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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