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F Marks

Publications and source records attributed to F Marks.

At least 163 records · Page 9Linked to original sources
PubMed1986

Molecular cloning of sequences activated during multi-stage carcinogenesis in mouse skin.

Recombinant DNA techniques were employed to isolate sequences that were activated during multi-stage carcinogenesis in the skin of NMRI mice. Differential screening of 5000 cDNA clones made from poly(A)+ RNA of squamous cell carcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) and the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) resulted in the identification of 35 cDNA clones displaying a different hybridization signal. Eight cDNA clones, three of which proved to be identical, were used as probes in transfer hybridization experiments. These cDNA clones, designated pmal-1 to pmal-6, showed a strong signal with carcinoma RNA but either a weak or no signal with RNA from normal epidermis. Clone pmal-2 contained repetitive sequences as shown by Southern analysis resulting in a smeared RNA hybridization signal in RNA blots whereas the other five clones corresponded to discrete size classes of mRNA. Studies on the expression pattern of mal-1,-2 and -3 related sequences revealed transcriptional activation already in the benign papilloma stage of multi-step carcinogenesis.

Animals↗
PubMed1986

Generation of the arachidonic acid metabolite 8-HETE by extracts of mouse skin treated with phorbol ester in vivo; identification by 1H-n.m.r. and GC-MS spectroscopy.

After a single in vivo application of 20 nmol of the 'complete' tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or of the 'incomplete' promoter 12-O-retinoylphorbol-13-acetate (RPA) to the back skin of mice, a major arachidonic acid metabolite (AAMX) was found in cell-free epidermal preparations after addition of arachidonic acid (AA). Studies with cyclooxygenase and lipoxygenase inhibitors indicated that this metabolite is a lipoxygenase product. The metabolite has been purified by sequential use of t.l.c. and h.p.l.c. and identified as 8-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid (8-HETE) by means of GC-MS and 1H-n.m.r. spectroscopy. To assist in the characterization of AAMX, a reference mixture of 8-HETE and 9-HETE was used for which a complete structural analysis could be performed using one- and two-dimensional 1H-n.m.r. at 500 MHz. The production of 8-HETE has been shown to start with a lag phase of 3 h after TPA treatment and to reach a maximum after 24-48 h. Nonpromoting phorbol esters are 10-fold, the Ca2+-ionophore A 23187 100-fold, less efficient in inducing in vivo the lipoxygenase responsible for 8-HETE production. 3-Day-old neonatal mice cannot be induced to generate 8-HETE in response to TPA, whereas 8-day-old mice show an extremely strong response. Neither primary basal keratinocytes, isolated from TPA-treated mouse epidermis nor TPA-treated epidermal cell lines generate 8-HETE, indicating that the metabolite may not originate from these epithelial cells.

Animals↗
PubMed1986

Three-stage tumorigenesis in mouse skin: DNA synthesis as a prerequisite for the conversion stage induced by TPA prior to initiation.

Recent evidence shows that stage I of tumor promotion in NMRI-mouse skin induced by a single dose of 12-O-tetradecanoylphorbol-13-acetate (TPA) can be effected not only after initiation by 7,12-dimethylbenz[a]anthracene (DMBA) but also several weeks prior to initiation. In view of this partial inversion of the initiation-promotion sequence we proposed to replace the term 'stage I of promotion' by the term 'conversion'. The convertogenic effectivity of a single dose of TPA applied after DMBA can be suppressed in a rather characteristic and non-toxic fashion by hydroxyurea (HU). In the present study we asked whether HU might also interfere with the converting effectivity of a single dose of TPA given prior to DMBA. NMRI mice received a single dose of TPA 3 weeks prior to initiation by DMBA which was followed by twice weekly application of skin irritant 12-O-retinoylphorbol-13-acetate (RPA) in order to effect stage II of promotion. A single dose of HU given i.p. at different times after TPA was found to interfere with tumor formation exhibiting an almost complete inhibition if administered 18 h after TPA. This inhibition did not prevent a subsequent promotion by repetitive TPA treatment. The data indicate that conversion can be inhibited by HU in the same characteristic fashion regardless of whether TPA was administered after or prior to initiation. The data also support the autonomous character of the conversion process for which epidermal DNA synthesis appears to be obligatory.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1985

Partial inversion of the initiation-promotion sequence of multistage tumorigenesis in the skin of NMRI mice.

Alterations in NMRI mouse skin induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in "stage I of tumor promotion" are slowly reversible, and this reversibility has a half-time of 10 to 12 weeks. The tumor response observed in the course of an initiation-promotion experiment in vivo is independent of whether stage I of promotion occurs before or after initiation. Since the time interval between treatment with the promoter, and subsequent initiation can be extended up to at least 6 weeks, an enhancement of initiation because of promoter-induced cellular DNA synthesis seems to be unlikely. This result may be inconsistent with the two-stage model of tumor promotion because it indicates that in skin the existence of initiated cells is not required for the induction of cellular alterations that are essential for the stage of skin tumorigenesis called stage I of promotion.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1985

Partial purification and characterization of the calcium-dependent and phospholipid-dependent protein kinase C from chick oviduct.

The calcium-dependent and phospholipid-dependent protein kinase C is partially purified (2000-fold) from chick oviduct cytosol by chromatography on DEAE-cellulose, Sephadex G150 and (after affinity binding of the enzyme to phosphatidylserine-diolein liposomes) on Bio-Gel A-0.5m. The enzyme is activated by phosphatidylserine and calcium ions. Diolein increases the affinity of the enzyme for both cofactors and can be replaced by the tumour-promoting phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate. Phosphatidylserine can be replaced by unsaturated long-chain fatty acids. Quercetin and phloretin inhibit the kinase reversibly competing with ATP. Several oviduct proteins are found to be phosphorylated by the partially purified enzyme. One of the substrates, a 78-kDa protein, appears to be ovotransferrin.

Animals↗
PubMed1985

Cyclosporin A inhibits biological effects of tumor promoting phorbol esters.

The antilymphocytic and antiphlogistic agent cyclosporin A (CsA) inhibits in vivo various effects induced by the tumor promoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). These include the edema of the mouse ear, the alkaline phosphatase (AP) activity and the ornithine decarboxylase (ODC) activity in mouse epidermis as well as the generation of a specific arachidonic acid (AA) metabolite in mouse epidermis. AA metabolism in an epidermal cell-free system of mouse epidermis was not suppressed by CsA. According to thin layer chromatography the TPA-induced and as yet unidentified AA metabolite exhibits a polarity between that of 5-HETE and 12-/15-HETE. Studies with inhibitors indicate it to be a lipoxygenase product.

Animals↗
PubMed1985

Demonstration and characterization of 1,25-dihydroxyvitamin D3 receptors in basal cells of epidermis of neonatal and adult mice.

Nuclear and cytosolic receptors for 1,25-dihydroxycholecalciferol [1,25(OH)2D3] were demonstrated in the epidermis of neonatal and adult mice. The macromolecular binding protein sedimented at 3.5 S (sucrose density gradient) and was distinct from the 6.0 S binding protein for 25-hydroxycholecalciferol [25(OH)D3]. Analysis at different ionic strengths suggested the presence of unoccupied nuclear receptors. Digestion with proteases or nucleases, respectively, and inactivation with alkylating agents demonstrated that the binding macromolecule is a protein with SH groups at the active site. Binding of 1,25(OH)2D3 was specific and reversible. In neonatal mice KD was 1.6 X 10(-10) M for both cytosolic and nuclear fractions, binding capacity was 54 fmol/mg protein in the cytosolic and 108 in the nuclear fractions, respectively. The phenotypic expression of the 1,25(OH)2D3 receptor (dissociation constant, binding capacity) was identical in neonatal and adult epidermis. Half maximal displacement of 1,25(OH)2D3 was achieved with an 80-fold and 200-fold molar excess of 25(OH)D3 and 1-alpha-hydroxycholecalciferol [1(OH)D3], respectively. Using Percoll density gradient centrifugation, 1,25(OH)2D3 receptors could be localized in the basal cell fraction. DNA cellulose chromatography with 1,25(OH)2D3 receptor elution from DNA at 0.25 M KCl (linear gradient) points to a possible role in gene transcription. In mouse primary epidermal cell cultures, 1,25(OH)2D3, but not 25(OH)D3, 24,25(OH)2D3, and 1(OH)D3 influenced [3H]thymidine incorporation (at physiological concentrations); the magnitude of change depending on the concentration of 1,25(OH)2D3 and the time of incubation. These data demonstrate that skin is a target organ for the active vitamin D secosterol.

Animals↗
PubMed1985

Development of phorbol ester responsiveness in neonatal mouse epidermis: correlation between hyperplastic response and sensitivity to first-stage tumor promotion.

As compared with the adult state, neonatal mouse skin (strain NMRI) has a hyperplastic appearance which gradually changes into the mature type between postnatal day 3 and 10. Concomitantly, the late fetal and neonatal keratin polypeptide pattern is replaced by the mature pattern. As long as the adult type of epidermal differentiation is not sufficiently developed (i.e., prior to postnatal day 5), topical application of the phorbol ester tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes neither morphological alterations nor a measurable induction of cellular proliferation and ornithine decarboxylase activity. TPA application at day 7 evokes, however, (i) a hyperplastic reaction followed by a massive 'psoriasis-like' hyperkeratosis, (ii) an increase of ornithine decarboxylase activity and (iii) a restoration of the neonatal keratin polypeptide pattern. Multistage tumorigenesis experiments carried out with prenatally initiated mice show that during the early period of development (prior to postnatal day 5) mouse skin is also resistant to the effects of TPA as a stage I tumor promoter. Since both the hyperplastic response and the sensitivity to tumor promotion develops in a strictly parallel manner, reactions involved in the induction of epidermal hyperplasia are assumed to provide an important condition of stage I skin tumor promotion.

Animals↗
PubMed1985

Comparative effects of a complete tumor promoter, TPA, and a second-stage tumor promoter, RPA, on intercellular communication, cell differentiation and cell transformation.

The biological activities in vitro of the incomplete (second-stage) tumor promoter, 12-O-retinoyl phorbol-13-acetate (RPA), and the complete tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) were compared. The doses of TPA and RPA necessary to inhibit the specific binding of [3H]-phorbol-12,13-dibutyrate ([3H]PDBu) to BALB/c 3T3 cells (50% inhibition doses; ID50; 8-13 ng/ml) were very similar; however, RPA was less potent than TPA in inhibiting [3H]-PDBu binding to Friend erythroleukemia cells (FELC). Intercellular communication between BALB/c 3T3 cells, measured by transfer of microinjected fluorescent dye (Lucifer Yellow), was inhibited by RPA as well as by TPA; TPA was about five times more potent than RPA. RPA also inhibited FELC differentiation induced by hexamethylene bisacetamide (HMBA) but not the differentiation of a TPA-resistant clone. The dose-responses of these two compounds in inhibiting differentiation of both TPA-sensitive and resistant FELC were very similar. When TPA and RPA were compared in their promoting activity of in vitro cell transformation of BALB/c 3T3 cells initiated with 3-methylcholanthrene (MCA, 0.1 microgram/ml), both TPA and RPA significantly increased the yield of morphologically transformed foci, and RPA was approximately 10 times more potent than TPA. These results suggest that RPA and TPA share many common in vitro biological effects and that these in vitro studies do not allow us to delineate clearly the effect of a second-stage tumor promoter from that of complete tumor promoters such as TPA.

Animals↗
PubMed1985

Mechanistic aspects of the delay in the G2 phase of the cell cycle caused by tumor promoter 12-O-tetradecanoylphorbol-13-acetate in HeLa cells.

In order to gain an insight into the nature of the radiomimetic activity by which the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alters cell cycle parameters in HeLa cells, possibilities of modifying the TPA-induced G2 block and recovery from it were studied. TPA-induced G2 blockage was analysed by counting mitotic figures. It was not influenced by hydroxyurea (10(-3) M) thus indicating that it is independent of DNA synthesis. TPA-induced decrease of mitotic activity occurred faster than that caused by cycloheximide (10(-5) M) indicating that the TPA-sensitive transition point in G2 is closer to mitosis than that for cycloheximide. Superoxide dismutase, catalase, alpha-tocopherol, the radioprotector S-(2-aminoethyl)isothiuroniumbromide.HBr (AET), caffeine and indomethacin and eicosatetraynoic acid (ETYA), both inhibitors of oxygenases in the arachidonic acid cascade, were not capable of reducing the TPA-induced G2 response. Under certain conditions small concentrations of AET (10(-8) M) and ETYA (10(-8) M) appeared to improve recovery slightly. Mannitol and sorbitol, however, both hydroxyl radical scavengers at 0.1 M concentration reduced TPA-effectiveness to a large degree (0.1 M D- and L-mannose were ineffective). Dimethylsulfoxide (0.1 M), another hydroxyl radical scavenger, was ineffective.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1984

Calcium and phospholipid-dependent protein kinase activity in mouse epidermis cytosol. Stimulation by complete and incomplete tumor promoters and inhibition by various compounds.

Calcium- and phospholipid-dependent protein kinase (Ca, PL-PK) activity is detectable in mouse epidermis cytosol. It can be stimulated in vitro by complete and incomplete tumor promoters (12-0-tetradecanoylphorbol-13-acetate (TPA) and 12-0-retinoylphorbol-13-acetate (RPA], respectively. Effective inhibition of the enzyme activity is achieved with quercetin and phloretin, whereas the lipoxygenase and cyclooxygenase inhibitors nordihydroguaiaretic acid (NDGA) and esculetin show just weak or no inhibition. Quercetin inhibits the lipoxygenase and cyclooxygenase equally well as the Ca, PL-PK, whereas the strong Ca, PL-PK inhibitor phloretin is absolutely ineffective in inhibiting the lipoxygenase/cyclooxygenase. The application of these inhibitors in differentiating tumor promoter induced effects in vivo is proposed.

Animals↗
PubMed1984

A system for the study of epidermal G1-chalone in cell culture. The rat tongue epithelial cell line RTE2.

A pig-skin preparation enriched in epidermal G1-chalone when administered to cells of the rat tongue epithelial line RTE2 at concentrations of 3-300 micrograms/ml (dry mass) caused a 60% reduction in cell number. Three other cell lines showed essentially no growth inhibition during chalone treatment. The kinetics of chalone inhibition were similar to those observed in mouse epidermis in vivo. Five hours after the addition of chalone preparation in fresh medium a decrease in the rate of DNA synthesis was observed. Maximum inhibition at 12 h was followed by a subsequent increase in DNA synthesis, reaching control values again after 30 h. The inhibitory effect was dose-dependent up to 3 micrograms/ml. At higher concentrations the degree of inhibition remained constant at about 50% of the control up to 300 micrograms/ml. Removal of added chalone by changing the medium at the time of maximum inhibition gave rise to a complete recovery within 9 h. These results indicate a cell-line specific, non-toxic and reversible inhibitory effect of the chalone preparation which resembles that observed in the living animal. The RTE2 cell line may thus be considered to provide a highly sensitive experimental system suitable for more detailed studies on the mechanism of action of epidermal G1-chalone.

Animals↗
PubMed1984

Stimulation of alkaline phosphatase activity in mouse epidermis by tumor promoters.

Complete as well as incomplete (second stage) tumor promoters increase alkaline phosphatase (AP) activity in mouse epidermis in vivo but not in vitro. This indicates that the high level of AP in epidermis after application of a tumor promoter arises from an extraepidermal site, possibly from leaky endothelial cells of promoter-damaged blood vessels. Quercetin, which is known to stabilize the microvasculature as well as to inhibit tumor promotion, very effectively suppresses tumor promoter-stimulated AP activity. Quercetin is also a potent inhibitor of calcium, phospholipid-dependent protein kinase (Ca, PL-PK). Since inhibitors of other enzymes also suppressible by quercetin are ineffective, the damage of microvasculature and the increase in AP activity in epidermis might be Ca, PL-PK mediated effects of tumor promoters.

Alkaline Phosphatase↗
PubMed1984

The mouse ear edema: a quantitatively evaluable assay for tumor promoting compounds and for inhibitors of tumor promotion.

The induction of edema in the mouse ear has been established as a reliable in vivo assay for tumor promoting compounds and for inhibitors of tumor promotion. Apparently all skin tumor promoting compounds induce an edema, and all inhibitors of a tumor promoter-induced edema are most likely inhibitors of skin tumor promotion. No exception to this rule has been found as yet. Besides the application of this assay in the screening of compounds, the assay allows comparison of various compounds (e.g. complete and incomplete tumor promoters) with respect to their kinetics and dosage response in the induction of edema.

Animals↗
PubMed1984

Suppression of the first stage of phorbol 12-tetradecanoate 13-acetate-effected tumor promotion in mouse skin by nontoxic inhibition of DNA synthesis.

In order to evaluate the significance of epidermal cell proliferation for the first stage of skin tumor promotion, the effect of hydroxyurea (HU), an inhibitor of DNA synthesis, on tumor formation was studied. Mice initiated with 7,12-dimethylbenz[a]anthracene received a single dose of phorbol 12-myristate 13-acetate (PMA) in stage I of promotion, followed by twice weekly application of the irritant skin mitogen phorbol 12-retinoate 13-acetate in stage II. A single dose of HU given intraperitoneally at different times before or after treatment with PMA was found to interfere with tumor formation, exhibiting an almost complete inhibition if administered 18 hr after PMA--i.e., at the time of maximal DNA synthesis. The inhibition of tumor formation by HU in the two-stage promotion experiment did not prevent a subsequent promotion of cells by repetitive PMA treatment. This indicates that the inhibitory effect of HU was due neither to cytotoxicity (killing of initiated cells) nor to an interference with initiation. The data indicate that epidermal DNA synthesis is obligatory for PMA-induced first-stage promotion. The causal relationship between both events remains to be established.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1984

On the role of superoxide anion radicals in skin tumour promotion.

The effect of phorbol ester tumour promoters on the release of superoxide anion radicals .O2- by human peripheral leukocytes and the role of such radicals in tumour promotion of mouse skin was studied. No significant difference was found between complete [12-O-tetradecanoylphorbol-13-acetate (TPA)] as compared with incomplete [12-O-retinoylphorbol-13-acetate (RPA), 12-O-(2Z,4E,6,8)tetradecatetraenoylphorbol-13-acetate (Ti8), mezerein] tumour promoters upon induction of .O2- when measured by the reduction of ferricytochrome c. The semisynthetic phorbol esters 12-O-ethacrynylphorbol-13-acetate (EPA) and 4-O-methyl-TPA were less active, and phorbol diacetate, phorbol and ionophore A 23187 were found to be inactive in stimulating superoxide anion radicals. TPA-induced .O2- release from leukocytes was strongly inhibited by Cu(II)-(diisopropylsalicylate)2 (CuDIPS), and, to a lesser extent, by ethacrynic acid, nordihydroguaiaretic acid and quercetin. Retinoic acid exhibited only a moderate inhibitory effect. No .O2- release was observed in epidermal cell cultures upon TPA treatment. When analysed by the alkaline elution technique, TPA-induced .O2- release from leukocytes did not lead to measurable DNA damage in co-cultivated keratinocytes even in the presence of DNA repair inhibitors. In multi-stage-tumourigenesis experiments including two-stage promotion, retinoic acid, ethacrynic acid and CuDIPS were unable to inhibit tumour promotion in mouse skin when applied in combination with TPA in first stage promotion. gamma-Irradiation at a dose level shown to cause DNA damage in vitro could not replace TPA as a stage I-promoting agent. It is concluded that superoxide anion radicals--if related to promotion at all--may play a role in stage II rather than in stage I of mouse skin tumour promotion.

Animals↗
PubMed1984

The effects of high fat feeding during one cycle of reproduction consisting of pregnancy, lactation and recovery on body composition and fat pad cellularity in the rat.

The changes in adipose depot weight, cell size, cell number and body composition during pregnancy, lactation and recovery were studied in Osborne-Mendel rats fed standard or high fat diets. Rats were killed on day 21 of pregnancy, after 21 days of lactation, and after 21 or 22 days of a postlactational recovery period. Nonpregnant control groups were killed at the beginning and at the conclusion of the experimental period. The high fat-fed, mated group was always fatter than similarly treated animals fed standard diets throughout pregnancy and lactation. However, by the end of the recovery period, carcass composition of the animals fed high fat or standard diets and the nonpregnant groups were not statistically different. The weight of the parametrial, retroperitoneal and subscapular depots was higher in the high fat-fed animals at the end of the recovery period, and in the latter two pads, this increase was statistically significant. Thus, despite the extensive lipid mobilization that occurs during lactation, the high fat-fed animals appear to be predisposed to postpartum obesity.

Adipose Tissue↗