CHEMICAL CARCINOGENESIS PROGRAM AREA REPORT
September 1977 to March 1979

Program Coordinators: Dr. Takashi Sugimura
Dr. I. Bernard Weinstein


Administrative Report and Scientific Summary
Progress continues to be made in this program area through three basic groups of activities: Meetings, Seminars, and Conferences; Exchange of Scientists; and Exchange of Materials.

MEETINGS, SEMINARS, AND CONFERENCES
During this reporting period, a conference on the "Role of DNA Repair and Misrepair in Radiation and Chemical Carcinogenesis" was held in Kyoto, Japan, from December 12 to 14, 1977. This meeting was organized by Drs. A.C, Upton, T. Sugimura, R. Setlow, W. Troll, and I.B. Weinstein, and was held jointly with the First Radiation Biology Center International Symposium entitled "Molecular Mechanisms and Genetic Factors in Radiation Carcinogenesis." This second meeting was held in Kyoto, Japan, on December 15, 1977, and was organized by Drs. A.C. Upton, T. Sugahara, and H. Takebe.
The joint scheduling of these two meetings in-creased the number of speakers that could be invited, thus including several outstanding scientists from Japan, the United States, England, and Europe, with a total of about 60 participants. The names of the invited speakers and the programs of both meetings follow this Program Area Report
There is increasing recognition of the importance of DNA repair mechanisms in the carcinogenic process. Genetic and perhaps metabolic variations in the efficiency and fidelity of cellular DNA repair in response to damage inflicted by environmental carcinogens and mutagens are extremely important determinants of host susceptibility. Hereditary diseases like xeroderma pigmentosum readily demonstrate this principle. The symposia spanned a broad range of topics including human medical genetics, cytogenetics, mutagenesls and carcinogenesis, radiation biology, interactions with viruses, an d biochemical, enzymatlc, and molecular studies of DNA repair. Recent advances in research on the human heritable disorders xeroderma pigmentosum, xeroderma pigmentosum varients, Fanconi's anemia, ataxia telangiectasia, and Bloom's syndrome were reviewed. The emerging results pro-vide clues to the complexity of DNA repair mechanisms in mammalian cells. For reasons that are not known, numerous cases of xeroderma pigmentosum have been found in Japan; cell lines established from these cases are proving to be extremely useful in research studies. Drs. Maher and McCormick (Michigan State University) presented studies with xeroderma pigmentosum cell cultures indicating that they are more susceptible to mutagenesis by ultraviolet light and by certain chemical carcinogens than are normal cells.
Encouraging progress in the identification and purification of specific enzymes involved in DNA repair was also presented. Dr. Mutsuo Sekiguchi, Kyushu University, reported on the purification of a T4 phage enzyme that specifically excises thymine dimers. He also described an N-glycosidase enzyme that excises uracil residues from DNA. Dr. Stuart Linn, University of California at Berkeley, described purification of a human apurinic endonuclease. He also described preliminary experiments suggesting the existence of a novel "insertase" enzyme that re-inserts base residues into apurinic sites in DNA. Dr. M. Radman, Brussels; Dr. S.G. Sedgwick, National Institute for Medical Research, London; and Dr. W. Troll New York University, described the "SOS" mechanisms involved in phage activation and mutagenesis in bacteria. Evidence was presented that these responses to DNA damage, particularly bulky lesions in DNA, involve induction of an "error-prone" DNA polymerase and the action of proteases.
Dr. Sugimura presented recent results of investigations in Japan indicating that the protease inhibitor "elastatinal" inhibits mutagenesis in S. typhimurium and that the tryptophane pyrolysis products norharman and harman enhance mutagenesis in this bacterial system. He also reported that elastatinal inhibits in vitro transformation of Syrian hamster embryo cultures by MNNG. Evidence that the protease inhibitor antipain inhibits mutagenesis in E. coli was presented by Ryo H. Ichikawa and Sohei Kondo (Osaka). Dr. Troll described exciting studies indicating that proteases are involved in the inactivation of repressors in bacterial systems. He reviewed evidence that pro-teases may also play an important role in carcinogenesis. Dr. I. Bernard Weinstein, Columbia University, described the types of covalent adducts formed between DNA and activated forms of Benzo(a)pyrene and DMBA. He reviewed recent evidence that tumor promoters induce a number of phenotypic properties that resemble those seen in tumor cells and also inhibit cellular differentiation. Additional papers dealt with chromosomal changes and interactions between radiation, chemical carcinogens, and oncogenic viruses. The meetings ended with the presentation of a paper by Dr. H.P. Klinger, Albert Einstein College of Medicine, New York, describing the suppression of tumorigenicity in somatic cell hybrids and associated chromosomal correlations.
Participants from Japan and abroad found these two meetings extremely stimulating and productive. There is no doubt that the information ex-changed, the collaborations established, and the warm friendships made in Kyoto will greatly enhance advances in this important area of cancer research.
At a second conference sponsored by the U.S.-Japan Cooperative Cancer Research Program held at the East-West Center, University of Hawaii, March 26 to 28, 1979, 17 researchers met to discuss "Gene Derepression and Host-Tumor Relationships in Carcinogenesis." Chairmen at this conference were Drs. S. Weinhouse, T. Ono, and A. Ichihara. The agenda and minutes of the meeting follow this Program Area Report.

Exchange of Scientists
A very productive aspect of the Chemical Carcinogenesis Program Area has been that of scientist exchange. In the period covered by this Report, the following scientists visited their counterparts:
1. Dr. Yutaka Tsukada, from Hokkaido University School of Medicine, visited Dr. Van R. Potter's laboratory at the McArdle Laboratory, University of Wisconsin. Dr. Tsukada conducted studies on the induction of AFP synthesis and secretion in H35 hepatoma cells by dexamethasone and the effects of feeding alphanaphthylisothiocynate on AFP synthesis and gamma glutaml-transpeptidase.
2. Dr. Hiroyasu Esumi, National Cancer Center Research Institute, Tokyo, visited the laboratories of Dr. W. Troll New York University and Dr. L. Greenbaum, Columbla University. During August and September 1978, Dr. Esurni performed collaborative studies on the effects of tumor promoters on differentiation and on protease action. During his visit, he also participated in the Gordon Cancer Conference in August 1978, where he presented a paper on his research studies. In studies conducted with Dr. Troll at the Woods Hoie Marine Biology Laboratory, he found that a phorbol ester tumor promoter inhibits sea urchin development.
In addition, Dr. Esumi and Dr. L. Greenbaum continued their studies on the role of proteases in ascites fluid accumulation. 3. Dr. Kohtaro Koyama, National Cancer Center Research Institute, Tokyo, visited the Division of Biochemical Oncology, Fred Hutchinson Cancer Research Center, University of Washington, Seattle (host: Dr. Sen'ichiro Hakomori, September 1, 1977 to February 28, 1978). He analyzed the cell surface proteins, carbohydrates, and glycolipids in membranes of several variants of fibrosarcoma cell lines.
4. Dr. Lowell Greenbaum, Columbia University, visited Dr. T. Sugimura and other scientists in Japan for collaborative studies on the role of proteases in tumor ascites accumulation and prevention of ascites formation by protease inhibitors.
5. Dr. A. Hsie, Oak Ridge National Laboratories, visited Dr. T. Sugimura and his colleagues to discuss in vitro studies on mammalian cell mutagenesis and assays for carcinogens using this system. His visit assisted in the establishment -of the CHO mutagenesis assay in Japanese laboratories.
6. Dr. Veronica Maher participated in two meetings on DNA repair held in Kyoto in December 1977. Dr. Maher then visited several laboratories in Japan where she and Dr. Justin McCormick discussed studies that they have done demonstrating increased sensitivity to mutagenesis in xeroderma pigmen-tosum cultures and more general aspects of DNA damage and repair.
7. Dr. I. Bernard Weinstein, Columbia University, New York, was an exchange scientist in Japan from December 5 to 24, 1977. He participated in the U.S.-Japan Cancer Seminar on the "Role of DNA Repair and Misrepair in Radiation and Chemical Carcinogenesis," for which he was co-organizer, and the First Radiation Biology Center International Symposium on "Moiecular Mechanisms and Genetic Factors in Radiation Carcinogenesis" held in Kyoto on December 12 to 14 and December 15, 1977, respectively. During his stay in Japan, he presented research seminars on the subject of "Structural and Functional Changes in Nucleic Acids Modified by Activated Forms of Benzo (a) pyrene" and "Studies on the Mechanism of Action of Tumor Promoters in Cell Culture." Seminars on these subjects were presented at the two meetings in Kyoto described above, and at the National Cancer Research Center in Tokyo, the Cancer Institute in Tokyo, the Institute of Medical Sciences in Tokyo, and the University of Tokushima. He also visited numerous research laboratories in Japan to learn details of research projects in the areas of carcinogenesis and to discuss possible areas of collaborative research. The scientists with whom he had detailed discussions included Drs. T. Sugimura, S. Nishimura, Y. Kuchino,H. Kasai, N. Nemoto, S. Takayama, M. Muramatsu, H. Sugano, I. Matsushima, I. Hirono, N. Yamaguchi, H. Shimojo, Y. Ikawa, and T. Yamamoto in Tokyo; A. Ichihara and S. Ohtsuka in Tokushima; and S. Kondo, T. Sugahara, H. Takebe, M. Sekiguchi, and H. Yoshikura in Kyoto.
Dr. Weinstein found considerable interest in recent cell culture studies in his own laboratory and other laboratories in the United States on the mechanism of action of tumor promoters. He has subsequently sent samples of phorbol esters and related macrocydic diterpenes to Dr. T. Sugimura and his colleagues for studies on myeloid cell differentiation; to Dr. V. Ikawa for studies on the transformation of chick embryo fibroblasts by avian sarcoma virus; to Dr. A. Ichihara for studies on liver isozymes; and to Dr. T. Sugiyama, Department of Pathology, Kobe University, for studies on erythroid differentiation in DMBA induced eryihroleukemia cell lines.
The possible roles of natural products in tumor promotion were discussed in detail with Dr. Iwao Hirono at the Institute of Medical Sciences (IMS) in Tokyo and Dr. Hirata Hoshimasa, Department of Chemistry, Nagoya University. This subject could develop into a future area for productive collaborative studies between Japanese and U.S. scientists.

EXCHANGE OF MATERIALS
An extremely valuable aspect of the U.S.-Japan Cooperative Cancer Research Program in the area of carcinogenesis has been the generous gift from Japanese scientists of several highly novel and specific protease inhibitors to researchers in the United States and elsewhere. Drs. Walter Troll and Bernard Weinstein have supervised the distribution of these compounds to over 65 U.S. scientists, as well as five scientists in the United Kingdom, three in Canada, two in West Germany, three in Israel, two in Belgium, and one scientist each in Switzerland, Poland, South Africa, and the U.S.S.R. The availability of these materials has made it possible to advance our understanding of the role of proteases in phage induction, in mutagenesis, and in the carcinogenic process itself. The results have been of major interest to the fields of bacterial genetics, mutagenesis, and carcinogenesis. The rapid advances in our understanding of the role of proteases in normal and abnormal growth would not have been possible without these inhibitors and the data on their properties so generoushy provided by the Japanese scientists.
The U.S.-Japan Cooperative Cancer Research Program has also greatly stimulated the exchange of information and materials between U.S. and Japanese scientists in the areas of carcinogenesis and tumor promotion. Dr. Sugimura's laboratory has obtained evidence that croton oil enhances the induction of gastric cancer in rats and that phorbol esters influence the maturation of leukemia cells in culture. Drs. Sugimura and Takayama participated in a Symposium on Tumor Promotion organized by Drs. I. Bernard Weinstein and Walter Troll at Cold Spring Harbor in May 1978. Other exchanges of materials and information related to tumor promoters are described above in the Exchange of Scientists section.

Current and Future Activities, 1978 to 1979

CONFERENCE UNDER DISCUSSION
The following topics are under consideration for future conferences:
Epidemiologic and Medical Applications of Computer Technology
Eukaryotic DNA Replication
Carcinogenesis Studies in Human Cells and Tissues
Metabolic Epidemiology-Dietary, Genetic, Hormonal Factors; Natural Carcinogens; Variations in Host Metabolism
Interactions Between Chemical Carcinogens and Oncogenic Viruses in Cancer Causation.

EXCHANGE OF SCIENTISTS
The following two American scientists recently visited Japanese colleagues:
Dr. Ernst Borek, University of Colorado, visited the National Cancer Center Research Institute, Tokyo, in March 1979. The hosts were Y. Kuchino and S. Nishimura.
Project Title: "Alterations in Modified Bases in Hepatoma Transfer RNAs"
Dr. Dezider Grunberger, Columbia University, visited the National Cancer Center Research Institute, Tokyo, in November 1978. This visit was hosted by S. Nishimura.
Project Title: "Structure Studies on Transfer RNAs in Tumor Cells and the Effects of Nutritional Factors"

EXCHANGE OF RESOURCES (tentative)
The following chemical substances are being considered for exchange between the United States and Japan:
Mutagens from amino acid pyrohysates discovered in Japan that may be potential carcinogens in cooked foods. To be provided by Dr. T. Sugimura and colleagues and distributed to American scientists with the assistance of Dr. Kakunaga, National Cancer Institute, Bethesda.
Activated forms of polycyclic aromatic hydrocarbons synthesized by American scientists.
Phorbol esters and related macrocyclic plant diterpene tumor promoters to be provided by American scientists.
Continued supply of protease inhibitors from Japanese scientists.

ETIOLOGY PROGRAM AREA
The following subject areas are under discussion for future collaborative efforts in this program area: Epidemiology, chemical and radiation carcinogenesis, viral carcinogenesis, genetics and molecular oncology.
United States and Japanese principal advisors in each of the above areas are being discussed.

OBJECTIVES
Beginning in 1979, the major objective of the Second Five-Year Cooperative Cancer Research Program in the Carcinogenesis Program Area is to "identify carcinogenic factors and clarify mechanisms with a view to providing a fundamental basis for the prevention of human cancer."


CONFERENCE REPORTS

U.S. -Japan Cancer Seminar (Sponsored by the National Cancer Institute (U.S.A.) and the Japan Society for the Promotion of Science; coordinated by Drs. A. C. Upton, I. B. Weinstein, and T. Sugimura.)
ROLE OF DNA REPAIR AND MISREPAIR IN RADIATION AND CHEMICAL CARCINOGENESIS
Kyoto Grand Hotel, Kyoto, Japan
December 12-14, 1977

AGENDA
Monday, December 12
9:00 a.m. Welcome Dr. T. Sugahara
9:05 a.m. Opening Session Dr. T. Sugahara-Chairperson
Opening Remarks Dr. T. Sugimura
Introduction Dr. I. B. Weinstein
Session 1: DNA Repair Defects in Hereditary Diseases with High Incidence of Cancer
Drs. H. Takebe and D. Bootsma-Co-Chairpersons
9:45 a.m. 1. Repair Defect and Chromosomal Sensitivity in Xeroderma Pigmentosum Dr. M. S. Sasaki
10:15 a.m. Discussion
10:25 a.m. COFFEE BREAK
10:45 a.m. 2. Ataxia Telangiectasia: A Model Genetic Disorder Relating Radiogenic DNA Damage to Human Malignancy Dr. M. C. Paterson
11:15 a.m. Discussion
11:25 a.m. 3. Relationship between DNA Repair Defects and Skin Cancers in Xeroderma Pigmentosum Dr. H. Takebe
11:55 a.m. Discussion
12:05 p.m. LUNCH
Session 1: (continued)
Drs. Y. Fujiwara and V. M. Maher-Co-Chairpersons
1:15 p.m. 4. Postreplication Repair in UV-irradiated Xeroderma Pigmentosum (XP) and its Variant Cells and Skin Carcinogenesis Dr. Y. Fujiwara
1:45 p.m. Discussion
1:55 p.m. 5. Cell Killing and Mutation in Human Repair Defective Syndromes Dr. C. F. Arlett
2:25 p.m. Discussion
2:35 p.m. 6. Biological Evidence that DNA Repair Processes in Normal Human Cells Act to Reduce the Lethal and Mutagenic Effects of Exposure to Carcinogens Drs. V. M. Maher and J. J. McCormick
3:05 p.m. Discussion
3:15 p.m. COFFEE BREAK
3:35 p.m. 7. Enzymes that Recognize DNA Damages Induced by Ultraviolet Light and Certain Chemicals Dr. M. Sekiguchi
4:05 p.m. Discussion
4:15 p.m. 8. Enzymes for the Repair of Apurinic/Apyrimidinic Sites in Human Cells Dr. S. Linn
4:45 p.m. Discussion

Tuesday, December 13
Session 2: Genetic and Epigenetic Factors in Radiation and Chemical Carcinogenesis
Drs. T. Sugimura and I. B. Weinstein-Co-Chairpersons
8:30 a.m. 9. DNA Damage and Cancer Production by 4-hydroxy-aminoquinoline-1-oxide (4HAQO) after Partial Pancreatectomy (p.p.) in Rats Dr. Y. Konishi
9:00 a.m. Discussion
9:10 a.m. 10. Biological and Biochemical Effects of Various Carcinogens in Normal and XP Strains Drs. J. J. McCormick and V. M. Maher
9:40 a.m. Discussion
9:50 a.m. COFFEE BREAK
10:10 a.m. 11. Genetic and Epigenetic Factors in Chemical Carcinogenesis Dr. I. B. Weinstein
10:40 a.m. Discussion
10:50 a.m. 12. Enhancement and Suppression of Mutagenesis Dr. T. Sugimura
11:20 a.m. Discussion
11:30 a.m. LUNCH
12:30 p.m. Excursion

Wednesday, December 14
Session 2: (continued)
Drs. K. Yokoro and L. G. Lajtha-Co-Chairpersons
9:00 a.m. 13. Chemical Carcinogen Action and Repair at the Chromosomal Level Dr. T. Sugiyama
9:30 a.m. Discussion
9:40 a.m. 14. Induction of Endogenous Murine Leukemia Virus: Effect of Caffeine and Polycyclic Carbons Dr. H. Yoshikura
10:10 a.m. Discussion
10:20 a.m. 15. Radiation Induction of Endogenous Murine Leukemia Virus in vitro: Effect of Antipain on the Induction Frequency. (Discussion Paper) Dr. O. Niwa
10:30 a.m. Discussion
10:55 a.m. COFFEE BREAK
Session 3: Inducible Repair and Role of Misrepair in Carcinogenesis and Mutagenesis
Drs. M. Sekiguchi and S. Linn-Co-Chairpersons
11:15 a.m. 16. Post-replication Error-Prone Repair of DNA Damage Dr. M. Radman
11:45 a.m. 17. Genetic Control of UV Mutagenesis in Escherichia coli. (Discussion Paper) Dr. T. Kato
11:55 a.m. Discussion
12: 10 p.m. LUNCH
1:15 p.m. 18. Error-Free and Error-Prone DNA Repair in Escherichia coli Dr. S. Sedgwick
1:45 p.m. Discussion
Session 3: (continued)
Drs. S. Kondo and W. Troll-Co-Chairpersons
2:00 p.m. 19. Differential Antimutagenicities of Caffeine and Antipain (a Protease Inhibitor) in Escherichia coli Treated with Different Mutagens Drs. Ryo H. Ichikawa and S. Kondo
2:30 p.m. Discussion
2:40 p.m. 20. Inhibition of Tumor Initiation by Protease Inhibitors Dr. W. Troll
3:10 p.m. Discussion
3:20 p.m. 21. Caffeine Effects on Chemical Carcinogenesis Dr. T. Nomura
3:50 p.m. Discussion
Closing Session
Dr. T. Sugimura-Chairperson
4:15 p.m. Summary and Closing Remarks Dr. T. Sugahara
General Discussion
4:55 p.m. Adjournment


The First Radiation Biology Center International Symposium (Sponsored by the Radiation Biology Center, Kyoto University; organized by Drs. A. C. Upton, T. Sugahara, and H. Takebe.)
MOLECULAR MECHANISMS AND GENETIC FACTORS IN RADIATION CARCINOCENESIS
Kyoto Grand Hotel, Kyoto, Japan.
December 15, 1977

AGENDA
Thursday, December 15
9:00 a.m. Opening Remarks Dr. T. Sugahara
Session 1: Molecular and Cellular Aspects in Radiation Carcinogenesis
Drs. H. Tanooka and M. Radman Co-Chairpersons
9:10 a.m. 1. A Review of the Mechanism(s) for Induction of Error-Prone Repair Dr. S. Sedgwick
9:40 a.m. Discussion
9:50 a.m. 2. Persistence of Radiation-Induced Carcinogenic Damage in the Mouse Skin and its Modification by Caffeine Drs. H. Hoshino and H. Tanooka
10:20 a.m. Discussion
10:30 a.m. COFFEE BREAK
10:50 a.m. 3. Radiation-Induced Salivary Gland Tumor Dr. F. Hirose
11:20 a.m. Discussion
11:30 a.m. 4. On Cellular Aspects of Leukemogenesis Dr. L. G. Lajtha
12:00 noon Discussion
12:10 p.m. LUNCH
Session 2: Genetic Factors in Radiation Carcinogenesis
Drs. A. Tonomura and M. C. Paterson-Co-Chairpersons
1:20 p.m. 5. Chromosome Aberrations in General Population Dr. A. Tonomura
1:50 p.m. Discussion
2:00 p.m. 6. Cell Killing and Mutation in Human Repair Defective Syndromes Dr. C. F. Arlett
2:30 p.m. Discussion
2:40 p.m. 7. Repair of the Radlation-Induced Lethal and Mutational Damage in Cultured Chinese Hamster Cells Dr. M. Horikawa
3:10 p.m. Discussion
3:20 p.m. COFFEE BREAK
3:40 p.m. 8. Genetic Analysis of Mammalian Cells by Gene Transfer Systems Dr. D. Bootsma
4:10 p.m. Discussion
4:20 p.m. 9. Suppression of Tumorigenecity in Somatic Cell Hybrids Dr. H. P. Klinger
4:50 p.m. Discussion
5:00 p.m. Closing Remarks Dr. H. Takebe


CONFERENCE ON GENE DEREPRESSION AND HOST-TUMOR RELATIONSHIPS IN CARCINOCENESIS
East West Center, Honolulu
March 26-28, 1979

AGENDA
Monday, March 26
1. Abnormal Gene Expression in Tumors
9:00 a.m. Is There Any Relationship between Arachidonic Acid Metabolism and Tumor Production? Lawrence Levine, Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts
9:45 a.m. Chemistry and Expression of Alpha-Fetoprotein and Its Molecular Variants Erkki Ruoslahti, Division of Immunology, City of Hope National Medical Center, Duarte, California
10:30 a.m. COFFEE BREAK
10:45 a.m. Reexpression and Modulation of Developmental Alkaline Phosphatases in Tumor Cells William Fishman, La Jolla Cancer Research Foundation, La Jolla, California
11:30 a.m. Polypeptide Growth Factors Produced by Virus-transformed Cells and by Tumor Cells Joseph DeLarco (and George Todaro), Laboratory of Viral Carcinogenesis, National Cancer Institute, Bethesda, Maryland
12:15 p.m. LUNCH
2:00 p.m. Gene Expression in Primary Cultured Rat Hepatocytes Akira Ichihara, Institute of Enzyme Research, Tokushima University Medical School, Tokushima
2:45 p.m. tRNA Transglycosylase from E. Coli and Rat Liver: Novel Mechanism of Post-Transcriptional Modification of tRNA, and Its Implication in the Detection of Under-Modified Unique tRNA Species in Tumor Tissues Susumu Nishimura, National Cancer Center Research Institute, Tokyo
3:15 p.m. Ectopic Hormone Production in Human Tumors as an Abnormal Gene Expression Hiro Imura, Kyoto University Medical School, Kyoto

Tuesday, March 27
2. Initiation and Promotion
9:00 a.m. Studies on the Mechanisms of Action of Tumor Promoters I. B. Weinstein, Institute for Cancer Research, Columbia University, College of Physicians and Surgeons, New York, New York
9:45 a.m. Effects of Tumor Promoters on the Differentiation of Friend Leukemia Cells in Culture Tetsuo Ono, Tokyo Metropolitan Institute for Medical Science, Tokyo
10:30 a.m. COFFEE BREAK
10:45 a.m. Naturally Occurring Genotoxic and Non-genotoxic Carcinogens Takashi Sugimura, National Cancer Center Research Institute, Tokyo
11:30 a.m. Effects of Modifiers of Tumor Promotion on Differentiation of Leukemia Cells Moto Hozumi, Saitama Cancer Research Center, Saitama
12:15 p.m. LUNCH
3. Effects of Tumors on Host Metabolism
2:00 p.m. Alterations in the Enzymic Composition of Liver and Spleen in Response to Distant Neoplasms Olga Greengard, Department of Pediatrics, Mount Sinai Medical Center, New York, New York

2:45 p.m. 		Expression of the Fetal Isozyme of Pyruvate Kinase in Adult Non-neoplastic Liver Kenneth H. Ibsen, Department of Biological Chemistry, California College of Medicine, University of California, Irvine, California
3:30 p.m. General discussion

Wednesday, March 28
3. Effects of Tumors on Host Metabolism (continued)
9:00 a.m. A Factor Responsible for Increase in Ornithine Decarboxylase Activity in the Liver of Tumor-bearing Mice: Partial Purification and Characterization Takehiko Tanaka, Osaka University Medical School, Osaka
9:45 a.m. Effects of Tumor on Host Liver Nitrogen Metabolism Sidney Weinhouse, Fels Research Institute, Temple Medical School, Philadelphia, Pennsylvania
10:30 a.m. DNA Synthesis in Tumor-bearing Rats: Thymidine Kinase-stimulating Factor Setsuro Fujii, Institute of Protein Research, Osaka University Medical School, Osaka


MINUTES OF THE CONFERENCE ON GENE DEREPRESSION AND HOST-TUMOR RELATIONSHIPS IN CARCINOGENESIS
Honolulu, Hawaii March
26 to 28, 1979

Evidence from several areas of biology points to abnormalities of gene regulation as a common feature underlying many of the diverse phenotypic properties associated with the induction and maintenance of the neoplastic state. At a conference sponsored by the U.S.-Japan Cooperative Cancer Research Program held at the East-West Center, University of Hawaii, 17 researchers met to discuss these topics. The program was divided into three main segments: abnormal gene expression in tumors; initiation and promotion; and effects of tumors on host metabolism.
Dr. Lawrence Levine, Brandeis University, pointed out that several tumors synthesize and excrete large amounts of prostaglandin E2, and that prostaglandins and their metabolites are elevated in the plasma of tumor hosts. This is assumed to be responsible for hypercalcemia and bone-resorption. Dr. Levine found that a large number of carcinogens and promoters stimulate prostaglandin formation by activating phospholipase A, thereby releasing arachidonic acid, its precursor.
Dr. Alan Mufson, Columbia University, enlarged upon the role of arachidonic acid release and prostaglandin formation in tumor promoter activity. In contrast with carcinogens, which attach covalently to DNA, promoters are not mutagenic and their target is the cell membrane. At nanomolar concentrations, they alter cell morphology, alter cell surface components such as fucose-ghycopeptides and LETS protein increase saturation density, and increase production of plasminogen activator and ornithine decarboxylase. The same effects are enhanced in transformed cells. Phorbol esters and related tumor-promoting plant diterpenes reversibly inhibit cell differentiation, as demonstrated by a variety of experimental systems, and reduce binding of epidermal growth factor to tumor cells.
Dr. Tetsuo Ono, Tokyo Metropolitan Institute of Medical Science, described inhibitory effects of a variety of tumor promoters on differentiation of Friend erythroleukemia cells induced by hexa-methylene bisacetamide. Urea, phorbol esters, deoxycholate, and Tween-60 either inhibited or in-creased the inhibitory effect of phorbol ester in this system. Carcinogens such as urethane, DMBA, 4-NQO, and saccharin, considered to be weak carcinogens as well as promoters, did not inhibit differentiation.
Using another experimental system, Dr. Moto Hozumi, Saitama Cancer Research Center, reported that the differentiation of a mouse myeloid leukemic (MI) cell line to macrophages and granulocytes was triggered by a variety of substances, including sera, ascitic fluid, saliva, urlne, amniotic fluid, and various polynucleotides.
Differentiation of MI cells was also induced by inhibitors of promotion in mouse skin, such as glucocorticoides, some prostaglandins, cyclic AMP, pohy I-pohy C, interferon, and vitamin A. Differentiation induced by steroids was highly specific to certain structures, was dependent on binding to dexamethasone receptors, and was inhibited by low concentrations of phorbol esters and other tumor-promoting diterpenes.
Dr. Takashi Sugimura, National Cancer Center, Tokyo, provided another experimental system, the use of microbial species for detection of mutagenesis, to aid in the dissection of initiation and promotion. Among the striking findings reported were the discovery and chemical identification of new classes of highly potent carcinogens formed by pyrolysis of meat, fish, and individual amino acids such as tryptophan and glutamic acid. Dr. Sugimura classified tumorigenic agents as genotoxic (initiators) and non-genotoxic (promoters) and reported evidence of a novel concept: co-mutagenesis by two agents, neither of which is mutagenic individually.
One of the most striking examples of gene derepression in cancer is the expression of fetal gene products. Dr. Erkki Ruoslahti, City of Hope Medical Center, Duarte, California, described a new isolation method for!!!-feto-protein, a product of liver and germ cell neoplasms, based on use of interspecies cross-reactivity of antibody, to produce gram quantities. This protein shows amino acid sequence homology with albumin as well as similarities in binding fatty acids; but it contains carbohydrate, the amount and composition of which may differ according to its source. The tumor form appears to resemble that of the yolk sac rather than that of the liver. Dr. Ruoslahti made the important point that AFP is produced in non-hepatoproliferative conditions such as hepatitis, partial hepatectomy, early premalignant effects of carcinogens, and in certain hereditary conditions, such as tyrosinemia and ataxia telangiectasia. It also persists as a hereditary trait in BALB/C/J mice.
According to Dr. William Fishman, La Jolla Cancer Research Foundation, California, the developmental expression of the many alkaline phosphatase isozymes follows a definite pattern, which is re-expressed as an oncodevelopmental feature in specific tumors. Both Drs. Ruoslahti and Fishman dwelt on the expression of!!!-glutamyl peptidotransferase, an enzyme rapidly assuming importance as an early marker of neoplasia. According to Dr. Ruoslahti, the activity of this enzyme is not correlated closely with AFP production. Dr. Fishman, who found this enzyme in lung and colon tumors, emphasized its embryonal expression in testes, blastocysts, and trophoblasts.
Dr. Susumu Nishimura, National Cancer Center, Tokyo, described another example of abnormal gene regulation resulting in novel isoprotein species in tumors, namely isoaccepting tRNAs, which have Q base adjacent to the anticodon region. The Q base, by the action of a transglycosylase, can be replaced with quanine; this reaction provides a method for detection of these unique tRNAs. Such Q base-containing tRNAs are present in a wide variety of tumors and, except for regenerating liver, are absent from normal tissues. Other tRNAs specific to tumors are under study.
With the rapid advancement of technology of cell culture, much new information is now available to assess the needs and requirements for growth of transformed cells and their relation to the endogenous synthesis of growth factors. Dr. Joseph DeLarco, National Cancer Institute, Bethesda, reported on the work of Dr. George Todaro's laboratory which has shown that the serum-free medium in which MSV-transformed cells are grown contains a factor that stimulates growth in cells arrested by serum depletion or confluence. This sarcoma growth factor (SGF) competes with the epidermal growth factor receptor in radioim-munoassay, and it is assumed that this is responsible for the apparent loss of EGF receptor in those cells. This substance, with a MW of 10,000, is one of several similar growth factors, separable by gel filtration.
Dr. Akira Ichihara, Tokushima University, found that in contrast with established liver cell lines, rat hepatocytes in primary culture, with the addition of insulin and dexamethasone, retained such adult liver functions as synthesis and secretion of liver proteins, glycogenolysis by glucagon, induction of tyrosine aminotransferase by dexamethasone, and of NADPH-generating dehydrogenases, DNA synthesis and polyamine formation by insulin. These cells did not proliferate, but over one week they gradually lost these functions, assumed an altered morphology, and enhanced activities of glycolytic enzymes.
Dr. Hiro Imura, Kyoto University School of Medicine, discussed another example of abnormal gene regulation, the ectopic production of pohypep-tide hormones by tumors of nonendocrine origin. An anahysis of extracts of ACTH-producing tumors revealed the existence of "heavy" ACTH, together with smaller fragments with!!!-endorphin and!!!-LPH activity. In some instances ACTH activity was accompanied by calcitonin and chorionic gonadotropin. Production of multiple hormones by a single tumor was not uncommon, and experimental data suggest the formation of a large molecule precursor which splits into ACTH and smaller MW polypeptide hormones.
The presentations of participants who discussed host-tumor relationships brought additional support for the formation and release of bioactive substances as expressions of abnormal gene regulation in cancer. Dr. Olga Greengard, Mount Sinai Medical Center, New York, reported that rats bearing a wide variety of neoplasms display striking alterations of liver enzyme activities, particularly in glycokinase and ornithine aminotransferase. These alterations are reversed with eradication of the tumor.
Dr. Kenneth Ibsen, University of California, Irvine, found similar effects on the isozymes of pyruvate kinase in livers of mice bearing Ehrlich ascites tumor, and partialhy purified what appears to be an active RNA-protein complex from these cells. A similar investigation, reported by Dr. Takehiko Tanaka, Osaka University Medical School, revealed a substance in Ehrilch ascites cells which lowers ornithine decarboxylase activity in liver, spleen, and kidney of treated mice. As little as 1 vg of a 70-fold purified preparation was active by i.p. injection in normal mice. It is a heat-labile, alkall-stable protein with MW > 300,000, and is in-activated by 10 mM dithiothreitol, 5 M urea, pronase, or glycosidase, but is stable to DNAse, RNAse, or neurarninidase.
Dr. Sidney Weinhouse, Fels Research Institute, Philadelphia, described work from his laboratory demonstrating a highly selective, reciprocal relationship between carbamyl phosphate synthetase (CPS-I) activity and urea synthesis in livers and tumors of rats bearing transplantable hepatomas. When these activities were high in the tumors, they were correspondingly low in the host liver. The existence of a humoral factor fostering homeostasis of urea production is suggested, although cross-circulation experiments in parabiotic rats failed to demonstrate effects of a tumor in one partner on the liver of the other normal partner.
Dr. Setsuro Fujii, Osaka University, following up previous reports of increased DNA synthesis in normal tissues of tumor bearing animals, found enhanced incorporation of thymidine kinase in liver and spleen of rats bearing the Yoshida or AH 130 sarcoma. This was accompanied by increased liver activities but decreased thymus activities of thymidine kinase, TMP kinase, and DNA polymerase. The responsible factor was partially purified. Dr. Fujii also described an inhibitor of ribonucleotide reductase in livers of normal rats which is altered in tumor bearers.

The proceedings of this conference attest to a growing recognition that a common feature underlying many of the phenotypic properties of cancer is an abnormality of gene regulation manifested by misprogramming of protein syn-thesis. This breach of the ordinarihy regid control of gene activity in differentiated tissues accounts for the production of growth factors, oncofetal proteins, alterations of isoenzyme composition, and probably also the many varied effects of tumors upon host enzyme activities and metabolism. Consistent with this conception is the large and growing body of evidence that promoters function as inhibitors of differentiation in a variety of experimental systems.


Respectfully submitted,

Sidney Weinhouse
Professor of Biochemistry