ABSTRACT
Recent studies suggest that erythropoietin plays an important role in the process of neoplastic transformation and malignant phenotype progression observed in malignancy. To study the role of erythropoietin and its receptor (EPOR) on the response of cancer cells in vitro, we used two solid tumor cell lines, namely the human malignant glioma cell line U87 and the primary cervical cancer cell line HT100. All experiments were done with heat-inactivated fetal bovine serum in order to inactivate any endogenous bovine erythropoietin. The expression of the EPOR in these cells was confirmed with immunoblot techniques. The addition of exogenous recombinant human erythropoietin (rhEPO) induces the cancer cells to become more resistant to ionizing radiation and to cisplatin. Furthermore, this rhEPO-induced resistance to ionizing radiation and to cisplatin was reversed by the addition of tyrphostin (AG490), an inhibitor of JAK2. Our findings indicate that rhEPO result in a significant, JAK2-dependent, in vitro resistance to ionizing radiation and to cisplatin in the human cancer cells lines studied in this report.
Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Drug Resistance, Neoplasm , Erythropoietin/therapeutic use , Glioma/pathology , Radiation Tolerance , Uterine Cervical Neoplasms/pathology , Animals , Cattle , Erythropoietin/metabolism , Female , Glioma/drug therapy , Glioma/radiotherapy , Humans , Janus Kinase 2 , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Radiation, Ionizing , Receptors, Erythropoietin/metabolism , Recombinant Proteins , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/radiation effects , Tyrphostins/pharmacology , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/radiotherapyABSTRACT
Ku is a heterodimer of M(r) 70,000 and M(r) 86,000 subunits. It binds with strong affinity to DNA ends and is indispensable for nonhomologous DNA end joining (NHEJ) and V(D)J recombination. In this study, we investigated whether down-regulation of the Ku86 gene, by 2'-O-methoxyethyl/uniform phosphorothioate chimeric antisense oligonucleotides (ASOs), increases the sensitivity of the DNA-protein kinase catalytic subunit (PKcs)-proficient human glioma cell line (M059K), and its isogenic DNA-PKcs-deficient counterpart (M059J), to ionizing radiation and anticancer drugs. Transfection of these cell lines with 200 nM Ku86 antisense ASOs was associated with a specific decrease in Ku86 mRNA levels (IC(50) <25 nM; n = 3) and a concomitant rapid decrease (<10% of control) in Ku86 protein expression. Moreover, transfection of M059K cells with Ku86 antisense ASOs markedly increased cell death after treatment with ionizing radiation, bleomycin, and etoposide. However, no sensitization to the DNA cross-linking agents chlorambucil and cisplatin was observed after Ku86 antisense transfection. As expected, transfection of M059J cells with Ku86 antisense ASOs did not result in any sensitization to ionizing radiation, bleomycin, or DNA cross-linking agents, but there was a 2-fold increase in sensitivity to etoposide. Thus, our results indicate that antisense ASOs targeted against Ku86 may increase the efficacy of radiotherapy and DNA-damaging agents in tumor treatment. Furthermore, Ku86 antisense ASOs may be used to create a temporal knockout in different human cell lines to further investigate the biological roles of Ku86.
