Allan H. Conney - US grants

oxidation; meeting /conference /symposium; drug /agent; microsomes; sterols; liver pharmacology; prostaglandins; enzyme induction /repression; fatty acids; cytochrome P450; hepatotoxin; steroids;

The overall objective of the proposed research program is to provide new information that will increase our understanding of cancer causation and provide new approaches for the prevention of cancer. The proposed research is concerned with: (1) identification of proximate and ultimate mutagenic and carcinogenic metabolites of polycyclic aromatic hydrocarbons and related nitrogen- containing heterocyclic compounds in our environment. An important aim of this research is to enhance our understanding of poorly explored aspects of structure-activity relationships of diol epoxides from polycyclic aromatic hydrocarbons and nitrogen-containing heterocytes. (2) mechanisms of carcinogenesis and mutagenesis by optically active R, S, S, R bay-region diol epoxides of polycyclic aromatic hydrocarbons and nitrogen-containing heterocycles. In this research, we will use R, S, S, R bay-region diol epoxides (ultimate mutagens and carcinogens) and their closely related optically active but biologically less active or inactive S, R, R, S enantiomers from several polycyclic aromatic hydrocarbons and nitrogen-containing heterocycles as probes for identifying cellular targets that are important for mutagenesis. We will determine (a) the effect of optically active bay-region diol epoxide enantiomers on DNA base sequences in the coding region of the endogenous hypoxanthine phosphoribosyltransferase gene of Chinese hamster V-79 cells and (b) effects of optically active bay-region diol epoxide enantiomers on the fidelity of DNA polymerase (a) possible protein target for ultimate carcinogens). (3) inhibitory effects of naturally occurring dietary constituents and their derivatives on chemical carcinogenesis and ultraviolet light-induced carcinogenesis. In this research, we will attempt to identify potentially useful inhibitors of carcinogenesis and to use these inhibitors as tools for better understanding of the carcinogenic process. We will evaluate (a) potential antagonists of bay-region diol epoxides of polycyclic aromatic hydrocarbons. (b) potential inhibitory effects of curcumin, carnosol, rosemary and other dietary plant substances on chemical carcinogenesis and ultraviolet light-induced carcinogenesis and (c) potential inhibitory and/or stimulatory effects of vitamin D3, 1alpha,25-dihydroxyvitamin D3 and calcium on chemical carcinogenesis and ultraviolet light-induced carcinogenesis. Possible mechanisms of action will be evaluated.

The overall objective of the proposed research program is to provide new information that will increase our understanding of cancer causation and provides new approaches for the prevention of cancer. The proposed research is concerned with: (1) identification of ultimate mutagenic and carcinogenic metabolites of polycyclic aromatic hydrocarbons and related nitrogen-containing heterocyclic hydrocarbons in our environment. An important aim of this research is to enhance our understanding of poorly explored aspects of structure-activity relationships of diol epoxides from environmentally important polycyclic aromatic hydrocarbons and their related nitrogen- containing heterocycles. (2) mechanisms of carcinogenesis and mutagenesis by optically active R,S,S,R bay-region diol epoxides of polycyclic aromatic hydrocarbons and their nitrogen-containing analogs. In this research, we will use R,S,S,R bay-region diol epoxides (ultimate mutagens and carcinogens) and their closely related optically active but biologically less active or inactive S,R,R,S enantiomers from several polycyclic and azapolycyclic aromatic hydrocarbons as probes for identifying cellular targets that are important for mutagenesis and carcinogenesis. We will determine the effects of optically active bay-region diol epoxide enantiomers on DNA base sequences in the coding region of the endogenous hypoxanthine phosphoribosyltransferase (HPRT) gene of Chinese hamster V-79 cells. We will also evaluate the mechanisms and possible in vivo significance of our recent observation of dose-dependent differences in the profile of mutations for an R,S,S,R bay-region diol epoxide in V-79 cells. We will determine whether the kinds of mutations that occur in the p53 and ras genes in tumors from animals the receive a high dose of the diol epoxide are the same or different from the mutations that occur in tumors from animals that receive a low dose of the carcinogen. An important goal of our research is to compare the mutation profiles of optically pure R,S,S,R bay-region diol epoxides in V-79 cells in vitro with the mutation profiles of these compounds in vivo. (3) inhibitory effects of naturally occurring dietary constituents and their derivatives on chemical carcinogenesis and ultraviolet light-induced carcinogenesis. In this research, we will attempt to identify potentially useful inhibitors of carcinogenesis and to use these inhibitors as tools for better understanding athe carcinogenic process. We will evaluate (a) potential inhibitory effects of curcumin, carnosol, ursolic acid, rosemary, tea and other dietary plant substances on chemical carcinogenesis and ultraviolet light-induced carcinogenesis, (b) potential inhibitory and/or stimulatory effects of 1alpha, 25-dihydroxyvitamin D3 on chemical carcinogenesis and ultraviolet light-induced carcinogenesis and (c) effects of chemopreventive substances on the growth and progression of skin papillomas. Possible mechanisms of action will be evaluated.

In previous studies, we found that oral administration of green or black tea inhibits ultraviolet light (UV)-induced carcinogenesis in mice. In the present proposal, we plan to investigate the effects of orally administered green and black tea, as well as their constituents (-)-epigallocatechin gallate (EGCG) and caffeine, on mechanistically important UV-induced early changes in mouse skin. A major focus will be to test the hypothesis that tea administration upregulates UV-induced increases in wild-type p53 and downstream effectors of p53. We plan to pursue the following specific aims: 1. Determine the effects of oral administration of green and black tea to SKH-1 mice on the time course for effects of single or multiple low- dose exposures to UV on (a) wild-type p53 expression, (b) expression of p300, ARF and MDM-2 (modulators of p53 stability and function), (c) p21(WAF1/CIP1) expression, (d) bromodemyuridine incorporation into DNA, (e) epidermal thickening (hyperplasia), (f) formation of apoptotic sunburn cells and (g) expression of apoptosis related proteins (BAX, BCL-2, Fas, Fas ligand and caspases) that are downstream effectors of p53. The formation of thymine dimers, oxidized bases and strand breaks in DNA will also be determined. If green and black tea are effective, we will determine the effects of the tea constituents EGCG and caffeine on the parameters that are modulated by tea. 2. Determine the effects of oral administration of green or black tea on the formation and elimination of UV-induced persistent patches of p53 positive cells. These patches will be sequenced to determine the effects of tea administration on the profile of p53 mutations. If tea alters the profile of UV-induced mutations, we will evaluate the effects of EGCG and caffeine. 3. Test the importance of wild-type p53 and Bax for the stimulatory effect of green tea on UV-induced increases in apoptotic sunburn cells and for the inhibitory effect of tea on UV-induced carcinogenesis by studies in p53(-/-) and Bax(-/-) knockout mice.

The objective of the proposed research is to reveal fundamental relationships between chemical structure and mutagenic/carcinogenic activity, and to provide insight into the molecular mechanisms by which carcinogens after DNA, cause mutations and result in cancer. Using several site-specifically adducted oligonucleotides, we will compare how the structure, conformation and position of the DNA adduct affect the fidelity, processivity and efficiency of DNA polymerases in vitro. We will also utilize these oligonucleotides in an in vivo model to analyze how these characteristics of an adduct affect the induced mutation. These oligomers contain the exact sequence context of two in vivo hotspots for (+)-BPDE (an ultimate carcinogenic metabolite of benzo[a]pyrene) and have the (+)-BPDE modification on the purine base either at or next to the hotspot. Nonadducted and (-)-BPDE (a weak or noncarcinogenic enantiomer) adducted oligomers will also be prepared for comparative studies. The specific aims are to: 1. Study the mutagenic effects of site-specific adducts by the primer extension reaction with the replicative DNA polymerases from human and the lesion bypass polymerase from yeast. We will a) Prepare site-specifically adducted 59- mer templates. b) Determine the effects of BPDE adduction on the fidelity, processivity and efficiency of DNA polymerases. c) Compare the effects of BPDE stereochemistry on DNA polymerase activities. d) Investigate effects of adduct position on DNA polymerase activities. 2. Study the mutagenic effects of site-specific adducts in vivo by transfection assays in mammalian cells. We will a) Construct several eukaryotic expression vectors so that each contains the entire coding region of the hprt gene and a single site-specific (+)- or (-)-BPDE adduct at or next to a hotspot in the hprt cDNA sequence. b) Determine the mutagenic effect of the site-specific adduct in vivo by transfecting these hprt expression vectors into V-79 cells. We will then analyze mutations in stable transformants and compare them with mutations obtained from our earlier random mutagenesis studies in V-79 cells. We will subsequently examine how the adduct position and chirality affect the outcome of the mutation formation.

DESCRIPTION (provided by applicant): The present proposal for preclinical studies with 12-O-tetradecanoylphorbol-13-acetate (TPA) will: 1. Identify and characterize combinations of TPA and other potential therapeutic agents that may enhance differentiation and therapeutic efficacy and overcome potential resistance to TPA-induced differentiation in the treatment of myeloid malignancies. 2. Examine the hypothesis that the effects of TPA, all-trans retinoic acid (ATRA), 1 alpha,25-dihydroxyvitamin D3 or butyrate alone or as synergistic combinations are mediated through changes in the levels and translocation of PKC alpha, PKC beta and PKC delta, and on the expression of mRNAs for PKC alpha, PKC beta and PKC delta in TPA-sensitive HL-60 and TPA-resistant HL-525 cells. We will also determine the effects of selective inhibitors of PKC beta and PKC delta as well as the expression of dominant negative PKC isoforms in HL-60 cells on the differentiation response of these cells to TPA alone or together with potential synergistic agents. The effects of overexpression of constitutive PKC isoforms on the action of TPA alone to cause differentiation in TPA-resistant HL-525 cells will also be determined. 3. Study the hypothesis that TPA may have therapeutic efficacy for inducing cytotoxicity in human prostate cancer cells. Specifically, we will determine the effects of TPA alone or in combination with (a) differentiating agents (ATRA, 1alpha, 25-dihydroxyvitamin D3, and butyrate), (b) inhibitors of NF-kappaB (BAY 11-7082) or (c) cytotoxic drugs (taxol, taxotere, mitoxantrone and estramustine) on proliferation and apoptosis of cultured human prostate cancer cell lines. We will test the hypothesis that levels of constitutive NF-kappaB activity determine sensitivity of these cells to TPA-induced cytotoxicity. The effects of TPA alone or in combination with synergizers on the levels and translocation of PKC alpha PKC beta and PKC delta and on the expression of these genes in prostate cancer cells will be evaluated. 4. Determine the in vivo effects of TPA alone, or in combination with other agents that are synergistic, on the growth of human prostate tumors in immunodeficient mice. Blood levels of TPA that are associated with inhibition of tumor growth will be determined. The effect of inhibitors of tumor growth on proliferation and apoptosis in the tumors will be determined.

DESCRIPTION (provided by applicant): Sunlight-induced skin cancer is the most prevalent cancer in the United States, and UVB is primarily responsible for these cancers. Earlier studies indicated that oral or topical administration of caffeine inhibits UVB-induced skin carcinogenesis in mice by enhancing apoptosis selectively in UVB-treated epidermis and in epidermal tumors but not in normal epidermis. These effects can occur by a p53 independent mechanism. Identification of mechanisms for the in vivo apoptotic effect of caffeine in UVB-treated epidermis and in UVB-induced tumors as well as the initiation of translational studies in humans are major goals of the present proposal. We plan to pursue the following specific aims: 1. Determine the effects of topical application of caffeine immediately after a single irradiation with UVB on the time course for UVB-induced apoptosis and for UVB-induced activation of the ATR/Chk1 signal transduction pathway in the epidermis of wild type mice, p53 knockout mice, ATR and Chk1 dominant negative mice and Chk1 haploinsufficient mice. The possibility that caffeine enhances the inappropriate initiation of mitosis early in the cell cycle will also be determined. 2. Determine the effects of topically applied caffeine on apoptosis and the ATR/Chk1 signal transduction pathway as well as the possibility of inappropriate early initiation of mitosis in UVB-induced tumors and in areas of the epidermis away from tumors. 3. Initiate a pilot study to determine the effects of topical application of caffeine immediately after a single irradiation with UVB on UVB-induced apoptosis as well as the levels of wild type p53 and the ATR/Chk1 signal transduction pathway in the epidermis of human skin. Since caffeine is ingested by large segments of the population in coffee, tea, soft drinks and chocolate, research on mechanisms of the potential anticancer effects of caffeine may have broad human significance for cancer prevention. The proposal also includes the first human studies on a path that may lead to a novel way of preventing sunlight-induced skin cancer in humans.

[unreadable] DESCRIPTION (provided by applicant): Sunlight-induced skin cancer is the most prevalent cancer in the United States. In earlier studies we treated SKH-1 mice with UVB twice a week for 20 weeks. These initiated mice have no tumors, but they have a high risk of developing tumors during the next several months (high risk mice). Topical applications of caffeine 5 days a week to these high risk mice inhibited carcinogenesis and selectively increased apoptosis in the tumors but not in areas of the epidermis away from the tumors. Our leading hypothesis is that caffeine induces apoptosis by inhibition of the ATR/Chk1-dependent G2/M cell cycle checkpoint leading to premature mitosis and cell death. In preparation for translational studies on the effects of caffeine in humans, we evaluated the tumorigenicity of four commercial moisturizing creams that are commonly used by the public. All four had tumorigenic activity when applied topically for several months to high risk mice. To overcome this obstacle, we developed a novel Custom Blend cream that lacks tumorigenic activity and can be used for translational studies in humans. We plan to pursue the following specific aims: 1. Determine the effects of topical applications of caffeine in our newly developed non-tumorigenic Custom Blend cream on carcinogenesis in UVB-pretreated high risk SKH-1 mice and study the effects of the treatment on apoptosis, proliferation and the ATR/Chk1 signal transduction pathway in focal hyperplastic areas of the epidermis and in tumors. The effects of oral administration of caffeine will also be studied. 2. Determine the primary targets or early events for the action of caffeine by studying its effect on key molecular markers based on Aim 1 in epidermal focal hyperplastic areas and tumors after short term treatment (e.g. with topically applied caffeine for 3 hr, 6 hr, 24 hr, 48 hr, 7 days, or 14 days). 3. Initiate a translational study to determine the effects of topical applications of caffeine on apoptosis, proliferation and the ATR/Chk1 pathway in actinic keratoses in human skin. Key molecular markers identified by research in Aim 1 will be determined in biopsies of large keratoses before and after two weeks of topical applications of caffeine or placebo. PUBLIC HEALTH RELEVANCE:Our proposed mechanistic studies on the effects of caffeine on ultraviolet light-induced cancer formation are important steps on the road to the development of a novel approach for the prevention of sunlight-induced skin cancer. Our studies on the effects of caffeine may also have importance for individuals ingesting caffeine-containing beverages and chocolate. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Sunlight-induced nonmelanoma skin cancer is the most revalent cancer in the United States with more than one million cases per year (almost as many cases as for all of the other cancers combined), and the number of these cancers has been increasing in recent years. The reasons for this increase are not known. Although most non-melanoma skin cancers are cured by surgical removal, deaths from these cancers continue to occur. We have developed a novel animal model that resembles sunlight-induced skin cancer in humans who receive heavy exposure to sunlight early in life and who develop skin cancer later in life in the absence of additional heavy sunlight exposure. In this model, SKH-1 mice are treated with low doses of UVB for 20 weeks and UVB irradiation is stopped. These mice have no tumors, but they have a high risk of developing skin tumors over the next several months in the absence of further UVB irradiation (high risk mice), and these mice can be used for evaluating tumorigenic and chemopreventive agents. In a recent study, we found that topical applications of several different commercially available moisturizing creams to UVB-pretreated high-risk mice, in the absence of further UVB irradiation, enhanced the formation of skin tumors (tumor-promoting effect). (See Lu et al in J. Invest. Dermatol. 129: 468-475, 2009.) We hypothesize that these tumorigenic commercial creams increase inflammation, stimulate proliferation, and inhibit apoptosis in the epidermis prior to tumor formation and in tumors of UVB pretreated high-risk mice. We also hypothesize that topical applications of sodium lauryl sulfate, mineral oil and petrolatum (substances present in the tumor-promoting moisturizing creams that we studied and in many other home care products) will be tumorigenic in UVB-pretreated high-risk mice. Our specific aims are: 1. Analyze the effects of topical applications of tumor-promoting moisturizing creams and their constituents on inflammation, proliferation, and apoptosis in the epidermis of SKH-1 mice pretreated with UVB for 2 weeks (short-term studies). 2. Evaluate the effects of topical applications of tumor-promoting moisturizing creams in UVB pretreated high risk mice on inflammation, proliferation, and apoptosis in the epidermis prior to tumor formation and in tumors and areas of the epidermis away from tumors in tumor-bearing mice (mechanism studies utilizing stored paraffin blocks). 3. Evaluate the tumor-promoting activity of topical applications of sodium lauryl sulfate, mineral oil and petrolatum in UVB-pretreated high-risk mice. PUBLIC HEALTH RELEVANCE: The possibility that commonly used moisturizing creams, cosmetics and other topical home care products contribute to the high and increasing incidence of nonmelanoma skin cancer in the United States (more than one million cases per year) is an important issue requiring careful mechanistic and epidemiology studies. The present proposal attempts to identify tumorigenic components in commonly used moisturizing creams that may increase UVB-induced carcinogenesis and to assess potential mechanisms for the tumorigenic effects of these creams and their constituents. Our study will be helpful for determining biomarkers of risk that can be used for designing epidemiology studies, and we anticipate that our research will also result in the design of safer moisturizing creams, cosmetics and other topically used home care products.