The tumor promoter TPA enhances benzo[a]pyrene and benzo[a]pyrene diolepoxide mutagenesis in Big Blue mouse skin.

The Big Blue mouse was used to investigate the role of cell proliferation in mutation fixation in the mouse back skin model of carcinogenesis. Phorbol 12-myristate 13 acetate (TPA) was applied to the dorsum of Big Blue mice to manipulate cell proliferation, and benzo[a]pyrene (BaP) or BaP-diolepoxide (BPDE) was applied to produce premutagenic DNA damage. Mutations in the lacI transgene of skin DNA were measured. BaP and BPDE elevated mutant frequency, DNA adducts, and cell damage over untreated and acetone-treated mice. BPDE-DNA adducts peaked within 30 min of exposure and DNA adducts, formed after application of both BaP and BPDE, declined rapidly with time. As the dose of BaP increased (4 to 64 microg), DNA adducts, mutant frequency, and cell damage increased in a dose-dependent manner. TPA applied after BaP and BPDE further increased mutant frequency, DNA adducts, and cell damage, while variably affecting mitotic index and other measures of cell proliferation. TPA became less effective at increasing mitotic index as the dose of BaP increased, although all measures of cell proliferation, taken together, increased. The most effective production of DNA adducts and mutations occurred when the carcinogen was applied simultaneously with or within 1 hr of TPA. Mutations induced by BPDE were predominantly base substitutions: of these base substitutions, 35% were G:C --> A:T transitions, and 36% were G:C --> T:A and 29% G:C --> C:G transversions. Approximately 88% of all mutations and 100% of base substitutions were at G:C sites; 60% of all mutations and 70% of the base substitution mutations occurred at CpG sites. A:T --> G:C transitions were not found. All of the single-base deletions were at G:C base pairs.

Sites of UV-induced phosphorylation of the p34 subunit of replication protein A from HeLa cells.

Exposure of mammalian cells to UV radiation alters gene expression and cell cycle progression; some of these responses may ensure survival or serve as mutation-avoidance mechanisms, lessening the consequences of UV-induced DNA damage. We showed previously that UV irradiation increases phosphorylation of the p34 subunit of human replication protein A (RPA) and that this hyperphosphorylation correlated with loss of activity of the DNA replication complex. To characterize further the role of RPA hyperphosphorylation in the cellular response to UV irradiation and to determine which protein kinases might be involved, we identified by phosphopeptide analysis the sites phosphorylated in the p34 subunit of RPA (RPA-p34) from HeLa cells before and after exposure to 30 J/m2 UV light. In unirradiated HeLa cells, RPA-p34 is phosphorylated primarily at Ser-23 and Ser-29. At least four of the eight serines and one threonine in the N-terminal 33 residues of RPA-p34 can become phosphorylated after UV irradiation. Two of these sites (Ser-23 and Ser-29) are known to be sites phosphorylated by Cdc2 kinase; two others (Thr-21 and Ser-33) are consensus sites for the DNA-dependent protein kinase (DNA-PK); the fifth site (Ser-11, -12, or -13) does not correspond to the (Ser/Thr)-Gln DNA-PK consensus. All five can be phosphorylated in vitro by incubating purified RPA with purified DNA-PK. Two additional sites, probably Ser-4 and Ser-8, are phosphorylated in vivo after UV irradiation and in vitro by purified DNA-PK. The capacity of purified DNA-PK to phosphorylate many of these same sites on RPA-p34 in vitro implicates DNA-PK or a kinase with similar specificity in the UV-induced hyperphosphorylation of RPA in vivo.

Induction of interleukin-6 in the epidermis of mice in response to tumor-promoting agents.

Recent reports imply that several epidermal cytokines have a functional role in the tumor promotion stage of the multistage carcinogenesis model in mouse dorsal skin. In this report we describe studies to assess the role of interleukin-6 (IL-6) in tumor promotion. Promoting, as well as non-promoting, hyperplastic agents were found to induce IL-6, as measured by mRNA expression. Inhibitors of tumor promotion inhibited tumor-promoter-mediated IL-6 induction. However, when mice were injected with a neutralizing antibody specific to murine IL-6, there was no effect on tumor-promoter-mediated epidermal hyperplasia and dermal inflammation. These studies suggest that even though IL-6 is produced in the epidermis following tumor promoter application, it does not modulate epidermal hyperplasia and dermal inflammation. Our findings also stress the importance of assessing, in in vivo studies, the function of those cytokines suggested by in vitro experiments to have important roles in tumor promotion.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in mouse skin in response to tumor-promoting agents and modulates dermal inflammation and epidermal dark cell numbers.

In mouse dorsal skin multistage carcinogenesis models, tumor promotion can be mediated by chemical agents, but also by wounding or abrasion of the epidermis, suggesting that endogenous growth factors mediate this process. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one such factor that has been reported to be produced by keratinocytes in vitro, and has been suggested both to stimulate keratinocyte proliferation, and also to be a chemoattractant for neutrophils and macrophages. In this study we examined the expression and function of GM-CSF in mouse skin following the application of tumor-promoting agents. Both single and multiple applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in accumulation of GM-CSF mRNA in the epidermis. Various phorbol and non-phorbol ester tumor promoters were found to induce increases in epidermal GM-CSF mRNA levels commensurate with their relative tumor promoting capabilities. Fluocinolone acetonide (FA) and tosyl phenylalanine chloromethyl ketone (TPCK), inhibitors of tumor promotion, inhibited tumor promoter-mediated GM-CSF accumulation, whereas all-trans-retinoic acid (RA) enhanced the TPA-induced increase. The retinoic acid analogue RO-109359 which, unlike RA, does not have tumor promoting activity per se, inhibited the TPA-induced increase in epidermal GM-CSF mRNA levels. When an antibody specific to GM-CSF was administered prior to TPA, the promoter-induced dermal inflammation and increase in epidermal dark cell number were reduced, yet promoter-induced epidermal hyperplasia was not. These findings implied that elevation of GM-CSF levels plays an important role in chemically-mediated mouse skin tumor promotion and principally via effects on promoter-induced inflammation and increased epidermal dark cell number.