Blotting, Southern↗
PubMed1993

Biological consequences of overexpression of a transfected c-erbB-2 gene in immortalized human bronchial epithelial cells.

In order to examine the effects of the overexpression of c-erbB-2 (HER-2, neu) on human bronchial epithelial cells, a human c-erbB-2 expression vector was introduced into the simian virus 40 large T-antigen-immortalized human bronchial epithelial cell line BEAS-2B. Isolation of multiple clonal cell lines after selection revealed a wide range of expression of the gene product gp185erbB-2. While three of six clones tested expressed gp185erbB-2 at levels detectable by immunocytochemistry, only one, B2BE6, induced adenocarcinoma-like tumors in athymic nude mice. Both a nontumorigenic clone, B2BE2, and a tumorigenic clone, B2BE6, expressed comparable amounts of gp185erbB-2, which became phosphorylated on tyrosine in response to treatment with the c-erbB-2 ligands gp30 and p75. These data suggest that overexpression of c-erbB-2 in human bronchial epithelial cells can contribute to, but is not sufficient for, induction of tumorigenicity in this human model system.

Animals↗
PubMed1993

Altered chromosomal methylation patterns accompany oncogene-induced transformation of human bronchial epithelial cells.

Abnormal methylation of CpG island sequences on chromosomes 11p and 17p, and tumor phenotype-associated differential methylation of chromosome 3p loci have been described in human lung tumors (S.B. Baylin, J.W.M. Hoppener, A. de Bustros, P.H. Steenbergh, C.J.M. Lips, and B. D. Nelkin, Cancer Res., 46: 2917-2922, 1986; M. Makos, B.D. Nelkin, M. I. Lerman, F. Latif, B. Zbar, and S.B. Baylin, Proc. Natl. Acad. Sci. USA, 89: 1929-1933, 1992; A. de Bustros, B. D. Nelkin, A. Silverman, G. Ehrlich, B. Poiesz, and S. B. Baylin, Proc. Natl. Acad. Sci. USA, 85: 5693-5697, 1988). Using an in vitro model of lung tumor progression, we now show that these aberrant methylation patterns occur at different stages during cellular immortalization and oncogene-induced neoplastic transformation of normal human bronchial epithelial cells (NHBE). The CALC1 CpG island locus on chromosome 11p15.4 was essentially unmethylated in NHBE and simian virus 40 T-antigen immortalized NHBE (BEAS-2B cells) but became de novo methylated in 5 of 6 BEAS-2B derived cell lines that were transfected or infected with various oncogenes and in a spontaneously neoplastically transformed subline of BEAS-2B cells. By contrast, an additional CpG island locus, pYNZ22, at 17p13.3 became fully methylated following the immortalization of NHBE and was not further changed by oncogene-induced transformation. Finally, at a non-CpG island locus pYNZ86.1 on chromosome 3p14, different tumor phenotype-associated methylation patterns became apparent only after passage of the turmorigenic oncogene-transformed bronchial epithelial cell lines in athymic nude mice. Whereas cell lines derived from tumors with a non-small cell lung carcinoma-like phenotype were significantly hypomethylated relative to their parental cell lines, a cell line derived from a tumor with a more small cell lung carcinoma-like phenotype retained the methylation status of its parental cell line. The data indicate that altered DNA methylation patterns, including the de novo methylation of normally unmethylated CpG island sequences and demethylation of nonisland sequences, arise at different stages during immortalization and oncogene-induced neoplastic transformation of bronchial epithelial cells. These findings suggest that DNA methylation abnormalities accompany, or may play a role in, the genetic changes that occur during lung tumor progression.

Base Sequence↗