These data indicate that the expression of GDF3 increase the number of CD24 and CD44 double-positive cells during tumorigenesis. Expression levels of GDF3 in implant tumor cells We finally confirmed AP26113 in vitro that GDF3-transfected F1 and F10 cells continued to express GDF3 in implant tumors. RT-PCR analyses of excised tumors suggested that the transfected F1/F10 cells expressed the mRNA of GDF3 10 days after implantation although the levels of GDF3 mRNA decreased after 10 days compared to day 0 (Figure 6A). A negative control Soxl5 and a positive control β-actin were not affected

by GDF3 transfection. Protein expression of GDF3 in F1 and F10 cells was examined by Western blotting using antibody against GDF3. A representative blotting profile is shown in Figure 6B. The protein as well as mRNA check details amounts of GDF3 were similar in F1 and F10 cells (Figure 6A,B). The results infer that the GDF3 message is translated into functional protein in these tumor cells and forced expression of GDF3 are still minimally expressed 10 days after transfection in these cells. Figure 6 (A) RT-PCR analysis of the GDF3 message in F1/F10 cells. F1/F10 cells were transfected with the plasmid for expression of GDF3 (upper panel). Cells just before inoculation (indicated as 0 day) and cells isolated from tumors on day 10 after inoculation (indicated as day 10) were prepared and

adjust the cell numbers. These cells were lysed and total RNA was extracted from the lysates. RT-PCR was performed to detect GDF3 as well as Soxl5 (nagative control, center panel) and Rebamipide β-actin (positive control, lower panel). PCR cycles are 32 rounds, 3 times less in those shown in Fig. 2C,D (B) Cell lysate (day 0) was subjected to SDS-PAGE (left 10% gel, right 8% gel) followed by immunoblotting. Lower panel-

Commassie brilliant blue (CBB) staining of the blot. Upper panel- blots GDF3 band visualized by treating with anti-GDF3 mAb and then HRP-labeled 2nd Ab. No relevant band of GDF3 was detected by CBB staining. Discussion We have shown that GDF3 mRNA increased during tumorigenesis in mouse melanoma GSK690693 B16-F1 and B16-F10 cells. Although the genotypic and phenotypic differences of these sublines of the same cell line origin was described earlier [32], genes responsible for their tumorigenic difference have not been fully elucidated. We found that GDF3 overexpression promotes tumorigenesis of mouse melanoma by B16-F1 and B16-F-10 cells but not hepatoma by G1 or G5 cells. Moreover, ectopic expression of GDF3 increased CD24 expression in both B16-F1 and B16-F10 cells. Human GDF3 is primarily expressed in embryonal carcinomas, testicular germ cell tumors, seminomas, and breast carcinomas. However, the role of GDF3 in tumorigenesis has not been shown yet. This is the first report that establishes a positive role of GDF3 in tumorigenesis.