Figure 1.

Modulation of CDC25C splicing by doxorubicin in human breast cancer cell lines. (A) Schematic representation of CDC25A, CDC25B and CDC25C pre‐mRNA and alternatively spliced transcripts sequences. Colored rectangles correspond to alternatively spliced exons. Arrows show the localization of primers used to amplify the different CDC25A, CDC25B and CDC25C variants by semi‐quantitative RT‐PCR (see Materials and Methods for primers sequences). Amplicon length expected for each splice variant appears at the right of the figure. (B) MCF‐7 cells were treated with 1 μM of doxorubicin at the indicated times. RNA was subjected to semi‐quantitative RT‐PCR to detect CDC25A, CDC25B and CDC25C splice variants. PCR products are identified on the left and molecular weight markers are indicated on the right. The β‐actin gene was used as a standardizing control. (C), top, RNA from MCF‐7 cells treated with doxorubicin as indicated were subjected to Real‐Time quantitative RT‐PCR to evaluate the ratio between CDC25C5 and C1 variants. The results are expressed as $2^{-\Delta C_T}$ C5/$2^{-\Delta C_T}$ C1 (mean ± S.D.) of three independent experiments. 18S rRNA was used as an endogenous reference gene. ** Shows significant difference from control at p < 0.01 (Student's t test); bottom, schematic representation of primers (arrows) and TaqMan® probe (bold line) used to detect C1 and C5 variants. (D) CDC25C protein expression from MCF‐7 cells treated as indicated was examined by immunoblotting. α‐tubulin was used as a loading control. Molecular weights for each protein are indicated on the right. (E) The MCF‐7 multidrug‐resistant counterpart cell line Vcr‐R was treated with 2 μM of doxorubicin for 12 h. CDC25C splicing was studied by semi‐quantitative RT‐PCR as described above.