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Biomedical subjects

J F Lechner

Publications and source records attributed to J F Lechner.

At least 37 records · Page 2Linked to original sources
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

Transformation of human neonatal prostate epithelial cells by strontium phosphate transfection with a plasmid containing SV40 early region genes.

Neonatal human prostatic epithelial cells (NP-2s) were transfected by strontium phosphate coprecipitation with a plasmid (pRSV-T) containing the SV40 early region genes. The cells transfected with pRSV-T, but not the sham-transfected controls, formed rapidly growing, multilayered colonies within 2 weeks at a frequency of 1 x 10(-4) in a serum-free medium (P4-8F). In all, 28 colonies of transformed cells were isolated. Three of these have been cultured for a sufficient length of time to show that their growth potentials are well beyond that of the normal progenitor cells (NP-2s). There is also little or no indication of the culture "crisis" commonly seen in SV40-transformed cells in these transfected lines. All contain cytokeratins and SV40 T-antigen as revealed by immunofluorescence, have ultrastructural features of epithelial cells, and are pseudodiploid. None have produced tumors within 1 year after s.c. injection into nude mice. The transformed as well as the parental NP-2s cells require bovine pituitary extract for growth in serum-free medium and are stimulated by transforming growth factor beta 1 (TGF-beta 1) and epidermal growth factor in clonal growth assays. In contrast, a prostatic carcinoma cell line (PC-3) is inhibited by TGF-beta 1. This serum-free system and immortalized transfected clones will be useful for studying the action of putative prostatic carcinogens and tumor-promoting agents.

Cell Division↗
PubMed1989

Effects of serum, transforming growth factor type beta, or 12-O-tetradecanoyl-phorbol-13-acetate on ionized cytosolic calcium concentration in normal and transformed human bronchial epithelial cells.

Fluctuations in ionized cytosolic calcium ([Ca2+]i) are considered important signals for induction of growth or differentiation in mammalian cells. The resting concentrations of [Ca2+]i in normal and adenovirus 12-SV40 hybrid virus-transformed (BEAS-2B) human bronchial epithelial cells were 63 +/- 15 nM (SD) and 44 +/- 15 nM, respectively. Eight % calcium-free fetal bovine serum rapidly caused a significant increase in [Ca2+]i, while causing both cell types to undergo squamous differentiation. When treated with 8% calcium-free fetal bovine serum, a serum-sensitive subclone of BEAS-2B cells exhibited a higher elevation of [Ca2+]i than a serum-resistant (i.e., not stimulated to differentiate by serum) subclone. However, a serum component involved in the induction of squamous differentiation, transforming growth factor type beta, did not increase [Ca2+]i in either normal cells or BEAS-2B cells. 12-O-Tetradecanoyl-phorbol-13-acetate, an exogenous inducer of squamous differentiation and activator of protein kinase C, did not increase [Ca2+]i, but did attenuate serum-induced elevation of [Ca2+]i. These results suggest that while an increase in [Ca2+]i is associated with serum-induced squamous differentiation, a cytosolic ionized calcium signal is not required for the initiation of the squamous differentiation pathway induced by either transforming growth factor type beta or 12-O-tetradecanoylphorbol-13-acetate.

Blood Physiological Phenomena↗
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

Control of growth and squamous differentiation in normal human bronchial epithelial cells by chemical and biological modifiers and transferred genes.

The majority of human lung cancers arise from bronchial epithelial cells. The normal pseudostratified bronchial epithelium is composed of basal, mucous, and ciliated cells. This multi-differentiated epithelium usually responds to xenobiotics and physical injury by undergoing basal cell hyperplasia, mucous cell hyperplasia, and squamous metaplasia. One step of the multistage process of carcinogenesis is thought to involve aberrations in control of the squamous metaplastic processes. Decreased responsiveness to regulators of terminal squamous differentiation may confer a selective clonal expansion advantage to an initiated cell. We studied the effects of endogenous [e.g., transforming growth factor beta 1 (TGF-beta 1) and serum] and exogenous [e.g., 12-O-tetradecanoyl-13-phorbol-acetate (TPA), tobacco smoke condensate, and aldehydes] modifiers of normal human bronchial epithelial (NHBE) cell in a serum-free culture system. NHBE cells are growth inhibited by all of these compounds and induced to undergo squamous differentiation by TGF-beta 1 or TPA. In contrast, lung carcinoma cell lines are relatively resistant to inducers of terminal squamous differentiation which may provide them with a selective growth advantage. Chemical agents and activated protooncogenes (ras,raf,myc) altered the response to endogenous and exogenous inducers of squamous differentiation and caused extended cellular lifespan, aneuploidy, and/or tumorigenicity. The data suggest a close relationship between dysregulation of terminal differentiation pathways and neoplastic transformation of human bronchial epithelial cells.

Bronchi↗
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↗
PubMed1989

Replicative cultures of adult human and rhesus monkey liver epithelial cells.

Exploratory experiments indicate that media containing lipids, phosphoethanolamine, epidermal growth factor, insulin, cholera toxin, bovine pituitary extract, chemically denatured serum, and triiodothyronine will support replicative cultures of normal liver epithelial cells obtained from adult Rhesus monkey and human donors. In addition, we have extended the culture population doubling potential of the human liver epithelial cells by their transfection with a plasmid containing the SV40 virus T-antigen gene. The T-antigen gene-containing cells continued to express albumin through 40 population doublings. Finally, results of preliminary experiments suggest that it may be possible to induce human liver epithelial cells to undergo differentiation to hepatocyte-like cells either by injecting them into the spleen of an athymic nude mouse or by incorporating them into a collagen "tissue equivalent" matrix.

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

Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors.

Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.

Cells, Cultured↗
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

Human bronchial epithelial cells neoplastically transformed by v-Ki-ras: altered response to inducers of terminal squamous differentiation.

Many human bronchial adenocarcinomas have been shown to contain an activated Ki-ras oncogene (Rodenhuis et al., N. Engl. J. Med. 317 929-935, 1987). To test the hypothesis that activated Ki-ras may be causally related to human bronchial carcinogenesis, v-Ki-ras oncogene was transferred into an established human bronchial epithelial cell line, BEAS-2B, by infection with Kirsten murine sarcoma virus (Ki-MSV) or by transfection with a plasmid containing the transforming region of Ki-MSV. These cells formed poorly differentiated adenocarcinomas in athymic nude mice. Cell lines established from these tumors expressed v-Ki-ras p21 protein and were highly tumorigenic. Whereas serum or transforming growth factor beta 1 induced the BEAS-2B cells at clonal density to undergo growth arrest and squamous differentiation, BEAS-2B cells containing activated ras genes were unaffected by transforming growth factor beta 1 and were mitogenically stimulated by serum.

Animals↗
PubMed1987

Comparison of production of transforming growth factor-beta and platelet-derived growth factor by normal human mesothelial cells and mesothelioma cell lines.

Steady state mRNA levels for transforming growth factor beta, platelet-derived growth factor (PDGF) A-chain, and PDGF B-chain were measured in normal human mesothelial cells, SV40 large T-antigen expressing human mesothelial cells, and human mesothelioma cell lines. The mRNA expression level for transforming growth factor beta was similar in all three types of culture while normal human mesothelial cells secrete more transforming growth factor beta than do mesothelioma cell lines or T-antigen expressing mesothelial cells. In contrast, both PDGF A- and B-chain mRNAs are expressed at higher levels in mesothelioma cell lines than in normal human mesothelial cells. PDGF-like mitogenic activity was readily detectable in medium conditioned by a mesothelioma cell line and was undetectable in conditioned medium from normal cells. These results suggest the hypothesis that PDGF may be an autocrine growth factor in mesothelioma.

Cell Line↗