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Biomedical subjects

J D Laskin

Publications and source records attributed to J D Laskin.

At least 91 records · Page 5Linked to original sources
PubMed1990

Production of hydrogen peroxide by murine epidermal keratinocytes following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

The ability of murine epidermal cells to produce intracellular hydrogen peroxide was analyzed by flow cytometry and the measurement of 2',7'- dichlorofluorescin (DCFH) oxidation. Epidermal cells isolated from acetone-treated CD-1 mice for 24 h were relatively homogeneous in cell size and density and oxidized low levels of DCFH. However, following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of mice (10 micrograms; 24 h), two cytokeratin-positive populations of cells were identified that were heterogeneous with respect to size and density. These two TPA-derived cell populations oxidized levels of DCFH that were time and dose dependent and were between 2- and 10-fold higher than levels of DCFH oxidized by cells isolated from acetone-treated mice. The ability of catalase, the enzyme that detoxifies hydrogen peroxide, to suppress DCFH oxidation to control levels suggested that intracellular hydrogen peroxide was responsible for the enhanced rate of DCFH oxidation in epidermal cells isolated from TPA-treated mice. The ability of mouse epidermal keratinocytes to oxidize DCFH in response to TPA treatment was confirmed using a cloned keratinocyte cell line. These results suggest that specific subpopulations of keratinocytes produce elevated levels of intracellular peroxides following treatment with TPA either in vivo or in culture.

Animals↗
PubMed1990

Identification of epidermal cell subpopulations with increased ornithine decarboxylase activity following treatment of murine epidermis with 12-O-tetradecanoylphorbol-13-acetate.

Ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway, has been used as a marker for the hyperplasia that occurs following exposure of mouse epidermis to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Using flow cytometry in combination with polyclonal antibodies to ODC, we examined the levels of ODC-associated immunoreactive protein present within mouse epidermal cells at 4 and 24 h after a single topical application of TPA, as well as following chronic exposure to TPA and in papillomas. Basal levels of ODC-specific antibody binding were detectable in acetone-treated CD-1 mouse epidermis and were increased 3-fold at 4 h after TPA treatment. The amount of ODC antibody binding detected after exposure to 17 nmol TPA twice weekly for 3 weeks was similar to that detected within cells isolated from papillomas and was 2.5-fold higher than in cells isolated at 4 h after a single topical treatment of mice with TPA. These observations support the hypothesis that specific subpopulations of keratinocytes constitutively express high levels of ODC following chronic exposure to TPA. The novel method for ODC detection described in these studies provides a means to identify, isolate, and further characterize epidermal cells that may give rise to papillomas and carcinomas.

Animals↗
PubMed1990

Differentiation of U-937 histiocytic lymphoma cells towards mature neutrophilic granulocytes by dibutyryl cyclic adenosine-3',5'-monophosphate.

Treatment of U-937 cells with the cyclic nucleotide analog, dibutyryl cyclic adenosine-3',5'-monophosphate (dBcAMP) induced these cells to differentiate towards granulocytes. dBcAMP produced a dose- and time-dependent inhibition of U-937 cell growth reaching a maximum after 48-h treatment with 500 microM. At this concentration, dBcAMP had no effect on cell viability. Treatment with dBcAMP caused a rapid (within 24 h) decrease in the number of cells in the S phase of the cell cycle, with a concomitant increase in cells in the G0/G1 phase. dBcAMP also induced the appearance of f-met-leu-phe receptors on U-937 cells as well as the ability to produce hydrogen peroxide and superoxide anion. These data suggest that dBcAMP-treated U-937 cells were functionally mature. Using specific monoclonal antibodies and flow cytometry, we found that differentiated U-937 cells expressed the monocytic/granulocytic surface markers MY8 and MAC-1, but not the monocyte specific markers MO2 or MY4. In addition, dBcAMP-treated U-937 cells did not stain for nonspecific esterase, displayed less HLA-DR antibody binding than undifferentiated cells and appeared smaller and more granular. These are all characteristics of mature granulocytes. Taken together our studies indicate that differentiation of U-937 cells is not necessarily limited to the monocytic pathway of development.

Antibodies, Monoclonal↗
PubMed1990

Flow cytometric detection of ornithine decarboxylase activity in epidermal cell subpopulations.

Using flow cytometry in combination with membrane permeabilization techniques to enhance binding of antibodies with immunoreactive protein within the cytoplasm, we have developed a method to examine the ornithine decarboxylase (ODC) activity present within subpopulations of epidermal cells following acute and chronic exposure to the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). The method described has the sensitivity to detect basal levels of ODC as well as increases in ODC at early time points following treatment with TPA and has the additional advantage of allowing subpopulation identification and characterization.

Animals↗
PubMed1990

Failure of F-Met-Leu-Phe to induce chemotaxis in differentiated promyelocytic (HL-60) leukemia cells.

Treatment of HL-60 promyelocytic leukemia cells with dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) induced these cells to differentiate toward mature neutrophilic granulocytes (PMN). DbcAMP was found to produce a rapid time- and dose-dependent inhibition of HL-60 cell proliferation, reaching a maximum after 48 hr treatment of the cells with 250-500 microM. This was associated with morphologic alterations consistent with maturing PMN. Using immunofluorescence and flow cytometry, we found that dbcAMP-treated HL-60 cells expressed some, but not all, surface markers characteristic of mature phagocytes. Thus receptors for the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP); the monocyte/granulocyte markers My-8, Mo-1, and Leu-15; as well as the monocyte markers Mo-2 and My-4 were identified on the cells. In contrast, dbcAMP-treated cells did not express the PMN-specific antigen DL1.2. We also found that dbcAMP-treated HL-60 cells produced hydrogen peroxide in response to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). However, the magnitude of this response was significantly less than that observed in mature PMN. In contrast, fMLP-stimulated levels of hydrogen peroxide production were comparable in both cell types. In addition, despite the fact that dbcAMP-treated cells expressed fMLP receptors, they did not exhibit directed migration toward this chemotactic peptide. Taken together, these data suggest that dbcAMP-induced differentiation of HL-60 cells is incomplete.

Antigens, Surface↗
PubMed1989

Purification of tyrosinase to homogeneity based on its resistance to sodium dodecyl sulfate-proteinase K digestion.

A two-step method was used to purify enzymatically active tyrosinase to apparent homogeneity from B16/C3 mouse melanoma cells. The purification was based on our finding that tyrosinase was highly resistant to sodium dodecyl sulfate-proteinase K digestion. Following treatment with proteinase K, tyrosinase was separated from all detectable contaminants using DEAE-52 ion-exchange chromatography. Using this procedure, tyrosinase was purified to high specific activity and in yields greater than 50%. The purified enzyme was then used to generate high titers of specific polyclonal antibodies in rabbits. These antibodies were used to immunoprecipitate all tyrosinase isozymes from murine melanoma tumor extracts and cultured B16/C3 cells metabolically labeled with [35S]methionine.

Amino Acids↗
PubMed1989

Ethoxylated alcohol (Neodol-12) and other surfactants in the assay of protein kinase C.

Most commonly used surfactants were found to be inhibitors of partially purified rat brain protein kinase C at or above their critical micellar concentrations (CMC). These include sodium lauryl sulfate, deoxycholate, octyl glucoside, dodecyl trimethylammonium bromide, linear alkylbenzene sulfonate and Triton X-100. Several detergents, including the nonionic surfactants digitonin and Neodol-12 (ethoxylated alcohol), did not inhibit protein kinase C activity, even at concentrations greater than their CMC, while the anionic surfactant, AEOS-12 (ethoxylated alcohol sulfate), inhibited enzyme activity only slightly (less than 8%). Since these latter surfactants have little or no inhibitory effect on protein kinase C, they may be of value in solubilizing cells and tissues for the determination of enzyme activity in crude extracts. Among the detergents tested, sodium lauryl sulfate and linear alkylbenzene sulfonate significantly stimulated protein kinase C activity in the absence of phosphatidylserine and calcium. This was found to be dependent on the presence of histone in the protein kinase C assay. These detergents failed to stimulate protein kinase C activity when endogenous proteins in the partially purified rat brain extracts were used as the substrate. Our results indicate that activity of protein kinase C can be modified by the conditions of the assay and by the detergents used to extract the enzyme.

Alcohols↗
PubMed1989

Transplantation of B16/C3 melanoma cells into the brains of rats and mice.

The B16/C3 mouse melanoma cell line produces L-DOPA, catecholamines and melanin in tissue culture. Growth and development of these cells after transplantation into the rat and mouse brain were studied by immunocytochemical and histological techniques. The implanted cells were localized by prelabelling the cell nuclei with bisbenzimide, a fluorescent marker which binds to DNA. Following transplantation into rats, B16/C3 melanoma cells were found to survive for at least 4-6 weeks. These cells initially expressed tyrosinase and tyrosine hydroxylase immunoreactivity and in some cases contained catecholamines. After 3 weeks, the cytoplasm of the transplanted cells began to accumulate melanin; catecholamines and tyrosinase immunoreactivity were no longer detected. Ultimately the cells became round in shape and densely pigmented. Growth of the tumor in the rats was restricted and the implant was encapsulated within a glial sheath. There was evidence of an immune reaction to the tumor in that cells with Ia antigen immunoreactivity were present surrounding the graft. The rat hosts were not adversely affected by the presence of the tumor, nor did the tumor cell grafts alter rotational behavior consequent to unilateral substantia nigra lesions. In mouse hosts, however, the melanoma grew rapidly, was not encapsulated by glia and led to death of all animals. These data suggest that the tumor was not rapidly destroyed in rats, even though its growth was controlled through immunological mechanisms. Both trophic and immunological mechanisms may therefore be involved in the regulation of survival and differentiation of intracerebral grafts of tumor cells.

Animals↗
PubMed1989

Inhibition of epidermal growth factor receptor tyrosine kinase activity in A431 human epidermoid cells following psoralen/ultraviolet light treatment.

One of the more prominent clinical treatments for skin diseases such as psoriasis and vitiligo involves the use of a combination of psoralens and UV light, a procedure referred to as PUVA chemotherapy. This drug regimen markedly alters epidermal cell growth and differentiation. In many cell types, an early cellular event following treatment of cells with PUVA is inhibition of binding of epidermal growth factor (EGF) to its receptor. To examine the mechanism underlying this effect, we used A431 cells, a human epidermal cell line known to express large numbers of EGF receptors. We found that exposure of A431 cells to PUVA caused a dramatic inhibition of EGF-stimulated EGF receptor tyrosine kinase activity. Inhibition required intact cells and did not appear to be mediated by protein kinase C, because this inhibition was apparent in cells in which the enzyme was down-regulated by phorbol ester pretreatment and in cells treated with inhibitors of protein kinase C. Inhibition of tyrosine kinase activity by PUVA was distinct from other inhibitors of EGF receptor function in that it was associated with a rapid increase in the amount of phosphate incorporated into serine residues of the EGF receptor. This suggested that PUVA-induced serine phosphorylation may mediate EGF receptor kinase activity. These results demonstrate that alterations in EGF receptor function may contribute to the therapeutic efficacy of PUVA in photo-chemotherapy.

Cell Line↗
PubMed1988

Functional and biochemical properties of rat Kupffer cells and peritoneal macrophages.

Functional and biochemical techniques were used to further characterize heterogeneity between rat Kupffer cells and peritoneal macrophages. Both macrophage cell types were found to phagocytize antibody coated sheep red blood cells in a time-dependent manner. However, Kupffer cells were two to three times more phagocytic than were peritoneal macrophages. In contrast, the peritoneal cells released significantly more superoxide anion in response to the complement cleavage product, C5a and the phorbol ester tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate, and produced more hydrogen peroxide than did the liver macrophages. Both cell types responded chemotactically to C5a. These results suggest that macrophages may develop specialized functions depending on the needs of their local environment. Using one and two dimensional SDS-polyacrylamide gel electrophoresis, we also compared the production of newly synthesized proteins by Kupffer cells and peritoneal macrophages. In general, the macrophages were found to produce similar types and numbers of proteins with some exceptions. These included proteins that were unique to peritoneal macrophages and other proteins observed only in Kupffer cells. The production of these proteins in liver macrophages did not appear to correlate with levels of functional activation, but may be more related to the tissue origin of the cells.

Animals↗
PubMed1988

Effect of retinoid deficiency on keratin expression in mouse bladder.

Twelve to sixteen weeks following treatment of CF-1 mice with a vitamin A-deficient diet, characteristic signs of retinoid deficiency including body wasting, poor hair coat, altered gait, decreased mobility, and xerophthalmia were observed. Histological examination of tissue sections from these mice revealed dramatic changes in the urinary tract epithelium. The normal transitional epithelium was replaced by a stratified squamous epithelium that resembled hyperproliferative epidermis. Using two-dimensional gel electrophoresis, a number of new proteins were found to be synthesized in vitamin A-deficient bladder when compared to tissue from control bladders. Using antikeratin antibodies in immunoblot experiments, we found that at least some of the newly synthesized proteins were keratins. These proteins, which comprise the intermediate filaments of the cytoskeleton, are known to be specific markers of epithelial differentiation. Of particular interest was the appearance of a Mr 67,000 basic and Mr 61,000 acidic keratin pair, characteristic of terminally differentiating murine epidermal cells. Unexpectedly, several other keratins, previously associated only with hyperproliferative epidermis, were also expressed in the tissue. These results demonstrate that vitamin A deficiency in the mouse leads to the appearance of a squamous metaplasia in the urinary tract epithelium that is characterized by the expression of distinct epidermal keratins.

Animals↗
PubMed1988

Keratin polypeptide expression in mouse epidermis and cultured epidermal cells.

Adult mouse epidermis contains up to 11 distinct keratin polypeptides, as resolved by two-dimensional gel electrophoresis. These include both basic (Type II; 67-, 65-, 63-, 62-, and 60-kDa) and acidic (Type I; 61- to 59-, 54-, 52-, 49-, and 48-kDa) keratins that exhibit multiple isoelectric forms. Several, but not all, of these keratins, identified by immunoblotting, were found to be actively synthesized in the skin when assayed in short-term pulse-labeling experiments. When compared to the adult, newborn mouse epidermis expresses fewer keratin subunits. However, greater amounts of keratins associated with differentiated suprabasal cells and stratum corneum, which is more pronounced morphologically in the newborn, were identified. We also observed strain-specific differences in the expression of a Type I acidic keratin. This 61-kDa (pI, approx. 5.3) keratin was produced exclusively by the CF-1 mouse and, based on peptide mapping, appeared to be related to the acidic 59-kDa keratin that was identified in this strain as well as all other mouse strains. The 61-kDa keratin was not expressed in vitamin A-deficient animals, suggesting that its appearance may be related to a retinoid-dependent posttranslational modification. In comparison to keratin expression in vivo, primary mouse keratinocyte monolayer cultures maintained in low Ca2+ (less than 0.08 mM) did not express the terminal differentiation keratins of 67-kDa (basic) or 59-kDa (acidic), although enhanced synthesis of the 60-kDa (basic) and the 52-kDa and 59-kDa (acidic) keratins associated with proliferation were observed. In addition, a subpopulation of nonadherent cells was continuously produced by the primary keratinocyte cultures that expressed the 67-kDa (basic) keratin specific for terminal differentiation. When the keratinocyte cultures were induced to terminally differentiate with Ca2+, the overall pattern of keratin expression was not changed significantly. Taken together, these results provide further evidence for the variable nature of keratin expression in mouse epidermal keratinocytes under different growth conditions.

Aging↗
PubMed1987

Specific alterations in keratin biosynthesis in mouse epidermis in vivo and in explant culture following a single exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

Treatment of mouse skin with a single dose of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) causes dramatic alterations in the biosynthesis of specific epidermal keratins. TPA was applied either topically to the skin of mice or added to the culture medium of skin explants maintained in vitro. Twenty-four h after exposure, skin samples were pulse labeled with [35S]methionine, and epidermal proteins were extracted and resolved by two-dimensional gel electrophoresis. In whole animals, TPA caused a marked decrease in the biosynthesis of the Mr 67,000 (basic) and 59,000 (acidic) keratins, specific markers for suprabasal differentiation in the epidermis. In addition, the synthesis of an acidic keratin of Mr 48,000 and a basic keratin of Mr 62,000 was also decreased. Concomitantly, TPA caused an increase in the synthesis of a basic keratin of Mr 60,000 and acidic keratins of Mr 52,000 and 49,000, markers for proliferating cells and primary epidermal cell cultures. The normal pattern of keratin synthesis observed in explants from normal skin was similar to that observed in skin samples from animals treated with TPA, except that synthesis of the Mr 67,000 basic keratin subunit was maintained. The addition of TPA to culture medium containing the skin explants resulted in a dose-dependent decrease in the synthesis of this keratin. Furthermore, resulting patterns of keratinization were identical to epidermis treated with TPA in vivo. These results suggest that a single acute exposure to TPA alters normal differentiation of mouse epidermal cells in vivo while causing a pronounced basal cell hyperproliferation. This response can be reproduced following TPA exposure to skin explants in culture, suggesting that the changes in keratinization observed are independent of the animal. Specific keratins modulated during TPA-induced hyperplasia may serve as marker proteins for aberrant epidermal cell growth and differentiation leading to the development of neoplasia.

Animals↗
PubMed1987

Characterization of a photoalkylated psoralen receptor in HeLa cells.

Psoralens in combination with ultraviolet light are potent modulators of epidermal cell growth and differentiation. Responsive cell types contain specific, saturable, high-affinity binding sites for the psoralens. These binding sites become covalently modified by the psoralen molecule following ultraviolet light exposure. In the present studies the psoralen receptor, labeled with [3H]8-methoxypsoralen, was visualized in the cytoplasmic and plasma membrane fractions of HeLa cells following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor had an apparent molecular mass of approximately 22,000 daltons and was shown to be sensitive to protease, but not nuclease treatment. The radiolabeled receptor could not be visualized in nuclear extracts of cells. Covalent binding of the radioligand to the receptor protein was inhibited by excess unlabeled 8-methoxypsoralen, indicating that covalent psoralen-receptor binding was saturable. In addition, the covalently modified receptor was found to persist in cells for over 5 h. The presence of a cellular protein that exhibits specific affinity for the psoralens and becomes photoalkylated by these compounds, together with previous data showing that the psoralens have direct effects on the cell surface membranes, supports our model that some of the biological effects of photoactivated psoralens are receptor-mediated.

Binding, Competitive↗
PubMed1987

Induction of chemotaxis in mouse peritoneal macrophages by activators of protein kinase C.

Two activators of calcium and phospholipid dependent protein kinase (protein kinase C), the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), were compared as chemotactic agents for mouse peritoneal macrophages. Both of these compounds were found to induce chemotaxis in the macrophages to a similar extent in a time and dose dependent manner. Induction of chemotaxis was observed in the concentration range of 10-100 nM for TPA and 25-250 microM for OAG. Two structurally related synthetic sn 1,2-diacylglycerols, 1,2-dioctanoylglycerol (diC8) and 1,2-didecanoylglycerol (diC10), were also found to be chemotactic for macrophages, while monoacylglycerol (2-monoolein) was inactive. Of the diacylglycerols, OAG was found to be the most active followed by diC8 and diC10. In contrast to TPA, the synthetic diacylglycerols had no effect on superoxide anion release by the cells, suggesting that the mechanism of superoxide anion release by TPA in macrophages is distinct from chemotaxis. Phorbol-12,13-diacetate, a biologically inactive phorbol ester analog that inhibits the binding of TPA to its cellular receptors, inhibited macrophage chemotaxis induced by TPA, the synthetic diacylglycerols, and the complement fragment, C5a. Taken together, our results suggest that chemotaxis in macrophages may be mediated by activation of protein kinase C.

Animals↗
PubMed1987

Alterations in the expression of specific epidermal keratin markers in the hairless mouse by the topical application of the tumor promoters 2,3,7,8-tetrachlorodibenzo-p-dioxin and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate.

The potent toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes chloracne and acts as a tumor promoter in the hairless HRS/J mouse model. In the present study we characterized changes in mouse epidermal keratin expression following a single topical application of TCDD to the skin of hairless (hr/hr) and haired (hr/+) HRS/J mice. Morphologic changes and alterations in keratin biosynthesis following TCDD treatment were compared with those induced by the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Both TPA and TCDD induced dose-dependent epidermal hyperplasia in hr/hr and hr/+ mice and this was associated with altered keratin subunit expression. In hr/hr mice TCDD caused a pattern of keratin expression that was similar to TPA, characterized by a marked decrease in the synthesis of the Mr 67,000 (basic) and 59,000 (acidic) keratins that are specific markers for suprabasal differentiation in the epidermis. In addition, the synthesis of an acidic keratin of Mr 48,000 and a basic keratin of Mr 62,000 was also decreased. Concomitantly, TCDD caused an increase in the synthesis of a basic keratin of Mr 60,000 and acidic keratins of Mr 54,000, 52,000 and 49,000 that are normally observed in proliferating basal cells and primary epidermal cell cultures. In contrast, while TPA induced similar changes in keratinization in both the hr/+ and hr/hr mice, TCDD-induced hyperplasia in hr/+ mice was only associated with changes in keratin synthesis reflecting increased basal cell proliferation. These results demonstrate that a single application of TCDD to the skin alters the normal pattern of epidermal differentiation in the hr/hr mouse. Molecular events influencing the expression of the keratin genes associated with this process may be linked to the strain- and/or species-specific toxicity of TCDD.

Administration, Topical↗
PubMed1986

Tyrosinase isozyme heterogeneity in differentiating B16/C3 melanoma.

The B16/C3 murine melanoma is a pigmented tumor that is rich in the copper-containing enzyme, tyrosinase. This enzyme, which converts tyrosine to melanin precursors, is largely associated with membrane fractions of cells and exists in a number of discrete isozymic forms ranging in molecular mass from 58,000 to 150,000 daltons and pI from 3.4 to 5.2. One of these isozymes (Mr = 58,000, pI 3.4) has been purified to homogeneity. The purified enzyme catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and the conversion of L-DOPA to dopaquinone. Ascorbic acid, tetrahydrofolate, and dopamine can serve as cofactors in the hydroxylase reaction. The Michaelis constants for the purified enzyme were 7 X 10(-4) M for L-tyrosine and 6 X 10(-4) M for L-DOPA. The Vmax for L-DOPA was much greater than the Vmax for L-tyrosine indicating that tyrosine hydroxylation is rate-limiting in melanin precursor biosynthesis. Two putative copper chelators, phenylthiourea and diethyldithiocarbamide inhibited both the tyrosine hydroxylase and L-DOPA oxidase activities of the enzyme. Phenylthiourea was a noncompetitive inhibitor while diethyldithiocarbamide was a competitive inhibitor indicating that these agents act by different mechanisms. When digested with proteases and glycosidases, higher molecular weight forms of tyrosinase co-migrated with the purified enzyme in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the isozyme was derived from larger precursors. Thus, post-translational processing of tyrosinase may underlie isozyme diversity and this may be important in the control of melanogenesis in this tumor model.

Amino Acids↗