Figure 1.
Scheme of c-RET and RFP-RET cDNA constructs.
(A) c-RET cDNA. (B) RFP-RET cDNA. The sites of c-RET (A) and RFP-RET (B) primers for real-time PCR analysis are shown by arrows. SS, signal sequence; CAD, cadherinlike domain; CYS, cysteine-rich region; TM, transmembrane domain; TK1, tyrosine kinase domain 1; TK2, tyrosine kinase domain 2; aa, amino acids; RFM, RING finger motif; BB, B box; CC, coiled coil.
Figure 2.
Stage-dependent RFP-RET transcript expression levels in tumors from RET-mice.
(A) Levels of RFP-RET transcript expression in tumors of various sizes from RET-mice. Histopathologically benign and malignant tumors are shown by open and closed squares, respectively. (B) Levels of RFP-RET transcript expression (mean ± SD) in benign melanocytic tumors (open bar) and malignant melanoma (closed bar) from RET-mice. RFP-RET transcript levels measured by real-time PCR were adjusted by hypoxanthine guanine phosphoribosyl transferase (Hprt) transcript levels. Difference between expression levels of RFP-RET in benign melanocytic tumors and malignant melanoma from RET-mice was statistically analyzed by the Mann-Whitney U test. *, Significantly different (p<0.05) from the control.
Figure 3.
Stage-dependent c-Ret, Gfra1 and Gdnf transcripts expression levels in tumors from RET-mice.
(A, C, E) Levels of c-Ret (A), Gfra1 (C) and Gdnf (E) transcripts expression in tumors of various sizes from RET-mice. Histopathologically benign and malignant tumors are shown by open and closed squares, respectively. (B, D, F) Levels of c-Ret (B), Gfra1 (D) and Gdnf (F) transcripts expression (mean ± SD) in benign melanocytic tumors (open bar) and malignant melanoma (closed bar) from RET-mice. c-Ret (A, B), Gfra1 (C, D) and Gdnf (E, F) transcript levels measured by real-time PCR were adjusted by hypoxanthine guanine phosphoribosyl transferase (Hprt) transcript levels. Differences in expression levels of c-Ret (B), Gfra1 (D) and Gdnf (F) between benign melanocytic tumors and malignant melanoma from RET-mice were statistically analyzed by the Mann-Whitney U test. *, Significantly different (P<0.05) from the control.
Figure 4.
Levels of c-RET, GFRa1 and GDNF transcripts expression in primary-cultured normal human epithelial melanocytes (NHEM) and human malignant melanoma cell lines.
(A, B, C) Levels of c-RET (A), GFRa1 (B) and GDNF (C) transcripts expression in NHEM (lane 1) and 4 kinds of malignant melanoma cell lines (lanes 2–5; SK-Mel28, G361, MNT-1 and HM3KO). The transcript levels measured by real-time PCR were adjusted by TATA-box-binding protein (TBP) transcript levels. Differences in expression levels of c-Ret (A), Gfra1 (B) and Gdnf (C) between NHEM (lane 1; open bar) and malignant melanoma cell lines (lanes 2–5; closed bars) were statistically analyzed by the Kruskal-Wallis test. **, Significantly different (P<0.01) from the control.
Figure 5.
Levels of c-RET protein expression in human malignant melanoma cell lines.
The levels of c-RET protein expression were examined in c-RET-transfected NIH3T3 cells as a positive control (c-RET transfectant; lane 1), primary-cultured normal human epithelial melanocytes (NHEM; lane 2), G361 (lane 3), HM3KO (lane 4) and MNT-1 (lane 5) by immunoblotting analysis with anti-RET antibody after immunoprecipitation with anti-RET antibody.
Figure 6.
GFRa1 protein expression in human melanocytic cells.
GFRa1 protein expression was examined in primary-cultured normal human epithelial melanocytes (NHEM; A, B), G361 cells (C, D) and HM3KO cells (E, F) by immunocytochemistry with anti-GFRa1 antibody (A, C, E) using DAPI counterstaining (B, D, F).
Figure 7.
Augmentation of c-RET tyrosine kinase activity in HM3KO cells by c-RET ligand (GDNF).
Expression of c-RET protein (A, B) and phosphorylated tyrosine 905 in c-RET (C, D) in HM3KO cells in the absence (A, C) or presence (B, D) of GDNF were examined by immunocytochemistry with anti-c-RET and anti-phosphorylated tyrosine 905 in c-RET antibodies using hematoxylin counterstaining (A–D).
Figure 8.
Signal transduction molecules potentially sited downstream of c-RET in HM3KO cells.
Expression and phosphorylation levels of ERK and AKT in HM3KO cells before (0 min) and at 15 and 60 min after stimulation with GDNF (100 ng/ml) were examined by immunoblotting. Equality of protein amounts in each lane was confirmed by immunoblotting with anti-β-actin antibody.
Figure 9.
Proliferation of HM3KO cells treated with a solvent (0.1% of DMSO) (open circle in A, lane 1 in B), GDNF (100 ng/ml) (closed circle in A, lane 2 in B), SU5416 (5µM) (open square in A, lane 3 in B) and GDNF plus SU5416 (closed square in A, lane 4 in B) for 24 (A) and 48 hours (A, B) was examined by cell counting with trypan blue staining (A) and MTT assay (B). Difference between proliferation levels of DMSO-treated control cells and other cells was statistically analyzed by the Kruskal-Wallis test. *, Significantly different (P<0.05) from the control.