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S M Fischer

Publications and source records attributed to S M Fischer.

At least 55 records · Page 3Linked to original sources
PubMed1997

Effect of 12-O-tetradecanoylphorbol-13-acetate on inhibition of expression of keratin 1 mRNA in mouse keratinocytes mimicked by 12(S)-hydroxyeicosatetraenoic acid.

Differentiation of cultured keratinocytes is controlled by the calcium concentration of the medium and is marked by the expression of differentiation-specific keratins. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) alters the normal differentiation program and suppresses keratin (K) 1 expression. Based on reported similarities in the effects of TPA and the arachidonic acid metabolite 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), we hypothesized that 12(S)-HETE might suppress K1 expression in mouse keratinocytes. We also investigated the effect of pretreatment with 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE) because others have reported that 13(S)-HODE prevents 12(S)-HETE-induced events. In our study, 100 nM 12(S)-HETE mimicked the effect of 500 nM TPA in suppressing K1 mRNA expression within 24 h of calcium-induced differentiation. Pretreatment with 100 nM 13(S)-HODE blocked the 12(S)-HETE effect but not the TPA effect. A role for protein kinase C (PKC) was suggested for both TPA and 12(S)-HETE based on the loss of response with the PKC inhibitors bryostatin-1 or RO-31-8220. Both TPA and 12(S)-HETE stimulated keratinocyte PKC activity. Pretreatment with 13(S)-HODE blocked the 12(S)-HETE-induced increase in PKC activity. Immunoblotting showed that whereas TPA caused a rapid, partial translocation of the PKC alpha isozyme, it had no effect on the distribution of PKC delta. Conversely, 12(S)-HETE had no effect on the distribution of PKC alpha but caused a complete translocation of PKC delta. Pretreatment with 13(S)-HODE prevented 12(S)-HETE-elicited translocation of PKC delta. We conclude that 12(S)-HETE mimics the effect of TPA on K1 mRNA and that the effect is mediated through different isoforms of PKC.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1997

Changes in protein expression during multistage mouse skin carcinogenesis.

To directly compare the expression patterns of different proteins known to be altered during mouse skin carcinogenesis, serial sections of normal and hyperplastic skin and tumors from various stages of 7,12-dimethylbenz[a]anthracene-initiated, 12-O-tetradecanoylphorbol-13-acetate-promoted female SENCAR mice were examined by immunohistochemistry. In untreated, normal mouse skin, keratin 1 (K1) and transforming growth factor-beta1 (TGFbeta1) were strongly expressed in the suprabasal layers, whereas integrin alpha6beta4 was expressed only in basal cells and only moderate staining for transforming growth factor-alpha (TGFalpha) was seen. In hyperplastic skin, TGFalpha expression became stronger, whereas expression of another epidermal growth factor (EGF) receptor ligand, heparin-binding EGF-like growth factor (HB-EGF), was strongly induced in all epidermal layers from no expression in normal skin. Likewise, the gap-junctional protein connexin 26 (Cx26) became highly expressed in the differentiated granular layers of hyperplastic skin relative to undetectable expression in normal skin. Expression of cyclin D1 in the proliferative cell compartment was seen in all benign and malignant tumors but not in hyperplastic skin. Beginning with very early papillomas (after 10 wk of promotion), expression of alpha6beta4 in suprabasal cells and small, focal staining for keratin 13 (K13) were seen in some tumors. Later (after 20-30 wk), focal areas of gamma-glutamyl transpeptidase (GGT) activity appeared in a few papillomas, whereas TGFbeta1 expression began to decrease. Cx26 and TGFalpha staining became patchier in some late-stage papillomas (30-40 wk), whereas suprabasal alpha6beta4, K13, and GGT expression progressively increased and K1 expression decreased. Finally, in squamous cell carcinomas (SCCs), there was an almost complete loss of K1 and a further decline in TGFalpha, HB-EGF, TGFbeta1, and Cx26 expression. On the other hand, almost all SCCs showed suprabasal staining for alpha6beta4 and widespread cyclin D1 and K13 expression, whereas only about half showed positive focal staining for GGT activity.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

Multifactor regulation of prostaglandin H synthase-2 in murine keratinocytes.

Prostaglandin H synthase (PGHS) is the rate-limiting enzyme responsible for the formation of the prostaglandins from arachidonic acid. Prostaglandins (and other metabolites) elicit signals for inflammation, which is thought to be required for tumor promotion in the mouse skin carcinogenesis model. This study was designed to examine the effect of protein kinase C (PKC)-activating tumor promoters (4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA)), non-PKC-type promoters (anthralin, benzoyl peroxide, okadaic acid), and mitogens (epidermal growth factor (EGF)) on the levels of the constitutive (PGHS-1) and inducible (PGHS-2) forms of PGHS in murine keratinocytes. Northern analysis of mRNA isolated from cultures treated with TPA (1 microgram/mL) showed that a single treatment of TPA produced a sevenfold increase in PGHS-2 mRNA by 1 h that decreased by 6 h after treatment. PGHS-2 protein levels were elevated threefold by 3 h and remained elevated through 9 h. Downregulation of PKC with a second TPA treatment 15 h after the first resulted in diminished induction of PGHS-2 expression. Of the other promoters examined, anthralin (5 microM), benzoyl peroxide (10 microM), and okadaic acid (1 microM) induced PGHS-2 mRNA with different kinetics and to different extents. Additionally, the non-tumor-promoting phorbol ester analogue 4 alpha-12-O-tetradecanoylphorbol-13-acetate induced PGHS-2 mRNA significantly by 1 h, and this response remained elevated up to 6 h after treatment. Elevated PGHS-2 expression was also observed by 3 h in response to EGF (10 ng/mL) treatment. Collectively, these observations indicate that there are several different signaling pathways by which PGHS-2 can be upregulated in murine keratinocytes.

Animals↗
PubMed1996

Comparison of ultraviolet light-induced skin carcinogenesis and ornithine decarboxylase activity in sencar and hairless SKH-1 mice fed a constant level of dietary lipid varying in corn and coconut oil.

To investigate the effect of various levels of corn oil and coconut oil on ultraviolet (UV) light-induced skin tumorigenesis and ornithine decarboxylase (ODC) activity, Sencar and SKH-1 mice were fed one of three 15% (weight) fat semipurified diets containing three ratios of corn oil to coconut oil: 1.0%:14.0%, 7.9%:7.1%, and 15.0%:0.0% in Diets A, B, and C, respectively. Groups of 30 Sencar and SKH-1 mice were fed one of the diets for three weeks before UV irradiation; then both strains were UV irradiated with an initial dose of 90 mJ/cm2. The dose was given three times a week and increased 25% each week. For Sencar mice (irradiated 33 wks for a total dose of 48 J/cm2), tumor incidence reached a maximum of 60%, 60%, and 53% for Diets A, B, and C, respectively, with an overall average of one to two tumors per tumor-bearing animal. For the SKH-1 mice (irradiated 29 wks for a total dose of 18 J/cm2), all diet groups reached 100% incidence by 29 weeks, with approximately 12 tumors per tumor-bearing mouse. No significant effect of dietary corn oil/coconut oil was found for tumor latency, incidence, or yield in either strain. The effect of increasing corn oil on epidermal ODC activity in chronically UV-irradiated Sencar and SKH-1 mice was assessed. Three groups of mice from each strain were fed one of the experimental diets and UV irradiated for six weeks. Sencar mice showed no increase in ODC activity until six weeks of treatment, when the levels of ODC activity in the UV-irradiated mice fed Diet A were significantly higher than those in mice fed Diet B or Diet C: 1.27, 0.55, and 0.52 nmol/mg protein/hr, respectively. In the SKH-1 mice, ODC activity was increased by the first week of UV treatment, and by three weeks of treatment a dietary effect was observed; ODC activity was significantly higher in mice fed Diet C (0.70 nmol/mg protein/hr) than in mice fed Diet A (0.18 nmol/mg protein/hr). Although there was no significant effect of dietary corn oil/coconut oil on UV-induced tumor incidence, the data indicate that chronically UV-irradiated hairless SKH-1 mice are more susceptible to UV-induced skin carcinogenesis than Sencar mice and that this susceptibility is correlated with increased in ODC activity, a parameter of cell proliferation.

Animals↗
PubMed1996

Arachidonate has protumor-promoting action that is inhibited by linoleate in mouse skin carcinogenesis.

Previous studies demonstrated a requirement for arachidonic acid metabolites in tumor development in mouse skin. The goal of this study was to determine whether the arachidonate content of epidermal phospholipids could be altered by increasing dietary levels of linoleate and whether specific metabolites of linoleate and arachidonate have dissimilar biological effects. In a series of tumor studies in which the quantity of dietary linoleate was incrementally increased, a slight reduction in phospholipid levels of arachidonate was observed that correlated with an increased phospholipid level of linoleate and a suppression in tumor yield. A comparison of the arachidonate lipoxygenase metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) with the 13-hydroxyoctadecadienoic acid (13-HODE) lipoxygenase metabolite of linoleate revealed that 12-HETE has biological activities that mimic the phorbol ester tumor promoters, whereas 13-HODE has antithetical effects. Specifically, 12(S)-HETE enhanced the activation of protein kinase C by phorbol esters, mimicked phorbol ester-induced adhesion of keratinocytes to fibronectin and mimicked phorbol ester repression of expression of a differentiation-related gene, keratin-1. 13-HODE blocked 12-HETE-induced cell adhesion and prevented 12-HETE-induced suppression of keratin-1 expression. Overall, these studies suggest that arachidonate and linoleate have opposing functions in the epidermis, particularly with regard to events involved in tumor development.

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

Tumor-promoting activity of 2,4-dinitrofluorobenzene.

The aim of this study was to determine whether the skin-sensitizing agent 2,4-dinitrofluorobenzene (DNFB) would elicit the same morphological and biochemical events that are characteristic of 12-O-tetradecanoylphorbol-13-acetate (TPA). While single applications of 0.1% or 0.2% DNFB produced only mild epidermal hyperplasia, multiple applications produced pronounced hyperplasia. Compared with TPA, a single application of DNFB produced small increases in ODC activity, although a second DNFB treatment produced a greater response. Both DNFB and TPA caused marked induction of ODC, c-fos and c-jun mRNA. Vascular permeability increased significantly in response to DNFB, such that after 15 hr the response was quantitatively the same as for TPA. Repeated TPA produced the same response as a single application, but repeated DNFB resulted in a response that was half that of TPA. In contrast to TPA, DNFB failed to activate partially purified protein kinase C (PKC), although it did cause transient down-regulation of activity 15 hr after treatment. The ability of DNFB to induce ODC activity, however, was unaffected by prior down-regulation of PKC. DNFB was also shown to promote tumors in initiated SSIN mice. Twice-weekly applications of 0.1% or 0.2% DNFB resulted in approximately 65% and 85% of the mice developing an average of 2.0 or 3.2 tumors each, respectively. These results demonstrate that DNFB elicits many of the same changes as TPA and that it does so in a PKC-independent manner.

Animals↗
PubMed1994

Signal transduction pathway(s) involved in phorbol ester and autocrine induction of interleukin-1 alpha mRNA in murine keratinocytes.

We investigated the signal transduction pathways leading to the 12-O-tetradecanoylphorbol-13-acetate (TPA)- and interleukin-1 alpha (IL-1)-induced IL-1 alpha mRNA in mouse keratinocytes. Induction of IL-1 alpha mRNA by TPA or IL-1 alpha was followed by increases in cell-associated IL-1 alpha protein measured by enzyme-linked immunosorbent assay. Although protein kinase C (PKC) was involved in TPA-induced IL-1 alpha mRNA, down-regulation of PKC did not block the induction of this gene by TPA. The autocrine induction of IL-1 alpha was not mediated through PKC or cAMP. IL-1 alpha did activate mitogen-activated protein kinase. Genistein, a tyrosine kinase inhibitor, blocked both IL-1 alpha-induced mitogen-activated protein kinase activation as well as IL-1 alpha mRNA expression. Genistein, at an unsaturating dose, plus a serine/threonine kinase inhibitor, H7, completely blocked the autocrine induction of IL-1 alpha suggesting that expression of this gene is regulated by tyrosine kinase(s) in combination or independently with serine/threonine kinase(s). In addition, both TPA and IL-1 alpha caused increases not only in the phosphorylation of c-Jun and c-Fos protein but also in the transactivating activity of AP-1 nuclear transcription factor. Neither TPA nor IL-1 alpha induced NF-kappa B binding activity, as assessed by electrophoretic mobility shift analysis. This study suggests that the activation of AP-1 may be a common event through which TPA and IL-1 alpha induce IL-1 alpha mRNA.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Transforming growth factor-beta 1 expression in Syrian hamster cheek pouch carcinogenesis.

The expression pattern of transforming growth factor-beta 1 (TGF-beta 1) during the stages of complete carcinogenesis in the hamster cheek pouch model was studied. The right cheek pouches of 18 male hamsters were treated with 0.5%, 7,12-dimethylbenz[a]anthracene (DMBA) for 16 wk. TGF-beta 1 was detected immunohistochemically in the resulting samples with two different polyclonal monospecific antibodies that recognize intracellular and extracellular forms of TGF-beta 1. In the normal cheek pouch, extracellular protein stained the corium strongly, but the reaction was not evenly distributed. As treatment progressed, the reaction increased in both area and intensity; the peak was reached at 8 wk. Intracellular TGF-beta 1 expression followed a similar pattern, with a peak at 4 wk of treatment. The results of northern blot analysis were concordant with the immunohistochemical results. Overexpression of TGF-beta 1 was also observed in the malignant tumors, but only the extracellular form of the protein was present; intracellular TGF-beta 1 was not detected in these tumors. The expression of TGF-beta 1 in this carcinogenesis model seems to have two formal stages, the first being an overexpression step as a reaction to the uncontrolled growth and the second being one in which tumors have no internal expression of TGF-beta 1 but in which external protein accumulates in the surrounding stroma. A possible explanation of this paradox may be that TGF-beta 1 has functions other than its growth-repressing activity.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1994

Altered expression of transforming growth factor-beta 1 mRNA and protein in mouse skin carcinogenesis.

Transforming growth factor (TGF)-beta 1, whose gene is located on mouse chromosome 7, has been proposed to be involved in skin carcinogenesis. In the study presented here, we demonstrated that single topical treatments with different types of tumor promoters, i.e., the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA, 2 micrograms); the non-protein kinase C activators anthralin (22.6 micrograms), benzoyl peroxide (20 mg), and cumene hydroperoxide (1.2 mg); the first-stage tumor promoters 4-O-methyl-TPA (500 micrograms) and A23187 (166 micrograms); and the second-stage tumor promoter mezerein (2 micrograms) produced transient induction of TGF-beta 1 mRNA in SSIN (inbred SENCAR) mouse skin. The time of maximum induction varied from 3 to 12 h; the relative extent of induction was ranked as cumene hydroperoxide > benzoyl peroxide > anthralin > TPA > 4-O-methyl-TPA > mezerein > A23187. These findings suggested that TGF-beta 1 mRNA induction is a common response of skin to several types of complete and stage-specific promoters; however, the extent of induction did not correlate with the reported hyperplastic activity of single applications of these promoters. We also demonstrated that TGF-beta 1 mRNA expression in papillomas of SENCAR mice generally correlated with expression levels of cyclin D1, another gene on chromosome 7, and with stage of tumor progression. TGF-beta 1 mRNA expression was constitutively elevated in most squamous cell carcinomas from either initiation-promotion or complete carcinogenesis protocols. Cell lines established from carcinomas also overexpressed TGF-beta 1 mRNA. Immunohistochemical staining of tissue sections of normal and TPA-treated skin revealed the presence of extracellular TGF-beta 1 protein in the dermis and intracellular TGF-beta 1 protein in the epidermis, especially in the suprabasal layers. The staining patterns of papillomas varied, with 62 +/- 13% of the tissue showing strong intracellular staining but only 25 +/- 8% of the connective tissue staining for extracellular TGF-beta 1. Variable staining patterns were also found in carcinomas; some areas stained heavily for both the intracellular and extracellular forms of TGF-beta 1. Overall, 28 +/- 6% of the tissue of the 12 analyzed carcinomas stained for the intracellular form and 18 +/- 5% for the extracellular form of TGF-beta 1.

Animals↗
PubMed1994

In vitro activation of mouse skin protein kinase C by fatty acids and their hydroxylated metabolites.

To understand how dietary fatty acids differentially modulate mouse skin tumorigenesis, the ability of specific fatty acids and their derivatives to activate murine epidermal protein kinase C (PKC) in vitro was investigated. Total PKC from untreated female SSIN mouse skin was partially purified and incubated with specific fatty acids at concentrations up to 300 microM in the presence of Ca2+ and phosphatidylserine. The cis-unsaturated fatty acids tested, ranging from 16:1 to 22:6, stimulated PKC activity in a similar dose-dependent manner with an approximate threefold maximum increase over control. Neither the number of cis-double bonds nor the chainlength of these fatty acids affected their relative ability to activate PKC. trans-Fatty acids, with the exception of linoelaidic acid (t,t-18:2n-6), exhibited about half of the potency of their corresponding cis-isomers in stimulating PKC at the plateau concentration (200 microM) or lower. Substitutions close to the double bond on cis-fatty acids abolished their ability to activate PKC. The hydroxylated metabolites of arachidonic acid (20:4n-6) and linoleic acid (c,c-18:2n-6), i.e., the hydroxyeicosatetraenoic acids (HETE) and hydroxyoctadecadienoic acids (HODE), also activated mouse skin PKC in vitro, but only about half as effectively as did the respective parent fatty acids. The results suggest that both hydroxyl substitution and trans-configuration of HETE and HODE are responsible for their reduced ability to activate PKC. Overall the data suggests that the reduced skin tumor yield observed in mice fed diets high in c,c-18:2n-6 is not likely to be due to differences in the ability of c,c-18:2n-6 or 20:4n-6, or their metabolites, to activate PKC.

Animals↗
PubMed1994

Effects of type and amount of dietary fat on mouse skin tumor promotion.

In a previous study (Cancer Res 51, 907, 1991) in which we found an inverse relationship between quantity of dietary corn oil and saturated fat, in a constant 15% fat diet, on the tumor promotion stage of skin carcinogenesis, it was not clear whether one or both types of fat played a modulatory role. The purpose of the present study therefore was to compare the effect of 1) increasing corn oil in corn oil-only diets and 2) increasing saturated fat, with a constant level of 5% corn oil, on tumor promotion. In the first study, the effects of five levels of dietary corn oil (5%, 10%, 15%, 20%, and 25%) on the incidence and rat of papilloma and carcinoma development were determined in female Sencar mice fed these diets one week after initiation with 7,12-dimethylbenz[a]anthracene and three weeks before the start of promotion with 12-O-tetradecanoylphorbol-13-acetate. A papilloma incidence of 100% was reached first in the 5% corn oil group, at 10 weeks, followed by the 10% group at 13 weeks and the 15% and 20% group at 16 weeks. The highest corn oil group achieved a 90% incidence. There were marked differences in latency of carcinoma development among the diet groups. At Week 29, the cumulative carcinoma incidence was 56% and 32%, respectively, in the 5% and 10% corn oil groups, whereas the incidence in the two highest corn oil (20% and 25%) groups was only 8% and 4%, respectively. In the second study, the effects of diets containing 5% corn oil and increasing levels of coconut oil (5%, 10%, 15%, and 20%) on the incidence and rat of papilloma and carcinoma development were determined, as described above. No significant difference in latency or incidence of papillomas or carcinomas was noted among these saturated fat diet groups. It thus appears that higher levels of dietary corn oil are associated with a reduced cancer incidence in this model system.

Animals↗
PubMed1994

Interleukin-1 alpha mediates phorbol ester-induced inflammation and epidermal hyperplasia.

The topical application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to murine skin produces acute inflammatory and hyperplastic responses that have been associated with the promotion stage of skin carcinogenesis. It has been shown in a previous study that TPA induces the expression of the highly inflammatory cytokine, interleukin (IL) -1 alpha, in the epidermis of SENCAR mice. The goal of this study was to investigate the role of IL-1 alpha in several TPA-induced responses in skin. Topical application of TPA (1 microgram) enhanced the production of immunoreactive IL-1 alpha protein, primarily associated with the suprabasal keratinocytes. IL-1 alpha intradermally injected in the dorsal surface significantly increased (P < 0.001) vascular permeability at low concentrations (1-1000 microU) and increased (P < 0.001) inflammatory cell infiltration and epidermal hyperplasia at higher concentrations (10(3) U). TPA produced fourfold increases in vascular permeability as measured by Evans blue dye leakage; this effect was prevented by intradermal injection of anti-IL-1 alpha antibody (25-75 micrograms). Furthermore, injected anti-IL-1 alpha antibody significantly reduced (P < 0.001) TPA-induced inflammatory cell infiltration and epidermal hyperplasia. This study suggests that IL-1 alpha directly or indirectly mediates the inflammatory and hyperplastic responses elicited by topical treatment with TPA.

Analysis of Variance↗
PubMed1993

Modulation of interleukin-1 alpha mRNA expression in mouse epidermis by tumor promoters and antagonists.

Increased expression of interleukin-1 (IL-1) in skin elicits a variety of responses, including inflammation and epidermal hyperplasia, which are also characteristic events elicited by tumor promoters. The goal of this study was to investigate whether various classes of tumor promoters increase expression of IL-1 alpha and whether phorbol ester-induced IL-1 alpha expression can be blocked by antitumor promoters. Northern analysis of mRNA isolated from the dorsal skins of SENCAR mice treated with 1 microgram of 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA) showed that a single application of TPA produced a significant increase in IL-1 alpha mRNA at 6 h that decreased by 24 h after treatment. Two treatments of TPA at 48-h intervals induced, at 6 h, twice as much IL-1 alpha mRNA as one treatment. Of the other promoters tested, anthralin (22.6 micrograms), mezerein (2 micrograms), calcium ionophore A23187 (120 micrograms), and benzoyl peroxide (20 mg) induced IL-1 alpha mRNA with different kinetics and to different extents. On the other hand, the non-tumor promoting phorbol ester analogue 4 alpha-12-O-tetradecanoylphorbol-13-acetate had little effect on the expression of IL-1 alpha mRNA. The effects of various antitumor promoters on TPA-induced IL-1 alpha mRNA expression were also assessed. Fluocinolone acetonide, mepacrine, and 5,8,11,14-eicosatetraynoic acid were the most effective inhibitors, and each produced about 80% inhibition. Other antitumor promoters such as retinoic acid, N-tosyl-L-phenylalanine chloromethyl ketone, and butylated hydroxytoluene inhibited approximately 35%, 65%, and 50% of TPA-induced IL-1 alpha mRNA expression, respectively. Therefore, this study suggests a possible role of IL-1 alpha in the promotion stage of skin carcinogenesis.

Animals↗
PubMed1993

Association of protein kinase C activation with induction of ornithine decarboxylase in murine but not human keratinocyte cultures.

The goal of this study was to compare the response of mouse epidermal keratinocytes (MEKs) and human epidermal keratinocytes (HEKs) to 12-O-tetradecanoylphorbol-13-acetate (TPA) with respect to the activation and downregulation of protein kinase C (PKC), the expression of c-jun and c-fos, and the expression and induction of ornithine decarboxylase (ODC) activity. Keratinocytes from adult CD-1 mice and from discarded adult human skin were grown in primary culture in a high-calcium serum-free medium that supported proliferation and differentiation. Immunoblotting of freshly isolated and cultured MEKs and HEKs for isozymes of protein kinase C revealed that fresh HEKs contained PKC alpha, PKC beta, and PKC delta; no PKC gamma, PKC epsilon, or PKC zeta were detected. In fresh MEKs, PKC alpha, PKC beta, PKC delta, and PKC zeta were observed, but not PKC gamma or PKC epsilon. After 2 wk in culture, the isozyme profiles of MEKs and HEKs were similar except that PKC gamma was noticeably present in HEK cultures. Activation of partially purified total PKC by TPA was similar in freshly isolated and cultured MEKs and HEKs, indicating that the two species were similar in this regard and that 2 wk of culture did not alter this characteristic. When MEK and HEK cultures were treated with TPA for 3 h, less than 30% of the control level of PKC activity was detected, indicating that TPA-induced downregulation of PKC was similar in MEKs and HEKs. After treatment with TPA, MEK cultures produced a large induction of both c-jun and c-fos mRNA by 60 min, as determined by northern blot analysis, and a large induction of ODC mRNA and enzyme activity by 6 h. TPA treatment of cultured HEKs, however, did not induce ODC activity; in fact, less activity, compared with that of control cultures, was observed. Northern blot analysis also revealed no increase in c-jun, c-fos, and ODC mRNA in HEKs. However, c-jun and c-fos mRNA and both ODC mRNA and enzyme activity were induced in HEKs fed growth factors after several days of deprivation. This suggests that the lack of ODC induction by TPA in HEKs is probably due to species differences in downstream steps in PKC signal transduction.

Adult↗
PubMed1993

Modulation of phorbol ester-associated events in epidermal cells by linoleate and arachidonate.

To elucidate the events elicited by the skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which are modulated by linoleic acid (LA) and arachidonic acid (AA), the activity of these fatty acids in cultured mouse epidermal cells was compared. Approximately 94% of either exogenous radiolabelled fatty acid was incorporated into the total phospholipid pool over 15 h. The relative distribution among the phospholipid classes differed, however, such that approximately 70% of phospholipid-associated [14C]-LA was found in phosphatidylcholine, compared to approximately 30% for [14C]AA. Phosphatidylethanolamine and phosphatidylinositol/phosphatidylserine contained 17 and 13% of the phospholipid [14C]LA, and 34 and 30% of [14C]AA, respectively. Prostaglandin (PG) E2 production was low but similar in unstimulated cultures prelabelled with either [14C]LA or [14C]AA. However, in cultures treated with TPA (1.6 microM), [14C]AA-prelabelling resulted in approximately three times the amount of [14C]PGE2 compared with cultures prelabelled with [14C]LA. Cultured cells were found to contain significant delta 6 desaturase activity, which may enable conversion of LA to AA, and thus may account for the observed PGE2 production from [14C]LA treated cells. AA-Supplemented (1.6 microM) cultures supported approximately twice the induction of ornithine decarboxylase activity by TPA compared with cultures treated with 1.8 microM LA. Activation of partially purified protein kinase C was similar for either fatty acid tested over a 10-300 microM dose range. Overall, the results suggest that LA does not have the same biological activity as AA with regard to several TPA-associated events known to be important in skin tumor promotion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Dietary fatty acid modulation of events associated with mouse skin tumor promotion.

Increasing levels of dietary corn oil have been correlated with inhibition of 12-O-tetradecanoylphorbol-13-acetate-(TPA) promoted skin tumorigenesis in mice (Leyton et al. Cancer Res. 51, 907-915, 1991). This study was undertaken to assess the effects of dietary corn oil on several events associated with tumor promotion. Three semipurified diets containing 15% (wt/wt) total fat with increasing levels of linoleate (0.8%, 4.5%, and 8.4%) supplied by corn oil were fed to mice for at least four weeks. Although incorporation of linoleate into epidermal phosphatidylcholine increased with increasing amounts of dietary corn oil, the elongated desaturated product of linoleate, arachidonate, was similar or decreased slightly in mice fed the three diets. Minimal activity of delta 6-desaturase, the rate-limiting enzyme in the conversion of linoleate to arachidonic acid, was found in the epidermis compared with the liver, suggesting that linoleate is not converted to arachidonic acid in the skin. Subcellular distribution of protein kinase C was altered in mice fed 0.8% linoleate, where 69% of protein kinase C activity was in the cytosol compared with 78% and 74% for groups fed 4.5% and 8.4% linoleate, respectively. Activation of partially purified protein kinase C isolated from mouse epidermis by linoleate was significantly lower (p < 0.01) than that isolated by arachidonic acid. TPA-induced vascular permeability was significantly greater (p < 0.05), whereas hyperplasia 48 hours after TPA treatment was significantly lower, in mice fed the 8.4% linoleate diet. However, TPA induction of ornithine decarboxylase activity did not appear to be significantly modified by dietary linoleate. These data suggest that cellular processes associated with carcinogenesis are affected by the level of dietary linoleate.

Animals↗
PubMed1993

12-O-tetradecanoylphorbol-13-acetate-mediated systemic co-promotion in the murine skin multistage carcinogenesis protocol.

The systemic promoting and co-promoting activities of 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined in a murine skin multistage carcinogenesis protocol. The dorsal skins of female inbred SENCAR (SSIN) mice were initiated with 25 nmol of 7,12-dimethylbenz[a]anthracene (DMBA). Topical treatment of initiated dorsal skin with 20 mg of benzoyl peroxide (BzP) promoted the eventual development of 8-15 dorsal papillomas per mouse. Repeated application of 2 micrograms of TPA to the ventral skins of dorsally initiated mice did not promote the formation of dorsal tumors. However, the latency of dorsal papilloma development was significantly decreased in initiated mice treated with 0.4 or 2 micrograms TPA (ventral application) and promoted with BzP (dorsal application). The co-promoting effects of topically applied TPA could not be mimicked by administering the promoter i.v. Only 6/23 initiated, BzP-promoted SSIN mice developed squamous cell carcinomas (SCC) during a 61 week promotion period. In contrast, during the same time-frame 12/24 and 11/24 BzP-promoted mice cotreated with 2 or 0.4 microgram of TPA developed SCC respectively. Repeated application of 2 micrograms of TPA to the ventral skins of dorsally initiated mice resulted in the development of ventral tumors. Ventral tumor incidence and multiplicities could be dramatically reduced by housing the mice individually, as opposed to collectively, for a day following initiation. Collectively these findings suggest that TPA can function as a co-promoter in the murine skin multistage carcinogenesis model through a systemic mechanism. The systemic co-promoting activity may be mediated by a factor(s) produced by the skin in response to TPA exposure. Furthermore, inter-mouse transfer of topically applied initiator from one cutaneous site to another occurs as a consequence of the huddling habits of mice.

9,10-Dimethyl-1,2-benzanthracene↗