Tumorigenic and molecular characterization of novel phorbol ester-resistant and -sensitive lines of mice.

Two outbred lines of CD-1 mice were developed using males and females in an initiation (dimethyl-benz[a]anthracene; DMBA), promotion (12-O-tetradecanoylphorbol-13-acetate; TPA) challenge, posttumorigenesis breeding protocol. Our results indicate that the phorbol ester-resistive (PESTI) line developed tumors at a rate 4.1 times faster than the CD-1 parental line, while the phorbol ester-resistant (PERTI) line developed tumors at a rate 36 times slower than the CD-1 parents. The average number of tumors per mouse reached levels of 27.5 at 12 wk in the PESTI line, 0.1 at 16 wk in the PERTI line, and 6.7 at 16 wk in the CD-1 line. Biochemical tests showed that the PESTI line had both a high basal level and an enhanced epidermal ornithine decarboxylase (E.C. 4.1.1.17) response to TPA, the latter being nine times that of the PERTI line at their maximum dosages. An autoradiographic analysis of in vivo epidermal cell protein phosphorylation indicated marked differences in basal protein phosphorylation profiles (with high phosphate incorporation, PERTI, 112.7, 95.5, 64.4, 40.8, 18.6, 17.4, and 12.3 kDa; PESTI, 64.4, 40.8, 31.8, and 12.3 kDa) as well as TPA-dependent changes in these profiles (difference from basal levels, PERTI, 31.8 and 12.8 kDa; PESTI, 139.6, 126.3, 37.2, and 18.6 kDa). These heterogeneous profiles indicate strong genetic segregation of these protein kinase C target substrates. This dermatologically based selection and breeding process also resulted in cosegregating genetic changes in TPA binding constants (Kd) for brain protein kinase C, which were reversed for cytosolic and plasma membrane fractions in the PESTI (cytosolic, 0.50 nM; plasma membrane, 1.89 nM) and PERTI (cytosolic, 2.02 nM; plasma membrane, 0.51 nM) mice. Moreover, TPA-inducible phosphorylation of 43 and 110 kDa proteins in vivo in the brains of only the PERTI line mice suggests that the PESTI line has either high levels of endogenous phosphorylation or reduced expression of these proteins. In contrast, in vitro screening of brain cytosolic proteins indicated that both lines have a 120 kDa protein whose phosphorylation can be induced by TPA while the PESTI line has a 14 kDa protein whose phosphorylation regulatory controls differ from the PERTI line. Thus, these lines of mice provide experimental systems for defining the skin tumorigenesis process and possibly extending initiationpromotion observations and concepts to other tissues.