Phorbol ester-mediated association of protein kinase C to the nuclear fraction in NIH 3T3 cells

Treatment of intact NIH 3T3 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a rapid redistribution (stabilization) of protein kinase C to the particulate fraction. Part of the enzyme activity stabilized to the membrane fraction in response to TPA can be recovered associated with nuclear-cytoskeletal components. An apparently pure nuclear fraction prepared from NIH 3T3 cells was found to contain 25-30% of the total membrane-associated protein kinase C activity when isolated in the presence of Ca2+. In untreated control cells, most of this activity found with the nuclear fraction can be extracted by chelators. Phorbol ester (TPA) treatment of NIH 3T3 cells induces the tight association of protein kinase C to the nucleus; this tightly bound activity is not dissociable by chelators and can be recovered only by solubilization with detergent. Nuclei purified from untreated human promyelocytic leukemic HL-60 cells contain higher amounts of chelator-stable, detergent-extractable protein kinase C activity compared with control NIH 3T3 cells. However, TPA treatment of HL-60 cells does not enhance the amount of protein kinase C found tightly associated with the nuclear fraction. Immunohistochemical studies with polyclonal antibodies directed against protein kinase C further indicate that TPA treatment of NIH 3T3 cells does significantly enhance the amount of protein kinase C found tightly associated with the nucleus and cytoskeleton, whereas exposure of HL-60 cells to TPA does not appreciably alter the amount of protein kinase C observed to be associated with the nuclear fraction. The TPA-mediated association (activation) of protein kinase C to the nuclear and cytoskeletal fractions with NIH 3T3 cells is further supported by the enhanced phosphorylation of specific endogenous proteins noted when purified nuclei and cytoskeletal preparations are incubated with [gamma-32P]ATP. These results suggest that tumor promoters may induce association (activation) of protein kinase C with different subcellular components to alter the availability of endogenous substrates. This may result in differential responses by different cell types during exposure to tumor promoters.     

Analysis of the drug synergism between thymidine and arabinosyl cytosine using mouse S49 T lymphoma mutants

Summary The synergism between arabinosyl cytosine (araC) and thymidine is characterized using two mutant S49 T lymphoma cell populations with altered deoxyribonucleotide metabolism. AraC-1-6 cells are deficient in dCMP deaminase activity resulting in a secondary elevation of intracellular dCTP pools, whereas dGuo-200-l cells have a mutation in the M₁ subunit of ribonucleotide diphosphate reductase, which also results in elevation of dCTP levels. These two mutant cell populations are partially resistant to araC cytotoxicity as compared to the wild type cells. The resistance to araC is contributed to the elevation of dCTP levels in these mutants which prevent araC incorporation into the DNA due to feedback inhibition of deoxycytidine kinase. Addition of extracellular thymidine to dCMP deaminase deficient cells causes a decrease in dCTP levels and in parallel increase their sensitivity to araC. In contrast, extracellular thymidine does not reduce dCTP levels in the mutant cells with altered ribonucleotide reductase and no synergism between araC and thymidine is observed in these cells. The expansion of dTTP pools in the presence of thymidine is similar in the two mutants. These results suggest that the depletion of dCTP pools by thymidine is responsible for the synergistic action of thymidine on araC cytotoxicity and that dTTP does not directly enhance the incorporation of araC into the DNA of T lymphoma cells.     

Chemical carcinogens induce cadmium resistance and activate metallothionein genes in cadmium sensitive S49 mouse cells

Treatment of cadmium-sensitive (Cd^s) metallothionein-negativeS49 mouse cells with two direct-acting chemical carcinogens(N-ethylnitrosourea or N-acetoxy-2-acetylamino-fluorene) orwith u.v. radiation induced a large increase in phenotypicallystable cadmium-resistant (Cd^r) variants. In contrast, treatmentwith any of three agents which alkylate proteins (N-ethylmaleimide,iodoacetate, or phenylmethyl-sulfonyl fluoride) was withouteffect. Similarly, treatment with 2-acetylaminofluorene (a pre-carcinogen)or with 12-O-tetra-decanoylphorbol-13-acetate (a tumor promoter)did not result in an increase in Cd^r variants. Initial studiesindicate that in many variants the metallothionein-I gene, themetallo-thionein-II gene, or both have been activated. Thusthe induction of cadmium resistance in Cd^s cells is a potentiallyuseful system to explore the activation of quiescent genes bycarcinogens.     

Phorbol ester-mediated inhibition of growth and regulation of proto-oncogene expression in the human T cell leukemia line JURKAT

The expression and function of several proto-oncogenes were examined in a human acute T cell leukemia line, JURKAT, during phorbol ester-induced terminal differentiation. Treating JURKAT cells with the phorbol ester tetradecanoyl phorbol acetate (TPA) inhibited their proliferation and induced expression of the gene for the interleukin 2 receptor alpha chain (IL2R-alpha), consistent with previous reports. In unstimulated proliferating JURKAT cells, high levels of C-MYC, N-RAS, and BCL2 mRNAs were found that diminished rapidly following TPA-induced cessation of growth. In contrast, accumulation of mRNAs for the C-FOS, C-JUN, and EGR-1 genes increased markedly in TPA-treated cells and preceded the induction of IL2R-alpha mRNA. Expression of C-MYB, C-RAF-1, C-LCK, C-FYN, and C-FGR proto-oncogenes was relatively unchanged. To explore directly the function of two of these protooncogenes in regulating the growth of JURKAT T cells, we stably transferred C-MYC and BCL2 expression plasmids into these cells. Despite sustained expression of C-MYC, BCL2, or the combination of these protooncogenes, TPA continued to inhibit JURKAT cell growth and to induce IL2R expression. Thus, although C-MYC and BCL2 proto-oncogene expression correlated with proliferation in TPA-treated JURKAT cells, continuous over-expression of even the combination of these oncogenes was insufficient for abrogating the effects of TPA in these leukemic T cells. Because human lymphoid malignancies frequently contain chromosomal translocations that deregulate the expression of C-MYC and BCL2, our findings could have relevance for attempts to induce terminal differentiation of leukemic cells by in vitro exposure of patients' bone marrow cells to phorbol esters.     

ATP uptake by mouse lymphoma cells

Mouse lymphoma cells were shown to be unresponsive to prostaglandin E1 in terms of cAMP production. The endogenous ATP concentration was shown to be low. Addition of ATP in the presence of IMBX into the incubation medium resulted in elevation of the intracellular pool of ATP and the cells responded to PGE1 by increasing cAMP production. The ATP effect is specific and cannot be substituted by GTP. ATP analogue (AMP-PCP) can, however, produce a similar effect to ATP. The intracellular ATP concentration can be lowered by incubation with iodoacetate and potassium cyanide. This caused a drastic decrease of the cAMP level. Ethionine on the other hand had no effect on the intracellular ATP level.     

Phorbol ester induced 1,2-diacylglycerol accumulation and protein phosphorylation in C3H/10T1/2 mouse embryo cells

Within 10 min of addition of the tumor promoter 12-O-tetra-decanoylphorbol-13-acetate(TPA) to C3H/10T1/2 mouse embryo fibroblasts, there is a two-foldincrease in the level of cellular 1,2-diacylglycerol levelscompared to controls. This increase in 1,2-diacylglycerol isdependent on the concentration of TPA added to the cell culturemedium. The ability of macrocyclic diterpenes to induce 1,2-diacylglycerolaccumulation correlated with their tumor promoting activityexcept for mezerein. The accumulation of 1,2-diacylglycerolin response to TPA was not blocked by a concentration of cycloheximidesufficient to inhibit protein synthesis by 95%. These data supportour previous suggestion that TPA activates a phospholipase C.During the same time period, TPA increased protein phosphorylationin both quiescent and growing cells. Proteins of mol. wt. 50000, 45 000, 35 000 and 27 000 are markedly phosphorylated inresponse to TPA in both growing and quiescent cultures. Therelationship of these phosphorylated proteins to a Ca²⁺ phospholipidactivated protein kinase remains to be determined.     

Inhibition of phorbol ester-mediated phenotypic changes in cultured cells by hypoxanthine

Hypoxanthine induces the differentiation of certain transformedcells in vitro, so analyses were undertaken to determine whetherthis purine metabolite might influence the expression of transformedphenotypes induced in normal cells by chemical agents. Chinesehamster embryo cells and human skin fibroblasts in culture weretreated with the promoting agent phorbol-12,13-didecanoate (PDD)with or without prior treatment with 3-methylcholanthrene (MCA),and various phenotypic effects were monitored. Hypoxanthinewas found to inhibit significantly the formation of type IIIfoci and the increase in saturation density observed for Chinesehamster cells treated with MCA plus the phorbol ester. Inosineand the hypoxanthine analogue aUopurinol could also mediatethe effect on saturation density, while xanthosine could not.An increase in the saturation density of human skin fibroblasts,which can be induced by the phorbol ester alone, was also inhibitedby hypoxanthine. There was no significant effect on the growthrate or the intracelhilar nucleotide pools with hypoxanthine-treatedcells. The results suggest that a normal purine metabolite,hypoxanthine, can modulate the expression of transformed phenotypesinduced in vitro by the known tumor promoter PDD. These observationscould help in elucidating the cellular basis for promotion ofcarcinogenesis.     

Phorbol ester activation of epidermal protein kinase C from tumor promotion sensitive and resistant mouse strains

Calcium-stimulated, phospholipid dependent protein kinase C activity was found in cytosolic extracts of the epidermis of SENCAR, C57BL/6 and Ha/ICR mice. The enzymes were partially purified by DEAE-cellulose chromatography. There were no significant differences between the 3 strains in basal protein kinase C activity. The enzymes from all 3 strains were stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the absence of calcium, but no correlation was seen between susceptibility to tumor promotion and any protein kinase C activity parameter measured.     

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

In mouse skin in vivo the irritant and hyperplasiogenic tumorpromoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stronglyincreased the epidermal content of the cysteinyl leukotrienesLTC₄, LTD₄ and LTE₄, but not of leukotriene LTB₄. This effectwas completely suppressed by the selective leukotriene biosynthesisinhibitor MK-886. Intragastric administration of MK-886 preventedphorbol ester-induced ear edema, but not epidermal hyperproliferationand tumor promotion. These data indicate that leukotrienes areinvolved in the pro-inflammatory effects of the phorbol ester,whereas its hyperproliferative and tumor-promoting activitiesdo not depend on 5-lipoxygenase-catalyzed leukotriene formation.This action differs from several non-selective inhibitors oflipoxygenases that were found to inhibit tumor promotion hiinitiated mouse skin.     

Phorbol diester and epidermal growth factor receptors in 12-O-tetradecanoylphorbol-13-acetate-resistant and -sensitive mouse epidermal cells

Several cell variants have been isolated from promotable mouse JB6 epidermal cells which are resistant either to mitogenic stimulation at quiescence or to promotion of anchorage independence by 12-O-tetradecanoylphorbol-13-acetate (TPA). Such resistant variants would be expected to lack one or more steps in the TPA response pathway leading to mitogenesis or promotion of tumor cell phenotype. This report is concerned with determining whether resistance is attributable to lack of receptors for phorbol diesters or epidermal growth factor (EGF, a potential mediator) or to absence of receptor down modulation following ligand binding. The results show that neither lack of phorbol diester receptors nor absence of down modulation can be demonstrated in the TPA-resistant variants. The phorbol ester binding affinity is also not altered in the resistant variants. The presence of EGF receptors cannot be an absolute requirement for TPA promotion sensitivity since three of the TPA-promotable cell lines lack available EGF receptors. Lack of EGF receptors may account for TPA mitogen resistance in at least three of four resistant variants. The TPA-induced EGF binding decrease occurs in both sensitive and resistant variants. Thus, phorbol diester binding and receptor down modulation remain as possible required events in mitogenic and promotion responses to TPA. EGF receptors are clearly not necessary for TPA promotion of anchorage independence in JB6 cells but may mediate mitogenic stimulation of these cells by TPA.     

Adherence to osteopontin via alphavbeta3 suppresses phorbol ester-mediated apoptosis in MCF-7 breast cancer cells that overexpress protein kinase C-alpha

Overexpression of protein kinase C-alpha in MCF-7 breast cancer cells (MCF-7-PKC-alpha cells) results in anchorage-independent growth and increased tumorigenicity of these cells in nude mice. MCF-7-PKC-alpha cells, unlike their parental MCF-7 cells, are sensitized to apoptosis by phorbol esters. When adhered to osteopontin, a bone matrix protein, MCF-7-PKC-alpha cells were resistant to phorbol ester mediated apoptosis. Fluorescence-activated cell sorting revealed that osteopontin receptors, alphavbeta3 and alphavbeta5, are expressed on MCF-7-PKC-alpha cells and that both are used to adhere to osteopontin. Addition of an RGD-containing peptide inhibited survival of MCF-7-PKC-alpha cells exposed to phorbol ester and adhered to osteopontin. This indicated that an integrin was involved in the cell death suppression signal. Whereas, anti-alphavbeta5 antibody did not reduce survival of MCF-7-PKC-alpha cells adhered to osteopontin, anti-alphavbeta3 antibody could efficiently block suppression of apoptosis. Phorbol ester also induced increased expression of alphavbeta3 on MCF-7-PKC-alpha cells by upregulating expression of a second species of beta3 mRNA. This study suggests that breast cancer cells that have metastasized to bone may have a survival advantage resulting from interaction of alphavbeta3 on these cells with the bone protein osteopontin.     

Histiocytic lymphoma in the mouse

This investigation studied the occurrence, distribution and morphology; and the transplantation, ultrastructural and in vitro characteristics of histiocytic lymphoma in the BALB/c and C57BL/6 strains of mice. The disease was more common in aged females of both strains. The liver was the major organ involved in the males and the liver and uterus were commonly involved in the females. Subcutaneous transplants readily metastasized to the lungs. Ultrastructurally the neoplastic cells resembled histiocytes and did not contain microfilaments or a basement membrane. The results suggest that this neoplasm is histiocytic in origin.     

Tumorigenicity of the cyc- variant of the S49 murine lymphoma deficient in the Gs-alpha subunit of adenylate cyclase

S49 cyc- lymphoma cells contain a mutation resulting in loss of a functional guanine nucleotide regulatory protein rendering their adenylate cyclase refractory to most stimuli. S49 wild-type and cyc- clones were used in the present study to investigate the possible association of altered cAMP metabolism with tumorigenicity and metastatic potential. The S49 clones were implanted i.v., i.p., and intracerebrally in both athymic nude mice and syngeneic, immunocompetent BALB/c mice. Both S49 clones gave rise to tumors when inoculated into athymic mice, and no differences were observed in the tumorigenicity or metastatic potential of S49 wild-type and cyc- cells. Implantation of S49 clones in syngeneic BALB/c mice gave rise to few tumors except when administered intracerebrally, where wild-type cells were more tumorigenic than cyc- cells. This raises the possibility of differences in immunogenicity between the S49 clones. Analysis of cell lines derived from tumors grown in athymic mice showed that they retained the phenotype of the S49 clones used for inoculations. The results indicate that, despite differences in adenylate cyclase responsiveness, S49 wild-type and cyc- cells are both highly tumorigenic and metastatic.