Biomedical subjects

P Nettesheim

Publications and source records attributed to P Nettesheim.

At least 91 records · Page 5Linked to original sources

PubMed1987

Repopulation of denuded tracheas by Clara cells isolated from the lungs of rabbits.

An experimental approach was examined to study the growth and differentiation potential of different epithelial cell types isolated from the airways of adult rabbits. Clara cells isolated from the lungs of rabbits and a mixed cell population obtained from rabbit tracheas were injected (separately) into denuded rat tracheas which were then grafted into nude mice. Epithelial cells were obtained from rabbit tracheas by digestion of tracheal epithelium with proteases. The resulting cell suspension contained mucous cells, Claralike cells, ciliated cells, basal cells and a small number of inflammatory and unidentified cells. This mixed cell suspension was inoculated into rat tracheas denuded of their own epithelium which were then grafted subcutaneously onto the backs of nude mice. The tracheas were recovered two weeks later and were prepared for light and electron microscopy. These grafts were found to be lined by a tall columnar pseudostratified mucociliary epithelium, indicating that the rat tracheal grafts transplanted into nude mice supported the growth and differentiation of tracheal epithelial cells. Similarly Clara cells, isolated and purified to 85.9 +/- 2.8% (n = 9) purity from rabbit lungs by a combination of centrifugation and elutriation procedures, were inoculated into rat tracheas which were transplanted into nude mice. Two weeks later the grafts were recovered and prepared for histological and ultrastructural examination. These tracheas were lined with a low cuboidal epithelium reminiscent of bronchiolar epithelium. The epithelium was composed of ciliated cells and Claralike cells rich in smooth endoplasmic reticulum but containing only a few secretory granules. These results indicate that the tracheal graft model is suited to examine the differentiative potential of specific cell types isolated from the airways. Furthermore, they show that Clara cell isolates are able to establish an epithelium resembling bronchiolar epithelium in denuded tracheas while tracheal cells containing at least four different epithelial cell types are able to establish a mucociliary epithelium resembling the pseudostratified tracheal lining.

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PubMed1987

The differentiation capacity of Clara cells isolated from the lungs of rabbits.

The purpose of our studies was to determine the growth and differentiation potential of Clara cells isolated from rabbit lungs. The Clara cell preparations were enriched (80 to 85%) by density gradient-elutriation procedures and then were inoculated into rat tracheas denuded of their own epithelium. These tracheas then were transplanted subcutaneously on the backs of nude mice. For purposes of comparison, other denuded tracheas were inoculated with mixed epithelial cells obtained from rabbit tracheas by enzymatic procedures. Control tracheas were inoculated with cell-free media. At 2, 4, and 14 weeks after transplantation, the tracheal grafts were removed from the recipient nude mice and examined by light and electron microscopy. Tracheal grafts not receiving cell inocula contained no epithelial lining, and the tracheal lumens were filled with loose connective tissue. Tracheas inoculated with 2 X 10(4) mixed tracheal cells showed a columnar, pseudostratified epithelium composed of five cell types: (a) poorly-differentiated cells, (b) ciliated cells, (c) mucous cells, (d) Clara-like cells, and (e) typical basal cells. A very different epithelium was established in tracheas repopulated with Clara cell isolates. This epithelium, at all time points examined, was cuboidal, single layered (never pseudostratified), and lacked basal cells. The tracheal lumens were lined with ciliated and nonciliated cells. The latter showed typical features of mature Clara cells (i.e., electron dense granules and smooth endoplasmic reticulum). At 14 weeks, the same two cell types were present, and often they were located on ridges and furrows of the tracheal walls. Mixed tracheal cells inoculated into denuded tracheas gave rise to a normal-appearing pseudostratified mucociliary epithelium, whereas the Clara cells inoculated under identical conditions gave rise to a low cuboidal epithelium resembling that seen in normal bronchioles. Establishment of these two types of epithelial linings occurred in the presence of the same mesenchymal components. Thus, we conclude that Clara cells have considerable self-renewal capacity, and their differentiation potential appears to be quite narrow.

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PubMed1986

Changing responsiveness to all-trans retinoic acid of rat tracheal epithelial cells at different stages of neoplastic transformation.

Retinoic acid (RA) treatment of rat tracheal epithelial (RTE) cells, pre-exposed to the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), inhibited transformation in a dose-dependent manner. Treatment with RA at concentrations ranging from 3-33 nM reduced the MNNG-induced transformation frequencies by 13-81% and in some experiments by greater than 90%. RA treatment for only 3 days caused 65-75% inhibition of transformation; treatments of longer duration resulted in greater inhibition of transformation. Delaying the onset of RA treatment reduced its effectiveness, but even when RA treatment was delayed for 3 weeks following MNNG exposure, 60% inhibition still occurred. The inhibition of transformation appeared to be irreversible. The colony forming efficiency of cells isolated from transformed colonies 5 weeks after MNNG exposure was drastically reduced when the replated cells were treated with RA either 1 day or 4 days after plating, indicating that RA blocked cell replication. However, cells isolated from transformed colonies at later times after MNNG exposure were increasingly resistant to the antiproliferative effects of RA. The RA concentration causing 50% inhibition (RA-IC50) of colony formation was 0.1-0.3 nM for cells isolated from 3-5 week-old transformed colonies; it increased greater than 100-fold for cells isolated from 12-week-old transformants. Five established RTE cell lines also showed a much increased resistance to the antiproliferative effects of RA; two of these cell lines were even slightly stimulated in their colony forming ability by RA. The RA-IC50 of colony forming efficiency of normal RTE cells was also determined and compared to that of cells isolated from 5-week-old transformed colonies. Since the normal RTE cells require 3T3 feeder layers for clonal growth, both cell types were grown on feeders. For both cell types, the RA-IC50 was similar (150-300 nM); the requirement for relatively high RA concentrations was attributed in part to the rapid RA metabolism by the feeder cells. These experiments show that early RTE cell transformants are growth-inhibited by RA; however, they increasingly lose their sensitivity to the growth controlling effects of RA as they progress to a more advanced stage of transformation. The inhibition of tracheal cell transformation by RA is probably due to the antiproliferative effects of the retinoid.

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PubMed1986

Biosynthesis of prostaglandins by isolated and cultured airway epithelial cells.

The metabolism of arachidonic acid to prostaglandins and thromboxane by freshly isolated and cultured respiratory tract epithelial cells was examined by HPLC methods. Homogenates prepared from freshly isolated rat and rabbit tracheal epithelial cells did not convert arachidonic acid to prostaglandins. Rat tracheal epithelial cells however, did convert arachidonic acid to uncharacterized metabolites possibly hydroxyfatty acids. In contrast, rat tracheal epithelial cells grown in culture for 9 days acquired the capacity to convert arachidonic acid to PGE2 and related products while cultured rabbit tracheal epithelial cells converted arachidonic acid to TXB2. The conversion of arachidonic acid to PGE2 by rat tracheal cells was studied at various times in culture. The formation of PGE2 appeared to parallel the growth of the cultures. In contrast to freshly isolated rat tracheal cells, enriched rat Clara cell fractions were able to convert 14C-arachidonic acid to prostacyclin (PGI2) ans measured by HPLC analysis of its stable end product 6-keto PGF1 alpha. PGI2 was also the major metabolite of arachidonic acid produced by enriched rat alveolar type II cell fractions. PGF2 alpha, and hydroxyfatty acids were also formed. These results suggest that arachidonic acid metabolism differs in various types of respiratory tract cells and that maintenance of such cells in culture alters the pattern of arachidonic acid metabolism.

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PubMed1986

Biosynthesis and release of proteins by isolated pulmonary Clara cells.

The major proteins synthesized and released by Clara cells were identified and compared with those synthesized and released by mixed lung cells. Highly purified Clara cells (85.9 +/- 2.4%) and mixed lung cells (Clara cells 4%, Type II cells 33%, granulocytes 18%, macrophages 2.7%, ciliated cells 1.2%) were isolated from rabbit lungs, incubated with Ham's F12 medium in collagen/fibronectin-coated plastic culture dishes in the presence of 35S-methionine for periods of 4 and 18 hrs. Radiolabelled proteins were isolated from the cells and from the culture medium, electrophoresed on polyacrylamide gels in the presence of SDS under reducing conditions, and then autoradiographed. After 4 and 18 hr of incubation of the Clara cells the major radiolabelled cell-associated proteins were those with molecular weights of 6, 48, and 180 Kd. The major radiolabelled proteins released by Clara cells into the medium after 4 hrs of incubation had molecular weights of 6, 48, and 180 Kd, accounting for 42, 16, and 10%, respectively, of the total extracellular protein-associated radioactivity. After 18 hr of incubation the 6 and 48 Kd proteins represented 30 and 18% of the total released radioactivity, and the relative amount of the 180 Kd protein had decreased to 3%. With the mixed lung cells, the major proteins released into the medium had molecular weights of 6 and 48 Kd. Under nonreducing conditions the 6 Kd protein released by Clara cells had an apparent molecular weight of 12 Kd. Labelling isolated Clara cells with a mixture of 14C-amino acids also identified this low molecular weight protein as the major secretory product of the Clara cell. The 6 Kd protein did not label when the cells were incubated with 14C-glucosamine indicating that it was not a glycoprotein. These data demonstrate the release of several proteins from isolated Clara cells but the major protein had a molecular weight of 6 Kd.

Amino Acids↗

PubMed1986

In vitro transformation of primary rat tracheal epithelial cells by 5-azacytidine.

The ability of the hypomethylating agent 5-azacytidine to transform normal primary rat tracheal epithelial cells was determined. A single 24-h exposure to 5-azacytidine resulted in the transformation of rat tracheal epithelial cells at concentrations of the cytosine analogue ranging from 1 to 4 microM. Doses of 5-azacytidine that produced these enhanced growth transformants were nonmutagenic as measured by the induction of 6-thioguanine or ouabain resistance. In addition, the hypomethylating agent 5-azadeoxycytidine also transformed primary rat tracheal epithelial cells, whereas 6-azacytidine, a cytosine analogue which does not induce DNA hypomethylation, did not. Enhanced growth transformants resulting from 5-azacytidine exposure continued to progress in the absence of further treatment to give rise to variants with indefinite growth capacity which ultimately became neoplastic. These results indicate that hypomethylating agents can transform normal cells and suggest that changes in DNA methylation may occur during the initial stages of neoplastic transformation.

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PubMed1986

Morphological characterization of transformed colonies in rat tracheal epithelial cell cultures exposed to carcinogen.

The purpose of the studies described in this and the accompanying paper (D.J. Fitzgerald et al., Cancer Res., 46:4642-4649, 1986) was to define the transformed colonies of the rat tracheal epithelial cell transformation system (see P. Nettesheim and J.C. Barrett, CRC Crit. Rev. Toxicol., 12: 215-239, 1984) in terms of differentiation and growth characteristics. Exposure of low-density primary rat tracheal epithelial cell cultures to N-methyl-N'-nitro-N-nitrosoguanidine results in the development of growth-altered colonies of different sizes and morphologies, which can be readily scored 5 wk after treatment. These colonies were classified into four morphological types (I to IV) based on their light microscopic and ultrastructural features. Colonies designated as types I and II were small, had a low cell density, and were composed principally of large, pale-staining (Giemsa) flattened cells with a low nuclear/cytoplasmic ratio. Examination of the fine structure of these colonies revealed a monolayer of extremely attenuated cells containing well-developed Golgi complexes, endoplasmic reticulum, numerous secondary lysosomes, but few tonofilaments and desmosomes. Colonies of types III and IV were large, basophilic (Giemsa), high-density colonies, consisting primarily of closely packed, small, round cells with high nuclear/cytoplasmic ratio. Analysis of the fine structure of these colonies revealed two to four stratified layers of poorly differentiated cells and cells having features of keratinocyte differentiation, as evidence by abundant tonofilament bundles, well-developed desmosomes, and occasional keratohyaline granules. Differential cell counts were carried out at the ultrastructural level on pooled cells from each colony type (except type I); approximately 90% of the cells from type II colonies were large, electron-lucent cells, while this cell type comprised only 20 to 30% of the cells of colonies of types III and IV. The predominant cell type (approximately 60%) in colonies of types III and IV showed clear signs of keratinocyte differentiation. Type IV colonies contained a significantly larger proportion (greater than 20%) of small, poorly differentiated cells than type III (6%) and type II (1%) colonies. A combined autoradiographic and ultrastructural study revealed that the small, poorly differentiated cells were most active in synthesizing DNA and had a labeling index of 60%. Other cell types were far less frequently labeled, particularly the large, electron-lucent cells which had a labeling index of only 10%. Morphometric measurements on fixed and stained colonies disclosed that each colony type differs markedly in cell population size.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1986

Analysis of growth fractions and stem cell compartments in transformed rat tracheal epithelial cell colonies.

The purpose of the studies described here was to define the biological behavior of the various clonally transformed colonies observed in cultures of carcinogen-exposed rat tracheal epithelial cells. As described in the preceding paper (H. Kitamura et al., Cancer Res., 46: 4631-4641, 1986), these colonies fall into four morphologically distinct categories. In the studies reported here we found that type I colonies had the smallest growth fraction (7%) and contained the lowest frequency of clonogenic cells (approximately 10(-3)). Colonies of types II to IV had mean growth fractions of 21 to 28%, and the frequency of clonogenic cells was 2 to 5 X 10(-2) when measured under growth-permissive conditions (3T3 feeders). When the clonogenic cell assays were performed under selective conditions to identify cell variants which can grow without feeder support, the average frequency of such clonogenic cells in type I colonies was less than 4 X 10(-5) and in colonies of types II to IV, between 5 X 10(-4) and 10(-2). In type IV colonies, the total number of cells per colony increased 8-fold between 5 and 12 wk postcarcinogen, but the clonogenic cell compartment increased 42-fold; the compartment of variant clonogenic cells, which are able to replicate on plastic, increased 139-fold during the same period of time. This indicated that major changes in the self-renewal capacity of the clonogenic cells were taking place during this early stage of transformation. Examination of the daughter colonies produced by replating colonies of types I to IV revealed that clonogenic cells with different growth potential existed within the same parent colony. Comparison of transformed colonies of the same type showed a marked degree of heterogeneity in the sizes of growth fractions and clonogenic cell fractions. These studies further indicated that, within all colonies, including the most advanced transformants, the majority of the cells were nonreplicating, terminal cells, suggesting that, at least during early stages of transformation, the transformed characteristics were not transferred from parent to daughter cells. With the exception of type I colonies, most of the colonies recognizable at 5 wk after carcinogen exposure progressed with time and acquired the morphological characteristics of type IV colonies, which were the most transformed phenotype. We conclude that transformation of rat tracheal epithelial cells is an asynchronous process and that the morphologically distinct types of rat tracheal epithelial cell colonies represent different stages of the clonal evolution of transformants.

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PubMed1985

Biochemical characterization of mucous glycoproteins synthesized and secreted by hamster tracheal epithelial cells in primary culture.

Hamster tracheal epithelial cells growing on type I collagen gel synthesize and secrete high molecular weight glycoconjugates which elute in the void volume upon Sepharose CL-4B column chromatography. The presence of any proteoglycans in this void volume material was ruled out based on both enzymatic analysis and behavior on DEAE-ion exchange chromatography. Based on the incorporation of radioactive precursors, followed by strong acid hydrolysis or neuraminidase digestion, the material was shown to contain sialic acid, fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and sulfate. Complete susceptibility to papain digestion and reductive beta-elimination suggests that the material consists of O-linked glycoproteins. The identification of N-acetylgalactosaminitol in the beta-eliminated oligosaccharides confirms this notion. The molecular weight of the oligosaccharides following beta-elimination ranges from 4,000 to 15,000. We conclude that the high molecular weight glyconjugates produced by hamster tracheal epithelial cells in primary culture are mucous glycoproteins based on size, sensitivity to alkaline borohydride treatment, and monosaccharide composition. Further characterization of these mucous glycoproteins showed both size and charge microheterogeneity among molecules. Detailed structural analysis of oligosaccharides of these mucous glycoproteins is currently under way.

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PubMed1985

The toxic response of preneoplastic rat tracheal epithelial cells to 12-O-tetradecanoylphorbol-13-acetate.

The purpose of our studies was to examine the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on survival, proliferation, and differentiation of normal and preneoplastic rat tracheal epithelial (RTE) cells. Normal RTE cells obtained from 8-week-old rats were plated into medium containing TPA at concentrations ranging from 0.1 to 100 ng/ml. A dose-related increase in colony-forming efficiency (CFE) was observed at concentrations above 0.1 ng/ml. At 100 ng/ml the CFE was increased 5-fold above that observed in controls. Similar studies were carried out with immortal, preneoplastic RTE cells derived from carcinogen-exposed primary cultures. Upon exposure to 10 ng/ml of TPA, 9 out of 11 cell lines showed a marked decrease in CFE ranging from a 70 to a greater than 99% reduction. Two out of 11 cell lines showed only a minor reduction in CFE at this TPA concentration. Teleocidin B was as potent as TPA in causing inhibition of CFE in preneoplastic RTE cells; the potency of 4-O-methyl TPA was approximately 1/100 that of TPA and phorbol was virtually inactive. Further studies suggested that inhibition of clonal growth of the preneoplastic tracheal cells by TPA was due to rapid cell death. As early as 2 h after addition of TPA to the cultures, a loss of viable cells from the cultures as well as a reduction in CFE was observed. Morphological studies support the interpretation that a large number of cells was severely damaged within 2--4 h after TPA exposure. The formation of cross-linked envelopes (CLE), a measure of keratinocyte differentiation, was determined in cultures of preneoplastic RTE cells. TPA caused only minor increases in CLE formation indicating that the cells were not dying from TPA-induced terminal keratinocyte differentiation, but rather from TPA toxicity. Thus, preneoplastic RTE cells respond differently to TPA from normal RTE cells and also from normal and transformed keratinocytes of various origins. The role of TPA toxicity in promoting preneoplastic RTE cells to the neoplastic state is being investigated.

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PubMed1985

Production of arachidonic acid metabolites by macrophages exposed in vitro to asbestos, carbonyl iron particles, or calcium ionophore.

Consequent to asbestos deposition, alveolar macrophages (AM) accumulate at alveolar duct bifurcations where they phagocytize fibers. Because phagocytosis can stimulate the release of arachidonic acid (AA) metabolites, the possibility that secretion of these powerful mediators of inflammation might be induced by chrysotile asbestos was investigated in vitro. Rat AM were treated in vitro with chrysotile asbestos, and the cyclooxygenase products--prostaglandins, thromboxane B2 (TXB2), 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT)--and lipoxygenase products--leukotrienes (LT), hydroxyeicosatetraenoic acids (HETE)--secreted in the medium were isolated by high-performance liquid chromatography. Composition of the AA metabolites released was compared with that from those stimulated by the calcium ionophore A 23187 (20 microM) and by another particulate phagocytic stimulus, i.e., carbonyl iron beads. Calcium ionophore stimulation induced a marked release of various AA metabolites in the medium from both the cyclooxygenase pathway (HHT, TXB2, and PGE2, in decreasing quantities, respectively) and the lipoxygenase pathway (LTB4, 5-HETE, 12-HETE, and LTC4). The major product was LTB4. Treatment of the macrophages with asbestos fibers induced the release of a similar array of AA metabolites, although there were smaller amounts of LTC4 and 12-HETE, but increased quantities of PGF2 alpha. A time course study showed a steady increase in metabolite production for 1 h, followed by a plateau. In addition, the amount of metabolites released was dependent on asbestos concentrations. Phagocytosis of iron beads induced the secretion of the same metabolites as asbestos stimulation, but in larger quantities, probably reflecting the lack of cytotoxicity of the particle.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1985

Morphological changes in rat tracheal cells during the adaptive and early growth phase in primary cell culture.

The purpose of our studies was to determine the fate of different cell types present in early primary cultures of tracheal epithelial cells and, if possible, to elucidate the role they play in the establishment of the cultures. Epithelial cells were isolated from rat tracheas with 0.5% Pronase and were cultured on collagen-coated dishes as described previously. Light and transmission electron microscopic studies showed that the cell population harvested from rat trachea was composed of approximately 30% ciliated cells, 50% granule-containing cells and 20% undifferentiated cells (presumably basal cells). Upon seeding the tracheal cell suspensions into culture, approximately 40% of the cells attached. Cell attachment was virtually complete after 16 h. Roughly 60% of the cells attaching during the first 12 h were neither ciliated nor granulated, suggesting that undifferentiated cells played a major role in establishment of the early cultures. Between 20 and 35% of the cells attaching during this time were identified as granulated cells (mucous cells). Ciliated cells did not start to attach in significant numbers until 8 h after seeding. They never amounted to more than 8-12% of the attached cell population. After 12 h of culture, the cell population underwent a progressive loss of differentiation. The number of poorly differentiated cells (i.e. those showing neither cilia nor mucous granules) increased correspondingly. This loss of differentiation preceded the onset of DNA synthesis and cell growth which began at about 24 and 40 h, respectively. Continuous [3H]thymidine-labelling studies showed that at 48 h after the start of culture about 90% of all attached cells had entered DNA synthesis at least once. This finding is consistent with the interpretation that the ciliated cells are terminally differentiated cells and are probably the only part of the tracheal cell inoculum not participating in the growth of the cultures. At 72 h, the cultures (now in mid-log growth phase) were composed of uniformly undifferentiated cells lacking cilia and mucous granules. The cells nevertheless showed unequivocal epithelial characteristics such as tight junctions and desmosomes. The studies suggest that both basal and mucous cells are responsible for the establishment and growth of the rat tracheal epithelial cell cultures.

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PubMed1985

Quantitation of the rate of spontaneous generation and carcinogen-induced frequency of anchorage-independent variants of rat tracheal epithelial cells in culture.

The rate of spontaneous generation and the frequency of carcinogen-induced anchorage-independent variants of preneoplastic rat tracheal epithelial (RTE) cells in culture were quantitated. Anchorage-independent variants of different RTE cell lines arose spontaneously by a stochastic process at rates of 0.5 X 10(-4) to 5.4 X 10(-4) variants/cell/generation, as determined by fluctuation analyses. These variants were also induced by the mutagen N-methyl-N'-nitro-N-nitrosoguanidine with a frequency of approximately 10(-3) variants/surviving cell. The rates of spontaneous change and the frequencies of induction are the first reported for epithelial cells and are similar to some, although not all, rates and frequencies of change to anchorage independence for fibroblast-like cells in culture. In addition, these rates and frequencies are similar to those for mutations at some known gene loci. The induced frequency of this late change in neoplastic progression is, however, considerably lower than the frequency of induction of the initial, preneoplastic changes in RTE cells in culture (approximately 3 X 10(-2)/surviving cell). These quantitative determinations are useful in defining the mechanisms of late changes occurring during the progression of RTE cells to the neoplastic state.

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PubMed1985

Contrasting responses of normal and transformed rat tracheal epithelial cells to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

The data presented here are part of an ongoing effort to examine the response of rat tracheal epithelium to tumor promoting agents and to elucidate the mechanisms of tumor promotion in that epithelial tissue (see Steele, this volume). Previous studies indicated that airway epithelium is responsive to the tumor promoter TPA and that TPA can promote the tumor response in rat tracheal epithelium But what are the mechanisms involved? We have divided this question into two main elements: 1) Which stages of neoplastic development are affected by TPA, i.e., which preneoplastic cell populations are targets for TPA action resulting in the acceleration and enhancement of the process of neoplastic development? 2) What effects does TPA have on various preneoplastic cell populations and how do such effects result in promotion? The experiments discussed here relate to the second part of the question. They suggested that TPA elicits a marked cytotoxic response in stably transformed RTE cell variants (see Fig. 1). These preneoplastic cell variants are clearly different from untransformed RTE cells which are triggered into cell cycle as indicated by an increase in CFE. This difference between normal and transformed cells is of considerable interest in itself since it points to a fundamental, biochemical alteration in the transformed cells. Evidence exists that transformed RTE cell lines have TPA receptors and that at least some of the responses elicited by TPA exposure, such as the induction of ornithine decarboxylase activity, are receptor-mediated. Whether the cytotoxic response elicited by TPA is receptor-mediated is presently not known.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1985

Changes in stem cell populations of rat tracheal epithelial cell cultures at an early stage in neoplastic progression.

The development of transformed colonies and concomitant changes in proliferative and nonproliferative cell compartments were studied in rat tracheal epithelial (RTE) cell cultures following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Primary RTE cells were plated onto 3T3 feeder layers and treated with MNNG (0.25 micrograms/ml) or solvent. Seven days later, the feeder cells were removed to select for enhanced growth variants, which are the transformants of the RTE cell system, usually scored 5 weeks after carcinogen exposure. Most of the RTE cell colonies, which originally formed during the first 7 days of culture, disappeared within 2 weeks after feeder cell removal in control and MNNG-treated cultures. In control cultures, about 3% of the original colonies persisted, while in MNNG-treated cultures, a larger percentage (approximately 9%) of the colonies persisted. These percentages remained constant from 3 to 7 weeks. Based on colony size, cell density, and cell morphology, the persistent colonies were classified into transformed colonies (large colony size, high cell density, high nuclear:cytoplasmic ratio) and untransformed colonies (small size, low cell density, low nuclear:cytoplasmic ratio). In the MNNG-treated cultures, about 50% of all persistent colonies showed transformed morphology. Their frequency remained unchanged between 3 and 7 weeks of culture. In contrast, only 10 to 15% of the persistent colonies in control cultures showed transformed morphology at 3 weeks, but that proportion increased steadily between 3 and 7 weeks. These data suggest that, in control cultures, transformed colonies developed spontaneously as a function of time within untransformed colonies. Autoradiographic studies with [3H]thymidine showed that labeling indices in the early "normal" RTE cell colonies between Days 4 and 7 of culture were very high, ranging between 75 and 90%. In contrast, the labeling indices of persistent colonies, both those without and those with transformed morphology, were low, i.e., between 18 and 25%, indicating that a major proportion of cells was either noncycling or cycling very slowly. The relative compartment sizes of cells with stem cell characteristics and of cells with characteristics of transformed stem cells were estimated before and after transformed colonies appeared.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1985

In vitro transformation of rat tracheal epithelial cells: a model for the study of multistage carcinogenesis.

A multistage transformation assay has been developed using normal primary rat tracheal epithelial cells as targets for measuring the transforming activity of a variety of test substances. The assay is suited for quantitation of cell transformation and allows the study of effects of promoters as well as inhibitors of transformation at various stages of this multiphasic process. Studies with TPA and retinoic acid have shown that the first stage of neoplastic transformation of RTE cells cannot be enhanced by TPA but is inhibited by retinoic acid. However, TPA can enhance a later stage of transformation, the conversion of the ag- to the ag+ phenotype. The cellular and biochemical mechanism of the inhibitory effects of RA are under investigation.

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PubMed1984

Monoclonal antibodies directed against rat tracheal epithelial cells transformed in vitro.

Cloned hybridoma cell lines were obtained by fusion of murine myeloma cells with spleen lymphocytes of either F344 rats or BALB/c mice immunized against the malignant F344 tracheal cell line, 2-10-1. Monoclonal antibodies were selected for their ability to bind to the immunizing cell line and not to normal tracheal epithelial cells. Results from quantitative binding assays indicated that such monoclonal antibodies recognized six epitopes. Each epitope was detected on four other malignant tracheal epithelial cell lines, and was also expressed during early preneoplastic cell passages (i.e. before the cells acquired the ability to produce carcinomas in vivo). Quantitative differences in epitope expression between non-tumorigenic and tumorigenic passages of individual cell lines could not be detected for five of the epitope groups. However, two monoclonal antibodies, recognizing the same epitope, did show quantitative binding differences between non-tumorigenic and tumorigenic cell passages on three of the five cell lines tested. Our results show that carcinogen-altered tracheal cell populations can be distinguished from non-altered cells (by our current assay methods) by use of monoclonal antibodies. The suggest that the antigen expression is an early event associated with the transformation of rat tracheal epithelial cells in culture.

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PubMed1984

The effect of 12-O-tetradecanoylphorbol-13-acetate and other tumor promoters on the colony formation of rat tracheal epithelial cells in culture.

Normal primary rat tracheal epithelial (RTE) cells were isolated and exposed in culture to tumor promoters and irritants of diverse chemical classes. The phorbol derivative class of tumor promoters greatly stimulated colony forming efficiency (CFE) in culture. The efficacies of the agents tested were ranked in the order: mezerein greater than 12-O-tetradecanoyl-phorbol-13-acetate (TPA) greater than phorbol didecanoate greater than phorbol dibutyrate greater than phorbol dibenzoate greater than 4-O-methyl TPA greater than phorbol diacetate. The parent alcohol phorbol did not stimulate CFE under the conditions tested. The indole alkaloid tumor promoter teleocidin stimulated CFE at concentrations at least 10-fold lower than those required for TPA. Other irritants and non-phorbol ester tumor promoters such as anthralin, benzoyl peroxide, calcium ionophore A23187, and ethylphenyl propiolate were either inactive or reduced CFE. Phenobarbital marginally stimulated CFE at one concentration but reduced CFE at higher concentrations. Increases in CFE elicited by TPA and analogs were dependent upon the time of addition of TPA to the cultures. Maximum increases in CFE were observed when the cells were plated into medium containing TPA. If TPA was added 40 h after plating, stimulation of CFE did not occur. This 40 h time interval may represent a crucial period for the commitment of RTE stem cells to proliferation or differentiation. Whether the stimulation of colony formation seen in normal RTE cells exposed to phorbol derivatives also occurs in carcinogen-altered cells, thereby causing their proliferative expansion, remains to be determined.

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