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Biomedical subjects

F Marks

Publications and source records attributed to F Marks.

At least 73 records · Page 4Linked to original sources
PubMed1995

Differential expression of prostaglandin H synthase isozymes during multistage carcinogenesis in mouse epidermis.

An anti-tumor-promoting effect of indomethacin and related nonsteroidal anti-inflammatory drugs (NSAIDs) as well as the ability of the tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) to increase the level of prostaglandins in murine keratinocytes and mouse epidermis in vivo has been repeatedly documented. Here, the expression of prostaglandin H synthase (PGHS) isozymes, which are major targets of NSAIDs, was investigated in different stages of tumor development in mouse skin. Mouse epidermis in vivo constitutively expressed PGHS-1. PGHS-1 steady-state levels remained unchanged upon induction of acute or chronic epidermal hyperplasia by TPA and in papillomas and carcinomas generated by the initiation-promotion procedure, with 7,12-dimethylbenz[a]anthracene as initiator and TPA as promoter. Thus, the elevated prostaglandin level in the acute hyperplastic epidermis was very likely due to PGHS-2 induction. Repeated applications of TPA resulted in stationary hyperplasia and downregulation of PGHS-2 expression and prostaglandin levels, suggesting that the epidermis had adapted to the TPA stimulus. In papillomas and carcinomas, however, constitutive overexpression of PGHS-2 was found, with a large amount of prostaglandin E2 and prostaglandin F2 alpha. Keratinocyte cell lines corresponding to different stages of tumor development also constitutively over-expressed PGHS-2. Considered with inhibitor studies, these data suggest that PGHS-2 has a critical role in skin carcinogenesis. The anti-tumor-promoting effect of the PGHS inhibitor indomethacin is specifically reversed by prostaglandin F2 alpha, indicating that this prostaglandin type has a significant role in tumor development.

Amino Acid Sequence↗
PubMed1995

12-Lipoxygenase isoenzymes in mouse skin tumor development.

12-lipoxygenase-catalyzed arachidonic acid metabolism in normal and neoplastic mouse epidermis was assessed by cDNA cloning of the epidermal 12-lipoxygenases and by studying their expression patterns, enzyme activities, and product levels. Papillomas and squamous cell carcinomas induced by the initiation/promotion protocol contained 50- to 60-fold more 12-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) than normal epidermis. The ratio of S to R enantiomers was 9:1. This indicates that most of this eicosanoid was of enzymatic origin. Accordingly, cell-free preparations of the tumors exhibited about fivefold elevated 12-lipoxygenase activities. A papilloma-derived cDNA library was screened with human platelet-type 12-lipoxygenase cDNA probes. Two cDNA clones encoding the platelet-type and the leukocyte-type isoforms of murine 12-lipoxygenase were isolated, demonstrating the coexpression of the isoenzymes in the same tissue and species. When expressed in COS-7 cells, the recombinant enzymes showed the characteristic substrate selectivity and product profile, with the leukocyte-type enzyme metabolizing linoleic and arachidonic acid to 13-hydroxy-9,11-octadecadienoic acid and to 12- and 15-HETE, respectively, and the platelet-type enzyme oxygenating exclusively arachidonic acid to 12-HETE. In epidermis in vivo and in keratinocytes in culture, only the platelet-type 12-lipoxygenase (mRNA and protein) was detectable. In mouse epidermis both isoenzymes were induced transiently by phorbol esters. Most tumors showed constitutive overexpression of platelet-type mRNA, whereas leukocyte-type specific transcripts were detectable only in a few tumors. These data suggest that the platelet-type enzyme is the 12-lipoxygenase isoform of keratinocytes that is responsible for the generation of most of the 12-HETE found in neoplastic epidermis.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1995

Loss of protein kinase C delta from human HaCaT keratinocytes upon ras transfection is mediated by TGF alpha.

The spontaneously immortalized human skin keratinocytes HaCaT contain protein kinase C (PKC) alpha, -delta, -epsilon, and -zeta. All PKC isoenzymes except PKC zeta are down-regulated by TPA as well as by bryostatin. However, with PKC delta, bryostatin but not TPA was found to be much less effective at high concentrations than at low ones. PKC delta expression at the protein and mRNA level is significantly suppressed in HaCaT cells I-7 and II-4, which are transfected with mutated c-Ha-ras. The expression of the other isoenzymes remains essentially unchanged in the ras-transfected cells compared to normal ones. PKC delta is lost when growing HaCaT cells in a medium obtained from the cultivation of ras-transfected cells ("ras-conditioned" medium). The factor secreted into the medium by the ras-transfected cells that is responsible for this effect appears to be TGF alpha, since the action of ras-conditioned medium on PKC delta expression can be overcome by the addition of an anti-TGF alpha antibody. Moreover, treatment of HaCaT cells with TGF alpha suppresses selectively the expression of the PKC isoenzyme delta.

Bryostatins↗
PubMed1995

Mechanisms in tumor promotion: guidance for risk assessment and cancer chemoprevention.

In mouse skin, tumor development promoted by 'non-genotoxic' carcinogens is closely related to the wound response. In both cases endogenous factors such as cytokines and eicosanoids released primarily from 'activated keratinocytes' play a key role as mediators of inflammation and cellular hyperproliferation. The liberation of interleukin-1 alpha and arachidonic acid from human keratinocytes has been used as an in vitro parameter of irritancy. The results (from experiments with 15 different chemicals) being validated at present in a clinical study indicate a quantitative relationship between irritancy in vivo and mediator release in vitro. In the course of experimental skin carcinogenesis an overproduction of eicosanoids due to a constitutive overexpression of the corresponding enzymes (i.e. PGH synthase-II and 8- and 12-lipoxygenase) is observed. Enzyme inhibitors, for instance nonsteroidal antiinflammatory drugs (NSAIDs), exert a strong tumoristatic effect. Thus, the approach of multistage skin carcinogenesis provides a suitable animal model for a mechanistic evaluation and further improvement of chemopreventive measures such as the inhibition of colorectal tumor development in humans by NSAIDs ('aspirin effect').

Animals↗
PubMed1995

Vibratory acoustic stimulation and fetal gasping.

OBJECTIVE: Vibratory acoustic stimulation (VAS) has been used to shorten the duration of antepartum fetal heart rate monitoring. Questions have been asked regarding the possible effects of VAS on the fetus. VAS was used to investigate whether the acute effect of VAS is associated with gasping in the normal fetus. STUDY DESIGN: Thirty-seven normal patients at 37-42 weeks' gestation with singleton fetuses were studied. All had normal amniotic fluid volume on ultrasound. These 37 subjects had a five-minute baseline sonographic assessment of fetal breathing and body movements. A fetal gasp was defined as an isolated, slow, irregularly occurring, deep, inspiratory-type movement, or series of them, of the chest and abdomen. Three-second VAS was then delivered with an artificial larynx placed directly over the fetal head. Fetal breathing, body and gasping movements were assessed at the time of this stimulus. RESULTS: A significant increase in fetal body movements (P < .001), a significant decrease in fetal breathing (P < .003) and absence of gasping movements were noted. CONCLUSION: The results suggest that gasping does not occur in response to VAS in the normal, term fetus.

Acoustic Stimulation↗
PubMed1994

Partial purification of a type eta protein kinase C from murine brain: separation from other protein kinase C isoenzymes and characterization.

Various murine tissues were tested, by using a protein kinase C-eta-specific antiserum, for the expression of type eta protein kinase C. Brain was found to be the richest source of a type eta isoenzyme. Native protein kinase C-eta was partially purified from the cytosol of murine brain by chromatography on DEAE-Sepharose, hydroxyapatite and protamine-agarose. This procedure resulted in a separation of protein kinase C-eta from the other phorbol 12-myristate 13-acetate (PMA)-responsive isoenzymes (alpha, beta, gamma, delta, epsilon) and allowed, for the first time, characterization of the native enzyme. The protein kinase C of type eta from mouse brain is a phospholipid-dependent Ca(2+)-unresponsive protein kinase. Both PMA and bryostatin activate the kinase for phosphorylation of a substrate as well as for autophosphorylation. Various pseudo-substrate-related peptides are suitable as substrates for the eta-type kinase, peptide delta being the best and peptides eta and epsilon the poorest substrates. The enzyme is inhibited by staurosporine and staurosporine-related compounds, such as K252a and Gö 6976. However, protein kinase C-eta, like protein kinase C-delta, is around two orders of magnitude less sensitive towards Gö 6976 than are the Ca(2+)-responsive isoenzymes (alpha, beta, gamma). The eta-type protein kinase C exhibits an extreme tendency to lose its PMA-responsiveness. Consequently, purification of the enzyme to homogeneity has not yet been successful.

Alkaloids↗
PubMed1994

Tyrosine phosphorylation and stimulation of protein kinase C delta from porcine spleen by src in vitro. Dependence on the activated state of protein kinase C delta.

Native protein kinase C delta from porcine spleen is phosphorylated in vitro by the tyrosine kinase src and to a much smaller extent by fyn. The tyrosine phosphorylation of PKC delta is restricted to the activated state of the enzyme, i.e. it occurs only in the presence of an activator, such as TPA or bryostatin. Upon phosphorylation at tyrosine, the apparent molecular weight of PKC delta increases by 6 kDa. Phosphorylation by src induces a stimulation of PKC delta activity apparently exhibiting some substrate selectivity. Other PKC isoenzymes, such as cPKC (alpha, beta, gamma), are not phosphorylated by src or only to a very small extent. This phosphorylation is not dependent on TPA and does not cause an increase in activity and molecular weight of the enzyme.

Amino Acid Sequence↗
PubMed1994

Rottlerin, a novel protein kinase inhibitor.

Rottlerin, a compound from Mallotus philippinensis, is shown to inhibit protein kinases with some specificity for PKC. To some extent, the novel inhibitor is able to differentiate between PKC isoenzymes, with IC50 values for PKC delta of 3-6 microM, PKC alpha,beta,gamma of 30-42 microM and PKC epsilon,eta,zeta of 80-100 microM. Inhibition of PKC appears, at least in part, to be due to a competition between rottlerin and ATP. Among the protein kinases tested, only CaM-kinase III is suppressed by rottlerin as effectively as PKC delta. The chemical structure of rottlerin might serve as a basis for the development of novel inhibitors with improved selectivity for a distinct PKC isoenzyme, such as PKC delta, or for CaM-kinase III.

Acetophenones↗
PubMed1994

Elongation factor-2 kinase: effective inhibition by the novel protein kinase inhibitor rottlerin and relative insensitivity towards staurosporine.

The elongation factor-2 (eEF-2) is selectively phosphorylated by the eEF-2 kinase (calmodulin-dependent kinase III). This phosphorylation can be inhibited by calmodulin antagonists, such as CGS 9343B (IC50 = 4 microM). The novel protein kinase inhibitor rottlerin is shown to suppress eEF-2 phosphorylation with an IC50 of 5.3 microM. By contrast, the eEF-2 kinase is rather resistant towards the potent but non-selective protein kinase inhibitor staurosporine (IC50 > 50 microM) and thus can be differentiated from most other protein kinases that are suppressed by staurosporine in the nM range.

Acetophenones↗
PubMed1994

Keratinocyte-derived proinflammatory key mediators and cell viability as in vitro parameters of irritancy: a possible alternative to the Draize skin irritation test.

This study is aimed at the development of a cell culture assay which may supplement or replace the animal Draize skin irritancy test. Using human keratinocytes, the measurement of proinflammatory eicosanoid and interleukin-1 alpha release and of the impairment of cell viability have provided a suitable in vitro/in vivo correlation for at least three surfactants. The in vitro study has been extended using structurally unrelated, pharmacologically relevant compounds including ethanol, glycerol, cyclohexanol, acetone, benzoic acid, phenol, acrylamide, triethanolamine, Tween 80, sodium dodecyl sulfate, benzalkonium chloride, NiSO4, SnCl2, and ZnCl2. Time- and dose-response studies were used to establish half-maximal stimulatory (SC50) and inhibitory (IC50) as well as 10-fold stimulatory (ED10) concentrations for arachidonic acid release, cytotoxicity, and IL-1 alpha release, respectively. Based upon these values a similar ranking has been obtained for the mildly acting acetone, ethanol, glycerol, and Tween 80 and for the severely acting A23187, benzalkonium chloride, and sodium dodecyl sulfate. With respect to all other test compounds, substantial variation occurred indicating that all three test parameters provide a more complete characterization of the test compound's potency than a single endpoint. These data are ready to be validated by a controlled clinical study aimed at a qualitative and quantitative evaluation of the symptoms of skin inflammation in volunteers.

Animal Testing Alternatives↗
PubMed1994

Third colloquium on cellular signal transduction: cell-cycle signalling. German Cancer Research Centre Heidelberg, 14 January 1994.

The annual meeting of the DKFZ research programme Tumor Cell Regulation was originally conceived by its members as an internal forum for mutual information and progress discussion with their cooperating groups. However, because the attractive topics and contributions, these colloquia receive increasing attention also from non-members of the Tumor Cell Regulation programme and, this year, the number of the external participants exceeded even that of the internal members. We therefore anticipate this activity becoming a traditional event of wide interest.

Animals↗
PubMed1994

Effects of complete and incomplete tumor promoters on hair growth, angiogenesis, and tenascin expression in the skin of NMRI mice.

Although the phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12-O-retinoylphorbol-13-acetate (RPA) are almost equipotent inducers of an inflammatory and hyperplastic response in NMRI mouse skin, TPA acts as a converting or complete and RPA as a non-converting or incomplete skin tumor promoter. Bearing in mind the converting effect of skin wounding and TGF beta, we have addressed the question of a relationship between conversion and stromal events. For this purpose we have compared the effects of TPA and RPA on dermal angiogenesis, hair regrowth and the expression of tenascin in NMRI mouse skin in vivo. In the reticular part of the dermis, shaving alone induced angiogenesis peaking at day 14, concomitant with hair regrowth. While TPA exhibited a stimulatory effect on both parameters, RPA suppressed shaving-induced angiogenesis and delayed the onset of hair regrowth. Whether this difference in the phorbol ester effects being consistent with the working hypothesis is critical for conversion, remains to be shown. In the papillary dermis, however, RPA was more potent than TPA in inducing vascularization and tenascin expression. The kinetics of both responses corresponded to the time course of hyperproliferation induced by the phorbol esters in the overlaying epidermis, i.e. may be related to promotion proper, rather than conversion.

Animals↗
PubMed1994

Phorbol ester-induced leukotriene biosynthesis and tumor promotion in mouse epidermis.

In mouse skin in vivo the irritant and hyperplasiogenic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) strongly increased the epidermal content of the cysteinyl leukotrienes LTC4, LTD4 and LTE4, but not of leukotriene LTB4. This effect was completely suppressed by the selective leukotriene biosynthesis inhibitor MK-886. Intragastric administration of MK-886 prevented phorbol ester-induced ear edema, but not epidermal hyperproliferation and tumor promotion. These data indicate that leukotrienes are involved in the pro-inflammatory effects of the phorbol ester, whereas its hyperproliferative and tumor-promoting activities do not depend on 5-lipoxygenase-catalyzed leukotriene formation. This action differs from several non-selective inhibitors of lipoxygenases that were found to inhibit tumor promotion in initiated mouse skin.

Animals↗
PubMed1993

Activation of cytosolic phospholipase A2 by transforming growth factor-alpha in HEL-30 keratinocytes.

In the mouse keratinocyte line HEL-30 the epidermal mitogen transforming growth factor-alpha (TGF-alpha) stimulated the rapid release of arachidonic acid in a dose- and time-dependent manner. The liberation of arachidonic acid was due to the activation of a Ca(2+)-dependent cytosolic phospholipase A2 (cPLA2). The activation mechanism critically depended on a functionally active epidermal growth factor receptor tyrosine kinase and occurred independently of phospholipase C-mediated increases in cellular diacylglycerol and inositol 1,4,5-trisphosphate concentrations and protein kinase C activation. The activation included an increase in cytosolic PLA2 (cPLA2) activity and an association of the enzyme with the membrane fraction. Both activation steps apparently occurred in the presence of basal cytoplasmic Ca2+ concentrations. Moreover, cPLA2 or a closely associated protein was found to be phosphorylated on tyrosine upon TGF-alpha challenge of the cells. The data suggest that tyrosine phosphorylation is involved in the TGF-alpha-induced activation of cPLA2.

Animals↗
PubMed1993

Protein kinase C forms a complex with and phosphorylates the GTPase activating protein GAP: phosphorylation by PKC is dependent on tyrosine phosphorylation of GAP and/or a GAP-associated protein.

Protein kinase C (PKC) and the GTPase-activating protein GAP can be detected in immunoprecipitates of mouse epidermis and lung cytosol obtained with either anti-GAP or anti-PKC antisera. The PKC in the immune-complex phosphorylates the coprecipitated GAP protein. Moreover, purified recombinant GAP is phosphorylated in vitro by purified PKC. The efficacy of this phosphorylation appears to depend on the extent of tyrosine phosphorylation of GAP and/or a GAP-associated protein.

Animals↗