DIET, DNA METHYLATION AND OTHER EPIGENETIC EVENTS, AND CANCER PREVENTION RELEASE DATE: September 27, 2002 RFA: CA-03-016 National Cancer Institute (NCI) (http://www.nci.nih.gov/) The Office of Dietary Supplements (ODS) (http://www.nih.gov/icd/od) LETTER OF INTENT RECEIPT DATE: February 18, 2003 APPLICATION RECEIPT DATE: March 18, 2003 THIS RFA CONTAINS THE FOLLOWING INFORMATION o Purpose of this RFA o Research Objectives o Mechanism of Support o Funds Available o Eligible Institutions o Individuals Eligible to Become Principal Investigators o Where to Send Inquiries o Letter of Intent o Submitting an Application o Peer Review Process o Review Criteria o Receipt and Review Schedule o Award Criteria o Required Federal Citations PURPOSE The NCI and the ODS invite applications for new R01 and R21 grants which are focused on research leading to the elucidation of mechanism(s) by which dietary factors influence epigenetic processes as well as increasing the understanding of these processes in cancer prevention. The approach is to encourage collaboration between nutrition and epigenetic /DNA methylation experts to study bioactive food components with cancer preventative properties, and to examine key epigenetic events in cancer processes (i.e., carcinogen metabolism, cell division, differentiation, apoptosis) so that investigators can begin to establish linkages between epigenetics, methylation pattern, and tumor incidence/behavior. It is anticipated that the information gained will provide guidance for the development of dietary intervention strategies for cancer prevention. In addition to the present announcement, the NCI and the ODS announce a related initiative about diet, DNA methylation and other epigenetic events, and cancer prevention utilizing an additional funding mechanism: Competing Supplements for existing NCI awardees (http://grants.nih.gov/grants/guide/pa-files/PAR-02-175.html) which can extend the goals of active grants to include studies related to the impact of diet and nutrition on epigenetic events. Contact individuals listed under "INQUIRIES" of this RFA for further information. RESEARCH OBJECTIVES Background Cancer is a manifestation of both abnormal genetic and epigenetic events. The importance of epigenetic events is that it represents a mechanism by which gene function is selectively activated or inactivated. Actually DNA methylation is the covalent addition of a methyl group to the 5 position of cytosine within CpG dinucleotides and is a fundamental process that not only modulates gene expression, but is also key to regulating chromosomal stability. A variety of regulatory proteins including DNA methyltransferases, methyl-CpG binding proteins, histone- modifying enzymes, chromatin remodeling factors, and their multimolecular complexes are involved in the overall epigenetic process. Since epigenetic events are susceptible to change they represent excellent targets to explain how environmental factors, including diet, may modify cancer risk and tumor behavior. Abnormal DNA methylation patterns are a hallmark of most cancers, including those of high proportion in the United States i.e., colon, lung, prostate, and breast cancer. Preclinical and clinical studies provide intriguing evidence that part of the anticancer properties attributed to several bioactive food components, encompassing both essential and non-essential nutrients, may relate to DNA methylation patterns. There are four ways in which nutrients may be interrelated with DNA methylation. The first is that nutrients may influence the supply of methyl groups for the formation of S-adenosylmethionine (SAM). The second mechanism is that nutrients may modify utilization of methyl groups by processes including shifts in DNA methyltransferase activity. A third plausible mechanism may relate to DNA demethylation activity. Finally, the DNA methylation patterns may influence the response to a nutrient. These interactions are described in greater detail below. Global DNA Methylation Patterns Global hypomethylation, accompanied by region-specific hypermethylation, is a common characteristic among tumor cells. Global genomic hypomethylation has been linked to the induction of chromosomal instability. Hypermethylation is associated with the inactivation of virtually all pathways involved with the cancer process, including DNA repair (e.g., hMLH1, BRCA1, MGMT), cell cycle regulation (e.g., p16(INK4a), PTEN), inflammatory/stress response (e.g., COX- 2) and apoptosis (e.g., DAPK, APAF-1). Thus, evidence exists that variations in the degree or site of DNA methylation can lead to abnormal DNA repair and influence multiple cancer related genes and thereby influence the incidence and behavior of tumors. Stress including that resulting from dietary methionine/choline, folate, zinc, and selenium inadequacy, as well as excessive alcohol intake can lead to global DNA hypomethylation. Interestingly, the continuous feeding of a diet deficient in choline and methionine is recognized to lead to global DNA hypomethylation and cause hepatocellular carcinomas in rats in the absence of any exogenous carcinogen. Paradoxically, either deficient or excess dietary arsenic has also been shown to lead to global hypomethylation in rat liver. Treatment with excess retinoic acid can also lead to hypomethylation as recently shown in rat liver. Clinically, global DNA hypomethylation has been observed in lymphocytic DNA from individuals consuming inadequate folate. While limitations in the supply of methyl groups appear to be a common mechanism, available data suggest that other factors determining DNA methylation, including DNA methyltransferase (Dnmt), may be influenced by bioactive food components. Availability and Utilization of Methyl Groups SAM and S-adenosylhomocysteine (SAH), as components of methyl metabolism, are the substrate and product of essential cellular methyltransferase reactions. SAM is derived from an ATP-dependent transfer of adenosine to methionine via methionine adenosyltransferase and serves as the proximal methyl donor for most methylation reactions. Cellular methyl acceptors include phospholipids, proteins, histones, neurotransmitters, RNA and DNA. The formation of SAM requires a continuous supply of folate, methionine, choline, vitamin B12, vitamin B6, vitamin B2 and energy from the extracellular milieu. Polymorphisms in the methyl metabolism genes methionine synthase, methylenetetrahydrofolate reductase (MTHFR), and cystathione beta-synthetase affect concentrations of SAM and likely modify the susceptibility to cancer. Investigators are currently exploring the interaction between these polymorphisms, diet and DNA methylation. For example, subjects homozygous for the C677T polymorphism in the MTHFR gene exhibited a significantly lower level of methylated DNA but only under conditions of low folate status. The Dnmts are a family of enzymes that catalyze the transfer of methyl groups from SAM to cytosine residues in DNA. This produces 5-methylcytosine and SAH. Higher DNA methyltransferase activity has been observed in tumor cells compared to normal cells. Chronic dietary methyl deficiency also increases DNA methyltransferase activity, which may be an attempt to compensate for diminished SAM supply. Nutrient supply, therefore, appears to play a key role in regulating DNA methyltransferase activity. Selenium is a dietary trace element that is recognized as having potential anticancer properties. Interest in selenium as a prostate cancer preventative nutrient is showcased by the recent initiation of the SELECT trial by NCI (http://newscenter.cancer.gov/pressreleases/SELECT.html). While selenium is known to modify several aspects of the cancer process, the one that is most critical for bringing about a phenotypic change remains to be established. Interestingly, increased selenium concentrations have been found to inhibit Dnmt1 activity in vitro and decrease Dnmt1 protein expression in vitro. Consistent with these data, selenium deficiency leads to increased Dnmt1 protein expression in vitro. The effects of selenium on Dnmt activity suggest that dietary factors influencing methyl utilization may also modify DNA methylation patterns. Although the enzymes for direct methylation of DNA are well characterized, the demethylation process has been assumed to be a passive process. A mammalian gene that codes for a protein that can catalyze demethylation recently has been described, however other attempts have yielded variable results. Demethylation activity has been hypothesized to be higher in non-neoplastic compared to neoplastic cells. Loss of demethylation activity may account for some of the differences in methylation patterns between neoplastic and normal tissue. The role, if any, of bioactive food components in regulating the demethylation process remains to be established. Gene-Specific DNA Methylation Patterns Fluctuations in the degree of CpG island methylation are key to regulating functional promoters by modifying the binding of transcription factors and methyl-DNA binding proteins. Aberrant methylation of CpG islands on the promoter region may contribute to the progressive inactivation of growth- inhibitory genes resulting in the clonal selection of cells with growth advantage during cancer development. While methyl-deficiency leads to global DNA hypomethylation, it also simultaneously leads to gene-specific hypomethylation and hypermethylation. Preclinical studies reveal that consumption of a methyl deficient diet leads to hypomethylation of specific CpG sites within several genes including c-myc, c-fos and c-H-ras. This hypomethylation was accompanied by elevated levels of mRNA for these same genes. Folate depletion was shown to produce gene- specific, rather than global DNA hypomethylation in human nasopharyngeal carcinoma KB cells. Interestingly, these investigators also reported that folate depletion led to hypermethylation of a 5" sequence of the H-cadherin gene, which accompanied diminished mRNA expression. These data suggest that global DNA hypomethylation may incompletely predict the response to an individual dietary component. More probing studies are needed to characterize gene-specific changes brought about by bioactive food components. Epigenetic events occurring in utero can lead to persistent changes in gene expression and can be modified by the diet. For example, the diet provided to female mice of two strains during pregnancy modified the offsprings" hair coat color (increased agouti vs yellow) and DNA methylation patterns. Expression of the yellow hair color in these mice is recognized to be controlled by hypomethylation of the agouti long terminal repeat (LTR), which was hypermethylated by dietary supplementation of the maternal diet with zinc, methionine, betaine, choline, folate and vitamin B12. Expression of this yellow coat color is linked with increased risk of obesity, diabetes, and cancer. While long-term health implications remain to be determined, these studies demonstrate that feeding a methyl-supplemented diet increased levels of DNA methylation in the agouti LTR and increased the proportion of agouti to yellow mice. It should be noted that this effect occurred with control diets that are considered adequate for meeting nutritional needs. These data point to the likelihood that in utero nutrient exposures can lead to genomic imprinting in the offspring and potentially modify cancer risk. Invariably, increased fruit and vegetable consumption is associated with reduction in cancer risk. Evidence from a variety of sources suggests that flavonoids, including genistein may have merit as an effective deterrent of cancer. Dietary genistein has been shown to lead to changes in DNA methylation patterns in prostate tissue of C57BL/6J mice. After sequencing, it was determined that genistein consumption led to hypermethylation at 4 specific CpG islands, including a dexamethasone-induced product gene (D44443). Neonatal exposure to the phytoestrogens coumestrol and equol has been found to lead to specific gene hypermethylation in the c-H-ras proto-oncogene in pancreatic DNA. Data indicating that consumption of high fiber diets is accompanied by a reduction in estrogen receptor methylation in colon tissue from healthy subjects has also been noted. Hypermethylation of the promoter CpG islands is recognized to contribute to the loss of function of several tumor related genes, including estrogen receptor (ER). Overall, several studies illustrate that bioactive food components other than essential nutrients can influence DNA methylation processes. Cell Responsiveness to DNA Methylation Defects The ability of bioactive food components to reduce proliferation in normal cells has been reported to be less than in neoplastic cells. For example, selenium preferentially induces growth inhibition and apoptosis in prostate cancer cells, but not in normal prostate cells. Recently, the flavonoid apigenin has also shown selective growth inhibition in prostate cancer cells without affecting normal cells. It is unclear if these differences in sensitivity relate to epigenetic and DNA methylation processes. The gene encoding the pi class glutathione S-transferase (GSTP1) is known to be silenced by CpG island methylation in prostate cancer cells. It has been suggested that compensation for loss of GSTP1 activity is possible with inducers of general glutathione S-transferase activity and one such inducer is the isothiocyanate compound sulforophane found in cruciferous vegetables. That dietary factors might circumvent changes induced by aberrant DNA methylation patterns in cancer requires investigation. The short-chain fatty acid butyrate induces cell cycle arrest, differentiation and apoptosis in colon cancer cells, but often induces opposite effects in normal colonic epithelial cells. A stable transgenic cell line containing a lacZ reporter gene coupled with a hormone-inducible promoter was examined for reversal of methylation-mediated gene silencing. In the methylated form, the promoter is unable to induce lacZ expression and is insensitive to reactivation by the histone deacetylase inhibitors trichostatin A and suberoylanilide hydroxamic acid. In contrast, the short-chain fatty acids (including proprionic, butyric and valeric acid) were capable of overcoming methylation-mediated repression and restored lacZ expression to levels approaching that from an unmethylated promoter. Retinoic acid provides an example that some nutrients may also restore methylation patterns and gene expression. RA demethylated the retinoic acid receptor beta2 promoter in acute promyeocytic leukemia cells. This led to cellular differentiation and accumulation of retinoic acid receptor beta2 mRNA in these cells. It is unknown whether other bioactive food components also lead to DNA demethylation. Sensitizing tumor cells to the bioactive food component can also be accomplished by using demethylating agents, such as 5-aza-2"-deoxycytidine (5- aza-dC). This compound has been used as a therapeutic agent for certain cancers, although treatment-associated side effects preclude its general usefulness. Treatment of several cell lines with MLL (mixed-lineage leukemia or myeloid/lymphoid leukemia) translocations with 5-aza-dC increases the ability of all-trans retinoic acid or 1,25-dihydroxyvitamin D3 to cause differentiation. This study suggests that methylation patterns can influence the response to bioactive food components. These are intriguing results, but we need more. Other Epigenetic Events Emerging evidence indicates that various chromatin states such as histone modifications (acetylation and methylation) and nucleosome positioning (modulated by ATP-dependent chromatin remodeling machines) determine DNA methylation patterning. Additionally, various regulatory factors interacting with the DNA methyltransferases may direct them to specific DNA sequences, regulate their enzymatic activity, and allow their use as transcriptional repressors. Continued studies of the connections between DNA methylation and chromatin structure and the DNA methyltransferase-associated proteins, will likely reveal that many epigenetic modifications of the genome are directly connected. DNA hypermethylation is not likely to be an isolated layer of epigenetic control, but is likely to be linked to other pieces of the puzzle such as methyl-binding proteins, DNA methyltransferases and histone deacetylase. Our understanding of the degree of specificity of these epigenetic layers in the control of gene function remains incomplete. Histone modifications have recently generated excitement in epigenetic research, culminating in the `histone code" hypothesis. The idea is that the modified histone tails provide binding sites for chromatin-associated proteins, which in turn induce alterations in chromatin structure and other downstream events. In particular, for silent chromatin domains, histone H3 lysine 9 methylation by the Su(var)3-9 family of histone methyltransferases has been shown to play a key part in the formation of transcriptionally repressed chromatin by providing a high-affinity binding site for the chromodomain of the heterochromatic HP1 proteins. Recent evidence suggests that histone methylation has considerable epigenetic regulatory impact and is likely influenced by dietary methyl group sufficiency. Moreover, the recent finding that some histone methyltransferases function as tumor suppressors provides another rationale for continued exploration of the diet and epigenetic relationship in cancer prevention research. Acetylation of core histone tails is a conserved mechanism modulating gene expression. It is able to relax higher order chromatin, to promote factor binding to DNA and to recruit unique biological complexes that mediate downstream functions. A switch from inactive to active chromatin is often accompanied by histone hyperacetylation of critical sites in gene regulatory regions. The antagonistic activities of histone acetyltransferases and histone deacetylases control the nuclear steady-state balance of this covalent modification. In addition to any effect on methylation patterns, the short- chain fatty acid butyrate has been shown to induce histone hyperacetylation in vitro and inhibit histone deacetylase in vitro. Recent evidence demonstrates that butyrate promotes hyperacetylation of the upstream region of the RET gene (a proto-oncogene which encodes a tyrosine kinase receptor predominantly expressed in the developing embryo) and increases transcription of the gene. More probing studies are needed to characterize additional gene-specific acetylation changes brought about by bioactive food components. Calorie restriction is one of the best-documented and most potent experimental manipulations for decreasing tumor development in rodents and increasing longevity in diverse organisms. The mechanisms underlying the antitumorigenic effects of caloric restriction have not yet been elucidated. Nevertheless, there is great interest in translating the anticarcinogenic effects of caloric restriction into prevention strategies. Recent observations suggest that a protein important for heterochromatin formation, Sir2, is central for caloric- restriction induced longevity in animals. Any role for Sir2 in cancer prevention requires elucidation. Objectives and Scope This RFA seeks to promote innovative, preclinical and clinical research to determine how diet and dietary factors impact DNA methylation and other epigenetic processes involved with cancer prevention. Although much evidence exists that dietary components are linked to cancer prevention, the specific nutrients and sites of action remain elusive. Diet, in fact, has been implicated in many of the pathways of cancer, including apoptosis, cell cycle control, differentiation, inflammation, angiogenesis, DNA repair and carcinogen metabolism. These are also processes that are likely regulated by DNA methylation, and possibly other epigenetic events, which impact gene function. The objective of this RFA is to begin to address the following issues: how bioactive food components regulate DNA methylation or other epigenetic events for cancer prevention, if bioactive food components can alter DNA methylation or other aberrant epigenetic events and restore gene function, and if these components can circumvent or compensate for genes and pathways that are altered by epigenetic events. An important aim of this RFA is to encourage collaboration between nutrition and epigenetic/ DNA methylation experts to study bioactive food components with cancer preventative properties and to examine key epigenetic events in cancer processes (i.e., carcinogen metabolism, cell division, differentiation, apoptosis) in order to begin to establish linkages between epigenetics, methylation pattern, and tumor incidence/behavior. Thus, each application should demonstrate experience in nutrition and cancer prevention as well as DNA methylation or epigenetics. Studies should also link phenotypic changes to epigenetic alterations induced by specific essential and non-essential nutrients. The resulting information will be critical for optimizing effective dietary intervention strategies for cancer prevention. Investigators may choose from the full range of clinical and preclinical approaches. The focus should be on how individual dietary components influence DNA methylation and other epigenetic events and how this correlates with phenotypic change. Very little information currently exists about gene-specific changes in DNA methylation as influenced by bioactive food components, furthermore, very little information exists to adequately evaluate the specificity of individual nutrients, the impact of intakes/exposures, and any acclimation with time and/or tissue specificity. This RFA encourages the submission of novel approaches to unravel relationships between DNA methylation and other epigenetic events, diet, and cancer prevention. A variety of technologies to assess DNA methylation sequences may be utilized. These can be broadly classified into techniques that measure the overall content or distribution patterns of 5-methylcytosine (i.e., methylated CpG island amplification, methylation-sensitive restriction fingerprinting, differential methylation hybridization, and Restriction Landmark Genomic Scanning) and those that examine known gene sequences (i.e., methylation- sensitive single nucleotide primer extension, and combined bisulfite restriction analysis). The use of genetically engineered animal models including transgenic or gene knockouts are appropriate. The efficient utilization of molecular resources such as gene databases and bioinformatics may also be used to expedite identification of gene-specific methylation targets. The following are viewed as relevant examples for the development of the R01 and R21 applications: o Gene-specific changes in DNA methylation caused by excesses and limitations in bioactive food components. o Relationship between dietary induced global DNA hypomethylation and gene- specific hypermethylation. o Relationship between diet induced changes in DNA methylation and histone acetylation/methylation and control of gene function o Temporality in DNA methylation patterns as influenced by bioactive food components. o Bioactive food component regulation of DNA methylation/epigenetic processes, i.e., methylenetetrahydrofolate reductase, methionine synthase, DNA methyltransferases, demethylases (or various demethylation processes), methylcytosine-binding proteins, histone methyltransferases, histone aceyltransferases and deacetylases. o Linkages between DNA methylation or other epigenetic pattern and the anticancer properties of bioactive food components. MECHANISM OF SUPPORT This RFA will use the National Institutes of Health (NIH) research project grant (R01) and exploratory/developmental grants (R21) as award mechanisms. As an applicant you will be solely responsible for planning, directing, and executing the proposed project. Future unsolicited, competing-continuation applications based on this project will compete with all investigator- initiated applications and will be reviewed according to the customary peer review procedures. The anticipated award date is December 2003. This RFA uses just-in-time concepts. It also uses the modular as well as the non-modular budgeting formats (see http://grants.nih.gov/grants/funding/modular/modular.htm). Specifically, if you are submitting an application with direct costs in each year of $250,000 or less, use the modular format. Otherwise follow the instructions for non- modular research grant applications. FUNDS AVAILABLE NCI intends to commit approximately $2.5 million in FY 2004 to fund 7 to 10 grants in response to this RFA. The ODS intends to commit up to $800,000 in FY 2004 to fund 3 to 4 grants in response to this RFA. An R01 applicant may request a project period of up to 4 years. An R21 applicant may request a project period of up to 2 years and a budget for direct costs of up to $100,000 per year. Because the nature and scope of the proposed research will vary from application to application, it is anticipated that the size and duration of each award will also vary. Although the financial plans of NCI and the ODS provide support for this program, awards pursuant to this RFA are contingent upon the availability of funds and the receipt of a sufficient number of meritorious applications. At this time, it is not known if this RFA will be reissued. ELIGIBLE INSTITUTIONS You may submit (an) application(s) if your institution has any of the following characteristics: o For-profit or non-profit organizations o Public or private institutions, such as universities, colleges, hospitals, and laboratories o Units of State and local governments o Eligible agencies of the Federal government o Domestic or foreign o Faith-based or community based organizations INDIVIDUALS ELIGIBLE TO BECOME PRINCIPAL INVESTIGATORS Any individual with the skills, knowledge, and resources necessary to carry out the proposed research is invited to work with their institution to develop an application for support. Individuals from underrepresented racial and ethnic groups as well as individuals with disabilities are always encouraged to apply for NIH programs. WHERE TO SEND INQUIRIES We encourage inquiries concerning this RFA and welcome the opportunity to answer questions from potential applicants. Inquiries may fall into three areas: scientific/research, peer review, and financial or grants management issues: o Direct your questions about scientific/research issues related to diet and cancer prevention to: Dr. Sharon A. Ross Division of Cancer Prevention National Cancer Institute 6130 Executive Blvd., Room 3157 Bethesda, MD 20892 Telephone: (301) 594-7547 FAX: (301) 480-3925 Email: sr75k@nih.gov o Direct your questions about scientific/research issues related to dietary supplements to: Rebecca B. Costello, Ph.D. Office of Dietary Supplements Office of Disease Prevention, Office of the Director National Institutes of Health 6100 Executive Blvd., Room 3B01, MSC 7517 Bethesda, Maryland 20892-7517 Phone: 301-435-2920 Fax: 301-480-1845 Email: CostellB@od.nih.gov o Direct your questions about peer review issues to: Referral Officer National Cancer Institute Division of Extramural Activities 6116 Executive Boulevard, Room 8041, MSC 8329 Bethesda, MD 20892-8329 Telephone: (301) 496-3428 FAX: (301) 402-0275 Email: ncidearefof@mail.nih.gov o Direct your questions about financial or grants management matters to: Jane Paull Grants Administration Branch National Cancer Institute 6120 Executive Blvd., EPS-243 Bethesda, MD 20892 Telephone: (301) 496-2182 FAX: (301) 496-8601 Email: paullj.gab.nci.nih.gov LETTER OF INTENT Prospective applicants are asked to submit a letter of intent that includes the following information: o Descriptive title of the proposed research o Name, address, and telephone number of the Principal Investigator o Names of other key personnel o Participating institutions o Number and title of this RFA Although a letter of intent is not required, is not binding, and does not enter into the review of a subsequent application, the information that it contains allows NCI staff to estimate the potential review workload and plan the review. The letter of intent is to be sent by the date listed at the beginning of this document. The letter of intent should be sent to: Dr. Sharon A. Ross Division of Cancer Prevention National Cancer Institute 6130 Executive Blvd., Room 3157 Bethesda, MD 20892 Telephone: (301) 594-7547 FAX: (301) 480-3925 Email: sr75k@nih.gov SUBMITTING AN APPLICATION Applications must be prepared using the PHS 398 research grant application instructions and forms (rev. 5/2001). The PHS 398 is available at http://grants.nih.gov/grants/funding/phs398/phs398.html in an interactive format. For further assistance contact GrantsInfo, Telephone (301) 435-0714, Email: GrantsInfo@nih.gov. SPECIFIC INSTRUCTIONS FOR MODULAR GRANT APPLICATIONS: Applications requesting up to $250,000 per year in direct costs must be submitted in a modular grant format. The modular grant format simplifies the preparation of the budget in these applications by limiting the level of budgetary detail. Applicants request direct costs in $25,000 modules. Section C of the research grant application instructions for the PHS 398 (rev. 5/2001) at http://grants.nih.gov/grants/funding/phs398/phs398.html includes step-by-step guidance for preparing modular grants. Additional information on modular grants is available at http://grants.nih.gov/grants/funding/modular/modular.htm. USING THE RFA LABEL: The RFA label available in the PHS 398 (rev. 5/2001) application form must be affixed to the bottom of the face page of the application. Type the RFA number on the label. 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 title and number must be typed on line 2 of the face page of the application form and the YES box must be marked. The RFA label is also available at: http://grants.nih.gov/grants/funding/phs398/label-bk.pdf. All application instructions outlined in the PHS 398 application kit are to be followed, with the following requirements for R21 applications: 1. R21 applications will use the "MODULAR GRANT" and "JUST-IN-TIME" concepts, with direct costs requested in $25,000 modules, up to the total direct costs limit of $100,000 per year. 2. Although preliminary data are not required for an R21 application, they may be included. 3. Sections a-d of the Research Plan of the R21 application may not exceed 25 pages, including tables and figures. 4. R21 appendix materials should be limited, as is consistent with the exploratory nature of the R21 mechanism, and should not be used to circumvent the page limit for the research plan. Copies of appendix material will only be provided to the primary reviewers of the application and will not be reproduced for wider distribution. The following materials may be included in the appendix: o Up to five publications, including manuscripts (submitted or accepted for publication), abstracts, patents, or other printed materials directly relevant to the project. These may be stapled as sets. Surveys, questionnaires, data collection instruments, and clinical protocols. These may be stapled as sets. Original glossy photographs or color images of gels, micrographs, etc., provided that a photocopy (may be reduced in size) is also included within the 25 page limit of items a-d of the research plan. SENDING AN APPLICATION TO THE NIH: Submit a signed, typewritten original of the application, including the Checklist, and three signed, photocopies, in one package to: Center for Scientific Review National Institutes of Health 6701 Rockledge Drive, Room 1040, MSC 7710 Bethesda, MD 20892-7710 Bethesda, MD 20817 (for express/courier service) At the time of submission, two additional copies of the application must be sent to: Referral Officer Division of Extramural Activities National Cancer Institute 6116 Executive Blvd., Room 8041, MSC-8329 Rockville, MD 20852 (express courier) Bethesda MD 20892-8329 APPLICATIONS HAND-DELIVERED BY INDIVIDUALS TO THE NATIONAL CANCER INSTITUTE WILL NO LONGER BE ACCEPTED. This policy does not apply to courier deliveries (i.e. FEDEX, UPS, DHL, etc.) (http://grants.nih.gov/grants/guide/notice-files/NOT-CA-02-002.html) This policy is similar to and consistent with the policy for applications addressed to Centers for Scientific Review as published in the NIH Guide Notice http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-012.html. APPLICATION PROCESSING: Applications must be received by the application receipt date listed in the heading of this RFA. If an application is received after that date, it will be returned to the applicant without review. The Center for Scientific Review (CSR) will not accept any application in response to this RFA that is essentially the same as one currently pending initial review, unless the applicant withdraws the pending application. The CSR will not accept any application that is essentially the same as one already reviewed. This does not preclude the submission of substantial revisions of applications already reviewed, but such applications must include an Introduction addressing the previous critique. PEER REVIEW PROCESS Upon receipt, applications will be reviewed for completeness by the CSR and for responsiveness by the NCI and ODS program staff. Incomplete applications will be returned to the applicant without further consideration. And, if the application is not responsive to the RFA, CSR staff may contact the applicant to determine whether to return the application to the applicant or submit it for review in competition with unsolicited applications at the next appropriate NIH review cycle. Applications that are complete and responsive to the RFA will be evaluated for scientific and technical merit by an appropriate peer review group convened by the Division of Extramural Activities (DEA) at NCI in accordance with the review criteria stated below. As part of the initial merit review, all applications will: o Receive a written critique o Undergo a process in which only those applications deemed to have the highest scientific merit, generally the top half of the applications under review, will be discussed and assigned a priority score o Those that receive a priority score will undergo a second level review by the National Cancer Advisory Board. REVIEW CRITERIA The goals of NIH-supported research are to advance our understanding of biological systems, improve the control of disease, and enhance health. In the written comments, reviewers will be asked to discuss the following aspects of your application in order to judge the likelihood that the proposed research will have a substantial impact on the pursuit of these goals: o Significance o Approach o Innovation o Investigator o Environment The scientific review group will address and consider each of these criteria in assigning your application"s overall score, weighting them as appropriate for each application. Your application does not need to be strong in all categories to be judged likely to have major scientific impact and thus deserve a high priority score. For example, you may propose to carry out important work that by its nature is not innovative but is essential to move a field forward. (1) SIGNIFICANCE: Does this study address an important question about the role of (a) bioactive food components in DNA methylation or other epigenetic event involved with cell vulnerability to cancer or cellular responsiveness to cancer prevention? Do studies focus on how dietary components influence DNA methylation or other epigenetic event and how this correlates with phenotypic change. Will these research projects advance the field of nutrition and cancer prevention from observational to more probing studies? (2) APPROACH: Are the conceptual framework, design, methods, and analyses adequately developed, well integrated, and appropriate to the aims of the project? Does the applicant acknowledge potential problem areas and consider alternative tactics? (3) INNOVATION: Does the project employ novel concepts, approaches or methods? Are the aims original and innovative? Does the project challenge existing paradigms or develop new methodologies or technologies? (4) INVESTIGATOR: Is the investigator appropriately trained and well suited to carry out this work? Is experience in nutrition and cancer prevention as well as DNA methylation or epigenetics demonstrated in the application? Is the work proposed appropriate to the experience level of the principal investigator and other researchers (if any)? (5) ENVIRONMENT: Does the scientific environment in which the work will be done contribute to the probability of success? Do the proposed experiments take advantage of unique features of the scientific environment or employ useful collaborative arrangements (i.e., between nutrition and epigenetic /DNA methylation experts)? Is there evidence of institutional support? ADDITIONAL REVIEW CRITERIA: In addition to the above criteria, your application will also be reviewed with respect to the following: o PROTECTIONS: The adequacy of the proposed protection for humans, animals, or the environment, to the extent they may be adversely affected by the project proposed in the application. o INCLUSION: The adequacy of plans to include subjects from both genders, all racial and ethnic groups (and subgroups), and children as appropriate for the scientific goals of the research. Plans for the recruitment and retention of subjects will also be evaluated. (See Inclusion Criteria included in the section on Federal Citations, below) o DATA SHARING: The adequacy of the proposed plan to share data. o BUDGET: The reasonableness of the proposed budget and the requested period of support in relation to the proposed research. RECEIPT AND REVIEW SCHEDULE Letter of Intent Receipt Date: February 18, 2003 Application Receipt Date: March 18, 2003 Peer Review Date: June-July, 2003 Council Review: September 2003 Earliest Anticipated Start Date: December 2003 AWARD CRITERIA Award criteria that will be used to make award decisions include: o Scientific merit (as determined by peer review) o Availability of funds o Programmatic priorities. REQUIRED FEDERAL CITATIONS MONITORING PLAN AND DATA SAFETY AND MONITORING BOARD: Research components involving Phase I and II clinical trials must include provisions for assessment of patient eligibility and status, rigorous data management, quality assurance, and auditing procedures. In addition, it is NIH policy that all clinical trials require data and safety monitoring, with the method and degree of monitoring being commensurate with the risks (NIH Policy for Data Safety and Monitoring, NIH Guide for Grants and Contracts, June 12, 1998: http://grants.nih.gov/grants/guide/notice-files/not98-084.html). Clinical trials supported or performed by NCI require special considerations. The method and degree of monitoring should be commensurate with the degree of risk involved in participation and the size and complexity of the clinical trial. Monitoring exists on a continuum from monitoring by the principal investigator/project manager or NCI program staff or a Data and Safety Monitoring Board (DSMB). These monitoring activities are distinct from the requirement for study review and approval by an Institutional review Board (IRB). For details about the Policy for the NCI for Data and Safety Monitoring of Clinical trials see: http://deainfo.nci.nih.gov/grantspolicies/datasafety.htm. For Phase I and II clinical trials, investigators must submit a general description of the data and safety monitoring plan as part of the research application. See NIH Guide Notice on "Further Guidance on a Data and Safety Monitoring for Phase I and II Trials" for additional information: http://grants.nih.gov/grants/guide/notice-files/NOT-OD-00-038.html. Information concerning essential elements of data safety monitoring plans for clinical trials funded by the NCI is available: http://www.cancer.gov/clinical_trials/ INCLUSION OF WOMEN AND MINORITIES IN CLINICAL RESEARCH: It is the policy of the NIH that women and members of minority groups and their sub-populations must be included in all NIH-supported clinical research projects unless a clear and compelling justification is provided indicating that inclusion is inappropriate with respect to the health of the subjects or the purpose of the research. This policy results from the NIH Revitalization Act of 1993 (Section 492B of Public Law 103-43). All investigators proposing clinical research should read the AMENDMENT "NIH Guidelines for Inclusion of Women and Minorities as Subjects in Clinical Research - Amended, October, 2001," published in the NIH Guide for Grants and Contracts on October 9, 2001 (http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-001.html), a complete copy of the updated Guidelines are available at http://grants.nih.gov/grants/funding/women_min/guidelines_amended_10_2001.htm. The amended policy incorporates: the use of an NIH definition of clinical research, updated racial and ethnic categories in compliance with the new OMB standards, clarification of language governing NIH-defined Phase III clinical trials consistent with the new PHS Form 398, and updated roles and responsibilities of NIH staff and the extramural community. The policy continues to require for all NIH-defined Phase III clinical trials that: a) all applications or proposals and/or protocols must provide a description of plans to conduct analyses, as appropriate, to address differences by sex/gender and/or racial/ethnic groups, including subgroups if applicable, and b) investigators must report annual accrual and progress in conducting analyses, as appropriate, by sex/gender and/or racial/ethnic group differences. INCLUSION OF CHILDREN AS PARTICIPANTS IN RESEARCH INVOLVING HUMAN SUBJECTS: The NIH maintains a policy that children (i.e., individuals under the age of 21) must be included in all human subjects research, conducted or supported by the NIH, unless there are scientific and ethical reasons not to include them. This policy applies to all initial (Type 1) applications submitted for receipt dates after October 1, 1998. All investigators proposing research involving human subjects should read the "NIH Policy and Guidelines" on the inclusion of children as participants in research involving human subjects that is available at http://grants.nih.gov/grants/funding/children/children.htm. REQUIRED EDUCATION ON THE PROTECTION OF HUMAN SUBJECT PARTICIPANTS: NIH policy requires education on the protection of human subject participants for all investigators submitting NIH proposals for research involving human subjects. You will find this policy announcement in the NIH Guide for Grants and Contracts Announcement, dated June 5, 2000, at http://grants.nih.gov/grants/guide/notice-files/NOT-OD-00-039.html. A continuing education program in the protection of human participants in research in now available online at: http://cme.nci.nih.gov/ HUMAN EMBRYONIC STEM CELLS (hESC): Criteria for federal funding of research on hESCs can be found at http://grants.nih.gov/grants/stem_cells.htm and at http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-005.html. Guidance for investigators and institutional review boards regarding research involving human embryonic stem cells, germ cells, and stem cell-derived test articles can be found at http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-044.html. Only research using hESC lines that are registered in the NIH Human Embryonic Stem Cell Registry will be eligible for Federal funding (see http://escr.nih.gov). It is the responsibility of the applicant to provide the official NIH identifier(s)for the hESC line(s) to be used in the proposed research. Applications that do not provide this information will be returned without review. PUBLIC ACCESS TO RESEARCH DATA THROUGH THE FREEDOM OF INFORMATION ACT: The Office of Management and Budget (OMB) Circular A-110 has been revised to provide public access to research data through the Freedom of Information Act (FOIA) under some circumstances. Data that are (1) first produced in a project that is supported in whole or in part with Federal funds and (2) cited publicly and officially by a Federal agency in support of an action that has the force and effect of law (i.e., a regulation) may be accessed through FOIA. It is important for applicants to understand the basic scope of this amendment. NIH has provided guidance at http://grants.nih.gov/grants/policy/a110/a110_guidance_dec1999.htm. Applicants may wish to place data collected under this RFA in a public archive, which can provide protections for the data and manage the distribution for an indefinite period of time. If so, the application should include a description of the archiving plan in the study design and include information about this in the budget justification section of the application. In addition, applicants should think about how to structure informed consent statements and other human subjects procedures given the potential for wider use of data collected under this award. URLs IN NIH GRANT APPLICATIONS OR APPENDICES: All applications and proposals for NIH funding must be self-contained within specified page limitations. Unless otherwise specified in an NIH solicitation, Internet addresses (URLs) should not be used to provide information necessary to the review because reviewers are under no obligation to view the Internet sites. Furthermore, we caution reviewers that their anonymity may be compromised when they directly access an Internet site. HEALTHY PEOPLE 2010: The Public Health Service (PHS) is committed to achieving the health promotion and disease prevention objectives of "Healthy People 2010," a PHS-led national activity for setting priority areas. This RFA is related to one or more of the priority areas. Potential applicants may obtain a copy of "Healthy People 2010" at http://www.health.gov/healthypeople/ AUTHORITY AND REGULATIONS: This program is described in the Catalog of Federal Domestic Assistance No. 93.3393 (Cancer Cause and Prevention Research) and is not subject to the intergovernmental review requirements of Executive Order 12372 or Health Systems Agency review. Awards are made under authorization of Sections 301 and 405 of the Public Health Service Act as amended (42 USC 241 and 284) and administered under NIH grants policies described at http://grants.nih.gov/grants/policy/policy.htm and under Federal Regulations 42 CFR 52 and 45 CFR Parts 74 and 92. The PHS strongly encourages all grant recipients to provide a smoke-free workplace and discourage the use of all tobacco products. In addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain facilities (or in some cases, any portion of a facility) in which regular or routine education, library, day care, health care, or early childhood development services are provided to children. This is consistent with the PHS mission to protect and advance the physical and mental health of the American people. The Office of Dietary Supplements (ODS) was mandated by Congress in 1994 and established within the Office of the Director, National Institutes of Health (NIH). The Dietary Supplement Health and Education Act (DSHEA) [Public Law 103-417, Section 3.a] amended the Federal Food, Drug, and Cosmetic Act "to establish standards with respect to dietary supplements." This law authorized the establishment of the ODS.


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