Figure 4.

hGPR49-overexpressed HaCaT cells showed tumor formation in NOG mice. A: Tumor formation at 10 weeks (left panel) and 22 weeks (right panel). HaCaT cells overexpressing hGPR49 (1 × 10⁷ cells) were transplanted subcutaneously on the back of NOG mice on the right side. Cells with empty vector were also transplanted onto the back of the same mouse on the left side. Cells with hGPR49 formed tumors (closed arrows) in all ten mice, while only a nodule (open arrowheads) was formed in three mice with cells harboring empty vector. B: Measurement of tumor size. The diameters of tumors were measured every other week after transplantation. Apparent tumor formation was observed 10 weeks after transplantation. All ten tumors derived from GPR49-overexpressed cells continued to grow until 24 weeks, while three nodules derived from cells with empty vector did not show enlargement. C: Gene expression level of GPR49 in innoculated cells and in formed tumors was measured by QRT-PCR. Tumors derived from GPR49-overexpressed HaCaT cells showed high levels of hGPR49, about 200 times higher than the nodule with empty vector (bars = SD). D: Histological examination. Tumors derived from GPR49-overexpressed HaCaT cells showed aberrant proliferation and invasion. Keratinization was observed in some tumor nests and had features resembling squamous cell carcinoma (upper panels). The nodule derived from HaCaT cells with empty vector showed a cystic structure with keratinization and calcification (lower panels). Left panels: low magnification (scale bar = 1000 μm), Middle panels: high magnification (scale bar = 200 μm). Right panels: immunohistochemical staining of Ki-67. Significantly increased expression of Ki-67 coincident with nucleus is observed in tumors derived from GPR49-overexpressed HaCaT cells (scale bar = 200 μm).