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Biomedical subjects

F Marks

Publications and source records attributed to F Marks.

At least 145 records · Page 8Linked to original sources
PubMed1988

Bryostatin 1, an activator of protein kinase C, mimics as well as inhibits biological effects of the phorbol ester TPA in vivo and in vitro.

The macrocyclic lactone bryostatin 1 activates protein kinase C as effectively as the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Nevertheless, there are only certain TPA-effects that can be induced by bryostatin 1. These include stimulation of epidermal DNA synthesis and alkaline phosphatase activity in vivo as well as activation of the Ca2+-independent, phospholipid-requiring phosphorylation of an epidermal protein in a cell-free system. Various other TPA-effects in vivo and in vitro, which are not mimicked by bryostatin 1 can be inhibited by applying bryostatin 1 30 min prior to TPA. TPA-effects suppressible by bryostatin 1 include the Ca2+-dependent stimulation of arachidonic acid and prostaglandin E2 release, of ornithine decarboxylase (ODC) activity and ODC-mRNA expression and of transglutaminase activity in keratinocytes in vivo and/or in vitro and, in addition, Epstein-Barr virus induction in Raji cells. The same is true for the conversion step (first stage of promotion) of multistage carcinogenesis. In contrast to the TPA induction of arachidonic acid and prostaglandin E2 release and of transglutaminase activity, induction by the Ca2+-ionophore and by high Ca2+-shift, respectively, are not significantly inhibited by bryostatin 1. We suggest that bryostatin 1 might inhibit a specific 'Ca2+-component' of TPA action.

Animals↗
PubMed1988

Differential effects of phorbol esters on c-fos and c-myc and ornithine decarboxylase gene expression in mouse skin in vivo.

Hyperplasiogenic and tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate or 12-O-retinoylphorbol-13-acetate induce the sequential transient expression of the proto-oncogenes c-fos and c-myc and the ornithine decarboxylase gene in mouse skin in vivo. This sequence of biochemical events probably depends on an activation of protein kinase C by these agents. The non-irritant skin mitogens 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and ethyl phenyl propiolate do not increase the expression of these genes to a comparable extent. Thus, 12-O-tetradecanoylphorbol-13-acetate and 12-O-retinoylphorbol-13-acetate induce epidermal hyperproliferation by different biochemical mechanisms as do 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and ethylphenylpropiolate.

Animals↗
PubMed1988

Cytogenetic effects caused by phorbol ester tumor promoters in primary mouse keratinocyte cultures: correlation with the convertogenic activity of TPA in multistage skin carcinogenesis.

The effects of the convertogenic ('first-stage') tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the non-convertogenic ('second stage') tumor promoter 12-O-retinoyl-phorbol-13-acetate (RPA) and the non-promoting phorbol esters 4-O-MeTPA and 4-alpha-PDD on the chromosomes of mouse keratinocytes in primary cultures were investigated. In these target cells of tumor promotion TPA caused severe numerical and structural chromosomal aberrations, which were evident after two cell cycles and accumulated after multiple applications. Numerical aberrations were visible as hypo- and hyperdiploidy, with non-random loss or gain of specific chromosomes. The clastogenic effects were evident as simple alterations such as gaps and breaks, but more severe alterations such as tri- and quadriradial chromatid interchanges and ring chromosomes, as well as translocations could be observed. The structural aberrations were nonrandomly distributed in the genome and chromosomes no. 1, 2, 5, 6 and 18 were preferentially involved in rearrangements. In addition to the aneuploidogenic, clastogenic and recombinogenic effects induced by TPA, short treatment with this tumor promoter was efficient in producing cytogenetic equivalents of gene amplification, i.e. double minute chromosomes. The cytogenetic effects were not merely due to cytotoxicity since they occurred after a low TPA dose (10(-8) M) and did not considerably increase with a higher dose (10(-6) M). Moreover, at both dose levels cell cycle traverse of mouse keratinocytes was not drastically altered. In contrast, the non-convertogenic tumor promoter RPA and the non-promoting phorbol esters 4-alpha-PDD and 4-O-MeTPA (at the same dose level) did not cause any substantial chromosomal alterations. This discrepancy between the action of TPA and RPA suggests that effects which result in chromosomal alterations in the target cells may be critical for the conversion stage of skin tumor promotion. This conclusion is supported by experiments with substances such as antipain and eicosa-5,8,11,14-tetraynoic acid which inhibit both tumor induction in initiated skin and the cytogenetic alterations induced by TPA in cultured keratinocytes. These studies provide for the first time the possibility of differentiating between convertogenic and non-convertogenic tumor promoters in an in vitro assay using the target cells of mouse skin carcinogenesis.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1988

The epidermal G1-chalone: an endogenous tissue-specific inhibitor of epidermal cell proliferation.

An apparently macromolecular factor is isolated from aqueous skin extracts which inhibits DNA synthesis in vivo and in vitro with high efficacy (ID50 in vivo 0.2 pmol/g, in vitro 0.2 pM) and in a highly specific manner showing a point of attack in the late G1-phase of the cell cycle (epidermal G1-chalone). Preliminary characterization indicates an unusual highly amphipathic structure consisting of amino acids and carbohydrate. Despite its apparent molecular weight of approximately 10 kD the chalone is stable against denaturing agents and most enzymes, including proteases. An inverse correlation between chalone responsiveness of mouse epidermis in vivo and the development of hyperplasia due to injury indicates an important role of the factor in the regulation of tissue homeostasis. According to its physicochemical and biological properties the epidermal G1-chalone appears not to be related to other endogenous inhibitors of epidermal cell proliferation such as the pentapeptide pyroGlu-Glu-Asp-Ser-GlyOH and transforming growth factor beta (TGF beta).

Animals↗
PubMed1988

Ciclosporin inhibits phorbol-ester-induced hyperplastic transformation and tumor promotion in mouse skin probably by suppression of Ca2+/calmodulin-dependent processes such as phosphorylation of elongation factor 2.

This study deals with the mechanism of the inhibitory effect exerted by the immunosuppressant ciclosporin (CsA) on phorbol-ester-induced inflammation, epidermal hyperplasia and tumor promotion in mouse skin in vivo. This effect coincides with an inhibition of the phosphorylation of a 100-kilodalton protein (p100) in epidermal cytosol in vitro, which has been identified as elongation factor 2 (EF-2) of protein biosynthesis. Phosphorylation of EF-2 is dependent on Ca2+ and calmodulin, and inhibition of EF-2 phosphorylation by CsA is due to an interaction of CsA with calmodulin. The EF-2 phosphorylation system has a metabolic half-life of 1.5 h probably due to a rather rapid turnover rate of the EF-2 kinase. Since CsA inhibits specifically 12-O-tetradecanoylphorbol-13-acetate (TAP)-stimulated but not basal protein synthesis in epidermis, it is proposed that Ca2+/calmodulin-dependent phosphorylation of EF-2 is involved in the induction of the hyperplastic response by TPA and that CsA suppresses TPA effects by inhibition of EF-2-phosphorylation and perhaps other calmodulin-dependent processes. The potential applicability of calmodulin inhibitors in the treatment of hyperproliferative skin diseases is discussed.

Animals↗
PubMed1988

Pregnancy post-Stevens-Johnson syndrome: case report and review of the literature.

A pregnancy complicated by vaginal stenosis six years after diagnosis of Stevens-Johnson syndrome is described. The pathologic changes in the vagina have not previously been reported. The basic pathology of severe mucosal erythema multiforme was present, as well as ectasia of the superficial capillaries and small venules. The effect of the vaginal scars on the mode of delivery is discussed.

Adult↗
PubMed1987

The induction of ornithine decarboxylase by the tumor promoter TPA is controlled at the post-transcriptional level in murine Swiss 3T3 fibroblasts.

The expression of ornithine decarboxylase (ODC) mRNA after treatment of murine Swiss 3T3 cells with the tumor promoter TPA was studied. The induction of ODC mRNA was detectable after 20-40 min, peaked after 60-120 min and declined within 24 hrs. Using an in vitro nuclear transcription assay, we found that the polymerase II density on the ODC gene is not affected by TPA treatment. Additionally, we were able to detect stable ODC mRNAs in cycloheximide pretreated fibroblasts. These two different experimental approaches lead us to the interpretation that in Swiss 3T3 cells TPA controls ODC expression predominantly at the post transcriptional level by prolonging the half-life of ODC-mRNA.

Animals↗
PubMed1987

Events associated with mouse skin tumor promotion with respect to arachidonic acid metabolism: a comparison between SENCAR and NMRI mice.

NMRI and SENCAR, two stocks of mice commonly used in multistage skin carcinogenesis studies, were compared with respect to the effects of inhibitors of arachidonic acid metabolism for the following 12-O-tetra-decanoylphorbol-13-acetate (TPA)-elicited events: tumor promotion, DNA synthesis in vivo and in vitro, ornithine decarboxylase induction, and prostaglandin (PG) E2 synthesis. Previous work had shown that the cyclooxygenase inhibitor indomethacin enhanced TPA promotion in SENCAR mice. We report here that over the same dose range (50 to 200 micrograms) indomethacin caused a dose-dependent inhibition of promotion in NMRI mice. Significant reversal of this inhibition was achieved with concomitant application of 10 micrograms PGF2 alpha but not PGE2. DNA synthesis studies showed that low doses of indomethacin and flurbiprofen increased TPA-stimulated DNA synthesis in primary cultures from SENCAR mice; indomethacin suppressed this response in NMRI cultures. In vivo DNA synthesis studies showed the same pattern: indomethacin enhanced TPA-stimulated DNA synthesis in SENCAR mice but inhibited in NMRI mice. Other classes of inhibitors of arachidonate metabolism (i.e., the cyclooxygenase-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and phenidone and the phospholipase A2 inhibitor dibromoacetophenone) had inhibitory activity in vitro and in vivo in both stocks of mice. Indomethacin was found to inhibit TPA-induced ornithine decarboxylase activity to the same extent in both mice. Indomethacin was also very effective in inhibiting TPA-induced PGE2 synthesis in both stocks of mice. 5,8,11,14-Eicosatetraynoic acid and phenidone were likewise suppressive in both stocks of mice. It is concluded that the NMRI and SENCAR mice respond similarly to TPA with respect to promotion, DNA synthesis, ornithine decarboxylase induction, and PG synthesis. The difference appears to be in the degree of involvement of the lipoxygenase pathway.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1987

Early changes in the arachidonic acid metabolism of HeLa cells in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and related compounds.

In order to search for possible mediators involved in the transient radiomimetic effectiveness of TPA and related compounds early changes in the AA metabolism of HeLa cells prelabeled with 1-14C-AA have been analyzed. Maximum release of AA with different concentrations of TPA (3 X 10(-9) to 3 X 10(-5) M) was observed after 2-3 h treatment in the presence of 10% calf serum. Released AA was reincorporated by the cells after that period, a phenomenon which was largely abolished or delayed by cycloheximide. Reincorporation of released AA was observed in the presence of 10% fresh serum as well as with 0.5% BSA, and appears to be due to an induction of responsible enzyme(s) by the phorbol ester. The earliest metabolites of AA produced via the cyclooxygenase such as PGE2 and PGF2 alpha and via lipoxygenases such as 12-, and 15-hydroxyeicosatetraenoic acids appear in small amounts and after later time points. AA release exhibited a pluriphasic dose response to TPA with maxima at 3 X 10(-8) M and greater than or equal to 10(-5) M. Comparative dose response measurements with respect to AA release were established using various promoting skin mitogens which exhibited the following order of potency: TPA greater than teleocidin approximately equal to RPA greater than mezerein much greater than EPA greater than 4-O-Me-TPA. For reasons discussed it appears unlikely that AA, Prostaglandins, or hydroxyeicosatetraenoic acid products play a significant role as mediators of the radiomimetic effects of TPA in G2 of the cell cycle.

Arachidonic Acid↗
PubMed1987

Effects of the phorbolester TPA and of the ionophore A 23187 on phospholipase A2 and C activities in the mouse epidermal cell line HEL-30.

The 12-O-tetradecanoylphorbol-13-acetate (TPA)- or ionophore A 23187-induced release of 14C-arachidonic acid from prelabeled murine HEL-30 keratinocytes was studied in vitro. Starting 8 min after drug treatment, a linear increase in the arachidonic acid content in the extracellular medium was observed with a concomitant loss of label in cellular phosphatidylcholine and phosphatidylinositol, but not in phosphatidylethanolamine and phosphatidylserine. No increase in intracellular diacylglycerol and phosphatidic acid was observed. The TPA-induced arachidonic acid release was inhibited by fluocinolone acetonide. The results indicate a direct activation of phosphatidylcholine- and phosphatidylinositol-specific phospholipases A2 by TPA and A23187. Cells prelabeled with 3H-choline released choline, choline phosphate and CDP-choline upon TPA but not upon A 23187 treatment. This could indicate activation of a phospholipase C-type enzyme by the phorbol ester. However, concomitant generation of diacylglycerol and phosphatidic acid was not detected.

Animals↗
PubMed1987

Isolation, characterization, and in vitro cultivation of keratinocyte subfractions from adult NMRI mouse epidermis: epidermal target cells for phorbol esters.

Adult dorsal mouse epidermis (strain NMRI) was separated from dermis in thin-split sections by cold trypsinization. From the isolated keratinocytes four cell fractions (F1-F4) were obtained using discontinuous Percoll density gradient centrifugation. The fractions were characterized by light microscopy, by indirect immunofluorescence using specific lectins (Bandeirea simplicifolia and Ulex europaeus) and an antibody against the spinous 67-kDa keratin polypeptides, and by electrophoretic analysis of the keratin polypeptide patterns. The heavy fractions, F3 and F4, were identified as being derived from the basal cell layer, whereas the light fractions, F1 and F2, consisted mainly of suprabasal cells. The basal cells (F3 and F4) could be cultivated on plastic substratum coated with rat-tail collagen (4 X MEM, 10% FCS at 34 degrees C; plating efficiency 70-85%). Labeling of DNA with [3H]thymidine indicated that during the first 5 days of cultivation, basal cells ran through two cell cycles, after which the proliferative activity ceased due to terminal differentiation. The addition of the tumor promoter TPA led to a stimulation of DNA synthesis in confluent cultures of both F3 and F4 cells.

Animals↗
PubMed1987

Didemnin B inhibits biological effects of tumor promoting phorbol esters on mouse skin, as well as phosphorylation of a 100 kD protein in mouse epidermis cytosol.

The immunosuppressive agent Didemnin B (DB) inhibits biological effects that are induced by topical application of phorbol esters to mouse skin as measured by mouse ear edema and the alkaline phosphatase activity of mouse epidermis. Furthermore, DB suppresses the phosphorylation in vitro of an epidermal cytosolic protein (p100). In these respects DB behaves like CsA, another immunosuppressive compound. Didemnin, however, does not bind to recently described CsA-binding sites.

Animals↗
PubMed1987

Cyclosporin A inhibits phorbol ester-induced cellular proliferation and tumor promotion as well as phosphorylation of a 100-kd protein in mouse epidermis.

Mouse epidermis and HEL30 keratinocytes contain cellular binding sites for the immunosuppressive agent cyclosporin A (CsA). Phorbol ester-induced DNA synthesis and tumor promotion in mouse skin is strongly suppressed by CsA. The anti-tumor effect of CsA is not the consequence of destruction of initiated cells. CsA selectively inhibits the phosphorylation in vitro of a protein with the relative molecular mass of 100 kd in mouse epidermis and HEL30 cytosol. The CsA-suppressible phosphorylation in vitro does not depend on the addition of Ca2+, phospholipid and the phorbol ester TPA.

Animals↗
PubMed1987

Decreased protein kinase C activity in psoriatic versus normal epidermis.

To study the possible involvement of protein kinase C in psoriasis, we determined the activity of this enzyme in involved and uninvolved epidermis from psoriasis patients as well as in normal epidermis from controls. Protein kinase C activity was measured in the 100,000 g supernatants and in the detergent-solubilized particulate fractions of tissue homogenates after partial purification by DEAE-cellulose chromatography. The total enzyme activities per gram protein for normal, involved, and uninvolved epidermis were 127 +/- 11.9 mU/g, 64.7 +/- 8.6 mU/g, and 88.5 +/- 14.4 mU/g, respectively. The respective values for total enzyme activity per mg DNA were 7.04 +/- 0.48 mU/mg, 4.28 +/- 0.54 mU/mg, and 5.02 +/- 0.66 mU/mg. By both data bases, protein kinase C activity was statistically lower in the psoriatic skin biopsies than in those from control persons, whereas no significant difference was found between involved and uninvolved epidermis from psoriasis patients. We hypothesize that alterations in protein kinase C-mediated processes due to decreased protein kinase C activity may play a significant role in the pathophysiology of psoriasis.

Chromatography, DEAE-Cellulose↗
PubMed1986

A novel type of phorbol ester-dependent protein phosphorylation in the particulate fraction of mouse epidermis.

In a Triton X100-extract from the particulate fraction of mouse epidermis but also of other murine tissues, the phosphorylation of a protein with the relative molecular mass of 82,000 (p82) is found to be dependent on phosphatidyl serine and the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Unlike protein kinase C-catalyzed phosphorylation, p82 phosphorylation is neither observed in the presence of high concentrations of Ca2+ and phosphatidyl serine alone nor after addition of exogenous protein kinase C. Dioctanoylglycerol and the "incomplete" promoter 12-0-retinoylphorbol-13-acetate are also capable of stimulating p82 phosphorylation, whereas the non-promoting phorbol ester 4-0-methyl-TPA is at least 100-fold less active in this respect.

Animals↗
PubMed1986

Isolation, characterization and in vitro cultivation of subfractions of neonatal mouse keratinocytes: effects of phorbol esters.

Using discontinuous Percoll density gradient centrifugation, keratinocytes from neonatal mouse skin were separated into four fractions. The fractions were characterized by light microscopy, by indirect immunofluorescence using lectins (Bandeira simplicifolia and Ulex europaeus) or an antibody against bullous pemphigoid antigen, and by electrophoretic analysis of the keratin polypeptide patterns. Two fractions, F1 and F2, were identified as being derived from suprabasal layers and two fractions, F3 and F4, as originating from the basal cell layer. F3 and F4 cells could be cultivated on plastic substratum in the presence of 4 X minimum essential medium, 10% fetal calf serum at 34 degrees C with plating efficiencies of 80-85%. DNA labeling of cultures of F3 and F4 cells indicated that basal cells in fractions F3 and F4 exhibited different growth behaviour in culture. Confluent cultures of F3 and F4 cells responded to phorbol esters by increased DNA synthesis. This result contradicts the in vivo situation where neonatal mouse skin is resistant to the irritant and mitogenic effects of phorbol esters. The mitogenic effect of 12-O-tetradecanoylphorbol-13-acetate on F3 cells is not mediated by endogenous prostaglandin formation.

Animals↗