Cells Carcinogenesis Studies Using Cultured Human ...

Carcinogenesis Studies Using Cultured Human Tissues and Cells Curtis C. Harris Cancer Res 1983;43:1880-1883.

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Downloaded from cancerres.aacrjournals.org on October 29, 2012 Copyright © 1983 American Association for Cancer Research

(CANCER RESEARCH 43, 1880-1883, 0008-5472/83/0043-OOOOS02.00

April 1983]

Meeting Report

Carcinogenesis Studies Using Cultured Human Tissues and Cells Investigations of differentiation and carcinogenesis using cul tured human cells are yielding exciting results. These recent advances have been made possible by the productive interaction among investigators in several fields including cell biology, mo lecular biology, chemical carcinogenesis, viral carcinogenesis, etc., the development of new methods in molecular biology, and improved conditions to culture human tissues and cells. Progress in this area of cancer research was reviewed at an international conference sponsored by the Division of Cancer Cause and Prevention, National Cancer Institute, held at the Given Institute of Pathobiology, Aspen, Colo., September 16-21, 1982, with 100 invited participants. The Program Committee1 focused the conference on 7 major topics: (a) Growth and Differentiation of Human Cells; (b) Metabolism of Chemical Carcinogens, DMA Damage, and DMA repair; (c) Tumor Promotion and Transforming Growth Factors; (d) Cellular Lesions Caused by Chemical Car cinogens; (e) Cellular Lesions Caused by Physical Carcinogens; (f) Viral Oncogenesis; and (g) Laboratory-Epidemiology studies. After the opening remarks by Richard Adamson (National Cancer Institute, Bethesda, Md.), Barry Pierce (University of Colorado, Denver, Colo.) discussed both in vitro and in vivo approaches to the study of cellular interactions and intercellular control of tumor progression, which is a caricature of normal cellular development. James Rheinwald (Sidney Farber Cancer Center, Boston, Mass.) presented evidence that each type of epithelium has its own "fingerprint" of cytoskeletal proteins. Malignant transformation of the keratinocyte involves a lesion in the mechanism for commitment of the cell to terminal differentia tion and is frequently accompanied by changes in keratin gene regulation. Growth and invasiveness of primary tumor expiants on substrates of either extracellular matrix produced by rat smooth muscle cells or living cultures of human fibroblasts were discussed by Peter Jones (University of Southern California, Los Angeles, Calif). More than 80% of the most common solid childhood neoplasms (brain tumors, neuroblastoma, Wilms' tu mor, rhabdomyosarcoma, osteogenic sarcoma, etc.) grew on and, in some cases, invaded these substrates. Eric Stanbridge (University of California at Irvine, Calif.) pre sented data from studies of human cell hybrids on the role of differentiation in the control of tumorigenesis. Anchorage-inde pendent growth and other commonly used in vitro markers of transformed cells were not predictive of the hybrid cell's tumorigenie potential when transplanted into animal recipients. Produc tion of a 75,000 molecular weight phosphoprotein and human 1 Herman Autrup,

National Cancer Institute,

Bethesda,

Md.; Carmia Borek,

Columbia University College of Physicians and Surgeons, New York, N. Y.; Peter Cerutti, Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland; Allan Conney, Hoffman-La Roche, Inc., Nutley, N. J.; Joseph Fraumeni, Jr., Curtis Harris, and Takeo Kakunaga, National Cancer Institute; David Kaufman, University of North Carolina School of Medicine, Chapel Hill, N. C.; Donald King, Columbia University, New York, N.Y.; Peter Magee, Fels Research Institute, Philadelphia, Pa.; Takashi Sugimura, National Cancer Center Research Institute, Tokyo, Japan; Benjamin Trump, University of Maryland School of Medicine, Baltimore, Md.; Michael Waters, United States Environmental Protection Agency, Research Triangle Park. N.C.; and I. Bernard Weinstein, Columbia University, New York, N.Y. Received December 9, 1982; accepted January 11, 1983.

chorionic growth hormone were more reliable in vitro markers of cancer in these hybrids between HeLa and normal cells. The roles of metabolism of chemical carcinogens, formation of DNA damage, and DNA repair in carcinogenesis were reviewed in the second session (Chairperson, Peter Magee, Fels Research Institute, Philadelphia, Pa.). Metabolic activation and detoxifica tion pathways of procarcinogens and their major DNA adducts in cultured human tissue and cells are generally qualitatively similar to those found in experimental animals (Herman Autrup, Laboratory of Human Carcinogenesis, National Cancer Institute). However, wide quantitative interindividual differences are found in humans and other outbred animal species. Dezider Grunberger (Columbia University, New York, N. Y.) described the metabolism of benzo(a)pyrene and DNA adduct formation in cultured human epidermal keratinocytes. In contrast to benzo(a)pyrene, A/-hydroxy-A/-2-acetylaminofluorene, the proximate carcinogenic form of the hepatocarcinogen in rats, did not bind covalently to DNA. This finding was interpreted to demonstrate that metabolism of carcinogens and covalent binding to DNA in cultured human keratinocytes correlates well with the cell specificity exhibited by chemical carcinogens. New approaches to measure DNA dam age and repair were discussed by William Haseltine (Sidney Farber Cancer Institute) who emphasized the possible role of DNA structure in formation of specific DNA lesions including mutational "hotspots" not involving thymidinedimer-typeof DNA damage. Carcinogens may damage DNA by either direct action, i.e., attack of DNA by a reactive chemical or its metabolite, or indirect action, e.g., attack of DNA by active oxygen species formed by membrane active agents (Peter Cerutti, Swiss Institute for Ex perimental Cancer Research, Lausanne, Switzerland). An ex ample of "membrane-mediated chromosomal damage" was pre sented in which the tumor promoter, PMA,2 was shown to be clastogenic for human lymphocytes and to cause formation of lipid-peroxides and aldehydes, which were postulated clasto genic factors. Michael Sirover (Fels Research Institute) presented results suggesting that DNA repair pathways of normal human fibro blasts are induced prior to DNA replication as a protective mechanism to reduce mutagenesis and carcinogenesis, and cells from cancer-prone individuals may exhibit aberrations in regula tory mechanisms of DNA repair in the cell cycle. In the concluding presentation of this session, Richard Setlow (Brookhaven Na tional Laboratory, Upton, N. Y.) reviewed the topic of variations in DNA repair among people. Although a number of human genetic diseases, often cancer prone, such as XP, are considered to involve deficiencies in DNA repair, comparatively little infor mation is available concerning the basal level and variations in DNA repair found among individuals in the general population. Setlow argued that relatively small variations in DNA repair among "normal" people found in the few studies currently

2 The abbreviations used are: PMA, phorbol-myristate-acetate; pigmentosum; LTR, long terminal repeat; HBV, hepatitis B-virus.

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XP, xeroderma

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Meeting Report available could result in large variations in cancer risk among the apparently normal population. Tumor promotion and "transforming" growth factors were discussed in the third session (Chairperson, Michael Waters, Environmental Protection Agency, Research Triangle Park, N.C.). Takashi Sugimura (National Cancer Center Research Institute, Tokyo, Japan) described 2 new chemical classes of tumor pro moters: (a) indole alkaloids, e.g., teleocidin, isolated from Streptomyces; and (b) polyacetate compounds, e.g., aplysiatoxin, isolated from seaweeds. When compared to PMA, these new agents have similar promoting activity in mouse skin previously exposed to 7,12-dimethylbenz(a)anthracene and produce similar changes in cultured cells including induction of differentiation of human promelocytic leukemia cells (HL-60) and inhibition of dimethyl sulfoxide-induced differentiation of Friend mouse erythroleukemia cells. Differential response of premalignant human cells to the tumor promoters PMA and deoxycholic acid was studied using primary epithelial cell cultures of normal colon, adenomas, and colonie adenocarcinomas (Eileen Friedman, Memorial Sloan-Kettering Cancer Center, New York, N. Y.). In studies of premalignant cells from adenomas, tumor promoters enhanced the size of the proliferative cellular fraction, multilayering and clustering of the cells and secretion of plasminogen activator, and decreased intercellular communication. Benjamin Trump (University of Maryland School of Medicine, Baltimore, Md.) described the possible role of cellular ion regu lation in tumor promotion. Membrane changes frequently found in tumors include rapid ion influx of Na+ followed by Ca2+, formation of Superoxide radicals which further modify mem branes, modulation of cAMP and cGMP modification of the cytoskeleton, and alteration of cellular pH. Whether or not these changes are critical for the mechanism of tumor promotion is not as yet known, but animal and human tumors studied thus far have increased Na/K ion ratios and increased calmodulin/tubulin ratios. "Transforming" growth factors isolated from mice, rats, and humans were discussed by John Stephenson (Laboratory of Viral Carcinogenesis, National Cancer Institute). These single polypeptides, with a molecular weight of approximately 6800, show considerable amino acid sequence homology in different animal species and have distant sequence homology with epi dermal growth factor. They also bind to the membrane receptor for epidermal growth factor and cause normal rat or human cells to express reversible phenotypic changes, e.g., anchorage-in dependent growth. These results are consistent with, but do not prove, the autocrine hypothesis that some tumors may sustain unlimited growth by producing their own growth factors. The role of cellular lesions caused by chemical carcinogens was the topic of the fourth session (Chairperson, Donald King, Columbia University). Takeo Kakunaga (Laboratory of Molecular Carcinogenesis, National Cancer Institute) compared the pro gressive sequence of phenotypic changes in rodent versus hu man fibroblasts during in vitro Carcinogenesis. During the multi stage carcinogenic process, anchorage-independent growth is an early phenotypic change in human fibroblasts possibly reflect ing a mutagenic event. In contrast, this change usually occurs as a later temporal event in rodent cells. Since the majority of the human cells "transformed" by chemical, microbial, or physi cal agents have a finite in vitro lifespan and do not produce

progressively growing tumors in athymic nude mice, these cells can be considered to be "partially" transformed and the so far rare tumorigenic cell line as being "completely" transformed. Dr. Kakunaga also described a "completely" transformed KD cell in which a point mutation in 0-actin gene produced a mutant ÃŸactin with a shorter cellular half-life than the normal 0-actin. George Milo (Ohio State University, Columbus, Ohio) reviewed the in vitro transformation studies of human foreskin fibroblasts. He emphasized the increased sensitivity of the cells to transfor mation if they are exposed to carcinogens at the interface between d and S phase. This is also at the point in the cell cycle where high levels of calmodulin are found. Transformation of human pancreatic cells was described by Ismail Parsa (State University of New York at Downstate, New York). When pancreatic expiants were continuously exposed to A/-nitrosodimethylamine for 12 weeks, the xenotransplanted cells produced progressively growing carcinomas in athymic nude mice. Monoclonal antibodies with specificity for either acinar or ductal cell surface antigens are being utilized to study the pathogenesis of preneoplastic and neoplastic lesions. David Kaufman (University of North Carolina School of Medi cine, Chapel Hill, N. C.) described the clonal growth of stromal cells from human endometrium and modulation of their differen tiated state by hormones and nutritional factors. When these cells were exposed repetitively to A/-methyl-A/'-nitro-A/-nitrosoguanidine, they expressed 7-glutamyl transpeptidase activity and anchorage-independent growth. DNA damage, mutagenesis, and transformation were studied using fibroblasts from normal human and XP patients (Justin McCormick, Michigan State University, East Lansing, Mich.). When compared to normal fibroblasts, XP cells are deficient in excision DNA repair, and are both hypermutatable and hypertransformable, the latter measured by anchorage-independent growth. He concluded that anchorage-independent growth in this system is a mutational event inherited in a dominant manner with an optimal expression time of 8 to 12 population doublings. Preliminary results indicating the hypermutatability of fibroblasts from familial melanoma patients were also presented. Using human cell hybrids, William Benedict (University of Southern California) also presented data indicating that anchor age-independent growth is a dominant trait while tumorigenicity is a recessive trait. He emphasized the advantages of using cells for transformation studies from patients with genetically inherited susceptibility to cancer. The fifth session focused on cellular lesions caused by radia tion, metals, and oncogenic fibers (Chairperson, Paul Ts'o, Johns Hopkins University, Baltimore, Md.). Carmia Borek (Columbia University) compared the responses of rodent cells and human cells to either ionizing radiation or UV B light. Transformation (anchorage-independent growth) of the human fibroblasts was potentiated by /3-estradiol. Thyroid hormone was found to in crease in in vitro transformation (foci formation) of the rodent cells. Effects of asbestos or carcinogenic metals in human lung cells were described by John Lechner (Laboratory of Human Carcin ogenesis, National Cancer Institute). Using human epithelial cells grown in a chemically defined medium, nickel sulfate caused the appearance of phenotypically altered cells with a prolonged life span, aneuploidy, and marker chromosomes. Mesothelial cells were shown to be 10-fold more sensitive to the cytotoxicity of

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C. C. Harris asbestos (chrysotile > amosite > crocidolite) than were bronchial epithelial cells. Quantitative studies of cytotoxicity and mutagenicity of human fibroblasts were presented by John Little (Harvard School of Public Health, Cambridge, Mass.). He concluded that human cells are hypersensitive to low-dose rate irradiation as compared with rodent cells and suggested that rodent cells in some cases may not be an accurate model for radiation effects in human cells. The role of viruses in human carcinogenesis was discussed in the sixth session (Chairpersons: Theodore Puck, Eleanor Roo sevelt Institute for Cancer Research, Denver, Colo.; and Char lotte Friend, Mount Sinai School of Medicine, New York, N. Y.). George Vande Woude (Laboratory of Molecular Oncology, Na tional Cancer Institute) reviewed the evidence that a segment of viral genome (termed one gene) acquired from the host chro mosome is responsible for acute transforming activity of oncogenic viruses. He also presented data indicating that the U3 region of LTR sequences is responsible for activation of mos expression. According to this model, cellular sequences analo gous to LTR could cause the neoplastic phenotype by activating expression of adjacent genes with oncogenic potential. A. J. van der Eb (University of Leiden, the Netherlands) de scribed the transformation of rat cells by human adenovirus in which the transforming activity is localized in the early region 1 (E1), which contains 2 transcriptional units (E1a and E1b). Re sults were presented suggesting that E1a is responsible for cellular immortalization and regulates E1b transcription. These results indicate that E1b expression leads to morphological transformation and tumorigenicity. A human retrovirus associated with T-cell leukemia and lymphoma in Japan and the Caribbean was described by M. Sarngadharan (Litton Bionetics, Kensington, Md.) and Mitsuaki Yoshida (Cancer Institute, Tokyo, Japan). This C-type virus shares only distant homology with bovine leukemia virus and has an LTR similar to other animal retroviruses. In vitro transmission studies showed that this retrovirus infects normal cord blood lymphocytes and induces immortalized growth and partial or complete independence of exogenous T-cell growth factor in the infected cells. Carlo Croce (The Wistar Institute of Anatomy and Biology, Philadelphia, Pa.) described the chromosomal localization of the genes for immunoglobulins which occur at the breakpoints of the chromosomal lesions associated with Birkitt's lymphoma. Preliminary evidence was also presented that the translocated distal portion of chromosome 8 is the site of the human homolog of the myc oncogene. The recombination of replication-competent type C RNA vi ruses with evolutionary well-conserved and modified (e.g. , point mutations) cellular genes has given rise to acute transforming retroviruses. Stuart Aaronson (Laboratory of Cellular and Molec ular Biology, National Cancer Institute) reviewed the rapid de velopments in this field. Oncogenes and the transforming gene product of the Kirsten-Harvey family of viruses have been iden tified in human tumor cell lines and primary human carcinomas. Epstein-Barr virus has been associated with Burkitt's lym phoma, nasopharyngeal

carcinoma, and B-cell lymphomas

in

transplant allograft recipients. David Purtilo (University of Ne braska Medical Center, Omaha, Neb.) has extensively studied the role of host factors, in particular, the underlying immunoreg-

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ulatory T-cell defects in the pathogenesis of these tumors. These studies emphasize the importance of host factors in the putative oncogenic response to an ubiquitous virus that in the immunocompetent person leads to a self-limited disease, infectious mononucleosis. The role of HBV in human liver carcinogenesis was discussed by Sun Tsung-tang (Cancer Institute, Beijing, People's Republic of China) and William Robinson (Stanford University, Stanford, Calif). Liver carcinoma is one of the leading worldwide causes of cancer deaths, e.g., yearly mortality of 100,000 people in the People's Republic of China. Sun emphasized both its multifactoral etiology, including the interactive effects between HBV and mycotoxins, and the use of cell and expiant cultures of human fetal liver to study these interactions. Advances in the molecular biology of HBV were discussed by Robinson, who reported that virion core polypeptide levels in human hepatocarcinoma PLC/ PRF/5 cells were controlled by the methylation of the C-gene and that DNA extracted from this human hepatoma cell line could transform NIH 3T3 cells. Linkage of laboratory and epidemiological sciences was em phasized in the last session (Chairpersons: Donald Davies, Ham mersmith Hospital, London, United Kingdom; and Dante Scar pelli, Northwestern University Medical School, Chicago, III.). Ernst Wynder (American Health Foundation, New York, N. Y.) argued that the highest priority should be given to studies of the most significant factors, i.e., tobacco, alcohol, and nutritional elements, in causation of human cancer. The concept of normal versus optimal diet and the importance of health education of children in cancer prevention were discussed. Tobacco is now well recognized as the most important cause of cancer in the western world. Dietrich Hoffmann (American Health Foundation, Valhalla, N. Y.) discussed the carcinogens and cocarcinogens found in chewing tobacco and tobacco smoke. Carcinogenic A/-nitrosamines can be detected in saliva of snuff dippers. The potential for endogenous formation of carcinogenic A/-nitrosamines in cigarette smokers was indicated by the finding of elevated levels of W-nitrosoproline in the urine of some smokers. Metabolism studies in cultured human tissues and in rodents demonstrate that tobacco-specific W-nitrosamines are activated to their ultimate carcinogenic forms by similar mechanisms. Genetic variations in carcinogen metabolism may be probed using drugs (Jeffrey Idle, St. Mary's Hospital Medical School, London, United Kingdom). Variability of chemical carcinogen activation is one plausible mechanism by which carcinogen doseresponse curves in humans may be modulated by host factors. In addition to examples of wide interindividual variation in poly morphic oxidative pathways in humans, an animal model was presented in which the female DA rat was shown to be a "poor metabolizer" of aflatoxin B! or an antihypertensive agent, debrisoquine, and was relatively resistant to the carcinogenic action of aflatoxin Bi. In the human body, carcinogenic N-nitroso compounds can be formed by the interaction of precursors ingested in food or water or can be produced endogenously. Helmut Bartsch (International Agency for Research on Cancer, Lyon, France) presented a method for the quantitative estimation of endogenous nitrosation in humans by measuring A/-nitrosoproline, a noncarcinogenic compound, in the urine of people given a standard diet consisting of vegetable juice (source of nitrate) and L-proline. Vitamin C or

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Meeting Report E inhibited endogenous nitrosation. Pilot investigations of people at low or high risk of gastric cancer or esophageal cancer are ongoing. Similar to most human cancer, the etiology of esophageal carcinoma appears to be multifactoral. Hsia Chu-chieh (Cancer Institute, Beijing, People's Republic of China) discussed the possible interactions of nutritional factors, fungal infections, and mycotoxin contamination in food in the etiology of esophageal cancer. In China, patients with severe dysplasia or early esoph ageal carcinoma have higher titers of anil-Candida antibodies when compared to the general population. Using cultures of fetal and adult esophagi, the cytotoxic and hyperplastic effects of T2 toxin, a trichothecene produced by Fusarium fungus, were de scribed. Dean Mann (National Cancer Institute) described the clinical features of several familial cancers including hereditary cuta neous malignant melanomas. Laboratory studies demonstrating defects in both DMA repair and immune system were presented. The recent outbreak of Kaposi's sarcoma and an acquired im mune deficiency syndrome among homosexual men was dis cussed. These individuals have decreased helper/suppressor Tcell ratios. This alteration was also found in some nondiseased male homosexuals and appears to have a correlation with the use of the "recreational" drugs amyl and butyl nitrate. The availability of practical, sensitive assay systems for meas uring the frequency of somatic cell mutations in humans would have obvious biological, medical, and epidemiolÃ³gica! signifi cance. Mortimer Mendelsohn (Lawrence Livermore National Lab oratory, Livermore, Calif.) discussed various assay systems in cluding the measurement of mutant hemoglobin in RBC by monoclonal antibodies and high-speed flow cytometry.

A special address was given by Frank Ruddle (Yale University, New Haven, Conn.) who discussed gene mapping and DNAmediated gene transfer. Most of the cellular oncogenes have been mapped to specific human chromosomes, e.g., c-mos, cras", and c-sis, on chromosomes 9, 12, and 22, respectively. Transfer of HLA Class I genes into mouse cells and transfer of an interferon gene into mouse embryos were given as examples of the potential of DNA-mediated gene transfer. In his concluding remarks, Curtis Harris (Laboratory of Human Carcinogenesis, National Cancer Institute) emphasized that can cer, like most diseases, is the result of complex interactions between host and environment. Host factors, both acquired and genetic, determine in part an individual's oncogenic susceptibility to carcinogens and cocarcinogens. Epidemiological studies indi cate that differences in susceptibility among individuals range from 3-fold to more than 100-fold. Host factors have been traditionally studied by 2 approaches, i.e., clinical investigations and animal models. As a bridge between these 2 approaches, in vitro models using human tissues and cells are providing new insights in the interactive effects of carcinogens, cocarcinogens, and host factors. In addition, combined laboratory-epidemiology studies are offering new opportunities to investigate the bio chemical and molecular basis of host susceptibility factors. The proceedings of this conference will scon be published. Curtis C. Harris3 Laboratory of Human Carcinogenesis Division of Cancer Cause and Prevention National Cancer Institute Bethesda, Maryland 20205 3To whom requests for reprints should be addressed.

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