Conjugation of human topoisomerase 2A with small ubiquitin-like modifiers 2/3 in response to topoisomerase inhibitors: cell cycle stage and chromosome domain specificity

Type 2 topoisomerases, in particular the A isoform in human cells, play a key role in cohesion and sister chromatid separation during mitosis. These enzymes are thus vital for cycling cells and are obvious targets in cancer chemotherapy. Evidence obtained in yeast and Xenopus model systems indicates that conjugation of topoisomerase 2 with small ubiquitin-like modifier (SUMO) proteins is required for its mitotic functions. Here, we provide biochemical and cytologic evidence that topoisomerase 2A is conjugated to SUMO-2/3 during interphase and mitosis in response to topoisomerase 2 inhibitors and ‘‘poisons’’ (ICRF-187, etoposide, doxorubicin) that stabilize catalytic intermediates (cleavage complexes, closed clamp forms) of the enzyme onto target DNA. During mitosis, SUMO- 2/3–modified forms of topoisomerase 2A localize to centromeres and chromosome cores/axes. However, centromeres are unresponsive to inhibitors during interphase. Furthermore, formation of topoisomerase 2A–SUMO-2/3 conjugates within mitotic chromosomes strongly correlates with incomplete chromatid decatenation and decreases progressively as cells approach the metaphase-anaphase transition. We also found that the PIASy protein, an E3 ligase for SUMO proteins, colocalizes with SUMO-2/3 at the mitotic chromosomal cores/ axes and is necessary for both formation of SUMO-2/3 conjugates and proper chromatid segregation. We suggest that the efficacy of topoisomerase inhibitors to arrest cells traversing mitosis may relate to their targeting of topoisomerase 2A– SUMO-2/3 conjugates that concentrate at mitotic chromosome axes and are directly involved in chromatid arm separation. [Cancer Res 2008;68(7):2409–18