Wild type C57BL/6 mice (6 weeks old) were treated with propylene glycol (vehicle) or 4-NQO (100 µg/ml) in the drinking water for 10 weeks. Two weeks after the 4-NQO treatment was stopped, these 4-NQO treated mice were randomized (10 mice/group) to receive intraperitoneal (i.p.) injections with either 100 µl PBS or various drugs for another 15 weeks before being sacrificed. The dosages and frequencies of the drugs are described in the MATERIALS AND METHODS. The mice were then sacrificed, and the tongues were dissected out and photographed (10×). The grossly visible lesions on the whole tongues were graded in a double blinded manner. A, A brief diagram of the experimental protocol. B. Representative gross morphological observations of the mouse tongues and the gross tongue lesion grading system (10×). C. Average numbers of cancerous tongue lesions (multiplicity, i.e. number of lesions per mouse). A one way ANOVA test was used to analyze the differences in tongue lesion numbers among all treatment groups. Differences with a p value of < 0.05 (marked with an asterisk) between carcinogen treated and subsequently PBS treated mice (4-NQO/PBS) and mice in other treatment groups (4-NQO/5-Aza, 4-NQO/LDRA, 4-NQO/5-Aza+LDRA, and 4-NQO/HDRA) were considered to be statistically significant. D. The percentages of mice in each group with different grade lesions, including the control mice (not treated with carcinogen and various drugs) and the mice treated with the carcinogen 4-NQO followed by treatment with various drugs. The differences among the distributions of lesions of different grades in each treatment group were analyzed by using a Fisher’s Exact probability test. Differences with a p value of < 0.05 between carcinogen treated and subsequently PBS treated mice (4-NQO/PBS) and mice in other treatment groups (4-NQO/5-Aza, 4-NQO/LDRA, 4-NQO/5-Aza+LDRA, and 4-NQO/HDRA) were considered to be statistically significant, indicated by an asterisk.

Ninety minutes after the final drug injections, mouse serum samples were collected and the serum retinoid levels were analyzed by HPLC (3 mice per group) as described in the MATERIALS AND METHODS. The retention time of RA was 23.4 min. and ROL was eluted at 33.1 min. Rol Ac, retinyl acetate; RP, retinyl palmitate. A. HPLC tracing of retinoid standards. B. HPLC tracing of control mouse serum. C. HPLC tracing of serum from mice in 4-NQO/PBS group. D. HPLC tracing of serum from mice in 4-NQO/HDRA group. E. Serum retinol levels in mice from different treatment groups. The data were analyzed by using a one-way ANOVA test for multiple comparisons.

Wild type C57Bl/6 mice were treated with propylene glycol (vehicle) or 4-NQO (100 µg/ml) in drinking water for 10 weeks and then treated with various drug injections for 15 weeks. The dosages and frequencies of the drugs are described in the MATERIALS AND METHODS. Mice were sacrificed, and the tongues were fixed, embedded, sectioned, and stained with anti-PCNA, anti-p16, and anti-Cox-2 antibodies (four mice per group, 300×). Four to five representative areas of each mouse tongue section were photographed and analyzed. PCNA and p16 indices were determined by the number of these positive nuclei divided by the total epithelial cell nuclei in the areas. A, PCNA staining in mouse tongue regions without visible lesions (300×). a, tongue section from control mice; b, tongue section from 4-NQO/PBS treated mice; c, tongue section from 4-NQO/5-Aza treated mice; d, tongue section from 4-NQO/LDRA treated mice; e, tongue section from 4-NQO/5-Aza+LDRA treated mice; g, tongue section from 4-NQO/HDRA treated mice. B, The quantitation of PCNA indices in panel A. C, PCNA staining in mouse tongue regions with visible lesions (300×). a, tongue section from 4-NQO/PBS treated mice; b, tongue section from 4-NQO/5-Aza treated mice; c, tongue section from 4-NQO/LDRA treated mice; d, tongue section from 4-NQO/5-Aza+LDRA treated mice; e, tongue section from 4-NQO/HDRA treated mice; f, negative control, a mouse tongue section stained only with a secondary antibody. D. The quantitation of PCNA indices in panel C. E, p16 staining of mouse tongues (200×). F, The quantification of p16 indices in panel E. G, Cox-2 staining of mouse tongues (200×). a, tongue section from control mice; b, tongue section from 4-NQO/PBS treated mice; c, tongue section from 4-NQO/5-Aza treated mice; d, tongue section from 4-NQO/LDRA treated mice; e, tongue section from 4-NQO/5-Aza+LDRA treated mice; f, tongue section from 4-NQO/HDRA treated mice; g, negative control, a mouse tongue section stained only with a secondary antibody. The data were analyzed by using a one-way ANOVA test for multiple comparisons. Differences with a p value of < 0.05 (marked with asterisks, *, p < 0.05; **, p < 0.01; ***, p < 0.001) between carcinogen treated, and subsequently PBS treated mice and mice in other groups (4-NQO/5-Aza, 4-NQO/LDRA, 4-NQO/5-Aza+LDRA, and 4-NQO/HDRA) were considered to be statistically significant.

In normal tissue, the RARβ₂ gene is transcriptionally activated by the endogenous agonist RA and the RARβ₂ protein acts as a transcription factor in the presence of RA, leading to transcriptional activation of target genes, cell growth inhibition, differentiation, and normal apoptosis. In tumors, the RARβ₂ promoter is often methylated, preventing the synthesis of RARβ₂ mRNA and protein. Thus, RARβ₂ does not function in these tumor cells. Only when both 5-Aza and pharmacological doses of RA are added to tumor cells are the transcriptional activation of the RARβ₂ gene and the transcription factor activity of the RARβ₂ protein restored.

Wild type C57BL/6 mice were treated with 4-NQO (100 µg/ml) in drinking water (see “Materials and Methods”) for 10 weeks and then maintained for another 15 weeks. The mice were sacrificed, and the tongues were fixed, embedded, sectioned, and stained with H&E. A, Representative pictures of pathology (200×). a, control tongue; b, hyperplasia with marked hyperkeratosis; c, mild dysplasia; d, moderate dysplasia; e, severe dysplasia/carcinoma in situ; f, invasive carcinoma with tumor cells invading the skeletal muscle fibers of the tongue. B, Percentage of 4-NQO treated mouse tongue samples in each drug treatment group at different carcinogenesis stages. The data were analyzed by using a Fisher’s exact probability test. Differences with a p value of < 0.05 (marked with an asterisk) were considered to be statistically significant.

Wild type C57Bl/6 mice were treated with propylene glycol (vehicle) or 4-NQO (100 µg/ml) in drinking water for 10 weeks and then treated with various drug injections for 15 weeks. The dosages and frequencies of the drugs are described in the MATERIALS AND METHODS. Total RNA was extracted from all mouse tongues from each treatment group and specific mRNA levels were measured by real time RT-PCR. A, RARβ₂ mRNA (Average ± SD). B, Cox-2 mRNA (Average ± SD). C, c-Myc mRNA (Average ± SD). The data were analyzed by using a Wilcoxon rank sum test for multiple comparisons. Differences with a p value of < 0.05 (marked with an asterisk) between carcinogen treated and subsequently PBS treated mice (4-NQO/PBS) and mice in other groups (4-NQO/5-Aza, 4-NQO/LDRA, 4-NQO/5-Aza+LDRA, and 4-NQO/HDRA) were considered to be statistically significant.