The aim of these studies was to investigate the effect(s) of the promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the evolution of different types of 7,12-dimethylbenz(a)anthracene (DMBA)-initiated rat tracheal epithelial cells in vivo. As described previously, upon exposure to carcinogens, cells appear in the tracheal epithelium which are distinguishable from the majority of the epithelial cells by a markedly enhanced in vitro growth capacity.

In the present study, tracheal transplants were exposed in vivo to 35 µg DMBA for 2 weeks and subsequently to 100 µg TPA. Controls were exposed to DMBA alone, TPA alone, or blank pellets alone. Tracheal cells were harvested by enzymatic procedures at 0, 3, 6, 12, or 18 months after the end of exposure to DMBA and at the same time points after the beginning of exposure to TPA or control pellets and were assayed in vitro with the epithelial focus (EF) assay for the frequency of different types of EF-forming units. Control tracheas yielded <1 EF/104 viable cells harvested. Of these EFs, 10 to 28% were subculturable (EFs), and no EFs derived from control tracheas exhibited growth in soft agarose EFs,ag+). Exposure to TPA alone did not increase the yield of EF, EFs, or EFs,ag+ above control levels. Carcinogen exposure resulted in a 6- to 20-fold increase in yield of EF, a 2- to 3-fold increase in EFs, and a ≥15-fold increase in yield of EFs,ag+ above control levels. Neither the yield of EF nor the yield of EFs was affected by subsequent exposure to TPA. In contrast, there was a marked effect of subsequent TPA exposure on the maintenance and size of the cell pool giving rise to anchorage-independent offspring (EFs,ag+). By 3 months after DMBA exposure, 15% of EFs were EFs,ag+ in both the DMBA and DMBA-TPA exposure groups. However, in cultures established 12 months following exposure to DMBA alone, only 2% of EFss were EFs,ag+ following exposure to DMBA alone. In contrast, at this same time following exposure to DMBA and TPA, 18% of EFss were EFs,ag+. In a parallel two-stage carcinogenesis study with tracheal transplants, a significant enhancement of the DMBA-induced tumor response by TPA was observed. At 20 months after exposure, 4 and 37%, respectively, of DMBA- and DMBA-TPA-exposed tracheas developed invasive carcinomas.

In summary, it appears that initiation can be viewed as a series of complex cellular changes. With time, some of these changes are reversible. Exposure to TPA of cell populations initiated with low doses of DMBA results in the persistence of altered cell populations in the intact tissue. Without TPA treatment, some phenotypically altered cells appear to revert to a more normal state and/or fail to replicate.


Research sponsored jointly by the National Institute of Environmental Health Sciences under Interagency Agreement Y01CP 50200; National Cancer Institute Interagency Agreement Y01CP 90211; and the Office of Health and Environmental Research, United States Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation.

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