Serine phosphorylation-dependent coregulation of topoisomerase I by the p14ARF tumor suppressor.
p14ARF (ARF) and topoisomerase I play central roles in cancer and have recently been shown to interact. The interaction activates topoisomerase I, an important target for camptothecin-like chemotherapeutic drugs, but the regulation of the interaction is poorly understood. We have used the H358 and H23 lung cancer cell lines and purified recombinant human topoisomerase I to demonstrate that the ARF/topoisomerase I interaction is regulated by topoisomerase I serine phosphorylation, a modification that regulates topoisomerase I activity. Both cell lines express wild-type ARF and topoisomerase I proteins at equivalent levels, but H23 topoisomerase I, unlike that of H358 cells, is largely devoid of serine phosphorylation, has low activity, and complexes poorly with ARF. The ability of H23 topoisomerase I to complex with ARF can be restored by treatment with the serine kinase, casein kinase II. Consistent with these observations, we show that the response of H23 cells to camptothecin treatment is unaffected by changes in intracellular levels of ARF. However, in H358 and PC-3 cells, which express a serine phosphorylated topoisomerase I that complexes with ARF, ectopic overexpression of ARF causes sensitization to camptothecin, and siRNA-mediated down-regulation of endogenous ARF causes desensitization to camptothecin. These biological responses correlate with increased and decreased levels, respectively, of ARF/topoisomerase I complex and DNA-bound topoisomerase I. Thus, ARF is a serine phosphorylation-dependent coregulator of topoisomerase I in vivo, and it regulates cellular sensitivity to camptothecin by interacting with topoisomerase I. Certain cancer associated defects affecting ARF/topoisomerase I complex formation could contribute to cellular resistance to camptothecin.
Bandyopadhyay
Keya
K
Department of Cancer Cell Biology, Sidney Kimmel Cancer Center, San Diego, California, USA.
Lee
Casey
C
Haghighi
Ali
A
Banères
Jean-Louis
JL
Parello
Joseph
J
Gjerset
Ruth A
RA
D002166:Camptothecin / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung; D045744:Cell Line, Tumor; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D008175:Lung Neoplasms; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D025803:Tumor Suppressor Protein p14ARF / Q000502:physiology*
D025803:Tumor Suppressor Protein p14ARF; D010768:Phosphoserine; D012694:Serine; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin