Expression of a novel alternatively spliced UCP-2 transcript in osteogenic sarcoma

Development of chemoresistance is common in patients with osteogenic sarcoma (OGS); however, the underlying mechanism is largely unknown. Many anticancer drugs exert their therapeutic action by generating reactive oxygen radicals, which might be countered by the cancer cell through induction of uncoupling protein 2 (UCP-2). UCP-2 has been shown to be able to protect tumor cells from the cytotoxic actions of chemotherapeutic drugs. Because OGS is seldom completely cured by current chemotherapy regimens, we hypothesized that increased expression of UCP-2 underlies this phenomenon. The primary initial interest of our research was to evaluate the level of UCP-2 mRNA in OGS. The level of UCP-2 mRNA was determined by reverse transcriptase polymerase chain reaction (RT-PCR) comparing expression in normal-bone-derived specimens and OGS-derived specimens. Semiquantification of mRNA expression was achieved by radioactive RT-PCR. Nucleotide sequencing was performed using automated instruments. Interestingly, we failed to observe induction of UCP- 2 mRNA in OGS tumor specimens and OGS-derived primary cell lines compared to the expression level in normal bone. However, we found expression of a hitherto unknown UCP-2 transcript in eight of eight OGS-derived and one EWSderived cell lines and in nine of ten OGS biopsy specimens but in only one of six normal bone-derived specimens. Thus, tumor samples express both types (normal and the novel one) of UCP-2 mRNAs, whereas normal bone expresses only the wild-type form. Further experiments identified the novel mRNA species as an alternatively spliced UCP-2 transcript (termed UCP-2as). UCP-2as has a 22-nucleotide insertion from the 3′ end of intron 3 that introduces an early stop codon in exon 4, which theoretically can produce a protein 79 amino acids long. We have identified a hitherto unknown UCP-2 transcript. Expression of the novel transcript appears to be OGS-specific, implying a function advantageous to the tumor.