Protein kinase C mediated anti-proliferative glucocorticoid-sphinganine synergism in cultured Pollard III prostate tumor cells.

PURPOSE: Experimental effort focused on the growth inhibition of an androgen-resistant prostatic carcinoma, using pharmacological inhibition of protein kinase C (PKC) as the therapeutic target. MATERIALS AND METHODS: Studies were performed in cell culture using the Pollard (PA) III androgen-insensitive spontaneous rat prostate tumor cells, and the human prostate tumor lines, PC-3 and LnCaP. Pharmacological agents included steroid hormones and PKC modulators; measured parameters of tumor growth/function included cell number, PKC activity and sphingolipid metabolism. RESULTS: Triamcinolone (TA) and sphinganine synergized to inhibit the proliferation rate of PA III prostate tumor cells by converging through separate mechanisms to inhibit protein kinase C. At five days of cell culture, 0.1 microM TA reduced both the soluble and particulate forms of PKC in association with a 35-40% reduction in cellular proliferation. Exogenous sphinganine, a competitive inhibitor at the regulatory domain of PKC had no anti-proliferative effect at 1 microM, but in combination with TA synergized to reduce proliferation 80-90%, three days in advance of any detectable inhibitory effect of TA alone on cell number. TA produced no discernable stimulation of endogenous free sphingosine production as evidenced by the lack of an effect on the activity of neutral membrane sphingomyelinase or in the turnover of total cellular sphingomyelin. Phorbol esters, but not cell permeable diglycerides, prevented the TA + sphinganine effect suggesting that a stable long term PKC activation was required for reversal. Steroid specificity studies of the synergistic response revealed that while other glucocorticoids mimicked TA, aldosterone was less active and representatives of the three major classes of sex steroids were inert. Tests of sphinganine specificity demonstrated that calphostin C, a chemically unrelated inhibitor of the regulatory site of PKC, also produced a supra-additive interaction with TA. Ceramides (C2 & C6), which were closely related chemically to sphinganine but lacked affinity for the regulatory subunit of PKC, were inactive in this system. Analyses of the cellular specificity of the TA-sphinganine synergism using the human prostate carcinoma cell lines PC-3 and LnCap revealed a true synergistic growth inhibition in the glucocorticoid receptor positive PC-3 line and no significant interaction in the glucocorticoid receptor negative LnCap cells. CONCLUSIONS: TA-induced reduction of PKC concentration coupled with sphinganine antagonism of PKC activation contributed to in a synergistic growth inhibition of an androgen resistant prostatic carcinoma.

Humoral factor(s) in hypertensive rats trophic for cultured aortic smooth muscle cells.

Humoral factors in one-kidney, one-clip (1K,1C) hypertension in rats increase growth of smooth muscle cells cultured from rat arteries. To further characterize the plasma factor or factors involved, we prepared male rats with early, benign 1K,1C hypertension and paired them with one-kidney (1K) normotensive controls. In the presence of growth stimulated by background levels (1%) of fetal calf serum (FCS), plasma-derived serum (PDS), fresh or frozen, from 99 1K,1C rats differentially increased [3H]thymidine incorporation of growth-arrested rat aortic cells; increases were up to 93% more than those evoked by the paired 1K PDS and were concentration related (P < 0.01). However, there was no evidence for a differential effect of 1K,1C PDS in the absence of FCS nor of PDS after boiling. On the other hand, neither treatment of PDS with proteolytic enzymes nor charcoal absorption ablated this differential trophic effect. Thus this study provides evidence that the humoral factor(s) in hypertension are resistant to freezing, proteolysis, and charcoal absorption, but sensitive to boiling, and they require background levels of growth for expression.

Inhibition of proliferative activity of pulmonary fibroblasts by tetrandrine.

Tetrandrine, an herbal drug, has been employed in China to treat pulmonary fibrosis. To date, the mechanisms governing the antifibrotic action of tetrandrine are unknown. The present study employs a fibroblast mitogenic assay to determine whether tetrandrine directly inhibits the ability of fibroblasts to respond to stimulation by growth factors. The data indicate that tetrandrine blocks proliferation and the incorporation of tritiated thymidine into DNA by fibroblasts stimulated with human serum, PDGF plus plasma, FGF plus plasma, or TNF plus plasma. Since tetrandrine inhibits the response to a variety of growth factors, its action does not appear to involve the blockade of a specific stimulatory receptor. Tetrandrine is effective in inhibiting thymidine incorporation when added up to 6 hr after stimulation of quiescent cells, suggesting either that tetrandrine does not block the attainment of competence by fibroblasts or that its activity is not limited to blocking the attainment of competence by these cells. Growth factor-induced mitogenesis is also inhibited by nitrendipine, a calcium channel blocker, and by cytochalasin B, a microfilament blocker. However, tetrandrine treatment of fibroblasts neither results in the changes of morphology seen with cytochalasin B nor is limited to the early events of stimulus-response coupling. Therefore, the mechanism of action for tetrandrine is not identical to that for either cytochalasin B or nitrendipine. In summary, these results suggest that the antifibrotic action of tetrandrine may be mediated in part by direct inhibition of fibroblast proliferation normally associated with the development and progression of silicosis.

A procedure for the extraction and high resolution two-dimensional gel electrophoresis of total nuclear phosphoproteins from isotonically purified nuclei.

Methods are described for the extraction and preparation of total nuclear proteins for high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The conditions for protein extraction and preparation limit both protease and phosphatase activity, allowing application of this technique to the reliable analysis of changes in nuclear protein composition and nuclear protein phosphorylation as well as other forms of post-translational modifications. Unlike other procedures for 2-D PAGE analysis of nuclear proteins the technique allows solubilization and extraction of all nuclear proteins along with removal of nucleic acids which interfere with isoelectric focusing and autoradiography of 32Pi-labeled proteins. It avoids lengthy dialysis in which precipitation of nuclear proteins often occurs and does not require precipitation and resolubilization of nuclear proteins to obtain sufficient protein concentrations for 2-D PAGE analysis; often impractical steps in which complete resolubilization of all proteins is not possible. It produces high resolution 2-D PAGE analysis in which identification of even low abundance proteins can be made, based on isoelectric point and molecular weight, allowing comparison with other studies.

Effect of retinoic acid on the phosphorylation of endogenous proteins in HL-60 cells.

HL-60 cells were incubated with [32P]-Pi in order to label endogenous phosphoproteins in situ. These were then resolved via two-dimensional electrophoresis and autoradiograms were made from the gels. A comparison of autoradiograms made from retinoic-acid-differentiated cells with those made from control cells revealed a small number of phosphoproteins whose labeling was enhanced in differentiated cells. Incubating HL-60 cells with [35S]-methionine revealed that RA induced the synthesis of one of these proteins, (53 kDa, pl 4.8) although not to a degree sufficient to account for the increased phosphate labeling observed in differentiated cells. Difluoromethylornithine (DFMO), which arrests HL-60 cell proliferation without inducing differentiation, had no effect on endogenous protein phosphorylation. Treatment of DFMO-arrested cells with retinoic acid, however, increased the phosphorylation of the proteins and resulted in differentiation of the cells. Densitometric analysis of autoradiograms made from two-dimensional gels revealed that the phosphorylation of the 53-kDa, pl 4.8 protein was significantly elevated in cells exposed to RA for as little as 12 hours, i.e., before the cells were committed to differentiate and before a significant increase in the number of phenotypically mature cells was observed. It therefore appears that this protein may be an intermediate in the retinoic-acid-induced differentiation process.

Estradiol-progesterone-induced growth of the rat dorsolateral prostate: effects on protein phosphorylation.

The combination of estradiol and progesterone can affect growth of the rat dorsolateral prostate by a mechanism that appears to be independent from androgen-mediated activity. The effects of estradiol and progesterone on protein kinase activity and endogenous protein phosphorylation in the castrate rat dorsolateral prostate were compared with androgenic effects on these intracellular events. Differences in protein kinase activity and protein phosphorylation were found with each type of hormonal therapy. Changes in protein kinase activity and protein phosphorylation produced by estradiol and progesterone in the rat dorsolateral prostate were not found in the rat ventral prostate, which was not affected by these hormones in the 28 day length of this study.

Inhibition of human mammary carcinoma cell proliferation by retinoids and intracellular cAMP-elevating compounds.

Retinoids and cAMP-elevating agents markedly inhibited the proliferation of human mammary tumor cells. Their response has been previously correlated with the presence of estrogen receptor (ER) positivity. MDA-MB-231 cells were ER negative and insensitive to the antiproliferative effects of retinoids. However, their growth was markedly inhibited by agents that elevated intracellular cAMP levels, i.e., 8-bromo-cAMP, cholera toxin (CT), forskolin, and the phosphodiesterase inhibitor papaverine. The CT and forskolin inhibition of the ER-positive cells (MCF-7) was associated with an elevation of adenylate cyclase activity and intracellular cAMP levels; however, similar elevations in intracellular cAMP levels were not observed following CT or forskolin inhibition of MDA-MB-231 cells but only following the addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine.

Correlation between the induction of leukemic cell differentiation by various retinoids and modulation of protein kinases.

During retinoic acid induced differentiation of the human promyelocyte leukemia cell line HL60 and the human myeloblast cell line RDFD along the myeloid pathway, there is marked modulation of both the cyclic adenosine 3':5'-monophosphate dependent protein kinase and the phospholipid-sensitive calcium dependent protein kinase. In order to further assess whether these kinases are intimately associated with the differentiation process, we have correlated the modulation of these enzymes and phosphorylations of their substrates with the extent of differentiation induced by various retinoid derivatives. We observed that there was a direct relationship between the degree of differentiation of the two leukemic cell lines and the elevation of the cyclic adenosine 3':5'-monophosphate dependent protein kinase and phospholipid sensitive calcium dependent protein kinase activities. In addition, the increased phosphorylation of the various substrates of these enzymes also correlates with the degree of differentiation. These observations support the hypothesis that modulation of these protein kinases and phosphorylation of their substrates are necessary steps in the differentiation process.

Glycosyltransferase levels in familial polyposis coli.

Glycosyltransferase levels have been reported to be decreased in the tumor mucosa of adenocarcinoma of the colon. The purpose of this study was to determine if similar changes are present in the polyp mucosa of patients with Familial Polyposis Coli (FPC). The levels of eight glycosyltransferases were determined by measuring transfer of a radiolabeled sugar from a nucleotide sugar donor to a glycoprotein acceptor. The levels of four of the enzymes were significantly different in the mucosa of tumors and the polyp mucosa of patients with FPC as compared to the colonic mucosa of persons without known neoplastic disease. The changes were specific for these four enzymes and occurred to the same degree in tumor mucosa and the polyp mucosa. These changes in glycosyltransferase levels are a marker of the malignant transformation of the cell and since they occur in the histologically benign cells of FPC may serve as a key to understanding the neoplastic process.

Characterization and androgenic regulation of soluble protein kinases and protein phosphorylation in rat ventral prostate gland.

The soluble protein kinase activities for protamine and casein, the histone kinases modulated by cAMP or Ca2+ and phospholipid, as well as the phosphorylation patterns of endogenous proteins were measured in rat ventral prostates from normal adults, castrates, and dihydrotestosterone-treated castrates. In normal prostate, the ratio of cAMP-dependent type I and II kinases was approximately 1:5. After a 3-week period of castration-induced regression, the concentrations of both enzymes were increased, but on a total organ basis, type I was decreased to 56%, while type II was reduced to 20% of normal levels. Casein kinase activity in unfractionated cytosol was not significantly altered by castration but when partially resolved into type I and II enzymes, there appeared to be a selective reduction in the type I component. In contrast, the total organ activities of protamine kinase or Ca2+-activated, phospholipid-dependent kinase, two measures of protein kinase C enzyme, were significantly increased (64 and 71%, respectively) above sham controls in regressed organs of castrates. All of the castration-induced changes in protein kinases were restored toward normal by dihydrotestosterone treatment. Castration effects on protein kinase C and the cAMP-dependent kinases appeared to be manifest in the phosphorylation of endogenous proteins. Castration resulted in a qualitative shift in the cAMP-dependent phosphorylation patterns as measured by gel electrophoresis, with increases in four major bands and decreases in two others, whereas the Ca2+-activated, phospholipid-dependent phosphorylation patterns were all enhanced. It is concluded that the androgenic regulation of protein kinase C differed qualitatively from that of other kinases, and its activation upon withdrawal of the androgenic stimulus may be involved in autophagic mechanisms in the prostate.

The role of 13 cis-retinoic acid in the remission induction of a patient with acute promyelocytic leukemia.

The addition of retinoic acid to human promyelocytic leukemia cells in culture results in their differentiation to mature myeloid forms with acquisition of the differentiated phenotype, i.e., the ability to reduce nitroblue tetrazolium. A heavily pretreated patient with acute promyelocytic leukemia and residual malignant cells in his marrow after multiple courses of chemotherapy was given 13-cis-retinoic acid upon demonstration of both morphologic and functional differentiation of his leukemic cells by transretinoic acid in vitro. The patient achieved a complete remission and was maintained on 13-cis-retinoic acid for 1 year, when the patient relapsed with a population of cells that were resistant to retinoic acid-induced differentiation.

Calcium-activated, phospholipid-dependent protein kinase activity and protein phosphorylation in HL60 cells induced to differentiate by retinoic acid.

Treatment of human promyelocytic (HL60) cells with retinoic acid for at least 48 h causes differentiation to more mature myeloid forms. Prior to commitment of cells to the myeloid pathway there is a marked increase in cytosolic calcium-activated, phospholipid-dependent protein kinase activity. This increase does not result from an intracellular redistribution of the enzyme. Concomitant with the increased enzyme activity there is enhanced phospholipid-dependent phosphorylation of proteins of 29, 49, 52, 58, 68, 69, 120, 170, 200 and 245 kDa.

Cyclic AMP-dependent and -independent protein kinases and protein phosphorylation in human promyelocytic leukemia (HL60) cells induced to differentiate by retinoic acid.

The human leukemia cell line HL60 which resembles promyelocytes can be induced to differentiate to cells displaying features of the mature myeloid phenotype by a variety of agents including retinoic acid (RA) and agents that elevate intracellular adenosine 3:5 cyclic monophosphate (cyclic AMP) levels, e.g., 8-bromo-cyclic adenosine 3:5 monophosphate (8-Br-cyclic AMP), cholera toxin. Since most, if not all the effects of cyclic AMP, are mediated by adenosine 3:5 cyclic monophosphate-dependent protein kinase (cyclic AMP-dPK), we investigated the role of cyclic AMP-dPK and adenosine 3:5 cyclic monophosphate-independent protein kinase (cyclic AMP-iPK) in the induced differentiation of HL60 cells. Marked stimulation of cyclic AMP-dPK and cyclic AMP-iPK appears to be intimately involved with and specific for HL60 myeloid differentiation as evidenced by: (1) Stimulation of cyclic AMP-dPK and cyclic AMP-iPK early during HL60 myeloid differentiation and prior to phenotypic changes. (2) RA and dimethylformamide (DMF), agents that induce differentiation along the myeloid pathway, cause a marked increase in the type l cytosolic cyclic AMP-dPK and cyclic AMP-iPK (protamine kinase) while no such increases are noted in cells treated with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) which induces differentiation along the monocyte/macrophage pathway. (3) Both native polyacrylamide gel electrophoresis as well as photoaffinity labeling with 8-azido-cyclic AMP demonstrate marked increases in type l cyclic AMP-dPK in the cytosols of cells exposed to agents that induce myeloid differentiation but no increase in TPA-differentiated cells. (4) The appearance and disappearance of specific cyclic AMP-dependent and -independent protein phosphorylations are associated with the induced myeloid differentiated state.

Cyclic AMP in the regulation of exocytosis in the rat parotid gland. Evidence obtained with cholera toxin.

The effects of cholera toxin on rat parotid gland function were determined in order to further characterize the relationship between cyclic AMP and exocytosis in this tissue. Cholera toxin induced the release of alpha-amylase from rat parotid minces in vitro. This release was accompanied by an activation of adenylate cyclase, elevated cyclic AMP levels, an elevated protein kinase activity ratio, and changes in the degree of phosphorylation of three endogenous phosphoproteins. Two of the phosphoproteins became more phosphorylated upon cholera toxin stimulation while the phosphorylation of the other decreased. The effects of cholera toxin on endogenous phosphoprotein labelling appeared to mimic those of the beta-adrenergic agonist isoproterenol but were of a smaller magnitude. These results are consistent with cyclic AMP functioning as a major mediator of exocytosis in this gland exerting its effects, at least in part, via activation of cyclic AMP dependent protein kinase. The mechanism by which an increased cyclic AMP level results in the decreased phosphorylation of an endogenous phosphoprotein is not known.

The occurrence of carcinoma of the rectum following ileoproctostomy for familial polyposis.

Ileoproctostomy was performed in 32 patients (13 Female and 19 male), with polyposis coli ranging in age from 10 to 54 years. Seven patients (22%) developed cancer of the retained rectum with a median follow-up of 14 years. Two (20%) of ten patients, followed for 10 to 15 years, and three (50%) of six patients, followed for 15 to 20 years, developed rectal cancer. Rectal cancer developed in two of 14 patients who had their ileoproctostomy at 14 cm and in five of 18 patients who had their ileoproctostomy at a higher level, with a median followup of 7 and 11 years, respectively. Rectal cancer developed in two of 15 teenage patients undergoing ileoproctostomy and in nine of 17 patients aged 20 to 54 years. The present average ages of the two groups were 25 and 41 years, and the average age at which rectal cancer appeared was 40 years. Three of the patients who developed rectal cancer had numerous polypectomies over the years, and there was a tendency to develop tubulovillous and villous adenomas with a variable degree of atypia leading to carcinoma. One patient also showed a return to high levels of coprostanol and secondary fecal bile acids. Proctocolectomy, if acceptable, may be the treatment of choice; ileoproctostomy may mean that the patient eventually will undergo a proctectomy. The ileoanal endorectal pull-through procedure has a great deal to offer to these patients, and further study is necessary to evaluate this procedure.

Purification of plasma membranes from mouse parotid gland and membrane reorganization in response to isoproterenol.

Two highly purified plasma membrane fractions have been obtained from mouse parotid glands by a combination of differential centrifugation and isopycnic centrifugation in discontinuous sucrose gradients. The membranes were characterized by enzymic, chemical and morphological criteria. The effect of isoproterenol, which induces parotid acinar cells to proliferate, upon sialic acid and five different enzyme activities located in the plasma membrane phosphodiesterase (EC 3.1.4.1), Mg2+-ATPase (EC 3.6.1.4), leucine aminopeptidase (EC 3.4.1.1), protein kinase (EC 2.7.1.37) and sialyltransferase (EC 2.4.99.1), were quantified along the cell cycle. Plasma membrane sialic acid content falls 30% within 30 min and remains depressed for at least 6 h with the major restoration towards normal levels occurring between 12 and 16 h later. In contrast multiple daily isoproterenol injections lead to a more than 2-fold elevation of sialic acid content. Sialyltransferase activity rises 2-fold by 12 h after isoproterenol treatment and then rapidly falls. This enzyme has a pH optimum of 6.5, requires a divalent cation for activity and is inhibited by Triton X-100. Other enzyme activities showed markedly different changes after isoproterenol stimulation, either increasing, decreasing or remaining unaltered. These continuous functional modifications suggest an active role of the plasma membrane in the control of the proliferative cycle.

Glycosyltransferase activities and the differentiation of human promyelocytic (HL60) cells by retinoic acid and a phorbol ester.

The activities of five glycosyltransferases were measured following the induction of HL60 cells to differentiate to mature myeloid forms or to macrophages by the addition of retinoic acid or a phorbol ester, respectively. Gal-T-II, Fuc-T-I and (NeuAc-T-I) are all increased and Fuc-T-II decreased in activity upon treatment with RA. Gal-T-I and Fuc-T-II are decreased and Gal-T-II increased in activity upon with TPA treatment. The increases in enzyme activities with RA are measurable as early as 1 day but while Fuc-T-I and NeuAc-T-I are fully elevated at 2 days, Gal-T-II shows a biphasic rise with initial elevation by day 2 and a further rise at days 3 to 5. The rises in Gal-T-II are due to increases in the enzyme form present in uninduced cells.