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U Lichti

Publications and source records attributed to U Lichti.

At least 37 records · Page 2Linked to original sources
PubMed1987

Two types of transglutaminase in the PC12 pheochromocytoma cell line. Stimulation by sodium butyrate.

Transglutaminases are a class of enzymes capable of covalently cross-linking both intracellular and extracellular proteins. The activity of tissue transglutaminase is known to decrease precipitously following neoplastic transformation, and it has been hypothesized that transglutaminase may be involved in growth regulation. We have found that the differentiation promoter sodium butyrate is able to cause a marked increase in transglutaminase activity in PC12 pheochromocytoma cells in a time- and dose-dependent manner. This increased transglutaminase activity is associated with growth arrest, as well as with striking morphological changes including increased cell adhesion. The transglutaminase induced by sodium butyrate appears to be tissue transglutaminase, based on its cytosolic localization, thermal lability at basic pH, and elution profile on anion-exchange chromatography. Untreated PC12 cells contain only small amounts of transglutaminase which resembles epidermal transglutaminase, an enzyme previously described only in skin. In contrast to sodium butyrate, nerve growth factor did not stimulate tissue transglutaminase in PC12 cells, although it, too, caused growth arrest. It is hypothesized that transglutaminase may be involved in certain morphological changes accompanying cellular differentiation and neoplastic transformation, rather than in growth regulation per se.

Adrenal Gland Neoplasms↗
PubMed1987

Response of carcinogen-altered mouse epidermal cells to phorbol ester tumor promoters and calcium.

Primary cultures of mouse epidermal cells are induced to terminally differentiate when extracellular calcium levels are increased to more than 0.1 mM. After carcinogen treatment, cellular foci can be selected that resist this calcium signal to terminally differentiate. Calcium causes these foci to stratify; however, in contrast to normal epidermis, DNA-synthesizing cells in these foci are found in the suprabasal cell layers as well as in basal cells. Cell lines derived from these foci may be considered to be putative initiated cells. Three of these cell lines, designated 308, D, and F, have been characterized for their response to calcium and phorbol ester tumor promoters. The formation of cornified cells and the activity of epidermal transglutaminase were utilized as markers of epidermal differentiation. Neither calcium nor the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increased transglutaminase activity or cornification of any of the 3 lines. Proliferation was estimated by the [3H]thymidine labeling index, by incorporation of [3H]thymidine into DNA, and by a clonal growth assay. Unlike primary normal cultures, raising the calcium level of the medium did not markedly reduce the rate of proliferation of any of the 3 cell lines. In 2 of the lines, line 308 and line D, proliferation increased in response to TPA exposure. In line F, [3H]thymidine incorporation in confluent cultures was inhibited by TPA, while in cells plated at clonal densities, TPA was cytotoxic at doses of 5 ng/ml or higher. If these calcium-resistant epidermal cell lines correspond to initiated cells, their lack of sensitivity to the induction of terminal differentiation by TPA could account for their growth relative to normal cells. Those lines that also respond to stimulation of proliferation by TPA to a greater extent than normal cells would have a further growth advantage.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1987

Effect of retinoic acid on cornified envelope formation: difference between spontaneous envelope formation in vivo or in vitro and expression of envelope competence.

A large number of cross-linked envelopes form spontaneously when cell lines derived from chemically induced mouse skin papillomas are cultured in medium containing 1.2 mM calcium. This phenomenon is associated with high activity of the cross-linking enzyme, epidermal transglutaminase (TGase). The influence of retinoic acid (RA) on envelope formation was studied in detail in a papilloma cell line, PE. Retinoic acid (3 microM) completely blocked cornified envelope (CE) production but reduced TGase activity only 50%. A rabbit antiserum was produced against sonicated CEs isolated from newborn mouse skin. On Western blots of epidermal extracts, diffuse staining was observed for particulate proteins of suprabasal, but not basal, cells and similar immunoreactive material was absent from the cytosolic fraction of both cell layers. The antibody also recognized particulate proteins from PE cells induced to differentiate by calcium, but not from cells grown in the presence of high calcium and RA. The antiserum appears to recognize partially cross-linked CE precursor proteins judging by the diffuse staining, the molecular weight range of the proteins stained, and their origin in the particulate cellular fraction. Cross-linked envelopes could be induced in RA-treated PE cells by permeabilization with 0.75 M NaCl or 50 micrograms/ml A23187. However, this treatment failed to cause the appearance of proteins recognized by the antiserum. Preincubation of the antiserum with purified fragments of CEs from newborn mouse epidermis, but not with cross-linked envelopes from permeabilized, RA-treated PE cells, removed immunoreactivity. These results indicate that the cross-linked envelopes formed in RA-treated cells after permeabilization lack a set of proteins contained in CEs from stratum corneum and may even be composed of different proteins. Retinoic acid appears to prevent CE formation in part by inhibiting activation of epidermal TGase but in addition by influencing the synthesis of precursor proteins.

Animals↗
PubMed1986

Cultivation and characterization of cells derived from mouse skin papillomas induced by an initiation-promotion protocol.

Methods to culture cells from papillomas induced by an initiation-promotion protocol in SENCAR mice were developed, and the resultant cell lines have been characterized. Using Eagle's medium with 0.05 mM Ca2+ conditioned by dermal fibroblasts and supplemented with 1 ng/ml epidermal growth factor (EGF) in culture dishes coated with collagen and fibronectin, six cell lines (PA, PB, PC, PD, PE and PF) were established from separate pools of papillomas. When tested for tumorigenicity in nude mice by injection of a cell suspension or implantation of cells growing on a plastic liner, two of the lines (PC and PF) produced no tumors at any passage. In contrast, cells of the lines PA and PE produced highly differentiated squamous cell carcinomas from the earliest passage tested. The results with PB and PD were variable on tumorigenicity testing with some passages positive and others negative. When tested for responsiveness to Ca2+ (greater than 0.1 mM) as a differentiation stimulus, all lines responded. In the higher Ca2+ medium there was a 50-95% decrease in colony-forming efficiency, a slight decrease in [3H]thymidine incorporation (except for PA) and an increase in the number of cornified cells (except for early passage PF). Epidermal transglutaminase activity, a marker for terminal differentiation, was increased in the presence of medium with Ca2+ greater than 0.1 mM. However, unlike normal cells, only a fraction of the cells from each of the papilloma-derived cell lines terminally differentiated in response to Ca2+ while the remaining cells continued to proliferate, although at a slower rate. Responsiveness to phorbol ester tumor promoters was also examined in papilloma cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment increased colony forming efficiency, DNA synthesis and colony size in all lines in medium with either 0.05 mM Ca2+ or 1.2 mM Ca2+. TPA treatment also increased ornithine decarboxylase activity in all lines, even at the higher Ca2+ concentration, although normal keratinocytes respond only when grown in medium with low Ca2+. TPA treatment caused only a slight increase in the number of cornified cells and no increase in epidermal transglutaminase activity in papilloma cells while it causes 10-fold or greater increases in these differentiation markers in normal keratinocytes. Thus papilloma cells appear to differ from normal keratinocytes in their ability to maintain a proliferating population under conditions favoring terminal differentiation, their consistent proliferative response to phorbol esters under these same conditions, and their reduced sensitivity to phorbol ester-induced terminal differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Retinoic acid-induced transglutaminase in mouse epidermal cells is distinct from epidermal transglutaminase.

Elevated transglutaminase activity and formation of cornified envelopes are markers of terminal differentiation in mouse epidermal cells. Epidermal transglutaminase catalyzes cornified envelope formation and in cultured cells is inducible by calcium ion or phorbol ester tumor promoters. Retinoic acid also induces transglutaminase activity but inhibits cross-linked envelope formation. This apparent paradox might be resolved by the observation that the retinoic acid-induced transglutaminase appears to be either a different enzyme or a markedly altered form of the epidermal enzyme. The retinoic acid-induced transglutaminase is soluble in aqueous buffers, is thermolabile at pH 9.0, 37 degrees C, and elutes from an anion exchange column at 0.4 M NaCl. In contrast, the epidermal enzyme is particulate and requires detergent for solubilization, is relatively thermostable, and elutes from the anion exchanger at 0.25 M NaCl. The retinoic acid-induced enzyme is probably identical with the "tissue" transglutaminase present in liver and in other cells. It is proposed that the transglutaminase induced by retinoic acid may play a role in the inhibition by retinoids of calcium and tumor promoter-induced differentiation.

Acyltransferases↗
PubMed1985

Inhibition of epidermal terminal differentiation and tumour promotion by retinoids.

Retinoids are physiological regulators of growth and differentiation for a number of epithelial tissues. In several of these, retinoids also act as pharmacological anticarcinogens. Retinoids are most effective as anticarcinogens in the post-initiation portion of carcinogenesis. In mouse skin, retinoids are inhibitors of phorbol ester-mediated tumour promotion and can cause regression of pre-existing benign tumours. Studies in vivo and in vitro have indicated that phorbol ester-mediated skin tumour promotion results from selective clonal expansion of initiated cells. We have proposed that the biological basis for selection resides in the induction of terminal differentiation in subpopulations of keratinocytes while other keratinocytes, including initiated cells, are stimulated to proliferate. Terminal differentiation is accelerated by phorbol esters through the induction of epidermal transglutaminase and consequent cornification. Retinoids inhibit terminal differentiation of keratinocytes. Retinoids also induce transglutaminase in epidermis, but they inhibit cornification. Recent results suggest a biochemical basis for this paradox. The phorbol ester-induced transglutaminase is primarily particulate but the retinoid-induced enzyme is cytosolic. The induced enzymes differ in kinetic parameters, thermal stability and in elution from ion-exchange columns. Induction of the retinoid enzyme is associated with suppression of the induction of transglutaminase by phorbol esters. The retinoid-induced epidermal transglutaminase could interfere with normal or promoter-induced differentiation by inappropriately cross-linking precursor proteins before their assembly at the cell periphery. This could explain one aspect of the inhibitory action of retinoids on tumour promotion.

Acyltransferases↗
PubMed1985

Similar effects of phospholipase C and phorbol ester tumor promoters on primary mouse epidermal cells.

Interaction of tumor promoting phorbol esters with specific high affinity receptors is probably essential for many of the biological responses elicited by these agents. Since diacylglycerols which can be produced enzymatically from phospholipids by phospholipase C are postulated to be the physiological ligands for the phorbol ester receptor, we have examined primary cultures of mouse epidermal basal cells exposed to phospholipase C (Clostridium perfringens) for several biological and biochemical responses characteristic of treatment with 12-O-tetradecanoyl-phorbol-13-acetate, the most potent phorbol ester tumor promoter. Formation of diacylglycerols by treatment with phospholipase C was demonstrated by the dose-dependent release of radioactive diacylglycerols in cells prelabeled with [3H]arachidonic acid. Treatment with phospholipase C at 0.05 units/ml for 30 min led to the morphological changes and to the reduction in epidermal growth factor binding (90%) associated with 12-O-tetradecanoylphorbol-13-acetate treatment. Continuous treatment at the same dose led to the induction of the enzymes ornithine decarboxylase and transglutaminase with a time course and extent similar to the inductions by 12-O-tetradecanoylphorbol-13-acetate. Treatment with phospholipase C at 0.1 enzyme unit/ml yielded substantial suppression of the binding affinity of phorbol-12,13-dibutyrate for its receptors without reduction in total number of binding sites, consistent with the production by phospholipase C of a competitive inhibitor of phorbol ester binding. Several diacylglycerols at concentrations of 250 microM and above effectively competed for phorbol-12,13-dibutyrate binding, reduced epidermal growth factor binding, and to a lesser extent induced ornithine decarboxylase and transglutaminase. These results support the hypothesis that diacylglycerols can act through the phorbol ester receptors and thus produce biological and biochemical responses similar to those of the phorbol esters.

Animals↗
PubMed1983

Regulation of epidermal transglutaminase activity and terminal differentiation by retinoids and phorbol esters.

A number of characteristics of phorbol ester-mediated mouse skin tumor promotion indicate that cell selection is the underlying biological process. Studies in vivo and in cultured mouse epidermal cells suggest that selection is based on heterogeneous responses of subpopulations of basal cells which can be induced to proliferate or differentiate in response to promoter exposure. Retinoids are effective inhibitors of tumor promotion in mouse skin but do not influence the proliferative response to phorbol esters. Since both retinoids and phorbol esters modify epidermal differentiation, the antipromoter action of retinoids could be related to differentiation responses. Retinoic acid induces transglutaminase activity in cultured mouse epidermal basal cells grown in less than 0.1 mM Ca2+. While this enzyme is associated with terminal differentiation in skin, retinoic acid paradoxically blocks the terminal differentiation of cultured cells. In contrast, phorbol esters and extracellular calcium greater than 0.1 mM induce both transglutaminase activity and terminal differentiation. Enzyme kinetic analyses indicate that the transglutaminases induced by all three inducers are the same enzyme. The increase in activity of transglutaminase by all three inducers requires RNA and protein synthesis. However, the time course of increase and decay of each activity differs for each inducer. A variety of biologically active retinoids induce transglutaminase activity, and their effectiveness correlates with their reported antipromoter activity. Exposures to both retinoic acid and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate are antagonistic resulting in less than additive induction. Induction kinetics with both inducers are more like those of retinoic acid than those of the phorbol ester. Simultaneous exposure to retinoic acid and 12-O-tetradecanoylphorbol-13-acetate protects the epidermal cell population from induced terminal differentiation and cell loss which is observed in response to the promoter alone. These results suggest that the antipromoting action of retinoids could be mediated by modification of phorbol ester-accelerated terminal differentiation through an effect on transglutaminase and cornification. This action of retinoids would block a critical aspect of cell selection involving loss of cells and subsequent regenerative hyperplasia, although simple hyperplasia may still occur.

Acyltransferases↗
PubMed1982

Genetic evidence that a phorbol ester tumor promoter stimulates ornithine decarboxylase activity by a pathway that is independent of cyclic AMP-dependent protein kinases in CHO cells.

Ornithine decarboxylase (ODC) inductions by cholera toxin and by the phorbol ester tumor promoter, TPA, were compared in wild-type Chinese hamster ovary (CHO) cells and in mutant cells having altered cyclic AMP-dependent protein kinase activity. The aim of these studies was to determine whether cyclic AMP-dependent protein kinase is involved in these inductions. The time course and the magnitude of ODC inductions by either 100 ng/ml cholera toxin or 100 ng/ml TPA were similar in wild-type cells with a maximum at 3-4 hours after treatment and a return to unstimulated levels by 8 hours. Induction of ODC by cholera toxin was suppressed more than 80% in the four protein kinase mutants studied (10215, 10248, 10260, and 10265), strongly implicating a cyclic AMP-dependent kinase step in the mechanism of induction. Similar results were found with the cyclic AMP analog 8-Br-cyclic AMP and the phosphodiesterase inhibitor, methyl-isobutylxanthine. The induction of ODC by TPA, on the other hand, was only partially inhibited (approximately 50%) in three of four mutants. Lower ODC activity in two mutants stimulated by cholera toxin or TPA whose kinetics were studied in more detail could not be ascribed to a reduced affinity (Km) of ornithine for the enzyme, but appeared to be due to reduced catalytic activity (Vmax) in the extracts. These results suggest that the induction of ODC by TPA proceeds by a mechanism which is only partially dependent on an intact cyclic AMP-dependent protein kinase activity.

Animals↗
PubMed1982

Divergent responses in epidermal basal cells exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

Mouse epidermal basal cells can be selectively cultivated in medium with 0.02 to 0.09 mM Ca2+ and can be induced to differentiate by medium containing 1.2 mM Ca2+. Basal cell cultures were studied to determine if all cells in this population responded identically to the skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Studies on the induction of the enzyme epidermal transglutaminase by TPA demonstrated a 2- to 4-fold increase in activity within 12 hr of exposure. This activity increase paralleled morphological differentiation in approximately 50% of the basal cell population, and differentiating cells sloughed from the culture dish within 24 to 48 hr as transglutaminase activity returned to basal levels. The cells which remained were resistant to induced differentiation by 1.2 mM Ca2+ medium, in that they failed to demonstrate increased transglutaminase activity or decreased thymidine incorporation, both characteristics of control basal cells induced to differentiate by 1.2 mM Ca2+. Cells remaining after a single exposure to TPA did not respond to a second exposure with an induction of transglutaminase if the interval between exposures was 4 days. TPA-pretreated cells did not undergo a transient decrease in thymidine incorporation (characteristic of control cells) when exposed to TPA a second time but instead were directly stimulated to proliferate by the phorbol ester, indicating that such cells were not refractory to the promoter. When the treatment-free interval after TPA was extended from 4 to 10 days, transglutaminase inducibility was restored in basal cells to either TPA or 1.2 mM Ca2+ as inducers. These results indicate that heterogeneity exits within the epidermal cell population and that exposure to phorbol esters induces differentiation in some cells, while stimulating proliferation in others. Such heterogeneous responses would cause a selective redistribution of the epidermal cell population and could lead to clonal expansion of initiated cells.

Animals↗
PubMed1981

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate induces ornithine decarboxylase in proliferating basal cells but not in differentiating cells from mouse epidermis.

The cultivation of mouse epidermal cells in medium of reduced calcium concentration (0.02--0.1 mM) selects for basal cell growth. Elevation of medium calcium levels above 0.1 mM results in rapid and well defined differentiative changes. This model was utilized to determine which cell type in mouse epidermis responds to the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), by an induction of the enzyme ornithine decarboxylase (ODC). Previous data had shown that TPA induces ODC in primary mouse epidermal cells only during the first 36 hr after plating in medium containing 1.44 mM Ca2+. In contrast, the induction in cells grown in low calcium medium was 2--10-fold greater, and inducibility persisted for at least 4 weeks. The greater inducibility of ODC in low calcium cells is not paralleled by increased thymidine incorporation after TPA treatment, probably because these cells are already proliferating at a maximum rate. When low calcium cells grown in 0.07 mM Ca2+ medium were switched to 1.2 mM Ca2+, there was a rapid loss of ODC inducibility. These results strongly suggest that the basal cells of the epidermis constitute the major target cells for the induction of ODC by TPA. The induction of ODC by ultraviolet light was not enhanced by growth of cells in low calcium medium, indicating that extracellular calcium concentration per se does not determine ODC inducibility. When epidermal cells grown in 1.2 mM or 0.07 mM Ca2+ medium were exposed to both UV light and TPA, there was a significant synergistic effect of combined treatment over the sum of each individual response, suggesting that factors in addition to differentiation determine the extent of ODC induction.

Animals↗
PubMed1981

Initiator and promoter induced specific changes in epidermal function and biological potential.

Mouse epidermal basal cells can be selectively cultivated in medium with a calcium concentration of 0.01--9.09 mM. Terminal differentiation and sloughing of mature keratinocytes occur when the calcium concentration is increased to 1.2--1.4 mM. When basal cell cultures are exposed to chemical initiators of carcinogenesis, colonies of cells that resist calcium-induced differentiation evolve. Likewise, basal cells derived from mouse skin initiated in vivo yield foci that resist terminal differentiation. This defect in the commitment to terminal differentiation appears to be an essential change in initiated cells in skin and is also characteristic of malignant epidermal cells. This model system has also provided a means to determine if basal cells are more responsive to phorbol esters than other cells in epidermis and to explore the possibility that heterogeneity of response exists within subpopulations of basal cells. The induction of the enzyme ornithine decarboxylase (ODC) was used as a marker for responsiveness to phorbol esters. ODC induction after exposure to 12-0-tetradecanoylphorbol-13-acetate (TPA) in basal cells is enhanced 20-fold over the response of a culture population containing both differentiating and basal cells. When basal cells are induced to differentiate by increased calcium, responsiveness to TPA is lost within several hours. In basal cell cultures, two ODC responses can be distinguished. After exposure to low concentrations of TPA or to weak promoters of the phorbol ester series, ODC activity is maximal at 3 hr. With higher concentrations of TPA, the ODC maximum is at 9 hr. These results are consistent with the presence of subpopulations of basal cells with differing sensitivities to TPA. Other studies that use the enzyme epidermal transglutaminase as a marker for differentiation support this conclusion. In basal cell culture TPA exposure rapidly increases transglutaminase activity and cornified envelope development, reflecting induced differentiation in some cells. As differentiated cells are sloughed from the dish, the remaining basal cells proliferate and become resistant to induced differentiation by 1.2 mM calcium. These data provide additional evidence of basal cell heterogeneity in which TPA induces one subpopulation to differentiate while another is stimulated to proliferate and resists a differentiation signal. Tumor promoters, by their ability to produce heterogeneous responses with regard to terminal differentiation and proliferation, would cause redistribution of subpopulations of epidermal cells in skin. Cells that resist signals for terminal differentiation, such as initiated cells, would be expected to increase in number during remodeling, Clonal expansion of the initiated population could result in a benign tumor with an altered program of differentiation. In skin, benign tumors are the principal product of 2-stage carcinogenesis. Subsequent progression to malignancy may involve an additional step, probably a genetic alteration, that is independent of the tumor promoter.

Animals↗
PubMed1981

Differential retinoic acid inhibition of ornithine decarboxylase induction by 12-O-tetradecanoylphorbol-13-acetate and by germicidal ultraviolet light.

Several retinoids including retinoic acid effectively inhibit phorbol ester-mediated tumor promotion and ornithine decarboxylase (ODC) induction in mouse epidermis. To understand better the possible cellular site of action of retinoids, the inhibitory action of retinoic acid on the induction of ODC was compared for two distinctly different inducers, namely, 12-O-tetradecanoylphorbol-13-acetate (TPA) and germicidal ultraviolet light (UV), in primary mouse epidermal cell cultures. It was found that the induction of ODC by TPA is almost completely prevented by 0.1 to 1 microM retinoic acid while the induction by UV is only moderately inhibited. Maximum inhibition is achieved by treating cells continuously with retinoic acid from 4 hr after plating, although pretreatment or simultaneous treatment relative to either inducer is almost as effective. When added after the inducer, retinoic acid loses its effectiveness as an inhibitor more rapidly for TPA induction than for UV induction of ODC. The differential inhibition of enzyme induction cannot be accounted for by selective retinoid inhibition of DNA, RNA, or protein synthesis either alone or in concert with TPA or UV. Other agents known to modulate the induction of ODC by TPA (fluocinolone acetonide, tosyl-L-lysylchloromethane, and local anesthetics) do not act differentially on UV induction. These agents possibly act at transcription or translation, both of which are required for ODC induction by TPA or UV. The preferential inhibition by retinoic acid of ODC induction by TPA is interpreted to result from specific interference at a unique and early site of interaction of TPA with the cell.

Animals↗