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Biomedical subjects

B I Gerwin

Publications and source records attributed to B I Gerwin.

At least 19 recordsLinked to original sources
PubMed1992

p53 and Kirsten-ras mutations in human mesothelioma cell lines.

Twenty cell lines from 17 individuals with malignant mesothelioma have been examined for p53 alterations by direct sequencing of genomic DNA, by evaluation of mRNA expression levels, and by immunocytochemical analysis of p53 protein expression in comparison with normal human pleural mesothelial cells. The results of this study show p53 abnormalities in cell lines from 3 individuals. These include 2 point mutations and one null cell line. Interestingly, while both cell lines with point mutations exhibit high levels of p53 protein, normal mesothelial cells as well as 12 of the mesotheliomas evaluated express low but significant levels. In addition, sequencing of K-ras at codons 12, 13, and 61 reveals wild-type sequence in all 20 mesothelioma cell lines. The capacity to induce tumors in athymic nude mice did not correlate with the presence of a p53 mutation or elevated p53 protein levels. These data suggest that neither p53 alteration nor K-ras activation constitutes a critical step in the development of human mesothelioma.

Animals↗
PubMed1992

Mutant p53 can induce tumorigenic conversion of human bronchial epithelial cells and reduce their responsiveness to a negative growth factor, transforming growth factor beta 1.

Loss of normal functions and gain of oncogenic functions when the p53 tumor suppressor gene is mutated are considered critical events in the development of the majority of human cancers. Human bronchial epithelial cells (BEAS-2B) provide an in vitro model system to study growth, differentiation, and neoplastic transformation of progenitor cells of lung carcinoma. When wild-type (WT) or mutant (MT; codon 143Val-Ala) human p53 cDNA was transfected into nontumorigenic BEAS-2B cells, we observed that (i) transfected WT p53 suppresses and MT p53 enhances the colony-forming efficiency of these cells, (ii) MT p53 increases resistance to transforming growth factor beta 1, and (iii) clones of MT p53 transfected BEAS-2B cells are tumorigenic when inoculated into athymic nude mice. These results are consistent with the hypothesis that certain mutations in p53 may function in multistage lung carcinogenesis by reducing the responsiveness of bronchial epithelial cells to negative growth factors.

Animals↗
PubMed1992

Human mesothelioma cells and asbestos-exposed mesothelial cells are selectively resistant to amosite toxicity: a possible mechanism for tumor promotion by asbestos.

To determine if asbestos exposure could contribute to mesothelial cell carcinogenesis by selection and/or expansion of an initiated cell population, we compared normal human pleural mesothelial cells to either human mesothelioma cell lines or mesothelial cells transfected with cancer-related genes for sensitivity to amosite fibers in vitro. Neither normal nor mesothelioma cells were directly stimulated to replicate or increase DNA synthesis by any of the asbestos exposure conditions tested. The potential selective effect of asbestos exposure was demonstrated by a differential sensitivity of normal mesothelial cells and mesothelioma cells to amosite: for example, up to 20-fold higher concentrations of amosite fibers were required to inhibit replication of mesothelioma cell lines than normal mesothelial cells. In addition, a significant resistance (4-fold) to amosite toxicity was observed for SV40 immortalized mesothelial cell lines that had previously been selected in vitro for resistance to asbestos. SV40 immortalized cells that have become tumorigenic after transfection with either Ha-ras or PDGF A-chain genes were not significantly more resistant to the cytotoxic effects of amosite than primary normal cells, and the primary cells were equally sensitive to amosite as mesothelial cells that were only immortalized by SV40. The sensitivity of normal mesothelial cells to asbestos does not appear to be simply a result of general fragility of the mesothelial cells, since similar levels of hydrogen peroxide and silica were cytotoxic for normal mesothelial cells and mesothelioma cell lines. Because mesothelioma cells have a greater resistance to asbestos cytotoxicity than normal mesothelial cells, we hypothesize that a differential resistance to cell killing by asbestos fibers in vivo may result in a selective expansion of an initiated or transformed cell population and thus contribute to the carcinogenesis process. Since tumorigenicity and asbestos resistance occur independently of one another in genetically altered mesothelial cell lines, genotypic and phenotypic alterations that lead to tumorigenic conversion may not be the same changes that provide resistance to cell killing by asbestos.

Asbestos↗
PubMed1991

A human bronchial epithelial cell strain with unusual in vitro growth potential which undergoes neoplastic transformation after SV40 T antigen gene transfection.

Bronchial epithelial cells were cultured from an individual with no evidence of malignant disease. These cells, designated HB56B, had a greatly extended in vitro life-span, being able to undergo 50 passages and 200 population doublings in contrast to the usual 3 to 4 passages and 20 to 30 population doublings characteristic of normal human bronchial epithelial cells. HB56B cells had karyotypic evidence of an amplified region on the short arm of chromosome II. Unlike normal bronchial epithelial cells, which undergo terminal squamous differentiation in vitro in response to fetal bovine serum, HB56B cells were only minimally affected by serum. These cells were readily established as an immortalized cell line, HB56B/5T, following transfection with a plasmid containing SV40 early region DNA. HB56B cells were non-tumorigenic in athymic nude mice, but HB56B/5T cells within a few passages of transfection with the SV40 plasmid formed tumors of which 28/37 regressed. HB56B cells may offer an experimental system for the study of proliferation, differentiation, and senescence control in human bronchial epithelial cells.

Adult↗
PubMed1991

Oncogenes and tumor-suppressor genes.

The functional role of oncogenes in human lung carcinogenesis has been investigated by transfer of activated oncogenes into normal cells or an immortalized bronchial epithelial cell line, BEAS-2B. Transfection of v-Ha-ras, Ki-ras, or the combination of myc and raf into BEAS-2B cells produced tumorigenic cell lines, while transfection of raf or myc alone produced nontumorigenic cell lines. In addition to studying the pathogenic role of oncogenes, we are attempting to define negative growth-regulating genes that have tumor-suppressive effects for human lung carcinomas. Our strategy to identify tumor-suppressor genes involves loss of heterozygosity studies, monochromosome-cell fusion, and cell-cell fusion studies. Loss of heterozygosity studies have revealed consistent allelic DNA sequence deletions on chromosome 17p in squamous cell carcinomas, while large cell carcinomas and adenocarcinomas retained this locus. Mutations in p53, a tumor-suppressor gene located on chromosome 17p, have been observed. Cell-cell hybrid clones produced from fusion of nontumorigenic BEAS-2B cells with tumorigenic HuT292DM cells generally are nontumorigenic. The mechanistic role of the known tumor-suppressor genes Rb-1 and p53 in the development of human lung carcinomas is being investigated in this epithelial cell model of human bronchogenic carcinogenesis.

Animals↗
PubMed1990

Expression of chromosomal proteins HMG-14 and HMG-17 in transformed human cells.

The relation between cellular phenotype and expression of chromosomal high mobility group proteins 14 and 17 (HMG-14 and HMG-17) has been examined in human cell lineages. Quantitation of HMG-14 and HMG-17 mRNA in several human cell lines revealed differences in both the steady state mRNA level and in the ratio of HMG-14 to HMG-17 mRNA. Analysis of phenotypically distinct derivatives of human bronchial epithelial cells revealed small differences between both the steady state mRNA levels and the relative amount of these proteins among the clonal variants. The effect of myeloid differentiation on the mRNA level of HMG-14 and HMG-17 was examined in the human promyelocytic leukemia cell line HL-60 following treatment with several granulocytic and monocytic differentiating agents. The ratio of HMG-17 mRNA to either HMG-14 or histone H4 mRNA varied among the cell phenotypes suggesting that phenotype switching may result in detectable alterations in the expression of the HMG-14 and HMG-17 genes. The data suggest that, although the ratio of HMG-14 to HMG-17 mRNA varies among human cell lines, these variations are relatively small.

Blotting, Northern↗
PubMed1990

Asbestos-related malignant mesothelioma: growth, cytology, tumorigenicity and consistent chromosome findings in cell lines from five patients.

Seven mesothelioma cell lines were established from clinical specimens from five patients with asbestos-related malignant pleural mesothelioma. The cells in culture show either epithelial or mixed epithelial/fibrosarcomatous growth with an average doubling time of 30 h. Giant multinucleated cells are common in all the cell lines, as well as long thin microvilli on the cell surfaces. All cell lines were cytokeratin positive and they stained negatively for monocyte-macrophage markers. All seven cell lines and one long-term tissue culture from a sixth mesothelioma patient were characterized cytogenetically. Karyotype analyses revealed complex structural and numerical abnormalities, primarily involving chromosome 1, 4, 5, 6, 7, 8, 9, 11, 12, 13 and 22. An excess of chromosome material of the short arm of chromosome 5 was seen consistently in six cell lines and in the long-term culture. In cell lines from four patients, changes in chromosome 13, mainly monosomy 13, were observed. The marker chromosomes observed in the early passages were conserved and few additional changes appeared in later passages. Six of the cell lines tested for tumorigenicity in athymic nude mice were weakly positive.

Adult↗
PubMed1990

TGF-beta 1 modulation of urokinase and PAI-1 expression in human bronchial epithelial cells.

Normal human bronchial epithelial (NHBE) cells respond to signals initiated by the binding of transforming growth factor-beta type 1 (TGF-beta 1) to its surface receptors by activating pathways that result in terminal squamous differentiation. By use of both normal and SV40 T-antigen-immortalized cells, it was found that treatment with TGF-beta 1 transiently increases mRNA levels for urokinase (uPA) and plasminogen activator inhibitor type 1 (PAI-1) approximately 5- and 50-fold, respectively, within 4 h. In NHBE cells, PAI-1 protein is increased by TGF-beta 1 in both extracellular matrix and medium. The net effect of TGF-beta 1 on plasminogen activator activity in the medium was a 50% reduction as measured by a caseinolytic assay. A T-antigen-immortalized bronchial epithelial cell line that does not undergo squamous differentiation in response to TGF-beta 1 but binds this growth factor did not respond to TGF-beta 1 by modulation of either uPA or PAI-1 expression. Comparison of human bronchial epithelial, pleural mesothelial, and lung fibroblastic cell strains indicated that the epithelial cells have a constitutively higher ratio of uPA to PAI-1 mRNA expression. These data suggest that modulation of pericellular proteolysis in bronchial epithelial cells in response to TGF-beta 1 represents a significant biological change in their pericellular environment. The induction of uPA and PAI-1 expression in human bronchial epithelial cells may be related to the ability of the cell to undergo squamous differentiation in response to TGF-beta 1. These observations identify specific changes in gene expression that may serve as markers for the differentiation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Bronchi↗
PubMed1990

Cell density governs the ability of human bronchial epithelial cells to recognize serum and transforming growth factor beta-1 as squamous differentiation-inducing agents.

Sparse (75 to 2000 cells/cm2) density cultures of normal human bronchial epithelial cells uniformly undergo terminal squamous differentiation when incubated in medium containing serum (fetal bovine serum [FBS]) or transforming growth factor beta-1 (TGF-beta 1). It was found that the cell density of the culture affects the probability that a cell will respond to these differentiation-inducing agents. Thus whereas irreversible inhibition of DNA synthesis occurs in sparse cell-density cultures within 24 hours after exposure, only a transient (less than 36 hours) depression in DNA synthesis was seen in high (more than 10,000 cells/cm2) density cultures. In addition, although phase microscopic image analysis revealed that virtually all of the cells displayed a squamous morphology within 1 hour after exposure to FBS or TGF-beta 1, observations made 48 to 72 hours later showed the presence of clusters of small prolate spheroid-shaped cells surrounded by many involucrin-positive squamous-appearing cells. Only the small cells were capable of DNA synthesis and cell division as determined by autoradiography and time-lapse photomicrographic images. These replicating cells immediately undergo squamous differentiation if they are subcultured and reinoculated at low cell density and incubated in medium supplemented with FBS or TGF-beta 1. Therefore the probability that a human bronchial epithelial cell will be refractive to FBS- or TGF-beta 1 induced terminal squamous differentiation is solely a function of the cell density of the culture.

Autoradiography↗
PubMed1989

Tumorigenicity of human mesothelial cell line transfected with EJ-ras oncogene.

We performed this study to determine whether human mesothelial cells are capable of undergoing neoplastic change in vitro and to observe their interaction with the activated c-Ha-ras (HRAS1) oncogene EJ-ras, which has a role in the development of many malignant human tumors. Mesothelial cells are presumed to be the progenitor cells of malignant mesothelioma, a cancer strongly correlated with asbestos exposure. Previously, we established a non-tumorigenic cell line, MeT-5A, from normal human mesothelial cells after transfection with a plasmid containing the simian virus 40 (SV40) early-region genes. In the present study, we performed transfection of a plasmid containing the EJ-ras gene and the neomycin-resistance gene into these cells and selected a population resistant to G418, a neomycin analogue. Cells from this cell line formed rapidly growing sc tumors in NIH Swiss athymic nude mice, but untransfected with the vector DNA and selected for G418 resistance formed no tumors. The tumors formed by EJ-ras-transfected cells were established in vitro, and cells from these tumor cell lines exhibited a characteristic altered morphology. The cells had the same isoenzyme phenotype as the parent cells, and they expressed the mutant EJ-ras p21 protein. This first demonstration of malignant transformation of human mesothelial cells in vitro may permit molecular analysis of mesothelial carcinogenesis.

Animals↗
PubMed1989

Differential responses to growth factors by normal human mesothelial cultures from individual donors.

A significant interindividual variation in the growth rates is found in normal cultured human mesothelial (NHM) cells derived from different donors. This variation is observed when the mesothelial cells are incubated in medium containing serum and when the potencies of several separate growth factors are measured by using defined media. Depending on the donor, gamma-interferon and interleukin-2 can be toxic, have no effect, or stimulate the growth rate of NHM cells. Cultured NHM cells can be induced to multiply by growth factors that are released by activated macrophages. Thus, interindividual variation in NHM cell growth control could play a role in the pathogenesis of mesothelioma for a person exposed to asbestos.

Adult↗
PubMed1989

Establishment of a human in vitro mesothelial cell model system for investigating mechanisms of asbestos-induced mesothelioma.

Normal human mesothelial (NHM) cells were transfected with a plasmid containing SV40 early region DNA. Individual colonies of transformed cells from several donors were subcultured for periods of 5 to 6 months and 60 to 70 population doublings (PDs) before senescence, in contrast to a culture lifespan of approximately 1 month and 15 PDs for NHM cells. One such culture, designated MeT-5A, escaped senescence and has been passaged continuously for more than 2 years. These cells had a single integrated copy of SV40 early region DNA in their genome, expressed SV40 large T antigen, and exhibited features of mesothelial cells including sensitivity to the cytotoxic effects of asbestos fibers. One year after injection subcutaneously or intraperitoneally in athymic nude mice, these cells remain nontumorigenic, and therefore are a potential model system for in vitro fiber carcinogenesis studies.

Animals↗
PubMed1988

Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes.

Normal human bronchial epithelial cells were infected with SV40 virus or an adenovirus 12-SV40 hybrid virus, or transfected via strontium phosphate coprecipitation with plasmids containing the SV40 early region genes. Colonies of morphologically altered cells were isolated and cultured; these cells had extended culture lifespans compared to normal human bronchial epithelial cells. All cultures eventually underwent senescence, with the exception of one which appears to have unlimited proliferative potential. Colonies arising after viral infection were screened for virus production by cocultivation with Vero cells; only viral nonproducer cultures were analyzed further. The cells retained electron microscopic features of epithelial cells, and keratin and SV40 T-antigen were detected by indirect immunofluorescence. All of the cultures were aneuploid with karyotypic abnormalities characteristic of SV40-transformed cells. No tumors formed after s.c. injection of the cells in nude mice. These cells should be useful for studies of multistage bronchial epithelial carcinogenesis.

Adenoviruses, Human↗
PubMed1988

Dissimilar peptide growth factors can induce normal human mesothelial cell multiplication.

Quiescent normal human mesothelial (NHM) cells will undergo one round of DNA synthesis when they are incubated in a defined medium consisting of LHC basal medium supplemented with hydrocortisone, insulin, transferrin, and one of the following peptide mitogens: epidermal growth factor; transforming growth factor beta (1 or 2); platelet derived growth factor (a,b heterodimer or b,b homodimer); fibroblast growth factor (acid or basic forms); interleukin 1 (alpha or beta forms); interleukin 2; interferon gamma; interferon beta; or cholera toxin. However, sustained cell multiplication does not occur unless the medium contains hydrocortisone, insulin, transferrin, any one of the above-listed peptide growth factors and high density lipoproteins. Growth can be increased twofold if the medium contains certain combinations of these mitogens and high density lipoproteins. The finding that NHM cells can respond to a broad spectrum of growth factors supports the possibility that an autocrine mechanism may be part of the mechanism that leads to transformation of these cells by asbestos.

Cell Adhesion↗
PubMed1988

Effects of palytoxin or ouabain on growth and squamous differentiation of human bronchial epithelial cells in vitro.

The effects of the non-12-O-tetradecanoylphorbol-13-acetate type tumor promoter palytoxin on human bronchial epithelial cells was studied in an in vitro serum-free culture system. Unlike the results of previous studies with another tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, palytoxin did not induce squamous differentiation of normal bronchial epithelial cells and was equally cytotoxic for normal human bronchial epithelial cells, a human lung tumor cell line, and human bronchial epithelial cells immortalized by infection with adenovirus 12-SV40 hybrid virus (BEAS-2B cells). Palytoxin did not induce a change in free cytosolic Ca2+ concentration of BEAS-2B cells. The effect of palytoxin on the c-myc mRNA steady state level in BEAS-2B cells was studied: 1 pM palytoxin increased the steady-state level at 12 and 18 h. Furthermore, the induction was accompanied by an increase in [3H]thymidine uptake. Because palytoxin binds to (Na+ + K+)ATPase, the effects of ouabain were compared to the effects of palytoxin. A ouabain-resistant cell line was as sensitive to the growth inhibitory effect of palytoxin as the parent ouabain-sensitive cell line, suggesting different binding sites to the (Na+ + K+)-ATPase for palytoxin and ouabain. Ouabain also increased the steady-state level of c-myc gene expression, but earlier than palytoxin, and the increase in the level of c-myc mRNA was accompanied by a drop in DNA synthesis. These results suggest that palytoxin does not act by growth stimulation, differential cytotoxicity or terminal differentiation of normal versus neoplastic cells which are proposed mechanisms of tumor promotion.

Acrylamides↗
PubMed1988

Human bronchial epithelial cells with integrated SV40 virus T antigen genes retain the ability to undergo squamous differentiation.

Human bronchial epithelial cells transformed by either DNA virus infection (SV40 or Adenovirus 12-SV40 hybrid virus) or transfection with the SV40 large T antigen gene were studied for their ability to undergo squamous differentiation when exposed to 12-O-tetradecanoylphorbol-13 acetate (TPA), transforming growth factor-beta 1 (TGF-beta 1), or fetal bovine serum (FBS), agents that induce the squamous differentiation of normal human bronchial epithelial cells. Squamous differentiation occurred in all ten T-antigen-positive cell cultures when they were exposed to either FBS or TGF-beta 1, but none differentiated when exposed to TPA. From one cell line, designated BEAS-2B, two subclones were isolated, one of which was induced to undergo squamous differentiation by FBS, and a second that failed to undergo squamous differentiation and was mitogenically stimulated when exposed to serum. These phenotypically different subclones provide a new in vitro cellular system for delineating the mechanism(s) of human bronchial epithelial cell squamous differentiation in response to FBS or TGF-beta 1.

Antigens, Viral↗
PubMed1988

Functional organization of the murine leukemia virus reverse transcriptase: characterization of a bacterially expressed AKR DNA polymerase deficient in RNase H activity.

The functional organization of the murine leukemia virus reverse transcriptase was investigated by expressing a molecular clone containing AKR MuLV reverse transcriptase-coding sequences in Escherichia coli. A purified preparation of the expressed enzyme (pRT250 reverse transcriptase) consisted primarily of a 69-kilodalton protein that has normal levels of murine leukemia virus polymerase activity but 10-fold-reduced levels of RNase H compared with the viral enzyme. The deficit in RNase H activity was correlated with the absence of 60 to 65 amino acids normally present at the carboxyl end of murine leukemia virus reverse transcriptase. The results provide additional experimental evidence for the localization of polymerase and RNase H domains to the N- and C-terminal regions of reverse transcriptase, respectively.

AKR murine leukemia virus↗