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P Nettesheim

Publications and source records attributed to P Nettesheim.

At least 55 records · Page 3Linked to original sources
PubMed1993

Growth regulation of primary rat tracheal epithelial cell cultures by endogenous transforming growth factor-beta s.

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to secrete transforming growth factor-beta (TGF beta) and to be growth inhibited by exogenous TGF beta. The purpose of the present studies was to determine whether the endogenous TGF beta(s) were regulating the growth of RTE cell cultures and, if so, which isoforms were involved. Neutralizing antibodies specific to TGF beta 1 and TGF beta 2 were added to cultures, and their effects on several growth parameters were measured. Addition of antibodies to early cultures (day 1), resulted in 1.8- and 3-fold increases in colony formation and cell number, respectively, above control IgG-treated cultures. Antibody dose-response experiments revealed that TGF beta 2 was the predominant isoform inhibiting early RTE cell growth. The antibody treatments resulted in similar stimulation of early growth at low and high seeding densities, suggesting that the endogenous TGF beta s were acting locally. Anti-TGF beta 1 treatment of cultures at various stages of growth resulted in 1.2-1.7-fold increases in DNA synthesis above controls, whereas anti-TGF beta 2 treatment resulted in increased DNA synthesis only in early and late cultures (1.7- and 2.5-fold, respectively), but not during midlogarithmic growth. Continuous treatment with a combination of both antibodies resulted in increased growth and decreased exfoliation in early cultures, but had no effect on the slow down of growth in late cultures. Thus endogenous TGF beta s inhibited primarily early growth and contributed to, but did not appear to be responsible for, plateau of growth in late stage cultures. Antibody treatment of secondary cultures resulted in 4-70-fold increases in colony formation, depending on the age of the primary cultures when replated, indicating that endogenous production of both TGF beta 1 and TGF beta 2 greatly inhibits the subculturability of primary RTE cells. Other experiments suggested that cholera toxin enhances RTE cell growth in part by counteracting the inhibitory effects of endogenous TGF beta s.

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PubMed1993

Phorbol ester and epidermal growth factor enhance the expression of two inducible prostaglandin H synthase genes in rat tracheal epithelial cells.

Previous studies from our laboratory suggested that phorbol 12-myristate 13-acetate (TPA) stimulates prostaglandin E2 (PGE2) production by inducing de novo synthesis of prostaglandin H synthase (PHS) in a rat tracheal cell line. We report here an extension of this work to further elucidate the mechanisms by which TPA (and epidermal growth factor) stimulates PGE2 production. We used the rat tracheal cell line EGV6, which has a lower basal level of PGE2 production and responds to TPA and EGF stimulation with a much greater increase in PGE2 synthesis than the previously used cell line, Incubation of EGV6 cultures with TPA or EGF resulted in a time- and dose-dependent increase in PGE2 synthesis up to 40-fold and 6-fold, respectively. Serum also stimulated PGE2 synthesis, while bombesin, retinoic acid, and bacterial lipopolysaccharide did not. PHS protein levels in microsomal preparations from the cells were estimated by Western analysis. Antibodies raised against murine PHS-2 cross reacted with the EGV-6 PHS while several antibody preparations that react with PHS-1 from ram or mouse reacted poorly with the cellular preparation. TPA treatment increased the de novo synthesis of PHS-2 while dexamethasone treatment reduced the response to TPA. Northern blot analysis of mRNA from EGV6 cultures using a ram PHS cDNA revealed a 2.8- and a 4.5- to 4.9-kb (designated 4.9 kb) transcript. Treatment with TPA or EGF increased the expression of both transcripts and this effect was further enhanced by cyclohexamide. To further define the PHS mRNA species of EGV6 cells, two well-characterized murine PHS cDNA probes were used. The constitutive murine PHS cDNA probe hybridized only with the 2.8-kb transcript, and the inducible murine PHS cDNA hybridized only with the 4.9-kb transcript. The rates of induction as well as degradation of the 4.9-kb PHS mRNA were much more rapid than those of the 2.8-kb mRNA species. Dexamethasone partially inhibited the induction of both PHS transcripts by TPA. Southern analysis of genomic EGV6 DNA indicated the presence of two distinct PHS genes in these cells. Taken together these findings indicate that two PHS genes are expressed in rat tracheal epithelial cells. In contrast to the PHS genes expressed in murine (and chicken) fibroblasts in which only the gene coding for the larger mRNA species is transcriptionally regulated, in the rat tracheal cells both genes are positively regulated by TPA and EGF and downregulated by glucocorticoids.

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PubMed1993

Enhanced growth potential of cultured rabbit tracheal epithelial cells following exposure to N-methyl-N'-nitro-N-nitrosoguanidine.

To establish a standardized model for the transformation of rabbit airway epithelial cells, we attempted to transform rabbit tracheal epithelial (RbTE) cells in culture with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). RbTE cells, harvested by enzymatic digestion from male New Zealand white rabbits, were plated onto feeder layers of irradiated 3T3 cells. Control cells proliferated exponentially during the 2nd week of culture and reached the plateau phase by the 3rd week. Cells exposed to MNNG (0.1 microgram/ml) proliferated in a fashion similar to the control cells, except that there was some delay before proliferation began. The clonogenic activity of RbTE cells rapidly decreased in parallel with the increase in cell population equally in the control and MNNG groups. During the late plateau phase, cells exposed to MNNG regained clonogenic activity, and this compartment size expanded with time, whereas the clonogenic activity in control cultures remained below the detectable level. In RbTE cell cultures exposed three times to 0.1 microgram/ml MNNG, large, persistent and proliferating colonies emerged at a frequency of 1-3 x 10(-2) among the surviving clones, whereas all the control cultures eventually became senescent. The MNNG-induced alteration in the growth potential of RbTE cells, i.e., the extended lifespan, and the maintenance and even expansion of clonogenic activity, was similar to that of transformed rat tracheal epithelial cells. However, no immortal cell line could be established from these growth-altered RbTE cells. We therefore concluded that the growth-altered RbTE cells were partially transformed.

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PubMed1993

Phenotypic marker expression during fetal and neonatal differentiation of rat tracheal epithelial cells.

The expression of phenotypic markers was examined during fetal and neonatal differentiation of rat tracheal epithelial (RTE) cells. The rat counterpart of human keratin 18 was predominantly found in columnar cells in the adult trachea. It was detected in the primordial tracheal epithelium first seen on gestational day (GD) 12 (term = 21.5 days). Staining intensity gradually increased, and by GD 17 it was principally localized to the apical portion of the epithelium. The rat counterpart of human keratin 19 was barely detectable in the trachea on GD 13 but became abundant in almost all RTE cells on and after GD 19. Morphologically and immunocytochemically identifiable secretory and ciliated cells appeared on GD 18. Ciliated cell number slowly rose while secretory cells increased dramatically on GD 19 through postnatal day 1. The secretory granule antigens detected by monoclonal antibodies RTE 9 and 11 were rare in the adult trachea but were highly expressed in virtually all of the perinatal secretory cells. In contrast, the epitope detected by monoclonal antibody RTE 12, which was present in all adult tracheal surface secretory cells, did not appear until postnatal day 1 and slowly increased. These results demonstrate marked shifts in the biochemical composition of secretory cells during development and postnatal maturation. For the above-mentioned molecules, a similar expression pattern was observed during epithelial regeneration in tracheal grafts (Am. J. Respir. Cell Mol. Biol. 1992; 7:30-41). Pseudo-stratification of the epithelium and basal cells was first observed on GD 20. Keratin 14, which is confined to basal cells in the normal adult trachea, was not present in the nascent basal cells but appeared after postnatal day 1. In contrast to the present results, during epithelial regeneration in tracheal grafts keratin 14 appeared before markers of highly differentiated secretory or ciliated cells. Thus, the biochemical sequence of cellular differentiation during regeneration did not precisely recapitulate development.

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PubMed1993

Rat tracheal epithelial cell differentiation in vitro.

In vitro culture conditions enabling rat tracheal epithelial (RTE) cells to differentiate to mucociliary, mucous, or squamous phenotypes are described. Medium composition for rapid cell growth to confluence in membrane insert cultures was determined, and the effects of major modifiers of differentiation were tested. Retinoic acid (RA), collagen gel substratum, and an air-liquid interface at the level of the cell layer were required for expression of a mucociliary phenotype which most closely approximated the morphology of the tracheal epithelium in vivo. Large quantities of high molecular weight, hyaluronidase-resistant glycoconjugates, most likely mucin glycoproteins, were produced in the presence of RA when the cells were grown with or without a collagen gel and in submerged as well as in interface cultures. However, extensive ciliagenesis was dependent on the simultaneous presence of RA, collagen gel, and an air-liquid interface. When RA was omitted from the media, the cells became stratified squamous and developed a cornified apical layer in air-liquid interface cultures. This phenotype was accompanied by loss of transglutaminase (TGase) type II and keratin 18 and expression of the squamous markers TGase type I and keratin 13. The ability to modulate RTE cell phenotypes in culture will facilitate future studies investigating molecular regulation of tracheal cell proliferation, differentiation, and function.

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PubMed1993

Characterization of a third transforming growth factor beta 1 transcript in rat tracheal epithelial cells.

It has been previously reported (R. W. Steigerwalt et al., Mol. Carcinog., 5:32-40, 1992) that primary cultures of rat tracheal epithelial (RTE) cells and immortalized RTE cell lines produce three mRNA transcripts [2.5, 1.9, 1.4 kilobases (kb)] which hybridize to a murine transforming growth factor beta 1 (TGF-beta 1) complementary DNA probe. In this report, we show that the 1.9- and 1.4-kb transcripts are not detectable by Northern analysis of resting adult trachea but are induced in regenerating tracheal grafts and tumors formed from transformed RTE cells. Northern analysis of the TGF-beta 1 transcripts with subclones of the murine complementary DNA demonstrate that the 1.4-kb transcript lacks much of the 5' untranslated region (UTR). RNase protection analysis was used to map the transcriptional start site of the 1.4-kb transcript to within 30-40 base pairs of the first ATG codon. No differences in the coding or 3' UTR were detected between the 1.4-kb and the 2.5-kb transcripts. Although RTE cells produce a 1.9-kb TGF-beta 1 mRNA, we were unable to detect a previously reported unique 3' UTR, which we found to be almost identical to a rat mitochondrial ATPase sequence. Because the 1.4-kb transcript is missing most of the long GC-rich 5' UTR, it may be translated at a different rate than the 2.5- and 1.9-kb transcripts, or it may code for an intracellular form of TGF-beta 1. The 1.4-kb transcript has been observed under several conditions of injury or stress and, therefore, may be an important component of the TGF-beta 1 response to these conditions.

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PubMed1992

Changes in responsiveness of rat tracheal epithelial cells to transforming growth factor-beta 1 with time in culture.

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to be highly sensitive to growth inhibition by transforming growth factor-beta 1 (TGF beta 1) when treated within 1-2 days after plating. The purpose of the present studies was to examine the effects of TGF beta 1 on the growth of RTE cells as a function of time in culture. We found that the sensitivity of RTE cells to growth inhibition by TGF beta 1 decreased dramatically as the cultures aged. The IC50 for inhibition of colony forming efficiency was 0.18 pM when TGF beta 1 was added 24 h after cell plating. When TGF beta 1 treatment was begun on day 5 of culture, the IC50 was 3-4 pM as measured by inhibition of growth (cell number) and DNA synthesis. However, when TGF beta 1 was begun on day 19, the IC50 was 65 pM or greater than 500 pM, depending on whether inhibition of growth or DNA synthesis, respectively, was measured. TGF beta 1 accelerated cell death, as measured by exfoliation of cells, and inhibited cell proliferation. The decrease in responsiveness to TGF beta 1 in late cultures was shown to be dependent on culture age as well as on cell density. No evidence was found for inactivation or degradation of the added TGF beta 1 by the late stage cultures. Cells subcultured from late stage primary cultures remained less responsive to TGF beta 1 than subcultured cells from early cultures. Similar to its effect on proliferation, TGF beta 1 down-regulated the expression of two proliferation-related genes, c-myc and transforming growth factor-alpha, in early but not late RTE cell cultures. On the other hand, fibronectin expression was increased by TGF beta 1 by about twofold at both early and late times in culture. This indicates that the changes in TGF beta 1 responsiveness with time in culture are selective, apparently affecting primarily proliferation-related events.

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PubMed1992

Epidermal growth factor dependence and TGF alpha autocrine growth regulation in primary rat tracheal epithelial cells.

We have examined dependence of primary rat tracheal epithelial (RTE) on exogenous epidermal growth factor (EGF) and determined whether a TGF alpha autocrine pathway is operating in these cells. Primary RTE cells plated in serum free media (SFM) without EGF and bovine pituitary factor (BPE) show little proliferation compared to cultures propagated in media containing EGF/BPE (CSFM). Removal of EGF/BPE shortly after plating, however, results in significant proliferation, although plateau cell densities are reduced and cell morphology is significantly altered compared to cells propagated in CSFM. Addition of EGF and/or BPE to cultures propagated in SFM minus EGF/BPE restores maximum cell density. The concentration of TGF alpha peptide in media conditioned by cells propagated without EGF/BPE is lower than the concentration in the media of CSFM cultures. TGF alpha mRNA and protein levels are also significantly lower in cells late in culture compared to logarithmically growing cells regardless of the presence or absence of EGF/BPE. The proliferation of primary RTE cells propagated without EGF/BPE is inhibited by neutralizing TGF alpha antiserum and by a tyrphostin compound that blocks TGF alpha/EGF receptor tyrosine kinase activity. These results indicate that primary RTE cells utilize TGF alpha as an autocrine growth factor and that the autocrine pathway is regulated as a function of growth state of the cells. However, this pathway does not provide growth autonomy to primary RTE cells, since cultures remain dependent on exogenous EGF/BPE for sustained proliferation.

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PubMed1992

Transformed rat tracheal epithelial cells exhibit alterations in transforming growth factor-beta secretion and responsiveness.

The purpose of our studies was to define abnormalities in the transforming growth factor-beta (TGF-beta) system of transformed rat tracheal epithelial (RTE) cells that might cause their abnormal growth behavior. We found that many, but not all, of the transformed cell lines were hyporesponsive or unresponsive to the growth inhibitory effects of TGF-beta 1. Scatchard and receptor cross-linking analyses indicated that loss of TGF-beta 1 responsiveness of transformed cells was probably not due to changes in receptor number or affinity, or to changes in expression of the three TGF-beta-binding protein subtypes. Transformed cells were found to secrete far less TGF-beta-like activity (less than 1/10) than primary cells. Cultured normal and transformed RTE cells expressed three TGF-beta 1 transcripts of 2.5, 1.9, and 1.4 kb. In contrast, rat kidney tissue, a rat embryo fibroblast cell line, and a rat liver cell line expressed only the typical 2.5-kb mRNA transcript commonly reported in the literature. In spite of the marked differences in TGF-beta secretion between normal and transformed cells, their levels of TGF-beta 1 mRNA expression were similar. This suggests a change in the posttranscriptional regulation of TGF-beta 1 expression. TGF-beta 2 message was not detected in either normal or transformed RTE cells in culture. These findings are consistent with the hypothesis that the abnormal growth behavior of transformed RTE cells is at least in part due to disturbances of the TGF-beta system.

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PubMed1992

The effect of sodium selenite on cell proliferation and transformation of primary rat tracheal epithelial cells.

The effects of sodium selenite (Na2SeO3) on cell proliferation and the development of preneoplastic transformed variants were studied in primary cultures of rat tracheal epithelial cells. Results revealed a biphasic effect of Na2SeO3 on cell proliferation: at concentrations between 6 x 10(-8) and 6 x 10(-6) M, it stimulated and at concentrations of approximately 2 x 10(-5) and above it inhibited cell proliferation (presumably due to toxicity). Nontoxic concentrations of Na2SeO3 (6 x 10(-8) -6 x 10(-7) M) significantly reduced the spontaneous transformation frequency. Transformation induced by the tobacco-specific nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was effectively inhibited by nontoxic as well as toxic concentrations of Na2SeO3. Treatment of cultures with Na2SeO3 after cessation of NNK exposure, i.e. during the selection period, also significantly reduced the transformation frequency. These experiments show that the inhibition of transformation by Na2SeO3 is not the result of an antiproliferative effect. They further indicate that the inhibitory effect occurs even when the chemical treatment occurs during the 'postinitiation' phase. Thus the inhibition of transformation by Na2SeO3 cannot solely be explained by its effects on drug metabolism.

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PubMed1992

Expression of "cell-type-specific" markers during rat tracheal epithelial regeneration.

Previous studies showed that Griffonia simplicifolia I-isolectin B4 (GS I-B4) and monoclonal antibodies (mAbs) against keratin 14 labeled basal cells in the adult rat trachea while other mAbs specifically stained secretory and/or ciliated cells. We used these "cell-type-specific" markers to study cellular differentiation during tracheal epithelial regeneration. Denuded tracheal grafts were inoculated with rat tracheal epithelial cells and were implanted in syngeneic hosts. Marker expression was correlated with the appearance of morphologically defined cell types. At 4 days, the epithelium was squamoid, one to three cell layers thick, and was apparently composed of a single morphologic cell type. Because this cell did not exhibit distinguishing features of any mature tracheal cell, we provisionally termed it the "poorly differentiated cell" (PD cell). PD cells expressed keratin 14 and GS I-B4 binding sites; they contained glycogen and had lipid droplets but did not react with secretory or ciliated cell-specific mAbs. At 7 days, areas of the epithelium were pseudostratified and secretory cell-specific markers were present at the apex of differentiating columnar cells; ultrastructurally, these cells resembled secretory cells in adult tracheas. Simultaneously, a few preciliated and ciliated cells appeared that expressed a ciliated cell-specific epitope. No cells were observed coexpressing secretory and ciliated cell markers. Basal cells also became recognizable on day 7. These expressed keratin 14 and GS I-B4 binding sites throughout the study. Newly appearing secretory and ciliated cells also expressed these two markers initially but lost them gradually as the mucociliary epithelium matured. In the tracheal graft model of epithelial regeneration, the PD cells were pivotal intermediates from which all differentiated cells developed. Basal cells continued to express the same markers as PD cells, which were gradually lost in secretory and ciliated cells as they acquired new sets of specific epitopes.

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PubMed1991

Role of TGF alpha and its receptor in proliferation of immortalized rat tracheal epithelial cells: studies with tyrphostin and TGF alpha antisera.

We have shown in the present study and in studies reported previously that preneoplastic and neoplastic rat tracheal epithelial (RTE) cell lines express TGF alpha and do so regardless of the mechanism by which they were transformed. In order to determine whether TGF alpha is an autocrine growth regulator of immortalized RTE cells, we have examined the function of TGF alpha/EGF receptors and the growth requirements for TGF alpha in these cells. The level of immunoprecipitated TGF alpha/EGF receptor protein in immortalized RTE cells was similar to or less than levels in primary RTE cells, indicating that chemically induced transformation of RTE cells does not involve overexpression of TGF alpha/EGF receptors. Scatchard analysis of TGF alpha/EGF receptors in the neoplastic EGV5T cell line revealed the presence of high-affinity (Kd = 0.4 nM) and low-affinity (Kd = 9.8 nM) binding sites. A tyrphostin TGF alpha/EGF receptor tyrosine kinase inhibitor decreased in a dose-dependent manner the proliferation as well as EGF-induced autophosphorylation of the TGF alpha/EGF receptor of transformed RTE cells. The inhibitory effect of tyrphostin on proliferation and receptor kinase activity was attenuated in late log and plateau phase cultures. The phosphotyrosine content of several other EGF-dependent and independent phosphoproteins was also decreased by the tyrphostin. Proliferation of transformed RTE cells was also inhibited when TGF alpha antisera was added to the media of growing cells. These data are consistent with the hypothesis that proliferation of transformed RTE cells involves autocrine regulation by TGF alpha and its receptor.

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PubMed1991

Critical variables controlling cell proliferation in primary cultures of rat tracheal epithelial cells.

The purpose of our experiments was to examine variables affecting early events in the establishment of rat tracheal epithelial (RTE) cultures as well as factors regulating long-term RTE cell growth. The experiments showed that when RTE cells were seeded into complete serum-free medium between 13 and 30% of the seeded cells attached. Of the seeded cells, only approximately 2% entered into DNA synthesis and underwent repeated cell divisions to form colonies containing greater than 20 cells. Coating the dishes with extracellular matrix components had little effect on cell attachment or colony forming efficiency (CFE). However, coating the dishes with fetal bovine serum markedly increased CFE. The media components bovine serum albumin and bovine pituitary extract were shown to be important in promoting cell attachment as well as CFE. Cholera toxin on the other hand had no effect on cell attachment but significantly increased CFE. These and other studies showed that cell attachment and cell proliferation are independently regulated. Studies on long-term culture growth indicated that the number of progeny produced per colony forming unit (CFU) is inversely proportional to the number of CFUs seeded. Inasmuch as the cultures did not become confluent under any of the culture conditions tested and media obtained from high density cultures were shown to be growth inhibitory, these findings suggest that a diffusible growth restraining factor is being produced by the cultures limiting clonal expansion. Experiments showing growth inhibitory effects of media conditioned by high cell density cultures support this interpretation. The putative factor reaches critical concentrations earlier in cultures seeded with high numbers of CFU than in cultures seeded with low numbers of CFU. Because the cultures are known to produce transforming growth factor-beta, this growth regulator probably plays a role in controlling RTE cell proliferation. However, it is likely than other events, such as depletion of growth factors from the media, also are significant in regulating the growth of the cultures.

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PubMed1991

Cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat tracheal epithelial cells.

The cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were studied by the assays of colony-forming efficiency (CFE), micronucleus formation (MN), and cell transformation in rat tracheal epithelial (RTE) cells both in vitro and in vivo. Liver S9, primary hepatocytes and RTE cells from normal and Aroclor-1254 induced rats were compared for bioactivation of NNK using Salmonella mutagenesis as the endpoint. Results from the in vitro experiments indicated that low concentrations of NNK (0.01-25 micrograms/ml) caused from 15% to greater than 100% increases in CFE of RTE cells. At high concentrations (100-200 micrograms/ml), NNK was significantly toxic to RTE cells. NNK treatment in vitro (50-200 micrograms/ml) increased MN frequency as much as 3-fold above background and significantly increased the transformation frequency (TF) in 4/5 (50 micrograms/ml) and 6/8 (100 micrograms/ml) experiments. The in vivo exposure of rats to NNK (150-450 mg/kg, given i.p.) resulted in a 60-85% reduction in CFE and a 3-5-fold increase in MN formation in RTE cells. In vivo treatment with cumulative doses of 150 and 300 mg/kg of NNK produced significant increases in TF of tracheal cells from 3/3 and 2/3 rats, respectively. Without activation, NNK was not mutagenic in Salmonella TA1535. The bioactivation of NNK to a mutagenic metabolite was achieved by incubation of NNK with liver S9 fraction from Aroclor-1254 induced rats or primary hepatocytes from both untreated and Aroclor-1254 pretreated rats. RTE cells did not produce sufficient quantities of mutagenic NNK metabolites to be detected by the Salmonella assay.

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PubMed1991

Properties of rat tracheal epithelial cells separated based on expression of cell surface alpha-galactosyl end groups.

We used Griffonia (bandeiraea) simplicifolia I (GS I) lectin and flow cytometry to isolate subsets of rat tracheal epithelial cells based on the presence or absence of cell surface alpha-galactosyl end groups. These fractions were designated GS I-positive and -negative, respectively. Ninety-eight percent of the cells in the GS I-positive fraction expressed cell surface alpha-galactosyl end groups; 95% had immunocytochemically detectable keratin 14-related protein (a basal cell marker) and 98% lacked alcian blue-periodic acid-Schiff (AB-PAS)-stained cytoplasmic granules. More than 90% of the GS I-positive cells had a high nuclear-to-cytoplasm ratio, had tonofilaments, and lacked organelles characteristic of other differentiated cell types; they were thus classified as basal cells. In bioassays, the GS I-positive fraction had a colony-forming efficiency greater than or equal to that of native tracheal cell suspensions, and the cells were able to repopulate denuded tracheal grafts with ciliated, secretory, and basal cells. More than 99% of the cells in the GS I-negative fraction lacked cell surface alpha-galactosyl end groups, 98% did not stain for keratin 14-related protein, 54% had significant numbers of AB-PAS-stained cytoplasmic granules, and 16% were identified as ciliated cells. The GS I-negative fraction had a lower colony-forming efficiency than the GS I-positive fraction but, it too, was able to repopulate denuded tracheal grafts with a complete mucociliary epithelium. These results show that both GS I-positive and -negative cells had the potential to proliferate and differentiate into the major tracheal cell types.

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PubMed1991

Cell type-specific lectin staining of the tracheobronchial epithelium of the rat: quantitative studies with Griffonia simplicifolia I isolectin B4.

We compared lectin staining patterns to cell population densities, as determined by morphological criteria in rat airways. Eight lectins were studied: Griffonia simplicifolia I isolectin B4 (GSI-B4), Arachis hypogaea (PNA), Wisteria floribunda (WFA), Glycine maximus (SBA), Dolichos biflorus (DBA), Helix pomatia (HPA), Ulex europaeus (UEA-1), and Maclura pomifera (MPA). Two of the lectins strongly stained morphologically distinct cell subpopulations. GSI-B4 stained basal cells, and MPA stained non-ciliated bronchiolar (Clara) cells. The specificity and sensitivity of GSI-B4 as a marker for basal cells was examined. In the trachea, 35% of all cells were GSI-B4 positive; 84% of these were basal cells, 7% were unidentified cells, 5% were serous/mucous cells, and 4% were ciliated, brush, or inflammatory cells. Comparison to cell population density data strongly suggested that all basal cells were GSI-B4 positive. The segmental bronchus was a transitional area; GSI-B4 positive basal cells were present in the region closest to the lobar bronchus but were absent in the distal region; instead, MPA-positive Clara cells appeared. When dissociated tracheal cells were obtained by pronase digestion, 43% were GSI-B4 positive. These results show that GSI-B4 is a sensitive and relatively specific marker for basal cells in the rat trachea which can be used to study dissociated epithelial cells.

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PubMed1991

Alterations in growth factor pathways in multistage carcinogenesis of rat tracheal epithelial cells.

The role of peptide growth factors in the process of multistage carcinogenesis of rat tracheal epithelial (RTE) cells was assessed by examining growth factor requirements and expression of growth factors and their receptors in normal and transformed RTE cells. Transformed RTE cell lines show decreased requirements for bovine pituitary extract, insulin and epidermal growth factor compared to primary RTE cells in culture. An autocrine role for TGF alpha in transformed RTE cells is suggested by data showing TGF alpha production and decreased proliferation in the presence of TGF alpha antisera and TGF alpha/EGF receptor kinase inhibitor. Therefore, decreased EGF requirements in transformed RTE cells could be explained by autocrine TGF alpha regulation. In contrast, no evidence for an insulin/IGF-I autocrine pathway could be detected in transformed RTE cells. These data indicate that multiple alterations in growth factor pathways occur in transformed RTE cells.

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