Inactivation of the quinone oxidoreductases NQO1 and NQO2 strongly elevates the incidence and multiplicity of chemically induced skin tumors

Abstract

The cytosolic quinone oxidoreductases NQO1 and NQO2 protect cells against oxidative stress by detoxifying quinones and preventing redox cycling. In this study, we used double knockout (DKO) mice deficient for NQO1 and NQO2 to investigate the role of these antioxidative enzymes in a two-stage model of inflammatory skin carcinogenesis. In this model, tumors are caused by exposure to topical carcinogen dimethylbenz(a)anthracene or benzo(a)pyrene (BP) followed by twice weekly application of proinflammatory phorbol 12-myristate 13-acetate. On this classic chemical carcinogenesis protocol, DKO mice showed a significantly higher skin tumor frequency and multiplicity compared with control wild-type or single knockout mice. Analysis of skin from wild-type and DKO mice exposed to BP for 6, 12, or 24 hours revealed a relative delay in the activation of p53, p63, p19ARF, and apoptosis in DKO mice, consistent with a negative modifier role for NQO1/NQO2 in carcinogenesis. Our findings offer genetic evidence of the significance of quinone oxidoreductases NQO1 and NQO2 in limiting chemical skin carcinogenesis.

Fig. 1

DMBA and BP induced skin tumor frequency and multiplicity. Wild type and DKO mice were exposed to indicated concentrations of DMBA or BP in a skin carcinogenesis model. The mice were analyzed for skin tumor frequency and multiplicity. A. DMBA-induced tumor incidence and tumor multiplicity/mouse. The table in left lower panel shows mice with tumors/total mice in the group. B. BP-induced tumor incidence and tumor multiplicity/mouse.

Fig. 2

Phenotype and histotype of DKO tumor induced by 200 nmol DMBA and comparison of tumor frequency and multiplicity among DKO, NQO1−/−, NQO2−/− and wild type mice. A. Gross appearance of skin tumors, which developed in DKO mouse. B. Histotype of tumor. microscopic sections of skin showing well differentiated squamous cell carcinoma (SCC) from 200 nmol DMBA-treated group. Islands of squamous malignant cells (black arrow) are seen in which there is nuclear atypia and apoptosis (white arrow). A high mitotic ratio is also seen. (Hematoxylin and eosin staining, original magnification: 20X). C. A comparison of DMBA and BP induced tumor incidence among wild type, NQO1−/−, NQO2−/− and DKO mice. D. A comparison of DMBA and BP induced tumor multiplicity among wild type, NQO1−/−, NQO2−/− and DKO mice.

Fig. 3

Immunohistochemistry analysis of induction of p63 in BP treated wild type and DKO mice. Dorsal skin of wild-type and DKO mice were exposed to acetone or 800 nmol BP. 6, 12 and 24 hours after BP treatment, the mice were euthanized and sections of treated skin removed by surgery. Skin samples were fixed in formalin, embedded in paraffin, and sections were cut. Sections were analyzed by immunohistochemistry with anti-mouse p63 antibodies. The p63 positive cells were counted from 15 fields from 3 mice in each group. Data presented as mean ± SD.

Fig. 4

Immunohistochemistry analysis of induction of p53 in BP treated wild type and DKO mice. Mice exposed to BP were analyzed by immunohistochemistry with anti-rabbit p53 antibody. The p53 positive cells were counted from 15 fields from 3 mice in each group. Data presented as mean ± SD.

Fig. 5

Immunohistochemistry analysis of alterations in ornithine decarboxylase (ODC), Bcl2, Bax and caspase 3 in BP treated wild type and DKO mice. Mice exposed to BP were analyzed by immunohistochemistry with anti-mouse ODC (A), Bcl2 (B), Bax (C) and caspase 3 (D) antibodies. 1-Wild type vehicle exposed; 2–4-Wild type exposed with BP for 6 hrs (2), 12 hrs (3) and 24 hrs (4); 5, DKO mice vehicle treated; 6–8, BP treated DKO mice for 6 hrs (6), 12 hrs (7), 24 hrs (8).

Fig. 6

Western analysis. Dorsal skin of wild-type and DKO mice were shaved, two days later, acetone or 800 nmol BP were applied on the shaved skin for indicated times. Skin samples from 3 mice in each group were combined. Samples from all groups were homogenized and analyzed by SDS-page and Western blotting. Western blots were probed with p53, p21, BCL-2, Bax, caspase 3, cleaved PARP, p19, c-Jun, ODC, NQO1, NQO2, and Actin antibodies.