Spermine-induced variations in the adenosine 5'-diphosphate ribosylation patterns of nuclear proteins from rat liver and hepatoma.

Rat liver and hepatoma nuclei were incubated in vitro with [3H]nicotinamide adenine dinucleotide to allow synthesis of a polymer of adenosine diphosphoribose subunits joined in an 1',2' ribose-ribose linkage. The addition of 1 mM spermine altered the adenosine 5'-diphosphate (ADP) ribosylation patterns of nuclear proteins in hepatoma, host liver, and regenerating liver. Spermine-treated nuclei showed a greater incorporation of ADP-ribose into H1 histones and nonhistone nuclear proteins with isoelectric points between pH 3.0 and 6.0 when separated on polyacrylamide gels. Conversely, a large reduction in ADP ribosylation was seen in core histones (H2A, H2B, and H3) from the same nuclei. The proportion of ADP-ribose incorporated into histones was reduced in the nuclei from proliferating cells relative to their respective control livers. These results imply that polyamines, which are higher in concentration in rapidly dividing cells, may elicit a regulatory function by causing the preferential ADP ribosylation of H1 histones, as well as the more acidic of the nuclear proteins.

Up regulation of the phorbol ester receptor-protein kinase C in HL-60 variant cells.

The human promyelocytic leukemia cell line HL-60 can be induced to differentiate into macrophage-like cells by nanomolar concentrations of phorbol esters. A phorbol ester-resistant variant R1B6 obtained by culturing HL-60 cells with increasing concentrations of 12-O-tetradecanoylphorbol-13-acetate, is reversibly resistant. These cells have been growing continuously in the presence of phorbol esters for more than 1 yr, but when the phorbol ester is removed, the cells gradually regain their sensitivity and express characteristics of macrophage-like cells upon readdition of phorbol ester. The concentration of phorbol ester receptors in R1B6 is about one-third that in the parental HL-60 cells. The reversion of the variants to sensitivity to phorbol esters is associated with the up regulation of the cytosol and membrane phorbol ester receptors. When partially purified, these receptor populations contain protein kinase C activity, in support of the identity of protein kinase C and the receptor. This study demonstrates that a phenotypic change in a clonal cell population correlates with the up regulation of the phorbol ester receptor-calcium-activated phospholipid-dependent protein kinase. This variant cell line is a useful model for analyzing the relationship between phorbol ester binding and protein kinase C during differentiation of HL-60 cells.

Specific high-affinity binding of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate to isolated nuclei and nuclear macromolecules in mouse epidermis.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) binds reversibly and with high affinity and specificity to nuclear macromolecules in mouse epidermis. The dissociation constants determined from Scatchard analysis of epidermal nuclei and nuclear macromolecules are 3.58 +/- 0.66 (S.E.) and 2.18 +/- 0.54 nM, respectively. The solubilization of TPA receptors from epidermal nuclei by DNase I was examined. Following a 20-min digestion at 22 degrees, more than a 2-fold increase in specific TPA binding was observed in the supernatant relative to non-nuclease-treated nuclei (0.71 versus 0.32 pmol/mg protein, respectively). Our data indicate that epidermal nuclei contain saturable and specific TPA-binding macromolecules and that these binding components may be associated with regions of chromatin that are preferentially susceptible to nucleolytic cleavage. These data suggest the existence of nuclear receptors for the phorbol ester tumor promoters. These observations may necessitate a more critical assessment of plasma membrane binding as the sole binding site responsible for triggering the multistep process of tumor promotion in mouse epidermis.

Inhibition of dihydroorotate dehydrogenase activity by brequinar sodium.

The novel anticancer drug candidate brequinar sodium (DuP 785, NSC 368390, 6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinoline- carboxylic acid sodium salt) was shown previously to be an inhibitor of dihydroorotate dehydrogenase, the fourth enzyme of the de novo pyrimidine biosynthetic pathway. Brequinar sodium inhibits the activity of this enzyme isolated from mammalian sources only but not those forms isolated from yeast or bacteria, which also use ubiquinone as the cofactor. Brequinar sodium also does not inhibit the activity of a soluble Zymobacterium oroticum dihydroorotate dehydrogenase which uses NAD+ as a cofactor. Brequinar sodium inhibits L1210 dihydroorotate dehydrogenase with mixed inhibition kinetics with respect to either the substrate (dihydroorotate) or the cofactor (ubiquinone Q6) with Ki' values in the 5-8 nM range. Our results suggest that brequinar sodium inhibits dihydroorotate dehydrogenase by binding to the enzyme at a unique site that is distinct from the dihydroorotate or the ubiquinone-binding site. This binding site appears to be unique to the mammalian enzyme, because brequinar sodium does not inhibit the yeast, Escherichia coli, or Z. oroticum forms of the enzyme.

Phospholipase C from human melanoma: purification and characterization of a phosphatidylinositol-selective enzyme.

Phospholipase C was purified from human melanoma grown as solid tumors in nude mice. The specific activity of the pure enzyme was approx. 100 mumol/min per mg; its apparent molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 150 kDa. The enzyme required calcium for activity and was activated by deoxycholate in the presence of the substrate phosphatidylinositol. The melanoma phospholipase C has a distinctly different substrate preference than those identified from normal tissues; it prefers phosphatidylinositol to phosphatidylinositol bisphosphate. The tumor enzyme was approx. 4-5-fold more active using phosphatidylinositol than phosphatidylinositol bisphosphate as the substrate.

Phospholipase C inhibitors: a new class of cytotoxic agents.

A series of nitrocoumarin and nitrochromene derivatives have been prepared and shown to inhibit the phosphatidylinositol-specific phospholipase C(PLC)(IC50 < 10 micrograms/mL) isolated from human melanoma. The inhibition of PLC by nitrocoumarin 4a was time-dependent and irreversible. The inhibition of PLC was shown to interfere with inositide metabolism in whole cells (IC50 = 4 micrograms/mL) in a manner consistent with their proposed mode of activity. Finally, the compounds were shown to be growth inhibitory to cultured melanoma cells (ID50 = 2 micrograms/mL), suggesting that PLC may be an attractive new target for chemotherapeutic intervention.

Triethylenemelamine: an initiator of two-stage carcinogenesis in mouse skin which lacks the potential of a complete carcinogen.

The dorsal skin of female CD-1 mice was treated with triethylenemelamine (TEM) to determine whether this agent acted either as a complete carcinogen or as an initiator of carcinogenesis. A dose of 0.01-1.0 mumol of TEM applied once a week for 32 weeks to the skin of the backs of mice did not produce any detectable tumors. A dose of 2.5 mumol applied once a week over the same period produced only a single papilloma in a group of 20 mice. However, when mice were treated with a single dose of 1 mumol of TEM followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) twice a week, 88% of the mice produced papillomas after 28 weeks. Using the same protocol, a single application of hexamethylmelamine (HMM), pentamethylmelamine (PMM), or melamine followed by promotion with TPA had no significant tumor initiating activity. These data suggest that TEM acts primarily as an initiator of two-stage carcinogenesis.

Effects of 12-O-tetradecanoylphorbol-13-acetate and mezerein on gamma radiation-induced DNA repair in resting bovine lymphocytes.

The present study was undertaken to determine the effect of the potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on gamma radiation-induced DNA repair in resting (Go) lymphocytes. Mezerein, a non-promoter but a co-mitogen in lymphocytes, was used as a control agent. It was previously proposed that a possible mechanism for the action of tumor promoters was through the inhibition of DNA repair processes. Our results indicate that TPA does not inhibit DNA repair following gamma irradiation of Go lymphocytes. These data support the hypothesis that the tumor promoting ability of TPA is not a result of impaired repair of potentially mutagenic lesions in DNA.

EZ-FIT: a practical curve-fitting microcomputer program for the analysis of enzyme kinetic data on IBM-PC compatible computers.

EZ-FIT, an interactive microcomputer software package, has been developed for the analysis of enzyme kinetic and equilibrium binding data. EZ-FIT was designed as a user-friendly menu-driven package that has the facility for data entry, editing, and filing. Data input permits the conversion of cpm, dpm, or optical density to molar per minute per milligram protein. Data can be fit to any of 14 model equations including Michaelis-Menten, Hill, isoenzyme, inhibition, dual substrate, agonist, antagonist, and modified integrated Michaelis-Menten. The program uses the Nelder-Mead simplex and Marquardt nonlinear regression algorithms sequentially. A report of the results includes the parameter estimates with standard errors, a Student t test to determine the accuracy of the parameter values, a Runs statistic test of the residuals, identification of outlying data, an Akaike information criterion test for goodness-of-fit, and, when the experimental variance is included, a chi 2 statistic test for goodness-of-fit. Several different graphs can be displayed: an X-Y, a Scatchard, an Eadie-Hofstee, a Lineweaver-Burk, a semilogarithmic, and a residual plot. A data analysis report and graphs are designed to evaluate the goodness-of-fit of the data to a particular model.

Characterization of phosphatidylinositol phospholipase C activity in human melanoma.

Phosphoinositide phospholipase C activity was investigated in human melanoma grown as solid tumor xenografts in nude mice. The enzyme was dependent on calcium for activity and was stimulated by the detergent deoxycholate. The pH optimum was 5.5 in the absence of detergent, and in the presence of deoxycholate two pH maxima were present, 5.5 and 7.2. Phospholipase C activity was inhibited by the sulfhydryl reagent dithionitrobenzoate with an IC50 in the micromolar range. Phospholipase C activity was distributed widely in mouse tissues. The enzyme showed a progressive increase in activity from heart, liver, lung, colon, spleen, to brain tissue. Mouse and human melanomas grown as solid tumors had higher phospholipase C activity than mouse brain. The relatively high activity of this enzyme in melanoma may suggest a biological role for phospholipase C in solid tumor growth.

Purification of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase from mouse epidermis.

Ornithine decarboxylase was purified at least 1500-fold from mouse epidermis pretreated with five consecutive doses of 12-O-tetradecanoylphorbol-13-acetate and 3-isobutyl-1-methylxanthine at 3- to 4-day intervals. Following DEAE-cellulose chromatography and ammonium sulfate precipitation, ornithine decarboxylase was purified further by affinity chromatography. Ornithine decarboxylase was then radioactively labeled by covalently binding [3H]-alpha-difluromethylornithine to the enzyme following polyacrylamide gel electrophoresis under non-denaturing conditions. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining of protein, a band was identified that corresponded to a molecular weight of approx. 56,000, coincident with a peak of radioactivity. This is the first study to purify ornithine decarboxylase from mouse epidermis.

IgE-dependent reactions to urologic catheter extracts by skin testing in latex-allergic patients.

BACKGROUND: Latex allergy is mediated by residual IgE-binding proteins found on latex products. While most reported reactions involve latex gloves, the allergenic potential of other latex containing devices is unknown. OBJECTIVE: To assess the allergenic potential of latex urologic catheters. METHODS: Two identical lots of urologic catheters were extracted in sterile saline and analyzed for latex protein content using sensitive ELISA and Western blot assays. The extracts were tested by skin prick testing in a population of 47 latex-allergic patients. RESULTS: Latex proteins were detected by ELISA assay, however, the Western Blot method was not sensitive enough to measure latex proteins in these catheter extracts. Skin prick testing using a standard latex reagent (Bencard) demonstrated the test population to be extremely sensitive to latex as 68% of the population reacted to a 1/100,000 dilution. Using undiluted catheter extracts, only 11% of these latex-allergic patients reacted to catheter lot A and 2% to catheter lot C, by prick skin test. Eight percent of the control patients had a positive latex prick skin test and none reacted to the catheter extract. CONCLUSIONS: The data suggest that processing and leaching of these specific latex urologic catheters resulted in a very low latex allergen content. The low prevalence of skin test reactivity in a population of latex-allergic patients suggests that these catheters would be an unusual cause of allergic reactions. It is not possible, however, to determine whether latex-allergic patients could safely use latex urologic catheters.