NAD(P)H:quinone oxidoreductase 1 deficiency and increased susceptibility to 7,12-dimethylbenz[a]-anthracene-induced carcinogenesis in mouse skin.

BACKGROUND: The phase II enzyme NAD(P)H :quinone oxidoreductase 1 (NQO1) catalyzes quinone detoxification, protecting cells from redox cycling, oxidative stress, mutagenicity, and cytotoxicity induced by quinones and its precursors. We have used NQO1(-/-) C57BL/6 mice to show that NQO1 protects them from skin cancer induced by the polycyclic aromatic hydrocarbon benzo[a]pyrene. Herein, we used NQO1(-/-) mice to investigate whether NQO1 also protects them against 7,12-dimethylbenz[a]anthracene (DMBA), where methyl substituents diminish primary quinone formation. METHODS: Dorsal skin of NQO1(-/-) or wild-type C57BL/6 mice was shaved. When tested as a complete carcinogen, DMBA (500 or 750 microg in 100 microL of acetone) alone was applied to the shaved area. When tested as a tumor initiator, DMBA (200 or 400 nmol in 100 microL of acetone) was applied to the shaved area; 1 week later, twice-weekly applications of phorbol 12-myristate 13-acetate (PMA)-10 microg dissolved in 200 microL of acetone-to the same area began and were continued for 20 weeks. Tumor development was monitored in all mice (12-15 per group). All statistical tests were two-sided. RESULTS: When DMBA (750 microg) was tested as a complete carcinogen, about 50% of the DMBA-treated NQO1(-/-) mice but no DMBA-treated wild-type mouse developed skin tumors. When DMBA (both concentrations) was used as a tumor initiator, NQO1(-/-) mice developed larger tumors at a greater frequency than their wild-type littermates. Twenty-three weeks after the first PMA treatment in the tumor initiator test, all 30 NQO1(-/-) mice given 400 nmol of DMBA had developed skin tumors, compared with 33% (10 of 30) of treated wild-type mice (P<.001). CONCLUSIONS: NQO1(-/-) mice are more susceptible to DMBA-induced skin cancer than are their wild-type littermates, suggesting that NQO1 may protect cells from DMBA carcinogenesis.

Deregulated expression of c-Myc depletes epidermal stem cells.

The beta-catenin/TCF signaling pathway is essential for the maintenance of epithelial stem cells in the small intestine. c-Myc a downstream target of beta-catenin/TCF (ref. 2), can induce differentiation of epidermal stem cells in vitro. To determine the role of c-Myc in epidermal stem cells in vivo, we have targeted expression of human MYC2 to the hair follicles and the basal layer of mouse epidermis using a keratin 14 vector (K14.MYC2). Adult K14.MYC2 mice gradually lose their hair and develop spontaneous ulcerated lesions due to a severe impairment in wound healing; their keratinocytes show impaired migration in response to wounding. The expression of beta1 integrin, which is preferentially expressed in epidermal stem cells is unusually low in the epidermis of K14.MYC2 mice. Label-retaining analysis to identify epidermal stem cells reveals a 75% reduction in the number of stem cells in 3-month-old K14.MYC2 mice, compared with wildtype mice. We conclude that deregulated expression of c-Myc in stem cells reduces beta1 integrin expression, which is essential to both keratinocyte migration and stem cell maintenance.

NAD(P)H:quinone oxidoreductase 1 deficiency increases susceptibility to benzo(a)pyrene-induced mouse skin carcinogenesis.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that catalyzes the metabolic detoxification of quinones and their derivatives. This protects cells against quinone-induced oxidative stress, cytotoxicity, and mutagenicity. C57BL6 NQO1-/- mice, deficient in NQO1 RNA and protein, were generated in our laboratory. To investigate the role of NQO1 in chemical carcinogenesis, the dorsal skin of NQO1-deficient (NQO1-/-) and wild-type (NQO1+/+) mice were treated with a single dose of benzo(a)pyrene, followed by twice weekly applications of phorbol-12-myristate-13-acetate. The NQO1-/- mice showed a much higher frequency of skin tumor development when compared with their wild-type littermates. Interestingly, the male NQO1-/- mice were slower to develop skin tumors than their NQO1-/- female littermates. Histological analysis of the NQO1-/- tumors showed proliferative activity. These results demonstrate that NQO1 acts as an endogenous factor in protection against benzo(a)pyrene carcinogenicity.

Targeted expression of c-Myc in the epidermis alters normal proliferation, differentiation and UV-B induced apoptosis.

c-Myc overexpression has been associated with several types of human cancers. To study the role of c-myc in epidermal differentiation and carcinogenesis, a transgenic mouse model was created to overexpress c-Myc in the epidermis. Human c-myc 2 cDNA was subcloned into a 6.5 kb mouse loricrin expression vector, ML.myc2. This loricrin promoter primarily directs expression in the epidermis in both proliferating and differentiated keratinocytes. On day 4, ML.myc2 transgenic pups develop a hyperkeratotic phenotype, which progressively worsens until day 7. Upon histological analysis, both hyperplasia and hyperkeratosis were evident. Bromodeoxyuridine (BrdU) incorporation revealed that transgenic mice had a threefold increase in the number of proliferating cells as compared with a normal littermate. Proliferative cells in the ML.myc2 epidermis were also found to be suprabasal, suggesting an inhibition of terminal differentiation in keratinocytes. Inhibition of terminal differentiation by c-Myc overexpression was further suggested by aberrant expression of differentiation markers, keratin 1, keratin 6, loricrin, and filaggrin in ML.myc2 transgenic mice. Interestingly, ML.myc2 keratinocytes exhibit a reduced sensitivity to UV-B induced apoptosis, in vivo. In vitro studies reveal the reduced sensitivity of ML.myc2 keratinocytes to UV-B irradiation is growth factor dependent. These findings provide evidence that overexpression of c-Myc in the epidermis induces proliferation, inhibits terminal differentiation and decreases the sensitivity of keratinocytes to UV-B induced apoptosis.