Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis

Type 2 topoisomerases, in particular the α 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 2α 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 2α localize to centromeres and chromosome cores/axes. However, centromeres are unresponsive to inhibitors during interphase. Furthermore, formation of topoisomerase 2α–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 2α–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]

Mammalian topoisomerase IIα (topo IIα) plays a vital role in the removal of topological complexities left on DNA during S phase. Here, we developed a new assay to selectively identify sites of catalytic activity of topo IIα with subcellular resolution. We show that topo IIα activity concentrates at replicating heterochromatin in late S in a replication-dependent manner and at centric heterochromatin during G2 and M phases. Inhibitor studies indicate that this cell cycle-dependent concentration over heterochromatin is sensitive to chromatin structure. We further show that catalytically active topo IIα concentrates along the longitudinal axis of mitotic chromosomes. Finally, we found that catalytically inert forms of the enzyme localize predominantly to splicing speckles in a dynamic manner and that this pool is differentially sensitive to changes in the activities of topo IIα itself and RNA polymerase II. Together, our data implicate several previously unsuspected activities in the partitioning of the enzyme between sites of activity and putative depots.

Background/Objectives: The management of moderate-to-severe psoriasis in patients with a recent history of internal malignancy is a clinical challenge, as systemic immunosuppressive therapies are often avoided because of concerns about cancer recurrence. While Psoralen and Ultraviolet A (PUVA) photochemotherapy remains a valuable non-immunosuppressive alternative, regional variations in therapeutic response are not well-characterized in this population. This study aimed to evaluate total and segmental Psoriasis Area and Severity Index (PASI) responses to PUVA in patients with chronic plaque psoriasis and recent internal organ malignancy. Methods: This prospective, single-center, real-world cohort study enrolled 20 adults with moderate-to-severe chronic plaque psoriasis and a recent (<5 years) diagnosis of internal organ malignancy in complete remission. Participants received oral PUVA three times weekly for up to 30 sessions. Primary and secondary outcomes included changes in total PASI, segmental PASI (head/neck, trunk, upper limbs, and lower limbs), and Dermatology Life Quality Index (DLQI) at baseline, completion of therapy, and 6 months post-treatment. Results: PUVA led to a significant reduction in mean total PASI from 18.6 ± 3.2 at baseline to 5.7 ± 6.0 at treatment completion (69% reduction; p < 0.001). However, regional responses differed significantly: the head and neck improved the most (80.4%), followed by the trunk (72.2%) and upper limbs (72.3%), while the lower limbs showed the weakest response (59.5%; p < 0.001). At baseline, trunk contributed the most to total PASI (38%), while post-treatment, lower-limb lesions accounted for approximately 47% of the remaining total disease burden, showing the highest contribution to total PASI of all body regions. At 6 months, the lower limbs remained the most affected area, with significantly lower improvement (52.9%) compared to other regions. Mean DLQI also improved significantly from 17.2 ± 2.8 to 5.6 ± 2.6 (p < 0.001). Conclusions: PUVA is an effective and safe treatment for patients with psoriasis and a recent history of malignancy. Nevertheless, lower-limb psoriasis is relatively recalcitrant and contributes disproportionately to residual disease burden and relapse. These findings support the use of regional PASI assessment to guide individualized management and clinical expectations in this complex patient group.