GENES DETERMINING STEM CELL SELF-RENEWAL AND COMMITMENT NIH GUIDE, Volume 21, Number 37, October 16, 1992 PA NUMBER: PA-93-006 P.T. 34 Keywords: Molecular Genetics Hematology National Heart, Lung, and Blood Institute PURPOSE The National Heart, Lung, and Blood Institute (NHLBI) announces the availability of a Program Announcement (PA) on the above subject. The purpose of this initiative is to encourage research aimed at providing an understanding of the genetic and molecular mechanisms responsible for controlling hematopoietic stem and progenitor cell self-renewal, commitment, and differentiation. 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 PA, Genes Determining Stem Cell Self-Renewal and Commitment, is related to the priority areas of hematologic disorders and bone marrow transplantation. Potential applicants may obtain a copy of "Healthy People 2000" (Full Report: Stock No. 017-001-00474-0) or "Healthy People 2000" (Summary Report: Stock No. 017-001-00473-1) through the Superintendent of Documents, Government Printing Office, Washington, DC 20402-9325 (telephone 202-782-3238). ELIGIBILITY REQUIREMENTS Applications may be submitted by foreign and domestic, for-profit organizations, public and private, such as universities, colleges, hospitals, laboratories, units of State and local governments, and eligible agencies of the Federal Government. Applications from minority individuals and women are encouraged. Awards in connection with this announcement will be made to foreign institutions only for research of very unusual merit, need and promise, and in accordance with PHS policy governing such awards. Foreign institutions are not eligible for First Independent Research Support and Transition (FIRST) Awards (R29). MECHANISM OF SUPPORT This PA will use the National Institutes of Health (NIH) individual research grant (R01) and FIRST (R29) awards. Applicants, who will plan and execute their own research programs, are requested to furnish their own estimates of the time required to achieve the objectives of the proposed research project. Up to five years of support may be requested. Because the nature and scope of the research proposed in response to this PA may vary, it is anticipated that the size of an award will also vary. Applications for R29 awards may request no more than $350,000 direct costs. Administrative adjustments in project period and/or amount of support may be required at the time of the award. All current policies and requirements that govern the research grant programs of the NIH will apply to grants awarded in connection with this PA. RESEARCH OBJECTIVES The purpose of this initiative is to encourage research aimed at providing an understanding of the genetic and molecular mechanisms responsible for controlling hematopoietic stem and progenitor cell self-renewal, commitment, and differentiation. The production of blood cells, a process called hematopoiesis, takes place in the bone marrow. Hematopoiesis begins with the most primitive, pluripotent hematopoietic stem cell which is believed to be present as only one of every 1,000 to 100,000 nucleated bone marrow cells. The stem cell can either self-renew or differentiate into myeloid or lymphoid stem cells, which in turn can further differentiate and mature, ultimately giving rise to all the circulating blood cells. Each of these complex hematopoietic pathways is under the influence of one or more hematopoietic growth factors (colony stimulating factors) or cytokines that enhance cellular proliferation and maturation and other substances that exert negative or inhibitory effects on the process. The past decade has witnessed the cloning and characterization of several hematopoietic growth factors. Many of these have already assumed a role in medical treatment. However, since these factors typically have more than one action, some of these actions may be undesirable in a given case. For example, some cytokines used to ameliorate chemotherapy-induced neutropenia may have the undesirable effect of stimulating the growth of tumor cells or of activating mature neutrophils. Hence, growth factors alone provide insufficiently precise control of the hematopoietic system. Growth factors are only a part of a complex system regulating hematopoiesis. For each growth factor there is a receptor, signal transducers, and responsive genetic elements. Many of these receptors have been characterized; some can be the target of therapeutic attack through molecules designed to compete with their natural ligands. Much current work focuses on signal transduction. Intervention via signal transducers may be complicated by the fact that some signal transducers are common to several pathways serving different functions. Studies in several animal and cell culture systems support the idea that tyrosine kinase receptors, Ras and protein kinase C are part of a common signaling pathway. Thus, identification of responsive genetic elements for growth factors may be the best approach to obtain the specificity required for therapeutic intervention. The hematopoietic stem cell and blood cell progenitors face a succession of "decisions," i.e., choices among alternative pathways. For example, between the quiescent stem cell and the mature neutrophil lie perhaps five such decisions: (a) whether to remain quiescent or to divide; (b) if to divide, whether to remain multipotential (self-renew) or to restrict potentiality; (c) if to restrict potentiality, whether to become a lymphoid or a myeloid stem cell; (d) if to become a myeloid stem cell, whether to become an erythroid, megakaryocytic, or granulocytic progenitor; and (e) if to become granulocytic, whether to become a neutrophilic, eosinophilic, or basophilic progenitor. What these decisions represent at the molecular level, which result in commitment of multipotent progenitors to differentiate along a given lineage, remains elusive. The hypothesis behind this initiative is that each such decision represents activation of a set of genes via a "master" gene. The purpose of this initiative is to identify the genes responsible for the particular alternative pathways selected. The concept of master genes in hematopoiesis has been advanced by studies using approaches in normal and transformed cell culture systems, isolation of multipotent cell lines derived from mouse bone marrow, and the use of hematopoietic cells transformed in vitro by oncogene-containing acute leukemia viruses. Alterations in gene expression can have profound effects on the growth and differentiation of hematopoietic cells. Thus, it is important to understand the mechanisms by which various genes are regulated during hematopoietic cell differentiation. There is some evidence to suggest that the decision for myeloid differentiation rather than for lymphoid differentiation may involve expression of the myb gene (1). Thus, myb may play a key regulatory role in myeloid differentiation and also play an important role in leukemogenicity (2). The decision for erythropoiesis rather than for granulopoiesis or megakaryocytopoiesis may involve expression of the ets gene. However, it was recently shown that the immature chick "erythroid" cells transformed by the E26 avian leukemia virus are in fact multipotent progenitors and can differentiate along at least three lineages (3). It is important to determine if mammalian counterparts exist and have similar functions, particularly in man. This approach was successful in the use of chicken and mouse genes to identify the human counterpart of a transcription factor gene (GATA) required for normal differentiation of erythroid cells (4). The gene for the GATA protein has been cloned which has led to the identification of a family of GATA proteins expressed in different tissues. Similarly, genes of the HOX 2 cluster of homeobox genes have been identified in human hematopoietic cell lines with erythroid potential, suggesting that these genes are involved in human hematopoietic cell differentiation. Overexpression of HOX 2.2 is associated with loss of erythroid features and an increase in certain myelomonocytic markers (5). This strategy can generally be applied to differentiation of other lineages, provided that several proteins specific for that lineage have been identified. The second approach, starting with a candidate gene, is also feasible. As discussed above, an attractive class of genes are the proto-oncogenes, many of which were discovered through their mutated form in tumor viruses. Their unmutated forms must perform important functions because they have been preserved over the course of eukaryotic evolution. A plausible normal function for them is the regulation of cellular proliferation and differentiation. Once a gene is selected for study, its role can be investigated by blocking its effect using antisense technology. This approach has been successfully used in several systems in which it provides a new strategy for inducing differentiation and also provides further insight into the molecular factors that govern the process of hematopoiesis (6,7,8). These approaches are intended to serve as examples and other novel approaches to address the molecular mechanisms for lineage commitment are encouraged. An attack upon these fundamental problems in hematopoiesis is now possible due to progress in isolating hematopoietic stem cells and defining the requirements for their culture. The hypothesis behind this initiative is that each decision made by a cell (to divide or not to divide; to divide and self-renew or commit, etc.) represents activation of a set of genes via a "master" gene. The purpose of this initiative is to identify these master genes by encouraging research aimed at providing an understanding of the genetic and molecular mechanisms responsible for controlling hematopoietic stem and progenitor cell self-renewal, commitment, and differentiation. STUDY POPULATIONS SPECIAL INSTRUCTIONS TO APPLICANTS REGARDING IMPLEMENTATION OF NIH POLICIES CONCERNING INCLUSION OF WOMEN AND MINORITIES IN CLINICAL RESEARCH STUDY POPULATIONS NIH policy is that applicants for NIH clinical research grants and cooperative agreements will be 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 should 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 compelling rationale should 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 should 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, 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 includes human biomedical and behavioral studies of etiology, epidemiology, (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 deferred until the information is provided. 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 NIH are required to address these policies. NIH funding components will not award grants or cooperative agreements that do not comply with these policies. APPLICATION PROCEDURES Applications are to be submitted on the grant application form PHS 398 (rev. 9/91) and will be accepted at the standard application deadlines as indicated in the application kit. The receipt dates for applications for AIDS-related research are found in the PHS 398 (rev. 9/91) instructions. FIRST Award applications must include at least three sealed letters of reference attached to the face page of the original application. FIRST Award applications submitted without the required number of reference letters will be considered incomplete and will be returned without review. Application kits are available at most institutional offices of sponsored research and may be obtained from the Office of Grants Inquiries, Division of Research Grants, National Institutes of Health, Westwood Building, Room 449, Bethesda, MD 20892, telephone (301) 496-7441. Section 2a on the face page of the application must be completed. Check "YES" to indicate the application is submitted in response to a program announcement. The title and program announcement number must be typed in Section 2a of the application as follows: "GENES DETERMINING STEM CELL SELF-RENEWAL AND COMMITMENT" NHLBI PA NUMBER: PA-93-006. The completed original application and five legible copies must be sent or delivered to -- Division of Research Grants National Institutes of Health Westwood Building, Room 240 Bethesda, MD 20892** REVIEW CONSIDERATIONS Although this is a National Heart, Lung, and Blood Institute PA, the National Institute of Diabetes and Digestive and Kidney Diseases also has an interest in the subject matter of this PA. Applications will be assigned to the most appropriate Institute on the basis of established Public Health Service referral guidelines. Applications will be reviewed for scientific and technical merit by study sections of the Division of Research Grants, NIH, in accordance with the standard peer review procedures. Following scientific-technical review, the applications will receive a second-level review by the appropriate National Advisory Council. AWARD CRITERIA Funding decisions will be made on the basis of scientific and technical merit of the proposed grant as determined by peer review, program needs and balance among research areas of the announcement, and the availability of funds. Awards in response to this PA will be made to foreign institutions only for research of very unusual merit, need, and promise, and in accordance with PHS policy governing such awards. INQUIRIES Written and telephone inquiries are encouraged. The opportunity to clarify any issues and questions from potential applicants is welcome. Inquiries regarding this request for applications may be directed to -- Dr. Helena O. Mishoe Cellular Hematology Branch Division of Blood Diseases and Resources National Heart, Lung, and Blood Institute Federal Building, Room 5A12 Bethesda, MD 20892 Telephone: (301) 496-5911 FAX: (301) 496-9940 For fiscal and administrative matters, contact: Ms. Jane R. Davis Chief, Blood Division Grants Management Section Division of Extramural Affairs National Heart, Lung, and Blood Institute Westwood Building, Room 4A15 Bethesda, MD 20892 Telephone: (301) 496-7257 FAX: (301) 402-1200 AUTHORITY AND REGULATIONS The programs of the Division of Blood Diseases and Resources, NHLBI, are described in the Catalog of Federal Domestic Assistance number 93.839. Awards will be made under the authority of the Public Health Service Act, Section 301 (42 USC 241) and administered under PHS grants policies and Federal regulations, most specifically 42 CFR Part 52 and 45 CFR Part 74. This program is not subject to the intergovernmental review requirements of Executive Order 12372. REFERENCES 1. Selvakumaran, M., Liebermann, D.A., Hoffman-Liebermann B. Deregulated c-myb disrupts interleukin-6- or leukemia inhibitory factor-induced myeloid differentiation prior to c-myc: role in leukemogenesis. (1992) Mol Cell Biol 12:2493-500. 2. Graf, T. Myb: a transcriptional activator linking proliferation and differentiation in hematopoietic cells. (1992) Curr Opin Genet 2:249-55. 3. Graf, T., McNagny, K., Brady, G., Frampton, J. Chicken "erythroid: cells transformed by the Gag- Myb-Ets-encoding E26 leukemia virus are multipotent. (1992) Cell 24:201-13. 4. Pevny, L., Simon, M.C., Robertson, E., Klein, W.H., et al. Erythroid differentiation in chimeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. (1991) Nature 349:257-60. 5. Shen, W. F., Detmer, K., Mathews, C. H., Hack, F. M., et al. Modulation of homeobox gene expression alters the phenotype of human hematopoietic cell lines. (1992) EMBO J 11:983-9. 6. Catlett, J. P., Leftwich, J. A., Westin, E. H., Grant, S., Huff, T. F. c-kit expression by CD34+ bone marrow progenitors and inhibition of response to recombinant human interleukin-3 following exposure to c-kit antisense oligonucleotides. (1991) 78:3186-91. 7. Wu, J., Zhu, J. Q., Zhu, D. X., Scharfman, A., et al. Selective inhibition of normal murine myelopoiesis in vitro: by a Hox 2.3 antisense oligodeoxynucleotide. (1992) Cell Mol Biol 38:367-76. 8. Collins, J. F., Herman, P., Schuch, C., Bagby, G. C. c-myc antisense oligonucleotides inhibit the colony-forming capacity of Colo 320 colonic carcinoma cells. (1992) J Clin Invest 89:1523-7. .
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