Carcinogenesis Studies in Human Cells and Tissues

[CANCER RESEARCH 38, 474-475, February 1978]

Meeting Report

Carcinogenesis researchers have striven for decades to metric and sorting methods, as well as density-gradient interpret data obtained from animal studies in terms of techniques, are now available and can supplement more their applicability to humans. The carcinogenic hazard of traditional cell culture methods, e.g., cell cloning and environmental chemicals has been determined primarily selective culture media. from studies with experimental animals and from retrospec In the second session significant advances in the expiant tive epidemiological investigations. An experimental ap culture of adult human tissues were discussed. Bronchus, proach to the direct study of Carcinogenesis in human pancreatic duct, bladder, uterine cervix, breast, colon, tissues and cells was recently developed to assess the: (a) esophagus, and aorta can all be cultured for weeks to mechanisms of Carcinogenesis in human cells, (o) variation months with maintenance of normal-appearing epithelium of carcinogenic susceptibility among individuals, and (c) (Benjamin F. Trump, University of Maryland; Herman Au- validity of the extrapolation of Carcinogenesis data from trup, NCI; and Sefton R. Wellings, University of California, experimental animals to the human situation. For stimula Davis). Chemically defined culture media have been devel tion of further progress in this new area of cancer research, oped for human bronchus, colon, and pancreatic duct, a workshop sponsored by the Carcinogenesis Program, which eliminates the biological variability due to serum NCI,1 was held at the Given Institute of Pathobiology, from studies of carcinogen interactions in these tissues. Aspen, Colo., August 15 to 19, 1977, with 30 invited partici Xenotransplantation, discussed in the third session, is pants. The program consisted of lectures, group discus required for the assessment of the neoplastic growth poten sions, and laboratory demonstrations. The workshop fo tial of cultured human cells exposed to chemical carcino cused on 5 major topics: (a) isolation and monolayer gens, since these cells obviously should not be inoculated culture of human epithelial cells, (o) expiant culture of into humans. The congenially athymic nude mouse accepts human tissues, (c) xenotransplantation, (d) metabolism of transplants of both fetal and adult human tissues. Matura chemical carcinogens, and (e) mutagenesis and neoplastic tion of xenotransplanted fetal tissues and the malignant transformation of human cells. growth of neoplastic cells were described by Carl Povlsen In the first session of the workshop, James Rheinwald (Pathology and Anatomy Institute, Kommunehospitalet, Co (Massachusetts Institute of Technology) described an in penhagen). The growth of xenotransplanted human atypical vitro system that permits cloning and serial cultivation and mammary lobules, presumably preneoplastic lesions, was differentiation of diploid human keratinocytes with a feeder dependent upon treating the athymic nude mouse with layer of irradiated mouse 3T3 cells. The addition of epider estrogen and progesterone (S. R. Wellings). mal growth factor tripled the culture lifetime of epidermal Metabolism of chemical carcinogens by human cells and keratinocytes from foreskins of newborn infants. The culti tissues was the third major topic of the workshop. Elliot S. vation of both normal and neoplastic human prostatic Vesell (Hershey Medical Center) reviewed the interindivid- epithelial cells was described by Edward Kaighn (Pasadena ual variation in metabolism of drugs and carcinogens. Foundation for Medical Research). The success of these Genetic factors, disease states, and the environment may studies was attributed in part to a new method of dispersing all alter the metabolism of xenobiotics. The metabolism of the prostatic epithelial cells by a 2-step procedure: (a) a carcinogenic polynuclear aromatic hydrocarbon, BP, and exposure of the cells to a hyperosmolar solution containing its interindividual variation have been extensively studied in a high concentration of potassium and (b) detachment of human tissues and cells. The activity of BP 3-monooxygen- the cells with ethylene glycol bis(ÃŸ-aminoethyl ether) N,N'- ase in liver biopsy specimens varied 30-fold (Olavi Pelko- tetraacetic acid i.e., EGTA. nen, National Institute of Child Health and Human Develop Cultured human and rat mammary epithelial cells were ment). The interindividual variation in binding levels of BP shown to require near physiological levels of estradiol, to DNA in cultured human colon and bronchus was even progesterone, and prolactin for cell maintenance (Douglas higher, i.e., 60- to 70-fold (H. Autrup and Curtis C. Harris, H. Janss, Frederick Cancer Research Program). Gordon NCI). Within an individual, binding levels of BP to cellular Sato (University of California, San Diego) presented evi DNA also vary among human tissues, e.g., bronchus, pan dence that the serum in cell culture medium can be re creatic duct, and colon. This variation among tissues may in part be due to different types of cytochrome P-450's, as placed by a mixture of hormones, transferrin, and putative hormones or growth factors and that each epithelial cell well as to the balance between activation and deactivation type may require a different mixture of hormones. pathways of carcinogen metabolism (0. Pelkonen). Methods to isolate epithelial cells were discussed by The many metabolic pathways of BP and the divergent Mortimer L. Mendelsohn (Lawrence Livermore Laboratory), pathways between microsomal preparations and intact cells Douglas H. Janss, and Gary D. Stoner (NCI). Flow cyto- were described by James K. Selkirk (Oak Ridge National Laboratory). The pathway of BP metabolism, leading to the major adduci formed with DNA in cultured human bron ' The abbreviations used are: NCI. National Cancer Institute; BP, chus, is similar to that found in cells of experimental benzo(a)pyrene. Received October 31, 1977; accepted November 16, 1977. animals (Shen Yang, Uniformed Services University of the

474 CANCER RESEARCH VOL. 38

Health Sciences). In addition, the major BP-DNA adduci presented both by Takeo Kakunaga (NCI), who used fibro formed in both cultured human bronchus and colon has blasts from buccal mucosa and the carcinogens 4-nitro- been identified as resulting from the frans addition of the 2- quinoline-1-oxide and A/-methyl-/V'-nitro-/V-nitrosoguani- amino group of guanine to position 10 of BP diol-epoxide I dine, and by Thomas J. Slaga (Oak Ridge National Labora (Alan Jeffrey, Columbia University). BP can also be metab tory), who used cells isolated from foreskin and the carcin olized by human arteries. Earl P. Benditi (University of ogen 3-methylcholanthrene-11,12-epoxide. In both reports Washington) reviewed these and other data that suggest the transformed human cells grew in semisolid media and the monoclonal and mutagenic origin of atherosclerotic were tumorigenic in athymic nude mice. plaques that may be considered benign tumors of the During 3 group discussion periods and the summary arterial wall. The metabolism of another polynuclear aro presentations by the session chairpersons, areas for future matic hydrocarbon, 7,12-dimethylbenz[a]anthracene, has study were identified. Culture media and methods for dis been studied in rat and human mammary epithelial cells in sociation of tissues have both been developed primarily for culture; the major metabolite has been identified as 8,9- fibroblasts. Evidence presented at the workshop suggests dihydro-8,9-dihydroxy-7,12-dimethylbenz[a]anthracene (D. that epithelial cells require both improved dissociation H. Janss). methods and more complex media than do fibroblasts. In addition to polynuclear aromatic hydrocarbons, other Specific markers for epithelial cells in vitro are also clearly chemical classes of carcinogens found in the environment needed. Studies of carcinogen activation and deactivation and/or tobacco smoke, e.g., W-nitrosamines, mycotoxins, in various experimental systems are complicated because and hydrazines, can be metabolized by cultured human the metabolism may differ with each level of organization, bronchus and colon (H. Autrup and C. C. Harris). One of i.e., microsomes versus cell versus tissue versus organism. these chemicals, aflatoxin B,, may be the most potent In addition, people may differ markedly in their ability to carcinogen known in experimental animals. Its major ad- activate and deactivate chemical carcinogens metabolically duct, formed with DNA incubated in vitro with either rat or because of variation of host biological factors. This varia human liver microsomes, has been identified as 2,3-dihy- tion should be considered in efforts to develop systems for dro-2-(/V7-guanyl)-3-hydroxyaflatoxÂ¡n B, (Gerald N. Wogan, the screening of chemicals for carcinogenic and/or muta Massachusetts Institute of Technology). genic activity. Test systems for screening may also not be The final group of presentations focused on studies of suitable as model systems for the study of molecular mech mutagenicity and neoplastic transformation. Helmut anisms of carcinogenesis. Both approaches are needed. Bartsch (International Agency for Research on Cancer) Finally, studies of neoplastic transformation, especially with observed a marked variation in mutation frequencies with epithelial cells, are hampered by the lack of sensitive and W-nitrosamines (Ames' Salmonella test) when liver surgical early occurring markers for transformed cells. specimens from different individuals were used for meta Although this field is in an early stage of development, it bolic activation. He also reported that N-nitroso-W-methyl- was evident from this meeting that many human cells and piperazine was 3 to 40 times more mutagenic with micro- tissues can now be maintained in a controlled experimental somes from human liver than it was with microsomes from setting and that mechanism studies in carcinogenesis are untreated rat liver. Ultimate chemical and physical carcino already being conducted in human cells. gens cause mutations in human diploid fibroblasts; the fre quency of mutations induced in the classical xeroderma Curtis C. Harris pigmentosum cells either by UV or by BP diol-epoxide I Umberto Saffiotti was significantly higher than the frequency found in normal Experimental Pathology Branch cells (Justin J. McCormick, Michigan State University). The Carcinogenesis Research Program rates of mutation caused by chemicals in human diploid National Cancer Institute fibroblasts were compared to presumed rates of neoplastic Bethesda, Md. 20014 transformation by Robert l. DeMars (University of Wiscon sin), who also discussed the hypothesis that neoplastic Benjamin F. Trump transformation of human cells may require 2 independent Department of Pathology mutations. University of Maryland Reports of successful neoplastic transformation by chem School of Medicine ical carcinogens of normal diploid human cells were then Baltimore, Md. 21201

FEBRUARY 1978 475

Curtis C. Harris, Umberto Saffiotti and Benjamin F. Trump

Cancer Res 1978;38:474-475.

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