(b) Transfectant clones lost normal cell morphology and presented aberrant microtubule overgrowth

(b) Transfectant clones lost normal cell morphology and presented aberrant microtubule overgrowth. the family of heterogeneous nuclear ribonucleoproteins (hnRNP) H/F as candidate splicing Daptomycin factors potentially responsible for impairedTPsplicing. Indeed, whereas parental cells lacked nuclear localization of hnRNPs H1/H2 and F, drug-resistant cells harbored marked levels of these splicing factors. Nuclear RNA immunoprecipitation experiments established a strong binding of hnRNP H1/H2 toTPpre-mRNA, hence implicating them inTPsplicing. Moreover, introduction of hCIT529I10 hnRNP H2 into drug-sensitive parental cells recapitulated aberrant TP splicing and 5-deoxyfluorouridine resistance. Thus, this is the first study identifying altered function of hnRNP H1/H2 in tumor cells as a novel determinant of aberrant TP splicing thereby resulting in acquired chemoresistance to TP-activated fluoropyrimidine anticancer drugs. Keywords:Drug Resistance, Gene Regulation, Leukemia, Protein Degradation, RNA Splicing == Introduction == Thymidine phosphorylase (TP),3also known as platelet-derived endothelial cell growth factor, catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose 1-phosphate (1). The frequent up-regulation of TP in solid tumors (2) facilitates chemotaxis of endothelial cells and angiogenesis (35) and was further shown to have an anti-apoptotic effect (6,7). Consequently, overexpression of TP was correlated with increased intratumoral microvessel density (7) and dismal patient prognosis (reviewed in Ref.8). Furthermore, the catalytic activity of TP is crucial for the cytotoxicity of fluoropyrimidine prodrugs, including capecitabine; the latter is a TP-activated oral fluoropyrimidine prodrug that is bioconverted to the mature chemotherapeutic agent 5-fluorouracil within malignant tumors where it exerts its cytotoxic activity (9,10). Capecitabine was designed to take advantage of the increased levels of TP observed in tumors as opposed to normal tissues, hence potentially allowing for selective cytotoxicity in malignant tumors (reviewed in Ref.11). Therefore, TP has recently gained a great deal of interest as a novel enzymatic mediator of the activity of fluoropyrimidine (pro)drugs that are bioconverted by TP to their active chemotherapeutic drug within malignant tumors. In this respect, antitumor and antiangiogenic activity of 6-(2-aminoethyl)amino-5-chlorouracil, a novel small molecule inhibitor of TP in combination with the vascular endothelial growth factor Trap, has been recently demonstrated Daptomycin in human small cell lung cancer xenografts (12). Previously, we have shown that exposure of the human monocytic/macrophage leukemia cell lines THP-1 and U937 to the anti-rheumatic drug sulfasalazine (SSZ) resulted in a complete down-regulation ofTPboth at the mRNA and protein levels (13). The complete loss of TP protein conferred a specific anticancer drug resistance phenotype to the fluoropyrimidine prodrug capecitabine by preventing the conversion of its intermediary metabolite 5-deoxyfluorouridine (5-DFUR) to 5-fluorouracil (9). Here, we show that the complete lack of TP protein in these SSZ-resistant tumor cells is due to unsplicing of the primary mRNA of TP, thereby leading to its nuclear accumulation (14) and/or premature translation termination (15). Furthermore, we identified the key splicing factors responsible for this impaired splicing as heterogeneous nuclear ribonucleoproteins (hnRNPs) H1 and/or H2. We recapitulated hnRNP Daptomycin H2-impaired splicing activity by its stable introduction into drug-sensitive parental cells that initially displayed intact splicing of TP. We further demonstrate that this impaired splicing confers drug resistance to the fluoropyrimidine 5-DFUR. == EXPERIMENTAL PROCEDURES == == == == == == Tissue Daptomycin Culture == Human monocytic/macrophage THP1 and U937 cell lines were maintained in RPMI 1640 medium (Invitrogen) containing 2.3 mfolic acid supplemented with 10% fetal bovine serum, 2 mmglutamine, 100 units/ml penicillin G, and 100 g/ml streptomycin sulfate (Biological Industries, Beth-Haemek, Israel) at 37 C in a humidified atmosphere of 5% CO2. Cells stably.