John J. Reiners

Fluorescence assay for per-cell estimation of cytochrome P-450-dependent monooxygenase activities in keratinocyte suspensions and cultures☆

An assay was characterized that facilitated per-cell estimation of cytochrome P-450-dependent monooxygenase activities in whole-cell suspensions and cultures of murine epidermal keratinocytes (MEKs). 7-Ethoxycoumarin O-deethylase (7-ECD), 7-ethoxyresorufin O-deethylase (7-ERD), and 7-pentoxyresorufin O-deethylase (7-PRD) activities were monitored by fluorescent detection of their products. MEKs were made permeable by a freeze-thaw cycle, and xenobiotic metabolism occurred in situ. Analyses of cultured MEKs were made with the cells attached to the culture dishes. Product formation was proportional with MEK cell number and assay time and was dependent upon a NADPH-generating system. The three monooxygenase activities were inhibited to various degrees, in a dose-dependent manner, by the P-450 inhibitors alpha-naphthoflavone and metyrapone. The number of MEKs obtained from a single mouse was sufficient for multiple analyses. The assay was also used to determine monooxygenase activities in whole-cell suspensions of rat hepatocytes. Constitutive per hepatocyte 7-ECD, 7-PRD, and 7-ERD activities were 357-, 96-, and 1926-fold greater, respectively, than the activities measured in suspensions of dorsal MEKs.

Phorbol ester - mediated suppression of cytochrome P450 Cyp1a-1 induction in murine skin: Involvement of protein kinase C

Epidermal 7-ethoxyresorufin O-deethylase (EROD) activity was elevated greater than 100-fold within 4 to 7 h of topical treatment of SENCAR mice with 100 nmol dibenz[a,c]anthracene (DB[a,c]A). Treatment of skin with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) 2 to 8 h prior to DB[a,c]A application suppressed induction by 80%. Suppression was dose-dependent over the range of 0.01 to 5 micrograms TPA (ID50 approximately 0.6 nmol). EROD activities in normal and TPA-treated epidermis paralleled steady state P450 CYP1A1 mRNA content. Analogs of TPA incapable of activating or down-regulating protein kinase C (PKC) did not suppress induction. Pretreatment of skin with sn-1,2-didecanoylglycerol, an activator of PKC which causes translocation but no down-regulation, did not suppress EROD induction. However, induction was suppressed by chrysarobin, an anthralin analog that causes PKC down-regulation in the absence of prior activation. These studies suggest that PKC participates in the processes associated with Cyp1a-1 induction and that TPA effects Cyp1a-1 induction through its down-regulation of PKC.

Differential expression of cytochrome P-450 in proliferating and quiescent cultures of murine lung epithelial cells

Expression of the cytochrome P-450 monooxygenase activity 7-ethoxyresorufin O-deethylase (7-ERD) was surveyed in proliferating and quiescent cultures of murine cell line C-10, a non-tumorigenic line of presumed alveolar type II origin. 7-ERD activities were undetectable in subconfluent/proliferating cultures but became detectable once the cultures had become confluent and their growth had arrested due to contact inhibition. Serum deprivation of subconfluent cultures resulted in a rapid inhibition of cell proliferation and the subsequent expression of 7-ERD. These results suggest that 7-ERD expression is regulated as a function of the proliferative status of C-10 cells.

DIFFERENTIAL MODULATION OF CONTACT HYPERSENSITIVITY AND DELAYED HYPERSENSITIVITY REACTIONS BY TOPICAL APPLICATION OF 12-O-TETRADECANOYLPHORBOL 13-ACETATE

Ear/footpad swelling following sensitization and challenge with 2,4-dinitrofluorobenzene (DNFB)/allogenic splenocytes (AS) were used to monitor the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on contact hypersensitivity (CHS) and delayed hypersensitivity (DHS) reactions, respectively. Topical treatment of dorsal or ventral SENCAR mouse skin 4x with 2 micrograms of TPA prior to sensitization of dorsal skin with DNFB suppressed attempts to induce CHS by subsequent challenge with DNFB. The adoptive transfer of splenocytes isolated from mice pretreated on the dorsum with TPA prior to dorsal sensitization with DNFB inhibited the development of CHS to DNFB in recipient mice. Conversely, topical treatment with TPA prior to s.c. sensitization with AS neither suppressed subsequent attempts to induce DHS, nor resulted in the generation of a splenocyte population capable of suppressing DHS reactions in adoptive transfer studies. Thus, promoting doses of topically applied TPA has differential effects on CHS and DHS reactions.

Antioxidant-Prooxidant Status of Murine Skin During the Ontogeny of Chemically-Induced Skin Cancer

Reduced/oxidized glutathione (GSH/GSSG) and the activities of catalase (CAT), Superoxide dismutase (SOD), glutathione peroxidase (GPX) and xanthine oxidase (XO) were quantitated in initiated SENCAR mice being promoted with 1 μg of 12-O-tetradecanoylphorbol-13-acetate (TPA). Within 24 hr of 4 or 10 applications of TPA epidermal SOD, CAT, GPX and XO specific activities were∼56, 39, 76 and 350% of the values measured in solvent-treated mice. SOD, CAT and XO activities in papillomas and squamous cell carcinomas (SCC) were∼15%, 30% and 175% respectively, of the activities in skins of age-matched, control mice. GPX activities were unaffected in papillomas but reduced 22% in SCC. Total epidermal GSHt (GSH+GSSG) and GSSG contents (nmol/g tissue) were not significantly altered 0.5, 4 or 24 hr after 1 or 4 applications of TPA. These studies suggest (a) that TPA orchestrates changes in enzymes involved in reactive oxygen metabolism to levels characteristic of the skin tumor phenotype; and (b) if TPA induces oxidative stress in vivo in murine epidermis, it cannot be detected by measurements of GSH and GSSG.

DNA-MEDIATED GENE TRANSFECTION INTO PRIMARY CULTURES OF ADULT MOUSE KERATINOCYTES

SummaryAn efficient and reproducible technique for the transfection of primary cultures of adult mouse keratinocytes has been developed. The procedure involves culturing the primary adult mouse epidermal cells at 32° C in an enriched media until they reach 70 to 95% confluency, followed by transfection with exogenous DNA in a low potassium environment. Using chloramphenicol acetyl transferase (CAT) transient gene expression assays and various strong viral promoter/CAT constructs, the transfection procedure was optimized for media formulation, plasmid DNA concentration, carrier DNA concentration, incubation temperature, incubation period, and cell density. Optimized parameters include the use of 6 µg plasmid DNA and 10 µg pUC19 carrier DNA per 60-mm tissue culture dish. Since primary keratinocytes undergo a well-characterized pattern of differentiation in vitro in response to extracellular calcium concentrations, this transfection procedure should provide a useful model in which to study both tissue- and differentiation-specific gene expression.