Decrease in the protein kinase C-catalyzed phosphorylation of an endogenous lung protein (Mr 36,000) following treatment of mice with the tumor-modulatory agent butylated hydroxytoluene.

Butylated hydroxytoluene (BHT) causes transient lung damage in mice, and it can either inhibit or enhance carcinogen induction of tumors in internal organs, such as urethan-induced lung adenomas. Since protein kinase C (Pk-C) may mediate the action of one class of tumor-modulatory agents, the phorbol esters which promote skin tumorigenesis, we are examining the hypothesis that Pk-C is involved in the modulatory effects of BHT on internal organs. Endogenous phosphorylation of a Mr 36,000 cytosolic protein (p36) with a pI of 5.7 was demonstrable in extracts from lung and spleen but not from brain or heart. Phosphorylation required the presence of both Ca2+ and phosphatidylserine, and phosphate was incorporated into seryl and threonyl residues but not into tyrosyl residues. This reaction thus has the characteristics of Pk-C-dependent catalysis. A single i.p. injection of BHT (400 mg/kg body weight) decreased p36 phosphorylation severalfold in both BALB/cByJ and A/J mice. This decrease correlated with the extent of BHT-induced lung damage with regard to both the time course following BHT administration and the dose dependence of BHT. All of the pulmonary effects of BHT are abolished if the mice are pretreated with cedrene, an inducer of drug-detoxifying enzymes. Such treatment with cedrene prevented any BHT-induced decrease in p36 phosphorylation. A decrease in Pk-C specific activity, as measured using histone as an exogenous substrate, which resulted upon BHT treatment may provide a mechanism for decreased p36 phosphorylation. The specificity of this toxicity-related effect of BHT is emphasized by the fact that urethan injection did not detectably affect the phosphorylation of any lung proteins. Both p36 phosphorylation and Pk-C specific activity increased as a function of postnatal age. Thus the extent of p36 phosphorylation was inversely related to the extent of lung cell proliferation in two different physiological states, postnatal growth and regenerative repair following BHT-induced toxic injury. A single BHT injection is sufficient to cause lung toxicity, tumor prophylaxis, or cocarcinogenesis, while tumor promotion requires chronic treatment. P36 phosphorylation also decreased when mice were given multiple BHT injections over a period of 5 weeks. These results are consistent with a hypothesis that decreased Pk-C-dependent phosphorylation of p36 is involved in lung tumor modulation by BHT.

Changes in cyclic adenosine 3':5'-monophosphate-dependent protein kinases during the progression of urethan-induced mouse lung tumors.

The cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases in lung adenomas are functionally different from those of normal lung. The relevance of this change to neoplastic conversion was examined by comparing tumor kinases with those obtained from the normal cell of origin and by studying the kinases at different stages of tumor growth. Lung tumors were collected from A strain mice at different times after a single injection of urethan. These tumors are predominantly of alveolar type two cell origin, and cAMP-binding proteins in extracts from isolated type two cells and from lung adenomas at various stages of tumor progression were compared. Both the incorporation of the cAMP photoaffinity analogue, cyclic 8-azidoadenosine 3':5'-[32P]monophosphate (8-N3-[32P]cAMP), into the regulatory subunits of the type I (RI) and type II (RII) cAMP-dependent protein kinases and the autophosphorylation of RII were similar in extracts from whole normal lung and from type two cells. Altered protein kinases are thus not characteristic of normal type two cells. Lung tumors showed a decrease in photodetectable RII which correlated in degree with tumor size and extent of anaplasticity. This decreased RII photolabeling during tumor growth was associated with increased RII autophosphorylation. In contrast, decreased RII photolabeling in extracts from neonatal lung is accompanied by a substantial decrease in RII autophosphorylation. The characteristics of RII during normal development thus clearly differ from those during neoplastic development. An increase in the amount of an Mr 37,000 proteolytic fragment derived from R-subunits was also noted as a function of tumor progression. DEAE-cellulose chromatography of tumor cytosol showed that the increase in the amount of Mr 37,000 protein was accompanied by increased subunit dissociation of the type I isozyme. The dissociated RI subunit has been shown to be more sensitive to cleavage by a Ca2+-dependent neutral protease than when RI was in the holoenzyme form. This protease is present in both normal lung and lung adenomas, and its activity increases during the later stages of tumor progression. A comparison of cAMP binding and the light-induced covalent incorporation of 8-N3-[32P]cAMP showed that, for both RI and RII, photoincorporation was about 75% as efficient as noncovalent binding. In contrast, although the Mr 37,000 fragment can be photolabeled with low concentrations of 8-N3-[32P]cAMP, noncovalent cAMP binding to the endogenous Mr 37,000 fragment could not be demonstrated with a standard filtration assay. Such altered cAMP binding characteristics following Ca2+-dependent proteolysis of R-subunits would all

Pharmacologic and genetic studies on the modulatory effects of butylated hydroxytoluene on mouse lung adenoma formation.

Food additive butylated hydroxytoluene (BHT) (CAS: 128-37-0; 2,6-di-tert-butyl-p-cresol) can modulate the formation of lung adenomas in mice treated with the carcinogen urethan (CAS: 51-79-6; ethyl ester carbamic acid). An ip injection of BHT administered 6 hours before a single urethan injection decreased the number of tumors formed, whereas six weekly BHT injections following a single urethan dose increased tumor multiplicity. Biotransformation of BHT was apparently required for both prophylaxis and enhancement. Pretreatment of mice with cedrene, which perturbs drug metabolism, prevented both of these BHT activities. Analogues of BHT with different substitutions at position 6 of the phenol ring also affected tumor formation. 2-tert-Butyl-4-methylphenol inhibited, not enhanced, adenoma formation. 2-tert-Butyl-4,6-dimethylphenol (BDMP) (CAS: 1879-09-0; 6-tert-butyl-2,4-xylenol); increased tumor number in A/J but not in the BALB/cByJ mouse strain; its prophylactic activity could not be measured since urethan injection following BDMP treatment was lethal. Thus the presence of an alkyl group at this site on the phenol ring was not required for prophylaxis but was necessary for tumor enhancement. These results were consistent with the possibility that different BHT metabolites were responsible for each effect. Adenoma formation was enhanced by BHT in four strains of mice with a U+ phenotype (susceptible to urethan-induced lung adenomas); these strains were A, BALB/cBy, SWR/J, and RIIIS/J. BHT had no such effect, however, in the U+ strain 129/J. A U+ B- phenotype (urethan inducible but unresponsive to BHT enhancement) also was found among the recombinant inbred lines originally derived from a cross between U+B+ BALB/-cByJ and U-/B- C57BL/6ByJ progenitor strains. This finding showed that the genes determining sensitivity to urethan and to BHT had recombined independently of each other. An inability to metabolize BHT was probably not involved in the B- phenotype, since BHT caused reversible lung damage in the 2 U+B- recombinant inbred lines and strain 129, and biotransformation was required for this effect. These U+B- mice thus can be used to characterize the lung toxic effects of BHT in the absence of its tumor-enhancing activity and will serve as valuable controls in studies on mechanisms of tumor enhancement.

Major effect on susceptibility to urethan-induced pulmonary adenoma by a single gene in BALB/cBy mice.

Fewer lung adenomas were induced by urethan in BALB/cBy mice than in the A/J or SWR/J mouse strains. When BALB mice were crossed with either of these more sensitive strains the response of the progeny to urethan was most easily explained by a single gene which regulates susceptibility, with the more resistant phenotype behaving as a dominant trait. C56BL/6J mice were more resistant to adenoma induction than were BALB mice; progeny obtained when these two strains were crossed resembled the BALB susceptibility phenotype. As an approach to understanding the mechanism of action of this gene, agents that modulate adenoma initiation and tumor promotion were tested in BALB mice and other strains. The number of adenomas in BALB mice were increased severalfold by multiple urethan injections, which presumably affect initiation, and by the use of butylated o-hydroxytoulene as a promoting agent. Tumor incidence in A-mice was increased 50% by each treatment; neither procedure caused tumors to appear in the resistant DBA/2J, C3H/-21BG, or C57BL/6J strains. No relationship was observed between the strain dependency of the lethal effects of multiple injections of these agents and the relative susceptibilities of these strains to adenoma induction. The role of certain host factors in the regulation of tumor susceptibility was also tested. Homozygosity for the beige (bg) mutation had no effect on tumor numbers in C57 mice, suggesting that natural killer cells, deficient in bg/bg mice, played no major role in determining adenoma susceptibility in this strain. No correlation was found between the susceptibility of various sublines to urethan-induced lung adenoma and the reported relative tumoricidal capacities of the peritoneal macrophages from these sublines.

Elution of the regulatory subunit of cAMP-dependent protein kinase Type I isozyme derived from epididymal fat within the type II isozyme chromatographic peak.

No cAMP-dependent protein kinase activity is found upon DEAE-cellulose chromatography of mouse fat extracts at the low salt concentration characteristic of the Type I isozyme. The RI detected in fat extracts by photoincorporation of the analog, 8-N3 [32P]cAMP, elutes within the high salt Type II isozyme peak. The multiple charge variants of this photolabeled RI which can be resolved by two-dimensional gel electrophoresis are similar to those of the histoptypically-related cultured cells, SV3T3 and 3T6, which do contain Type I kinase isozyme activity peaks. This high salt-eluting RI may be part of a Type I holoenzyme whose elution properties are altered by interactions with other substances present in the extract.