Full Text CA-92-22


NIH GUIDE, Volume 21, Number 32, September 4, 1992

RFA:  CA-92-22

P.T. 34

  Biochemistry, Proteins 
  Biology, Molecular 
  Biomedical Research, Multidiscipl 

National Cancer Institute

Letter of Intent Receipt Date:  October 2, 1992
Application Receipt Date:  December 8, 1992


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


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.


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.


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.


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



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)

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

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

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

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

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

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.



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

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

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


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


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-

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 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

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

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


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.


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

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


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.


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,

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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