John P. Durham

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.

The purification and characterization of plasma membranes and the subcellular distribution of adenylate cyclase in mouse parotid gland.

1. Plasma membranes have been purified 17-fold from mouse parotid gland homogenates prepared in hypertonic sucrose media using differential centrifugation. The method is fast and simple. The membranes were characterised by electron microscopy, enzyme composition and chemical composition. Further purification was achieved by isopycnic centrifugation in discontinuous sucrose gradients. 2. The purified membranes contain an adenylate cyclase activity which is stimulated by isoproterenol and fluoride. Only 50% of the total adenylate cyclase activity sedimented in the plasma membrane fraction. The rest of the activity resided in the crude nuclear and mitochondrial pellets. However, this adenylate cyclase activity was not associated with these organelles but with membrane fragments in the pellets. Purified nuclei did not contain adenylate cyclase activity. 3. Adenylate cyclase activity was also localised by electron microscopic cytochemistry. Besides being found at the plasma membrane, large amounts of adenylate cyclase were found in a small proportion of the vesicles within the acinar cells, which appeared to be secondary lysosomes. 4. Adenylate cyclase activities, under standard assay conditions, are proportional to the time of incubation and the concentration of enzyme. The enzyme requires both Mg-2+ and CA-2+ for activity. Isoproterenol increased activity 2-fold and this increase is abolished by beta-adrenergic blocking agents.

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 follow-up 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 poly-pectomies 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.

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.

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.

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.

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.

Androgen Induced Norepinephrine Release From Postganglionic Neurons Mediates Accessory Sex Organ Smooth Muscle Proliferation

PURPOSE Guinea pig seminal vesicle smooth muscle displays an initial androgen dependent, proliferative response during early puberty, followed by progression to an androgen resistant, amitotic state in adults. We determined the role of norepinephrine in androgen dependent pubertal proliferation and in the subsequent terminal differentiation of adult seminal vesicle smooth muscle. MATERIALS AND METHODS Guinea pig seminal vesicle provided a suitable model since its unique anatomy allowed clean harvest of smooth muscle without epithelium. Norepinephrine release from postganglionic adrenergic nerve terminals in seminal vesicle smooth muscle was measured using several techniques. Prazosin sensitive electrical field stimulation of contractile responses qualitatively assessed norepinephrine release. Norepinephrine release was quantified directly in vitro from incubated seminal vesicle smooth muscle minces and indirectly ex vivo from intact tissue using the endogenous seminal vesicle smooth muscle concentration ratio of 3,4-dihydroxyphenylglycol-to-norepinephrine (Sigma Chemical Co., St. Louis, Missouri). Norepinephrine mediated seminal vesicle smooth muscle proliferation was assessed by the time course relationships of androgen induced norepinephrine release, protein kinase C activation-depletion and increases in total DNA, the impact of in vivo reserpine induced norepinephrine depletion on protein kinase C activation-depletion and the mitogenic response, and the alpha1-adrenoceptor mediated mitogenic response in cultured seminal vesicle smooth muscle cells. RESULTS In prepubertal smooth muscle androgen induced norepinephrine release from postganglionic neurons. The effect was independent of preganglionic innervation. Increased norepinephrine release was concurrent with the onset of androgen induced protein kinase C activation-depletion and cellular proliferation. In vivo norepinephrine depletion to 1% or less of control values by chronic reserpine treatment selectively antagonized the androgen induced increases in smooth muscle DNA and protein kinase C down-regulation. Norepinephrine depletion by reserpine neither induced apoptosis nor altered cell number. Cell culture experiments demonstrated that alpha1-adrenoceptors mediated the proliferative response to norepinephrine. Together these findings indicate that increased norepinephrine release has an obligatory role in androgen dependent muscle cell proliferation during puberty. Terminally differentiated smooth muscle in adults was characterized by androgen resistance to elevated norepinephrine release and protein kinase C activation. CONCLUSIONS Androgen induced norepinephrine release from postganglionic neurons in seminal vesicle smooth muscle mediated the proliferative response that occurs in early pubertal development. Normal uncoupling of elevated norepinephrine release and protein kinase C activation-depletion may represent a key event in the normal terminal differentiation of accessory sex organ smooth muscle in adults.

Elevation of adenylate cyclase activity during leukemic cell differentiation

Exposure of the various human myeloid leukemic cell lines (HL60 and RDFD) to various compounds results in marked differentiation of the cells. This differentiation is associated with a marked increase in both basal and NaF-stimulated adenylate cyclase (AC) activity. The increase in AC activity occurs regardless of the differentiation inducer one has utilized (retinoic acid (RA), dimethyl formamide (DMF), hypoxanthine (HPX) or actinomycin D (actD) and is correlated with this process, as a variant of the HL60 cell (HL60-Blast) that does not differentiate upon exposure to the various inducers does not demonstrate this increase in AC activity. In addition, the differentiation process is associated with a rapid increase in intracellular cAMP within hours of adding the inducer, followed by a gradual decrease.

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.