J Mol Hist (2009) 40:177–181 179 Fig. 2 Histological ﬁndings of tongue in mice treated with 4NQO during 16 weeks. a Normal tongue with no histopathological changes 24 weeks after starting the experiment. b Mild dysplasia after 24 weeks. c Moderate dysplasia after 28 weeks. d Severe dysplasia after 28 weeks. e Invasive squamous cell carcinoma after 32 weeks. Original magniﬁcation 910 and severe dysplasia (Fig. 2d) could be detected. The ﬁrst evidence of squamous cell carcinoma was identiﬁed after 32 weeks (Fig. 2e). At the time point of 36 weeks 4 out of 6 animals that were sacriﬁced showed squamous cell car- cinoma at histopathological examination. At 40 weeks, the end of our experiment, all 6 tumors were squamous cell carcinomas (data not shown). Immunohistological ﬁndings Once all 6 mice bearing tumors at the 40 weeks time point were diagnosed with squamous cell carcinoma, immuno- histochemistry was performed. A comparison of Cyclin D1 and E-cadherin expression was made in squamous cell carcinoma detected after 40 weeks and mild dysplasia established after 24 weeks. Immunohistological evaluation was done in the tumors of our interest. Cyclin D1 expres- sion was investigated by assessing the nuclear staining of the antigen. Only nuclear staining was considered positive, while cytoplasmatic staining was regarded as negative. Mild dysplasia exposed predominantly basal cells expressing cyclin D1 (Fig. 3c). The stainability increased with the progression from dysplasia to carcinoma and the positive ratio was the highest in the squamous cell carci- noma (Fig. 3d). After 40 weeks, squamous cell carcinoma Fig. 3 Histopathology and immunohistochemistry staining with biomarkers cyclin D1 and E-cadherin. The left panel shows mild dysplasia after 24 weeks, the right panel shows squamous cell carcinoma after 40 weeks. Hematoxylin-eosin staining (HE) of mild dysplasia (a) and of squamous cell carcinoma (b) are shown. Cyclin D1 is primarily expressed in basal cells in mild dysplasia (c) and throughout the tumor and in tumor nests in squamous cell carcinoma (d). E-cadherin is expressed in the parabasal cell layer of the epithelium, but not on the surface of basal cells in mild dysplasia (e) and is partly lost in the invasive front of squamous cell carcinoma (f). Original magniﬁcation 94 showed a distribution throughout the tumor (Fig. 3d) as well as the basal cell layer and lower spinous cells. Staining of the tongue after 24 weeks showed that E-cadherin was strongly expressed in the parabasal cell layer, but not on the surface of basal cells (Fig. 3e). In the oral squamous carcinoma we noticed a loss of E-cadherin expression in the invasive front and positive staining of cancer cell nests (Fig. 3f). Tumor volume The initial measurement of a volume of exophytic tumor was done after 20 weeks following the start of the exper- imental procedure. By measuring the size of the tumor in three dimensions the volume was calibrated in mm3 by using the following formula: V = l 9 w 9 h. The volumes and ranges of each time point are shown in Fig. 4. After 20 weeks, the average volume was 6 mm3 with a range of 2–12 mm3. Gradually with time the volume increased and reached 30 mm3 at the 40 weeks time point. 123