Figure 1.
(A) Schematic of PAX3, FKHR, PAX3-FKHR and the predicted FKHR-PAX3 protein structures indicating the known functional domains. R: repressor; DBD: DNA binding domain; AD: activation domain. (B) Diagrammatic illustration of the exon-intron organization of human PAX3 gene, and the five alternatively spliced mRNAs that could result from processing of the FKHR-PAX3 primary transcript. PAX3c, PAX3d, and PAX3e use stop codons in intron 8, intron 9, and exon 10. PAX3g and PAX3h are truncated isoforms of PAX3d and PAX3e, respectively, that splice out exon 8. (C) Expression of FKHR-PAX3 transcript isoforms c, d, and e in ERMS (RD) and ARMS (RH4, RH28, RH30) cell lines as detected by RT-PCR and confirmed by Southern hybridization. Top panel: schematic indicates the positions of the FKHR-specific primer (F4) and the isoform-specific PAX3 PCR primer pairs, and the DNA probe spanning the FKHR-PAX3 fusion site used in the Southern analysis are indicated (not to scale). (D) Quantitative RT-PCR analysis of PAX3, FKHR, PAX3-FKHR, and FKHR-PAX3 expression in ARMS cell lines. The relative expression data are presented at two different scales on the Y-axis, high (left panel) and low (right panel) to compensate for the high levels of PAX3-FKHR expression. The relative expression level of PAX3/GAPDH in RH4 cells was assigned an arbitrary value of 1, and used as the reference to calculate fold change. (E) Nucleotide sequences of the cloned FKHR-PAX3 isoforms c, d, and e cDNAs. Top panel: schematic of the FKHR and the isoform-specific PAX3 primer pairs used to clone the full-length protein coding cDNA from RH30 cells. Primer location is approximate for illustrative purposes only. Sequence data is annotated by text in: Plain: 5’ FKHR-UTR; plain/italic: 3’ PAX3-UTR; Bold: protein coding sequences; bold/capital/underline: translation start codon; bold/capital/italic/underline: translational stop codons; underline: isoform-specific sequences.
Figure 2.
Immunodetection of in vitro and in vivo expressed FKHR-PAX3 protein.
(A) Left panel: schematic of deduced sizes and amino acid sequence variations for FKHR-PAX3 isoforms c, d, and e. Right panel: Autoradiographic image of S35-methionine labeled in vitro translated FKHR-PAX3 protein isoforms. (B) Verification of PAX3-specific C2 and FKHR-specific L27 antibodies in detecting in vivo expressed FKHR-PAX3 proteins. Top panel: diagrammatic illustration of the epitope locations within the FKHR-PAX3 protein recognized by L27 and C2 antibodies. Bottom panel: western blot detection of FKHR-PAX3 in whole cell extracts (30 μg) prepared from RD cells that were transiently transfected with control expression vector (lane1), FKHR-PAX3 isoform c (lane 2), and FKHR-PAX3 isoform d (lane 3) using C2 (left panel) and L27 (right panel) antibodies. (C) Western blot detection of the endogenously expressed FKHR-PAX3 in RH28 and RH30 cells by L27 and C2 antibodies. Protein extract from FKHR-PAX3 negative RH4 cells was included as negative control. n.s.: non-specific bands resulting from high amount of protein extracts used and long film exposure. (D) Effect of PAX3-FKHR knockdown on the endogenous level of FKHR-PAX3 in RH28 and RH30 cells. Whole cell extracts were prepared from cells that stably expressed the inducible PAX3-FKHR shRNA treated with DMSO or DOX for 48 hours, and analyzed for FKHR and FKHR-PAX3 expression. (E) Effect of MG132 (10 μM for 12 hours) and 5’-Aza-C (1 μM for 48 hours) on endogenous FKHR-PAX3 expression levels in RH28 and RH30 cells. (C-E) A total of 400 μg of protein extracts were used for the analyses. (D-E) Alpha-tubulin was used to normalize sample loading.
Figure 3.
FKHR-PAX3 preserved high level of PAX3-FKHR expression in myogenic cells.
(A) RH4 cells that do not express endogenous FKHR-PAX3 were transfected with empty vector (lane 1) or FKHR-PAX3 expression vector (lane 2). After continuous culture for 35 passages, cells were assayed for PAX3, FKHR, PAX3-FKHR, and FKHR-PAX3 expression by western blot. (B) Western blot analysis on the ability of FKHR-PAX3 to sustain high PAX3-FKHR expression. Stable clones of C2C12 cells expressing high levels of PAX3-FKHR were subjected to a second round of transfection to select for FKHR-PAX3 expression as described in Materials and Methods. The first confluent plate was designated as passage zero and used to generate early (< 10) and late (>35) passage cells. Cells were cultured at comparable density and passaged every three days. Immunoblot images were from four representative control and FKHR-PAX3 expressing lines. L27 antibody was used to detect FKHR-PAX3, and α-tubulin was used to normalize sample loading. Representative data from FKHR-PAX3 isoform c is shown.
Figure 4.
FKHR-PAX3 localized predominantly in the cytoplasm of cells.
(A) NIH3T3 and RH30 cells were transfected with vectors expressing wild-type FKHR-GFP, triple-mutant (T24A/S256A/S319A) FKHR-GFP, or FKHR-PAX3-GFP (FP3-GFP) by lipofection. Cells were maintained in low serum medium (0.5% FBS) overnight prior to the addition of DMSO or LepB (2 μM) for six hours. At the end of treatment, fluorescent microscopy (magnification: 200X) was used to visualize the GFP-tagged proteins as indicated. (B) Western analysis confirmed the cytoplasmic localization of the FKHR-PAX3 protein in cells. RH30 cells were transfected with FKHR-PAX3 and treated with or without LepB as described in (4A). MyoG and α-tubulin served as nuclear and cytoplasmic specific controls, respectively, to evaluate the fractionation protocol. L27 antibody was used to detect FKHR-PAX3. Representative data from FKHR-PAX3 isoform c is shown.
Figure 5.
FKHR-PAX3 did not transactivate promoters that were responsive to FKHR (IGFBP1, A) and PAX3-FKHR (e5, B; MyoG, C; PDGFαR, D).
C2C12 cells were transiently transfected with a total of 2 μg of DNA including 0.1 μg of LacZ, 0.5 μg of promoter-reporter (Luc or CAT as indicated), and 0.2 μg of the pcDNA3 vector or vector expressing the transcription factor by lipofection. After 48 hours, cells were harvested for LacZ, Luc, and CAT assays. Fold increase was calculated as the ratio of reporter activity from cells expressing the indicated transcription factor to the activity in cells transfected with the empty expression vector. Results were normalized to LacZ activity. The reporter activity in the presence of empty expression vector was assigned a value of 1. ND: statistically no difference. Representative data from FKHR-PAX3 isoform c is shown.
Figure 6.
FKHR-PAX3 promoted low-density cell proliferation and blocked terminal myogenic differentiation.
(A) Comparison of cell proliferation in NIH3T3, RD, RH4, and RH30 cultures with or without FKHR-PAX3 (FP3) expression. Cells were seeded in triplicate at 1 x 104 cells/well into a 24 well-plate. Proliferation was measured daily by counting the number of live cells (trypan blue-negative) over five days beginning a day after the initial seeding. Cell death was minimal in all experiments. (B) The effect of FKHR-PAX3 expression on low-density RD and RH30 growth. Cells were seeded in triplicate at 2 x 103 cells/well into a 24 well-plate. Cell growth was quantified daily using the WST-1 cell proliferation kit. (C) Top panel: Immunodetection of MyoG and MHC expression in proliferating (GM) and differentiated (five days, DM) C2C12 cells with or without FKHR-PAX3. Bottom panel: light (left panel, 100X magnification) and fluorescent (middle and right panels, (250X magnification) microscopic images of day-5 differentiated cells stained with MF20 antibody against MHC (middle) or with DAPI (right). (D) The effect of PAX3-FKHR knockdown on MyoG and MHC expression in control and FKHR-PAX3 expressing RH30 cells. Cell extracts were analyzed by western blot as in Figure 2D. (C-D) Alpha-tubulin was used to normalize sample loading. Representative data from FKHR-PAX3 isoform c is shown.
Figure 7.
FKHR-PAX3 enhanced anchorage-dependent and anchorage-independent colony growth.
(A) Photographic images of crystal violet blue stained cell colonies in the clonogenicity assay at the end of a 15-day growth (10% FBS). (B) Quantitative analysis of anchorage-independent soft agar colony formation under growth (10% FBS, top panel) and differentiation (2% HS, bottom panel) conditions. Inset: representative micrographic images of RD cells showing that FKHR-PAX3 (isoform c) enhanced both number and size of the colonies. (A-B) Assays were conducted as described in Materials and Methods. Representative quantitative data from FKHR-PAX3 isoform d is shown.
Figure 8.
FKHR-PAX3 selectively increased cell adhesion over cell movement and invasion.
(A) FKHR-PAX3 increased attachment of RD (top panel) and RH30 (bottom panel) cells to the indicated extracellular matrix components. (B) FKHR-PAX3 expression in RD (left panel) and RH30 (right panel) increased the strength of cell adhesion to culture dishes as indicated by a reduced sensitivity to trypsin. (C) Western blot analysis of the effect of ectopic FKHR-PAX3 expression on FAK phosphorylation in RD and RH30 cells. A total of 30 μg of whole cell extracts were analyzed. (D) FKHR-PAX3 reduced the migratory function in RD (top panel) and RH30 (bottom panel) cells as measured by scratch wound assay. Left panel: quantification of migratory index; right panel: representative micrographs of the scratched wound assays. (E) FKHR-PAX3 decreased the invasive potential of RD (left panel) and RH30 (right panel) as determined by Matrigel assay. ND: statistically no difference. (A-B, D-E) Assays were conducted as described in Materials and Methods. Representative data from FKHR-PAX3 isoform c is shown.
Figure 9.
FKHR-PAX3 induced early onset of tumor formation in nude mice xenograft model.
(A) RD (top panel) and RH30 (bottom panel) cells expressing empty vector or FKHR-PAX3 were injected intramuscularly into the hind legs of nude mice as described in Materials and Methods. Data points represent mean ± s.d. of tumor volume (mm3) of all injected mice at the indicted time points. Inset: an expanded view over the early tumor development period. (B) Dissecting microscopic images (magnification 25X) of lung organs of two representative mice from the control (left) and FKHR-PAX3 (right) group, showing clear evidence of more extensive tumor mass and infiltration metastasis in the control group (red dashed lines: margins surrounding the tumor mass; blue arrow heads: light-reflective artifacts). Representative data from FKHR-PAX3 isoform c is shown.