Differential expression of the splicing regulatory factor genes during two-step chemical transformation in a BALB/3T3-derived cell line, MT-5.

Although the alternative splicing of various genes is a common event in human tumors, the mechanisms behind it have not been characterized. We hypothesized that the expression of splicing regulatory factors would be changed during cellular transformation. Gene expression of three splicing regulatory factors, alternative splicing factor/splicing factor 2 (ASF/SF2), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and the 65 kDa subunit of U2 small nuclear ribonucleoprotein particles auxiliary factor (U2AF(65)), were examined by northern blotting in a two-step chemical transformation model. This in vitro model is composed of BALB/3T3 cells and a BALB/3T3-derived N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-initiated cell line (MT-5). MT-5 cells can be transformed on exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). ASF/SF2 mRNA levels were decreased 2-fold in both MNNG-initiated cells and TPA-induced transformed cells compared with the normal parental cells, whereas hnRNP A2 mRNA expression did not significantly change between these three types of cells. U2AF(65) mRNA levels were markedly increased ( approximately 4.7-fold) associated with progression of cellular transformation. Moreover, RT-PCR analysis showed that distinct forms of ASF/SF2 mRNA were present in the MNNG-initiated cells and TPA-induced transformed cells but not in the parental cells. These findings indicate that ASF/SF2 or U2AF(65) gene expression is altered during in vitro two-step chemical transformation. The data suggest that the differential expression of splicing regulatory factors is one cause of aberrant expression of alternatively spliced mRNAs encoded by various genes in tumor cells.