Inhibition of phorbol ester-caused synthesis of mouse epidermal ornithine decarboxylase by retinoic acid

The mechanisms by which topically applied retinoic acid to mouse skin inhibits tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced epidermal ornithine decarboxylase activity were analyzed. Retinoic acid inhibition of the induction of epidermal ornithine decarboxylic activity was not the result of nonspecific cytotoxicity, production of a soluble inhibitor of ornithine decarboxylase, or direct effect on its activity. In addition, inhibition of TPA-caused increased ornithine decarboxylase activity does not appear to be due to enhanced degradation and/or post-translational modification of ornithine decarboxylase by transglutaminase-mediated putrescine incorporation. We found that retinoic acid inhibits the synthesis of ornithine decarboxylase caused by TPA. Application of 10 nmol TPA to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase activity which was paralled by increased [3H]difluoromethylornithine binding and an increased incorporation of [35S]methionine into the enzyme. Application of 17 nmol retinoic acid 1 h prior to application of 10 nmol TPA to skin resulted in inhibition of the induction of activity which accompanied inhibition of [3H]difluoromethylornithine binding and [35S]methionine incorporation into ornithine decarboxylase protein as determined by the tube-gel electrophoresis of the enzyme immunoprecipitated with monoclonal antibodies to it. Inhibition of ornithine decarboxylase synthesis was not the result of the inhibitory effect of retinoic acid on general protein synthesis. The results indicate that retinoic acid possibly inhibits TPA-caused synthesis of ornithine decarboxylase protein selectively.     

Antitumor effects of vitamin A and inhibitors of ornithine decarboxylase in cultured neuroblastoma and glioma cells

The antiproliferative effects of vitamin A analogs and difluoromethyl ornithine, an irreversible inhibitor of ornithine decarboxylase, were evaluated in cultured neuroblastoma and glioma cells. Retinol and retinoic acid arrested the proliferation of neuroblastoma at concentrations of 50 μM; retinal was effective at 5 μM. Glioma cells were 5–10 fold less sensitive to all three analogs. A correlation existed between the inhibition of growth and the inhibition of ornithine decarboxylase in both cell lines treated with the retinoids. Difluoromethyl ornithine did not toally arrest tumor growth at concentrations of 5 mM; however, the antitumor effects were enhanced with the addition of retinol. An earlier and more complete cytostatic response was observed in glioma cells which had been treated with the combination of 100 μM retinol and 5 mM difluoromethyl ornithine than in cells which had been treated with either agent alone. These results show that tumors of neural origin are retinoid sensitive and suggest that a combination of vitamin A analogs and ornithine decarboxylase inhibitors may produce an increased chemotherapeutic response.     

Effects of retinoids and phorbol esters on the sensitivity of different cell lines to the polypeptide toxins modeccin, abrin, ricin and diphtheria toxin.

The effects of retinoic acid and 12-O-tetradecanoylphorbol 13-acetate on the sensitivities of a number of cell lines to the toxins modeccin, abrin, ricin and diphtheria toxin were studied. Retinoic acid and some other retinoids were found to protect a number of the cell lines against the toxins. HeLa cells that were protected bound much more retinoic acid than L-cells that were not protected. The tumour promoter 12-O-tetradecanoylphorbol 13-acetate was found to increase the sensitivity of cells to abrin, ricin and modeccin in the absence as well as in the presence of retinoic acid. Neither retinoic acid nor 12-O-tetradecanoylphorbol 13-acetate affected the extent of binding and pinocytotic uptake of toxins by the cells. Apparently retinoic acid and 12-O-tetradecanoylphorbol 13-acetate interfere with the entry of the toxins through the cell membrane.     

Short-term treatment with gamma interferon induces stable reversion of ras-transformed mouse fibroblasts.

Persistent revertants have been generated from NIH 3T3 cells transformed by an activated human Ha-ras gene after short-term gamma interferon treatment in the presence of the cardiac aminoglycoside ouabain. Normal fibroblastlike morphology and anchorage dependence are restored in revertants. Tumorigenicity in nude mice is abolished. The revertants continue to express high steady-state levels of the ras oncogene. Partial retransformation of reverted cells is induced after 5-azacytidine treatment or after infection with retrovirus vectors carrying the v-abl, v-fes, v-myc, or v-src oncogene. The revertants resist the transforming activities of the v-Ha-ras and v-mos oncogenes.     

Neoplastic transformation of normal and carcinogen-induced preneoplastic Syrian hamster embryo cells by the v-src oncogene.

The ability of cloned Rous sarcoma virus (RSV) DNA encoding the v-src oncogene to neoplastically transform normal, diploid Syrian hamster embryo (SHE) cells was examined. Transfection of RSV DNA into early passage SHE cells resulted in a low but significant number of tumors when treated cells were injected into nude mice. Tumors formed with a low frequency (two tumors out of ten sites injected) and only after a long latency period (14 weeks). In contrast to the normal SHE cells, several different carcinogen-induced preneoplastic immortal SHE cell lines were highly susceptible to transformation by the v-src oncogene to the neoplastic phenotype. Tumors formed with high efficiency and a short latency period (less than 3 weeks). Further studies were performed to determine the basis for the inefficient transformation of the normal SHE cells. NeoR clones isolated after cotransfection of SHE cells with pSV2-neo and RSV DNAs were neither morphologically altered nor immortal and did not contain detectable levels of the v-src gene product. These results suggest that neoplastic transformation by v-src DNA in the normal cells is initially suppressed. However, cells from a v-src-induced tumor expressed v-src RNA, and antibody to v-src protein precipitated from the tumor cells a 60,000-molecular-weight protein which displayed protein kinase activity. Karyotypic analyses confirmed that the tumor was derived from Syrian hamster cells and suggested that it was clonal in nature. These results indicate that the v-src oncogene was primarily responsible for neoplastic transformation of SHE cells. In contrast to the results with the v-src oncogene, our previous studies showed that v-Ha-ras oncogene alone is unable to induce neoplastic transformation of SHE cells. Furthermore, the v-myc oncogene was able to compliment v-Ha-ras to neoplastically transform SHE cells, while cotransfection with v-src plus v-myc did not increase the incidence of tumors.     

Inhibition of ornithine decarboxylase by retinoic acid and difluoromethylornithine in relation to their effects on differentiation and proliferation

Murine embryonal carcinoma F9 cells can be induced to differentiate by 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC). The differentiated phenotype is similar to that of retinoic acid (RA)-treated F9 cells. In contrast to F9 cells the differentiated cells secrete plasminogen activator and express keratin intermediate filaments. Both DFMO and RA reduce ornithine decarboxylase activity, polyamine levels and inhibit cell proliferation of F9 cells. These compounds also reduce ODC, polyamine levels and proliferation of mouse BALB/c 3T6 fibroblasts. RA inhibits the induction of ODC by insulin, serum and to a lesser extent that of epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of DFMO and RA can be distinguished by their response to putrescine. The induction of differentiation and the inhibition of cell proliferation by DFMO can be totally abolished upon the addition of putrescine, whereas the actions of RA are not affected at all. These results suggest that the inhibition of ODC and reduction of polyamines are not causal in the induction of differentiation and the inhibition of proliferation by RA.     

v-erbA cooperates with sarcoma oncogenes in leukemic cell transformation

The v-erbB, v-src, v-fps, v-sea, and v-Ha-ras oncogenes induce avian erythroid progenitor cells to self-renew in an erythropoietin-independent manner. These transformed erythroblasts retain both their capacity to differentiate into erythrocytes and their requirement for complex growth media. However, previous studies showed that erythroblasts transformed by v-erbB plus v-erbA (which by itself is not oncogenic) are blocked in differentiation and grow in standard media. Here we show that the introduction of v-erbA into erythroblasts transformed with v-src, v-fps, v-sea, or v-Ha-ras likewise induces a fully transformed phenotype. It also reduces the capacity of ts sea- and ts erbB-transformed erythroblasts to differentiate terminally in an erythropoietin-dependent manner after a temperature shift. Cooperativity involving v-erbA also occurs in vivo since chicks infected with a retroviral construct encoding v-erbA and v-src develop both acute erythroblastosis and sarcomas.     

Synergism of v-myc and v-Ha-ras in the in vitro neoplastic progression of murine lymphoid cells.

Murine bone marrow was either singly or doubly infected with retroviral vectors expressing v-myc (OK10) or v-Ha-ras. The infected bone marrow was cultured in a system that supports the long-term growth of B-lineage lymphoid cells. While the v-myc vector by itself had no apparent effect on lymphoid culture establishment and growth, infection with the v-Ha-ras vector or coinfection with both v-myc and v-Ha-ras vectors led to the appearance of growth-stimulated cell populations. Clonal pre-B-cell lines stably expressing v-Ha-ras alone or both v-myc and v-Ha-ras grew out of these cultures. In comparison with cell lines expressing v-Ha-ras alone, cell lines expressing both v-myc and v-Ha-ras grew to higher densities, had reduced dependence on a feeder layer for growth, and had a marked increase in ability to grow in soft-agar medium. The cell lines expressing both oncogenes were highly tumorigenic in syngeneic animals. These experiments show that the v-myc oncogene in synergy with v-Ha-ras can play a direct role in the in vitro transformation of murine B lymphoid cells.     

Different effects of TPA on two skin-derived cell lines: murine (HEL-30) and human (NCTC) epidermal cells

12-O-tetradecanoylphorbol-13-acetate (TPA) caused a rapid activation of protein kinase C in a murine (HEL-30) and in a human (NCTC) epidermal cell line. In HEL-30 cells, protein kinase C activation is followed by ornithine decarboxylase stimulation and cell proliferation, events inhibited by H-7, a specific inhibitor of protein kinase C. TPA in NCTC cells inhibited the basal ornithine decarboxylase activity and cell growth, whereas H-7 did not modify TPA effect. The response of NCTC cells was not due to direct toxicity of TPA. These data confirm that in murine epidermal cells, the proliferation induced by TPA is mediated by protein kinase C, whereas in a human skin-derived cell line these events are not or inversely associated.     

Novel revertants of H-ras oncogene-transformed R6-PKC3 cells.

Rat 6 fibroblasts that overproduce protein kinase C beta 1 (R6-PKC3 cells) are hypersensitive to complete transformation by the T24 H-ras oncogene; yet T24 H-ras-transformed R6-PKC3 cells are killed when exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) (W.-L. W. Hsiao, G. M. Housey, M. D. Johnson, and I. B. Weinstein, Mol. Cell. Biol. 9:2641-2647, 1989). Treatment of an R6-PKC3 subclone that harbors a T24 H-ras gene under the control of an inducible mouse metallothionein I promoter with ZnSO4 and TPA is extremely cytocidal. This procedure was used to isolate rare revertants that are resistant to this toxicity. Two revertant lines, R-1a and ER-1-2, continue to express very high levels of protein kinase C enzyme activity but, unlike the parental cells, do not grow in soft agar. Furthermore, these revertants are resistant to the induction of anchorage-independent growth by the v-src, v-H-ras, v-raf, and, in the case of the R-1a line, v-fos oncogenes. Both revertant lines, however, retain the ability to undergo morphological alterations when either treated with TPA or infected with a v-H-ras virus, thus dissociating anchorage independence from morphological transformation. The revertant phenotype of both R-1a and ER-1-2 cells is dominant over the transformed phenotype in somatic cell hybridizations. Interestingly, the revertant lines no longer induce the metallothionein I-T24 H-ras construct or the endogenous metallothionein I and II genes in response to three distinct agents: ZnSO4, TPA, and dexamethasone. The reduction in activity of metallothionein promoters seen in these revertants may reflect defects in signal transduction pathways that control the expression of genes mediating specific effects of protein kinase C and certain oncogenes in cell transformation.     

Induction of ornithine decarboxylase activity by 1-oleoyl-2-acetyl-glycerol in isolated mouse epidermal cells

1-Oleoyl-2-acetyl-glycerol induced a rise in ornithine decarboxylase activity in isolated epidermal cells in a concentration-dependent manner. The time course of the induction of ornithine decarboxylase by 1-oleoyl-2-acetyl-glycerol was similar to that by 12-O-tetradecanoylphorbol-13-acetate. A23187 did not enhance the enzyme induction caused by 1-oleoyl-2-acetyl-glycerol. Palmitoyl-DL-carnitine prevented the induction of the enzyme either by 1-oleoyl-2-acetyl-glycerol or 12-O-tetradecanoyl-phorbol-13-acetate. These results suggest that the activation of protein kinase C is an initial and essential event in the process of ornithine decarboxylase induction caused by 12-O-tetradecanoyl-phorbol-13-acetate.     

Phorbol ester tumor promoters induce epidermal transglutaminase activity

Epidermal basal cells in culture have low levels of epidermal transglutaminase, the enzyme responsible for the formation of the cross-linked envelope in differentiated cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and other active (but not inactive) phorbol ester skin tumor promoters induce transglutaminase activity. Sloughing of differentiated cells accompanies the rise in transglutaminase activity. Phorbol esters do not affect transglutaminase activity when added directly to cell lysates. Corticosteroids have little influence on transglutaminase induction by phorbol esters. Retinoic acid induces transglutaminase activity, but activity does not further increase when basal cells are treated with both retinoic acid and 12-O-tetradecanoylphorbol-13-acetate.     

Comparison of inhibition by a tumor promoter (12-O-tetradecanoylphorbol-13-acetate) of expression of the differentiated phenotype of chondrocytes in rabbit costal chondrocytes in culture with inhibition by retinoic acid

Both retinoids and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit expression of the differentiated phenotype by rabbit costal chondrocytes in culture, as judged by morphological changes and decreased sulfation of glycosaminoglycans (GAG). However, the inhibition of the differentiated phenotype of chondrocytes in TPA-treated cells is restored by parathyroid hormone (PTH), while the inhibition by retinoids is not [Takigawa et al. (1982) Mol. Cell. Biochem. 42, 145-153; Takigawa et al. (1983) Cell Differ. 13, 283-291]. In the present study, we examined the difference between TPA-treated chondrocytes and retinoic acid-treated chondrocytes to determine the mechanism of the restoration of the differentiated phenotype in de-differentiated cells treated with TPA. PTH increased the activity of ornithine decarboxylase [ODC; EC 4.1.1.17], a rate limiting enzyme of polyamine biosynthesis, and proteoglycan synthesis in chondrocytes pretreated with TPA as well as in normal chondrocytes. The maximal stimulations of ODC activity and GAG synthesis were observed 4 h and 24-36 h, respectively, after addition of PTH. The dose-response curve for ODC induction by PTH was parallel to that of PTH-stimulated proteoglycan synthesis both in TPA-treated chondrocytes and in normal chondrocytes. PTH also increased the intracellular cyclic AMP level after 2 min in TPA-treated cells as in normal cells. Addition of dibutyryl cyclic AMP (DBcAMP) induced ODC and restored proteoglycan synthesis in TPA-treated cells. The dose-response curve for induction of ODC by DBcAMP was parallel to that of DBcAMP-stimulated proteoglycan synthesis in both TPA-treated chondrocytes and normal chondrocytes. On the other hand, the increases by PTH in the intracellular cyclic AMP level, ODC activity, and proteoglycan synthesis were inhibited in chondrocytes pretreated with a combination of TPA and retinoic acid as well as in those pretreated with retinoic acid alone. TPA stimulated the syntheses of DNA and RNA in chondrocytes but did not increase the cyclic AMP level or ODC activity. PTH and DBcAMP inhibited the syntheses of DNA and RNA both in TPA-treated cells and in normal cells. These results suggest that ODC induction mediated by elevation of cyclic AMP plays an important role in re-differentiation of de-differentiated cells pretreated with these agents.     

Changes in inducibility of ornithine decarboxylase activity in differentiating human neuroblastoma cells

Human SH-SY5Y neuroblastoma cells could be induced to differentiate morphologically and biochemically in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA), or a combination of these two substances. The phenotypical changes induced by these substances differed, but one effect of both was an inhibition of the cell growth. Addition of TPA or RA to non-treated cells had no effect on the activation of ornithine decarboxylase (ODC, EC 4.1.1.17.), while a change to fresh medium stimulated the ODC to maximum activity after 4-6 h. The activity was not altered by the presence of RA in the fresh medium, but TPA partially inhibited the medium-stimulated ODC activity. Cells treated for 4 or 8 days with TPA or a combination of TPA and RA had a low ODC activity which could not be induced by fresh medium. However, RA-treated (and thus growth-inhibited) cells still responded to a change of medium by exhibiting an ODC activity of the same magnitude and duration as in medium-stimulated control cells. The results seem to suggest that the growth inhibition induced by TPA and RA, respectively, is mediated by different mechanisms.     

A human epidermal differentiation-specific keratin gene is regulated by calcium but not negative modulators of differentiation in transgenic mouse keratinocytes.

Keratins K1 and K10 represent the major differentiation products of the maturing epidermal keratinocytes. Primary epidermal cell cultures from newborn K1 transgenic mice containing a 12-kilobase human K1 genomic fragment were established in order to examine the expression of both human and mouse K1 in the presence of known modulators of epidermal differentiation. Elevated levels of Ca2+ in the culture medium induced both mouse K1 and human K1. Supplementing the medium with retinoic acid or 12-O-tetradecanoylphorbol-13-acetate or introducing a Harvey viral ras oncogene (v-rasHa) into the cells completely suppressed mouse K1 but not human K1. Our results suggest that: (a) the human 12-kilobase insert contains all the necessary cis-acting elements to respond to the Ca2+ signal, and (b) other cis-acting elements, not present within this insert, may function independently to regulate the response of K1 to retinoids, 12-O-tetradecanoylphorbol-13-acetate, and v-rasHa transformation. This transgenic model provides an approach to identify elements required for the regulation of an epidermal differentiation-specific gene.     

Enhanced papilloma formation in response to skin tumor promotion in transgenic mice overexpressing the human ornithine decarboxylase gene.

We have studied the induction of papilloma formation in response to skin tumor promotion in transgenic mice overexpressing the human ornithine decarboxylase gene and in their nontransgenic littermates. The transgenic animals displayed a basal epidermal ornithine decarboxylase activity that was nearly 20 times higher than in their nontransgenic littermates. A single topical application of 12-O-tetradecanoylphorbol-13-acetate induced a much more profound and longer-lasting increase in transgene-derived ornithine decarboxylase activity in comparison with the endogenous enzyme activity. Initiation of skin tumorigenesis with a single topical application of dimethylbenz[a]antracene followed by twice-weekly application of 12-O-tetradecanoylphorbol-13-acetate resulted in the appearance of first papillomas both in nontransgenic and transgenic animals by week 7. However, after 11 weeks of 12-O-tetradecanoylphorbol-13-acetate application, the number of papillomas per animal was almost 100% higher in the transgenic animals than in their nontransgenic littermates. These results indicate that an overexpression of epidermal ornithine decarboxylase confers a growth advantage on skin tumors in vivo.     

The c-Ha-ras oncogene and a tumor promoter activate the polyoma virus enhancer

A c-Ha-ras oncogene, to a lesser extent the c-Ha-ras proto-oncogene, and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate activate the inactive polyoma virus (Py) enhancer in a myeloma cell line and the partially active Py enhancer in NIH 3T3 fibroblasts, but have no effect on the active Py enhancer in LMTK- fibroblasts. In addition, c-Ha-ras can stimulate the inactive Py enhancer in embryonal carcinoma F9 cells. c-Ha-ras activation in embryonal carcinoma cells does not appear to involve reversal of "E1A-like" inhibition of the enhancer. We suggest that modulation of cellular enhancer activity could play a key role in tumorigenesis by oncogenes.