Biomedical subjects

T E Gray

Publications and source records attributed to T E Gray.

30 records · Page 2Linked to original sources

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.

Animals↗

PubMed1989

Arachidonic acid metabolism in normal and transformed rat tracheal epithelial cells and its possible role in the regulation of cell proliferation.

The objectives of our investigations were to characterize the profile of arachidonic acid metabolites produced by cultured rat tracheal epithelial cells, and to determine whether or not transformation of these cells causes major qualitative or quantitative changes in arachidonic acid metabolism and whether arachidonic acid metabolites play an important role in the regulation of proliferation of rat tracheal cells. Our studies showed that prostaglandin E2 was the only major prostanoid produced by normal and transformed rat tracheal epithelial cells. When stimulated with calcium ionophore A23187, 12-O-tetradecanoylphorbol 13-acetate, arachidonic acid, or serum, the cultures produced small amounts of thromboxane B2, prostaglandin F2 alpha, and hydroxyeicosatetraenoic acid(s) in addition to prostaglandin E2. Mitogenesis studies showed that none of the peptide growth factors tested stimulated either prostaglandin E2 production or DNA synthesis. Fetal bovine serum, on the other hand, stimulated both. 12-O-Tetradecanoylphorbol 13-acetate and arachidonic acid stimulated prostaglandin E2 production but caused no increase in DNA synthesis. Dexamethasone and indomethacin, inhibitors of phospholipase A2 and cyclooxygenase, respectively, significantly inhibited prostaglandin E2 production at concentrations as low as 10(-8) and 10(-9) M but did not inhibit DNA synthesis. It is concluded (1) that prostaglandin E2 is the major arachidonic acid metabolite of rat tracheal epithelial cells, (2) that transformation does not significantly alter arachidonic acid metabolism in these cells, and (3) that neither prostaglandin E2 nor other arachidonic acid metabolites play a significant role in mitogenic stimulation of rat tracheal epithelial cells.

Animals↗

PubMed1987

Site-directed mutagenesis reveals transition-state stabilization as a general catalytic mechanism for aminoacyl-tRNA synthetases.

Some aminoacyl-tRNA synthetases of almost negligible homology do have a small region of similarity around four-residue sequence His-Ile(or Leu or Met)-Gly-His(or Asn), the HIGH sequence. The first histidine in this sequence in the tyrosyl-tRNA synthetase, His-45, has been shown to form part of a binding site for the gamma-phosphate of ATP in the transition state for the reaction as does Thr-40. Residue His-56 in the valyl-tRNA synthetase begins a HIGH sequence, and there is a threonine at position 52, one position closer to the histidine than in the tyrosyl-tRNA synthetase. The mutants Thr----Ala-52 and His----Asn-56 have been made and their complete free energy profiles for the formation of valyl adenylate determined. Difference energy diagrams have been constructed by comparison with the reaction of wild-type enzyme. The difference energy profiles are very similar to those for the mutants Thr----Ala-40 and His----Asn-45 of the tyrosyl-tRNA synthetase. Thr-52 and His-56 of the valyl-tRNA synthetase contribute little binding energy to valine, ATP, and Val-AMP. Instead, the wild-type enzyme binds the transition state and pyrophosphate some 6 kcal/mol more tightly than do the mutants. Preferential transition-state stabilization is thus an important component of catalysis by the valyl-tRNA synthetase. Further, by analogy to the tyrosyl-tRNA synthetase, the valyl-tRNA synthetase has a binding site for the gamma-phosphate of ATP in the transition state, and this is likely to be a general feature of aminoacyl-tRNA synthetases that have a HIGH region.

Amino Acid Sequence↗

PubMed1987

The valyl-tRNA synthetase from Bacillus stearothermophilus has considerable sequence homology with the isoleucyl-tRNA synthetase from Escherichia coli.

We report the DNA sequence of the valS gene from Bacillus stearothermophilus and the predicted amino acid sequence of the valyl-tRNA synthetase encoded by the gene. The predicted primary structure is for a protein of 880 amino acids with a molecular mass of 102,036. The molecular mass and amino acid composition of the expressed enzyme are in close agreement with those values deduced from the DNA sequence. Comparison of the predicted protein sequence with known protein sequences revealed a considerable homology with the isoleucyl-tRNA synthetase of Escherichia coli. The two enzymes are identical in some 20-25% of their amino acid residues, and the homology is distributed approximately evenly from N-terminus to C-terminus. There are several regions which are highly conservative between the valyl- and isoleucyl-tRNA synthetases. In one of these regions, 15 of 20 amino acids are identical, and in another, 10 of 14 are identical. The valyl-tRNA synthetase also contains a region HLGH (His-Leu-Gly-His) near its N-terminus equivalent to the consensus HIGH (His-Ile-Gly-His) sequence known to participate in the binding of ATP in the tyrosyl-tRNA synthetase. This is the first example of extensive homology found between two different aminoacyl-tRNA synthetases.

Amino Acid Sequence↗

PubMed1987

In vitro analysis of multistage carcinogenesis.

Several key events in the multistep process of neoplastic transformation of rat tracheal epithelium (RTE) are described. Whether tracheal epithelium is exposed in vivo to carcinogenic agents or whether primary tracheal epithelial cells are exposed in vitro to carcinogens, initiated stem cells can be detected soon after the exposure by their ability to grow under selective conditions in culture. These initiated stem cells differ fundamentally from normal stem cells in their response to factors normally constraining proliferation and self-renewal. Thus, disruption of inhibitory control mechanisms of stem cell replication appears to be the first event in RTE cell transformation. While the probability of self-renewal (PSR) is clearly increased in initiated stem cells, most of the descendants derived from such stem cells differentiate and become terminal and do not express transformed characteristics. Progression from the first to the second stage of RTE cell transformation, the stage of the immortal growth variant (IGV), is characterized by loss of responsiveness to the growth-restraining effects of retinoic acid. In the third stage of neoplastic transformation, the stage during which neoplastic growth variants (NGV) appear, a growth factor receptor gene is inappropriately expressed in some of the transformants. Thus, it appears that loss of growth-restraining mechanisms may be an early event, and activation of a growth stimulatory mechanism a late event, in neoplastic transformation of RTE cells.

Animals↗

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.

Animals↗

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)

Animals↗

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.

Animals↗

PubMed1984

Studies on the action of tumour promoters and antipromoters on respiratory-tract epithelium.

Studies were carried out to examine the susceptibility of normal and initiated tracheal epithelial cells of rats to known tumour-promoting agents. The ability of normal rat tracheal epithelial cells to form colonies in cultures was enhanced markedly by addition of known tumour promoters to the medium. Several retinoids were shown to reduce the ability of these cells to form colonies in culture and to inhibit this effect of 12-O-tetradecanoylphorbol-13-acetate (TPA). A transformation assay of tracheal epithelial cells was used to study promotional effects of TPA in cultures initiated with N-methyl-N'-nitro-N-nitrosoguanidine. In this assay, four stages of transformation are recognized. TPA did not affect measurably the first two transformation stages, i.e., the development of transformed colonies (enhanced growth variants) and the 'immortalization' of enhanced growth variant-derived subcultures. However, treatment of cultures with TPA during the early post-initiation period resulted in a marked enhancement of the appearance of the third transformed phenotype, which is characterized by anchorage independence of growth. The findings of these in-vitro initiation-promotion studies paralleled, in all major respects, the results obtained in in-vivo - in-vitro studies. Tracheas exposed in vivo to initiator and promoter were shown to develop the same transformants observed in the in-vitro assay. TPA affected primarily the late anchorage-independence phenotype. Parallel tumour induction studies showed that TPA increased markedly the incidence of tracheal carcinomas following initiation with a low dose of 7,12-dimethylbenz[a]anthracene. The studies thus demonstrate that TPA is an effective tumour promoter for rat tracheal epithelium, causing an increase in tracheal carcinomas. They further suggest that the action of TPA on an early transformed cellular phenotype enhances the development of later phenotypes, including neoplastic cell variants.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1983

Quantitation of cell proliferation, colony formation, and carcinogen induced cytotoxicity of rat tracheal epithelial cells grown in culture on 3T3 feeder layers.

A cell culture system is described for the growth of rat tracheal epithelial (RTE) cells at clonal density. The system uses normal, early passage RTE cells grown on feeder layers of lethally irradiated 3T3 cells. The RTE cells have a high colony forming efficiency (5 to 10%) in culture, can be passaged up to 5 times, and are capable of more than 20 cumulative doublings per colony forming cell. The epithelial nature of the cells was confirmed by cell and colony morphology, immunoperoxidase staining of intracellular keratin, and cellular ultrastructural studies. The cytotoxic response of RTE cells to a variety of carcinogens, including a direct acting chemical carcinogen, a physical carcinogen, and a series of polycyclic aromatic hydrocarbons, was quantitated. A linear decrease in the logarithm of survival was observed with increasing doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), gamma-irradiation, 7,12-dimethylbenz(a)anthracene, and a diol-ep-oxide of benzo(a)pyrene. No toxicity was observed after treatment with benzo(a)pyrene or 3-methylcholanthrene over the concentration range examined. In contrast, phorbol ester tumor promoters stimulated cell growth markedly. Based on these and other studies, the RTE cell culture system represents a model system that will be useful for quantitative studies of epithelial cell growth, differentiation, and carcinogenesis.

Animals↗

PubMed1983

High frequency of carcinogen-induced early, preneoplastic changes in rat tracheal epithelial cells in culture.

To study the mechanisms of carcinogenesis, we have developed a system that uses normal cells from an environmentally and epidemiologically relevant tissue, respiratory epithelium. The induction of preneoplastic variants of epithelial cells in culture was quantitated on a per-cell basis following exposure of rat tracheal epithelial (RTE) cells in vitro to the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Following treatment of normal RTE cells, large colonies of altered cells exhibiting an enhanced growth potential under selective culture conditions were observed, while normal RTE cells ceased proliferation after several cell doublings. After further growth in culture, these altered cells acquired the ability to grow in semisolid medium and to produce squamous cell carcinomas when injected into nude mice. The induction of enhanced growth variants of RTE cells by MNNG occurred with a high frequency (greater than or equal to 2.6%/colony-forming cell). In addition, a linear dose-response curve with a slope of approximately 1 was observed when the logarithm of MNNG-induced transformation frequency was plotted versus the logarithm of MNNG dose. These results are consistent with a one-hit mechanism for induction of preneoplastic variants of RTE cells by MNNG. Similar frequencies and kinetics of induction of preneoplastic variants in other culture systems using diploid cells have been observed, suggesting a common mechanism for this early step in carcinogenesis. The RTE cell system will be useful for mechanistic studies of early as well as late changes in the development of neoplasia by epithelial cells.

Animals↗

PubMedDate not supplied

Ligand binding by fibroblast growth factor receptors investigated using chimeric receptor molecules.

In order to map in detail the ligand binding sites of fibroblast growth factor receptor 2 (FGFR2) and keratinocyte growth factor receptor (KGFR), we have generated receptor molecules that are chimeric within the membrane proximal sequence that varies between them. The chimeric molecules are found to bind aFGF with a greater than 5-fold difference in affinity, indicating that there is coupling between the chimeric regions with respect to aFGF binding. Further, binding of bFGF and KGF is abolished in the chimeras, showing that the binding site for these ligands requires the whole of the 48- or 50- amino acid variable sequence to be intact. Direct interactions between the different regions exchanged in the chimeras are most probably involved in forming KGF or bFGF binding sites.

3T3 Cells↗