Full Text CA-92-22 POSSIBLE ROLE OF METALLOTHIONEIN IN CARCINOGENESIS NIH GUIDE, Volume 21, Number 32, September 4, 1992 RFA: CA-92-22 P.T. 34 Keywords: Cancer/Carcinogenesis Toxicology Biochemistry, Proteins Biology, Molecular Biomedical Research, Multidiscipl National Cancer Institute Letter of Intent Receipt Date: October 2, 1992 Application Receipt Date: December 8, 1992 PURPOSE The Chemical and Physical Carcinogenesis Branch, Division of Cancer Etiology (DCE), National Cancer Institute (NCI), in collaboration with the Cancer Biology Branch, Division of Cancer Biology, Diagnosis, and Centers (DCBDC), NCI, and the Grants and Contracts Operations Branch, Division of Cancer Treatment (DCT), NCI, invites investigator-initiated research grant applications to elucidate the possible role of metallothionein (MT) in carcinogenesis. New and experienced investigators in relevant fields and disciplines may apply for funds to pursue this research. Historically, three major groups of scientists have contributed to the knowledge of MT: (1) toxicologists and physiologists interested in its role in heavy metal metabolism and detoxification; (2) protein chemists intrigued by its unusual structural features, and (3) molecular biologists interested in gene regulation and the use of MT promoter sequence for genetic engineering experiments. In order to better define the possible role of MT in carcinogenesis, this RFA seeks to encourage interactions among these major groups, other basic scientists, oncologists, and clinicians in multidisciplinary research projects. HEALTHY PEOPLE 2000 The Public Health Service (PHS) is committed to achieving the health promotion and disease prevention objectives of "Healthy People 2000," a PHS-led national activity for setting priority areas. This Request for Applications (RFA), Possible Role of Metallothionein in Carcinogenesis, is related to the priority area of cancer. Potential applicants may obtain a copy of "Healthy People 2000" (Full Report: Stock No. 017-001-00474-0 or Summary Report: Stock No. 017-001-00473-1) through the Superintendent of Documents, Government Printing Office, Washington, DC 20402-9325, telephone 202-783-3238. ELIGIBILITY REQUIREMENTS Applications may be submitted by domestic and foreign, for-profit and non-profit, public or private organizations, such as universities, colleges, hospitals, laboratories, units of State and local governments, and eligible agencies of the Federal Government. Applications from women and minority investigators are encouraged. MECHANISM OF SUPPORT Support of this program will be through the National Institutes of Health (NIH) traditional research project grant (R01). Applicants will be responsible for the planning, direction, and execution of the proposed project. The total project period for applications submitted in response to this RFA may not exceed four years. Awards will be administered under PHS grants policy as stated in the Public Health Service Grants Policy Statement, DHHS Publication No. (OASH) 90-50,000, revised October 1, 1990. This RFA is a one-time solicitation. Future unsolicited competitive continuation applications will compete with all other investigator-initiated research grant applications and be peer reviewed by a chartered study section in the Division of Research Grants (DRG), NIH. However, should the NCI determine that there is a sufficient continuing program need, a request for competitive continuation applications will be announced. Only recipients of awards under this RFA will be eligible to apply. FUNDS AVAILABLE Total costs of $1,500,000 per year for four years will be committed to fund applications that are submitted in response to this RFA. It is anticipated that 9 to 11 awards will be made. This funding level is dependent upon the receipt of a sufficient number of applications of high scientific merit. The earliest feasible start date for the initial awards will be July 1, 1993. Although this program is provided for in the financial plans of the NCI, the award of grants pursuant to this RFA is also contingent upon the availability of funds for this purpose. RESEARCH OBJECTIVES Background Metallothioneins are a family of low molecular weight, cysteine-rich, heavy metal-binding proteins. The mammalian forms have 61 amino acids, including 20 cysteine residues, but no aromatic amino acids or histidines (1). The metal content of purified MT is highly variable and depends on the organism, tissue, and history of heavy metal exposure. For example, MT isolated from human liver autopsy samples contains almost exclusively zinc (Zn), whereas MT from kidney contains substantial levels of cadmium (Cd) and copper. These differences probably reflect both the natural heavy metal exposure of the organs and the expression of different MT isoforms (2). The synthesis of MT in animals is induced in a variety of ways, including exposure to heavy metals, physiologic stress, and changes in endocrine status and requires increased transcription of MT genes (3,4). Our knowledge of MT degradation, however, is somewhat limited. In vivo experiments have suggested that the rate of MT turnover in the cytosol fraction is influenced by the specific metal bound to the polypeptide. The disappearance of 35S-labeled MT from the cytosol fraction reflects degradation of this protein by lysosomal proteases since apometallothionein (without metal), Zn-MT, and Cd-MT were rapidly degraded in lysosomal extracts (5). To date, our understanding of the function of MT has been focused on its role in metal transport, mineral nutrition, metal detoxification, and detoxification of other chemicals (6-8). Other possible functions suggested for MT include participation in embryonic development, cell differentiation and proliferation, and carcinogenesis (9-11). In an attempt to better understand the possible role of MT in carcinogenesis and to develop future research directions in this area, a workshop was sponsored by the Chemical and Physical Carcinogenesis Branch on November 18-19, 1991. The approach of the workshop was to focus only briefly on what is known and to concentrate on what is unknown, regarding MT and the cancer process. The notion that MT may play a role in carcinogenesis is formulated on the basis of two basic properties of this protein. First, MT genes are universally inducible. A variety of stress conditions, excess metals notwithstanding, has been shown to elicit expression of MT genes, which are otherwise expressed at a relatively basal level. Such a transcriptional response is typical of cellular defense and suggests a close relation with normal or abnormal growth. Second, MT is ubiquitous and highly conserved in its amino acid sequence wherever cysteine residues are found. These occur in motifs where cysteine pairs are separated by one or two non-cysteine amino acids (CXC, CXXC) (12). On a strictly molecular affinity level, Cd can displace Zn from Zn-fingers in DNA binding proteins (e.g., oncogenes, proto-oncogenes, tumor suppressor genes), thereby altering their activity or function within the cell. Disruption of the function of these proteins could result in an alteration in gene expression and thus in the promotion of cell growth and turnover (promotion). The displacement of Zn from these proteins could generate free radicals thereby contributing to Cd carcinogenesis. In this regard, MT could function to sequester Cd, thereby not permitting accumulation of free Cd within the cell, and thus limiting the displacement of Zn from these important proteins. The interaction of Cd with DNA binding proteins requires further investigation. In addition, such in vitro molecular research should provide important insight into whole cell and whole animal systems. The carcinogenic effect of Cd appears to be tissue specific and may be related to MT levels in the target organs. For example, the ventral prostate, a target site of Cd carcinogenesis in the rat, appears to be deficient in MT, thus apparently making it susceptible to carcinogenesis by Cd. Also, certain cells within the rat testes, and possibly the lung, appear to contain low levels of MT, and both of these tissues are clearly target sites for Cd carcinogenesis. Further, the ability to induce MT may be equally important in the carcinogenic process. Tissues able to respond rapidly to insult by producing MT may achieve protection from the carcinogenic effects (e.g., liver), whereas a "non-responsive" tissue (e.g., ventral prostate) may be highly susceptible to Cd carcinogenesis. Clearly, tissue differences in susceptibility to the carcinogenic effects of Cd exist (13,14). An investigation of the differences between these tissues should provide further insight into (1) a role of MT in Cd carcinogenesis and (2) a possible mechanism of Cd carcinogenesis. In addition, a species difference in susceptibility to Cd exists, particularly within the lung, and species comparisons may also provide answers to these questions. Most mammalian tissues contain a basal level of MT that may vary with age and type of tissue. Various localization techniques have demonstrated MT mainly in the cellular cytosol, but it is also present in the nucleus of hepatocytes in early development of the human fetus and in fetal and neonatal rat. Moreover, MT has been demonstrated in various types of human thyroid, testicular germ cell tumors, bladder transitional cell carcinomas, and salivary gland tumors. Both benign and malignant tumors express MT in the nucleus and/or cytoplasm and the staining for MT is more predominant in cisplatin- and radiation-resistant tumors than in those sensitive to chemotherapy. The functional role of MT in protection of tumor cell growth or in drug resistance is poorly understood. A number of factors may be involved in the development of drug resistance in tumor cells and expression of MT may be one of them. The importance of MT in this process appears to depend on the phenotype of the tumor and its cellular origin (15,16). Research Goals and Scope The objectives of this RFA are to encourage research designed to elucidate the possible role of MT in carcinogenesis. Specific topic areas that might be supported by the RFA include, but are not limited to: o Biological and toxicological roles of MT. Studies such as metal homeostasis, detoxification, transport, role in cell proliferation during the perinatal period, and involvement of Zn as a second messenger in signal transduction, as related to cellular normality; o Regulation of MT gene expression. Studies of metal induction in various tissues, during development, and organismal specificity in transgenic and model systems and in normal versus transformed cells; o Role of MT in tumor cell pathobiology. Studies to define the role of MT in tumor cell progression and metastasis and the types and staging of tumors that may or may not express excess MT. Studies directed at enhancing a rational basis for therapeutic intervention with metallic anticancer drugs; o Role of MT in cancer chemotherapy. Studies on the role of MT in tumor cell resistance to anticancer drugs, especially metal based drugs. Studies on the use of induction of MT in non-tumor tissue as an adjunct to reduce toxicity for metallic chemotherapeutics. Studies involving mechanisms by which MT synthesis could be specifically depressed in tumor cells to make them hypersusceptible to metallic chemotherapeutics; o Susceptibility factors in metal carcinogenesis. Studies assessing MT gene expression in target tissues of metallic carcinogens in rodents and molecular epidemiology of MT with special emphasis on target tissues of metallic carcinogens in humans (e.g., prostate, lung), and o Molecular interaction of MT with ligands (metals and anticancer drugs) including binding and exchange, and structural and dynamic studies. These research objectives cut across the traditional administrative divisions of the NCI. The Chemical and Physical Carcinogenesis Branch, DCE, is the issuing program. The Cancer Biology Branch, DCBDC, and the Grants and Contracts Operations Branch, DCT, are co-sponsoring programs. A joint funding plan will be developed after the review is completed. In addition, the National Institute of Environmental Health Sciences has an interest in the general topic of metallothionein, but not in the specific emphasis of this RFA, which is the possible role of metallothionein in carcinogenesis. STUDY POPULATIONS SPECIAL INSTRUCTIONS TO APPLICANTS REGARDING IMPLEMENTATION OF NIH POLICIES CONCERNING INCLUSION OF WOMEN AND MINORITIES IN CLINICAL RESEARCH STUDY POPULATIONS NIH and ADAMHA policy is that applicants for NIH/ADAMHA clinical research grants and cooperative agreements are required to include minorities and women in study populations so that research findings can be of benefit to all persons at risk of the disease, disorder or condition under study; special emphasis must be placed on the need for inclusion of minorities and women in studies of diseases, disorders and conditions which disproportionately affect them. This policy is intended to apply to males and females of all ages. If women or minorities are excluded or inadequately represented in clinical research, particularly in proposed population-based studies, a clear and compelling rationale must be provided. The composition of the proposed study population must be described in terms of gender and racial/ethnic group. In addition, gender and racial/ethnic issues must be addressed in developing a research design and sample size appropriate for the scientific objectives of the study. This information must be included in the form PHS 398 (rev. 9/91) in Sections 1-4 of the Research Plan AND summarized in Section 5, Human Subjects. Applicants are urged to assess carefully the feasibility of including the broadest possible representation of minority groups. However, the NIH recognizes that it may not be feasible or appropriate in all research projects to include representation of the full array of United States racial/ethnic minority populations [i.e., Native Americans (including American Indians or Alaskan Natives)], Asian/Pacific Islanders, Blacks, and Hispanics. The rationale for studies on single minority population groups should be provided. For the purpose of this policy, clinical research is defined as human biomedical and behavioral studies of etiology, epidemiology, prevention (and preventive strategies), diagnosis, or treatment of diseases, disorders or conditions, including but not limited to clinical trials. The usual NIH policies concerning research on human subjects also apply. Basic research or clinical studies in which human tissues cannot be identified or linked to individuals are excluded. However, every effort should be made to include human tissues from women and racial/ethnic minorities when it is important to apply the results of the study broadly, and this should be addressed by applicants. For foreign awards, the policy on inclusion of women applies fully; since the definition of minority differs in other countries, the applicant must discuss the relevance of research involving foreign population groups to the United States' populations, including minorities. If the required information is not contained within the application, the application will be returned. Peer reviewers will address specifically whether the Research Plan in the application conforms to these policies. If the representation of women or minorities in a study design is inadequate to answer the scientific question(s) addressed AND the justification for the selected study population is inadequate, it will be considered a scientific weakness or deficiency in the study design and will be reflected in assigning the priority score to the application. All applications for clinical research submitted to the NIH are required to address these policies. NIH funding components will not award grants or cooperative agreements that do not comply with these policies. LETTER OF INTENT Each prospective applicant is asked to submit, by October 2, 1992, a letter of intent that includes a descriptive title of the proposed research; the name, address, telephone and FAX numbers of the Principal Investigator; the names of other key personnel; the participating institution(s); and the number and title of the RFA in response to which the application may be submitted. Although a letter of intent is not required, is not binding, and does not enter into the review of a subsequent application, it contains information that is helpful in planning for the review. The letter of intent allows NCI staff to estimate the potential review workload and helps to avoid conflict of interest in the review. The letter of intent is to be sent to: Dr. Yung-Pin Liu Program Director, Carcinogenesis Mechanisms Chemical and Physical Carcinogenesis Branch Division of Cancer Etiology National Cancer Institute Executive Plaza North, Suite 700 Bethesda, MD 20892 Telephone: (301) 496-5471 FAX: (301) 496-1040 APPLICATION PROCEDURES The research grant application form PHS 398 (rev. 9/91) is to be used in applying for these grants. The application package is available at most institutional business offices; from the Office of Grants Inquiries, Division of Research Grants, National Institutes of Health, Westwood Building, Room 449, Bethesda, Maryland 20892, telephone 301-496-7441; and from the NCI Program Director named above. The RFA label available in the application form PHS 398 must be affixed to the bottom of the face page of the application. Failure to use this label could result in delayed processing of the application such that it may not reach the review committee in time for review. In addition, the RFA number and title must be typed on line 2a of the face page of the application form and the YES box must be marked. Submit a signed, typewritten original of the application, including the Checklist, and three signed, exact photocopies in one package to the DRG at the address below. The photocopies must be clear and single- sided. Division of Research Grants National Institutes of Health Westwood Building, Room 240 Bethesda, MD 20892** At the time of submission, two additional copies of the application must be sent to: Referral Officer Division of Extramural Activities National Cancer Institute Westwood Building, Room 838 Bethesda, MD 20892 If the applicant has an approved assurance covering the research (Multiple Project Assurance of Compliance for human subjects or Animal Welfare Assurance for animal subjects), the applicant should provide with the application certification of Institutional Review Board (IRB) approval if humans are involved, and verification of Institutional Animal Care and Use Committee (IACUC) approval if animals are involved. These reviews and approvals should occur prior to submission of the application, and the certifications and verifications should be submitted with the application. If animals or humans will be subjects of the research at performance sites other than the applicant organization, the applicant must identify in the application the assurance status of each participant. In cases where submission delays are unavoidable, follow the instructions on pages 12 and 13 of the PHS 398 application package. Failure to submit required certifications and verifications to the Scientific Review Administrator of the initial review group within 60 days of receipt of the application could result in deferral or rejection. Applications must be received by December 8, 1992. If an application is received after that date, it will be returned to the applicant. The DRG will not accept any application in response to this announcement that is essentially the same as one currently pending initial review, unless the applicant withdraws the pending application. The DRG will not accept any application that is essentially the same as one already reviewed. This does not preclude the submission of a substantial revision to an already reviewed application, but such an application must include an introduction addressing the previous critique. REVIEW CONSIDERATIONS Review Procedure Upon receipt, applications will be reviewed by the DRG for completeness. An incomplete application will be returned to the applicant without further consideration. Evaluation for responsiveness to the RFA is an NCI program staff function. Applications will be judged to determine how well they meet the goals and objectives of the program as described in the RFA. Applications judged non-responsive will be returned, but may be submitted as investigator-initiated research grant applications at the next unsolicited receipt date. Questions concerning the relevance of proposed research to the RFA are to be directed to the Program Director as named in INQUIRIES. If the number of applications is large compared to the number of awards to be made, the NCI may conduct a preliminary scientific peer review to triage applications on the basis of relative competitiveness and to eliminate those applications that are clearly not competitive. The NCI will withdraw from further competition those applications judged to be noncompetitive and notify the applicant and institutional business official. Those applications judged to be both competitive and responsive will be further evaluated according to the review criteria stated below for scientific and technical merit by an appropriate peer review group convened by the Division of Extramural Activities, NCI. The second level of review by the National Cancer Advisory Board will consider the special needs of the NCI and the priorities of the National Cancer Program. Review Criteria Applications responsive to this competitive solicitation will be reviewed in accordance with the criteria stated below: o scientific, technical or medical significance, and originality of the proposed research; o appropriateness and adequacy of the experimental design methodology proposed to carry out the research; o qualifications and research experience of the Principal Investigator and proposed staff and/or collaborators, particularly but not exclusively in the area of the proposed research as well as the level of commitment and time availability of each on the project; o documentation of the availability and adequacy of existing/proposed facilities, equipment and resources for all laboratories involved in the research, including adequate laboratory safety, biohazard and animal welfare practices and procedures as well as provisions for the protection of human subjects; and o appropriateness of the requested budget and timetable for completion relative to the proposed research. The review group will critically examine the submitted budget and will recommend an appropriate budget and period of support for each scored application. AWARD CRITERIA Awards will be made primarily on the basis of scientific merit but responsiveness to the RFA, overall program balance, and the availability of resources are important considerations that will be taken into account. INQUIRIES The Program Director welcomes the opportunity to clarify any scientific or programmatic issues or questions from potential applicants. Written and telephone inquiries concerning the objectives and scope of this RFA, and inquiries about specific proposed research are encouraged and are to be directed to Dr. Liu at the address provided in LETTER OF INTENT. Written and telephone inquiries of a budgetary, administrative, and/or policy nature are to be directed to: Ms. Jean Cahill Grants Management Team Leader Grants Administration Branch National Cancer Institute Executive Plaza South, Suite 243 Bethesda, MD 20892 Telephone: (301) 496-7800, ext. 47 FAX: (301) 496-8601 AUTHORITY AND REGULATIONS This program is described in the Catalog of Federal Domestic Assistance No. 93.393, Cancer Cause and Prevention Research. Awards are made under the authorization of the Public Health Service Act, Title IV, Part A (Public Law 78-410, as amended by Public Law 99-158, 42 USC 241 and 285) and administered under PHS grants policies and Federal Regulations 42 CFR 52 and 45 CFR Part 74. This program is not subject to the intergovernmental review requirements of Executive Order 12372 or Health Systems Agency review. References Kagi, J.H.R. and Schaffer, A. Biochemistry of metallothionein. Biochemistry 27: 8509-8515, 1988. Hamer, D.H. Metallothionein. Ann. Rev. Biochemistry 55: 913-951, 1986. Bauman, J.W., Liu, J., Liu, Y.P., and Klaassen, C.D. Increase in Metallothionein produced by chemicals that induce oxidative stress. Toxicol. Appl. Pharmacol. 110: 347-354, 1991. Cousins, R.J. Absorption, transport and hepatic metabolism of copper and zinc: Special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65: 238-309, 1985. Feldman, S.L., Failla, M.L., and Cousins, R.J. Degradation of liver metallothioneins in vitro. Biochem. Biophys. Acta 544: 638-646, 1978. Roesijadi, G. Metallothioneins in metal regulation and toxicity in aquatic animals. Aquatic Toxicology 22: 81-114, 1992. Bremner, I. Nutritional and physiological significance of metallothionein. In: J.H. R. Kaji (ed.), Metallothionein II, pp. 81-107. Basel: Birkhauser, 1987. Chernaik, M.L. and Huang, P.C. Differential effect of cysteine-to-serine substitutions in metallothionein on cadmium resistance. Proc. Natl. Acad. Sci. USA 88: 3024-3028, 1991. Waalkes, M.P. and Ward, J.M. Induction of hepatic metallothionein in male B6C3F1 mice exposed to hepatic tumor promoters: Effect on phenobarbital, acetaminophen, sodium barbital, and Di(2-ethylhexyl) phthalate. Toxicol. Appl. Pharmacol. 100: 217-226, 1989. Kontozoglou, T.E., Banerjee, D., and Cherian, M.G. Immunohistochemical localization of metallothionein in human testicular embryonal carcinoma cells. Virchows Archiv. A Pathol. Anat. 415: 545-549, 1989. Muller, T., Schuckelt, R., and Jaenicke, L. Cadmium/zinc-metallothionein induces DNA strand breaks in vitro. Arch. Toxicol. 65: 20-26, 1991. Huang, P.C., Cody, C., Cismowski, M., Chernaik, M., Rhee, I.K., and Lin, L.Y. Native and engineered metallothioneins. In: C. Klaassen, and K. T. Suzuki (eds.), Metallothionein in Medicine and Biology, pp. 87-101. Florida: CRC Press, 1991. Zeng, J., Vallee, B.L., and Kagi, J.H.R. Zinc transfer from transcription factor IIIA fingers to thionein clusters. Proc. Natl. Acad. Sci. USA 88: 9984-9988, 1991. Waalkes, M.P., Rehm, S., Riggs, C.W., Bare, R.M., Devor, D.E., Poirier, L.A., Wenk, M.L., Henneman, J.R., and Balaschak, M.S. Cadmium carcinogenesis in male Wistar[Crl:(W1)BR]rats: Dose-response analysis of tumor induction in the prostate and testes and at the injection site. Cancer Research 48: 4656-4663, 1988. Kelley, S.L., Basu, A., Teicher, B.A., Hacker, M.P., Hamer, D.H., and Lazo, J.S. Overexpression of metallothionein confers resistance to anticancer drugs. Science 241: 1813-1815, 1988. Kasahara, K., Fujiwara, Y., Nishio, K., Ohmori, T., Sugimoto, Y., Komiya, K., Matsuda, T., and Saijo, N. Metallothionein content correlates with the sensitivity of human small cell lung cancer cells lines to cisplatin. Cancer Research 51: 3237-3242, 1991. .
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