Development in Rat Tracheal Epithelium Effect of Carcinogen Dose on the Dynamics of Neoplastic

The aim of our studies was to analyze carcinogen dose effects on the development of "carcinogen-altered" preneoplastic epithelial cell populations in rat tracheas following ex posure to 7,12-dimethylbenz(a)anthracene. As described pre viously (Cancer Res., 39: 4003-4010, 1979), cells appear in the trachéalepithelium upon exposure to carcinogen which are distinguishable from the majority of the epithelial cells by a markedly enhanced in vitro growth capacity. In normal trachéal epithelium, most of the clonogenic cells have only a limited replicative capacity. After a limited number of population dou blings, trachéalepithelial cell cultures cease to proliferate and senesce. In contrast, cultures established from carcinogenexposed epithelium contain clonogenic cells which proliferate extensively, forming expanding epithelial foci (EF) in late pri mary cultures. These altered clonogenic units are designated EF-forming units (EFFU). They often escape senescence per manently, thus giving rise to epithelial cell lines. Some of these become neoplastic after repeated subculturing. There are clon ogenic units giving rise to subculturable progeny (EFFUS) and those giving rise to progeny which became anchorage inde pendent (EFFUs.agt). In the trachéal cell culture system, an chorage-independent growth is highly correlated with oncogenicity in vivo. In the present study, tracheas were exposed in vivo for 4 weeks to carcinogen doses ranging from 5 to 335 /¿gof 7,12'dimethylbenz(a)anthracene. Trachéalcells were harvested by enzymatic procedures at 0, 4, 16, and 52 weeks after the end of exposure and were assayed in vitro in the epithelial focus assay (EF assay) for the frequency of different types of EFFU. Control tracheas contained less than one EFFU per 10s cells. Exposure to 7,12-dimethylbenz(a)anthracene resulted in a 100to 500-fold increase. We were unable to detect any definitive carcinogen dose-response effect on the number of clonogenic cells forming EF or subculturable EF (EFFU or EFFUs), although there was some indication that the latter might be increasing with increasing dose. In contrast, there was a marked carcinogen dose effect on the frequency and rate of appearance of EFFUs,ag». For example, at 16 weeks after exposure, the frequencies of EFFUs.ag» (as the percentage of all EFFUs) were 4, 24, and 70% in the tracheas treated with 50, 162, and 335 /¿gof 7,12-dimethylbenz(a)anthracene, re spectively. The data suggest that the carcinogen dose affects either the rate of growth of EFFUs.ag», the cell compartment with neoplastic potential, or the conversion rate of EFFUS to EFFUs.ag». INTRODUCTION We described recently (17,18) a method for the identification and isolation of "carcinogen-altered" epithelial cells from tra cheas, esophagi, and lungs exposed in vivo to carcinogen. These altered cells appear soon after the target tissues have been exposed to carcinogen and can be found throughout the tumor latency period. They are considered to be preneoplastic, since, compared with unexposed cells, they have an increased likelihood of becoming neoplastic. These early preneoplastic cells can be identified because of the acquisition of a markedly increased in vitro growth capacity. Under culture conditions which are "nonpermissive" for most normal trachéalepithelial cells, they not only survive but proliferate rapidly, forming expanding EF2 at a time when the cultures of normal trachea! cells have ceased to proliferate and are senescing. Some of the epithelial colonies escape permanently from senescence (or terminal differentiation); they become "immortal" and can be propagated in vitro indefinitely. After repeated subculturing, some of the cell line cultures become neoplastically trans formed, producing invasive carcinomas upon inoculation into compatible hosts. The progenitor-progeny relationship be tween the cells of the EF and the malignant cells of the late immortalized cell cultures is, if not proven, strongly suggested by previous studies (9, 13, 17). This sequence of (a) increased in vitro growth capacity, (b) escape from senescence or ter minal differentiation, and (c) neoplastic transformation is by no means unique for trachéalepithelial cells. It has been observed in a number of epithelial and nonepithelial cell types after carcinogen exposure in vitro as well as in vivo (1, 5, 6, 8, 13, 15, 17, 18, 21), suggesting that it may represent an important series of events in neoplastic transformation. In an earlier publication (17), we described the EF assay and its use for the analysis of the cellular events of neoplastic development. With this assay, we were able to distinguish and quantitate 3 different "growth phenotypes" of carcinogen-al tered clonogenic cells: (a) the EFFU; (b) the EFFUS which permanently escapes senescence; and (c) the EFFUs.ag» which gives rise to anchorage-independent offspring. We showed that these 3 phenotypes of carcinogen-altered cells are de tectable in trachéal epithelium immediately after carcinogen exposure and that the number of subculturable and anchorageindependent focus-forming units increases with time, even in the absence of any further carcinogenic stimulus. The purpose of the present studies was to investigate the effect of carcinogen dose on the induction of different types of epithelial focus-forming cells and on the dynamics of their 1Research sponsored by USPHS Grant Y01-CP-90211 from the Division of Cancer Cause and Prevention. National Cancer Institute, and by the Office of Health and Environmental Research, United States Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation. Received February 24. 1982; accepted August 6, 1982. 2 The abbreviations used are: EF. epithelial foci; EFFU. epithelial focus-forming units; EFFUs, epithelial focus-forming unit(s) that ca¡ibe subcultured; EFFU...,» epithelial focus-forming unit(s) that grew in soft agarose; DMBA, 7,12-dimethylbenz(a)anthracene; HBSS, Hanks' balanced salt solution; SE, seeding efficiency; EF,, epithelial foci that can be subcultured.