Type II DNA Topoisomerases Cause Spontaneous Double-Strand Breaks in Genomic DNA

Type II DNA topoisomerase enzymes (TOP2) catalyze topological changes by strand passage reactions. They involve passing one intact double stranded DNA duplex through a transient enzyme-bridged break in another (gated helix) followed by ligation of the break by TOP2. A TOP2 poison, etoposide blocks TOP2 catalysis at the ligation step of the enzyme-bridged break, increasing the number of stable TOP2 cleavage complexes (TOP2ccs). Remarkably, such pathological TOP2ccs are formed during the normal cell cycle as well as in postmitotic cells. Thus, this 'abortive catalysis' can be a major source of spontaneously arising DNA double-strand breaks (DSBs). TOP2-mediated DSBs are also formed upon stimulation with physiological concentrations of androgens and estrogens. The frequent occurrence of TOP2-mediated DSBs was previously not appreciated because they are efficiently repaired. This repair is performed in collaboration with BRCA1, BRCA2, MRE11 nuclease, and tyrosyl-DNA phosphodiesterase 2 (TDP2) with nonhomologous end joining (NHEJ) factors. This review first discusses spontaneously arising DSBs caused by the abortive catalysis of TOP2 and then summarizes proteins involved in repairing stalled TOP2ccs and discusses the genotoxicity of the sex hormones

Hyperosmolarity Attenuates the Contraction of Rat Trachea Through the Inhibition of Phosphatidylinositol Response

Although hyperosmolarity associated with diabetes is known to attenuate contractile response of airway smooth muscle, intracellular mechanisms involved are not fully understood. We examined the effects of hyperosmolarity on carbachol (CCh)- and aluminum fluoride (AF)-induced contractile and phosphatidylinositol (PI) responses of rat trachea. In vitro measurements of isometric tension and [3H] inositol monophosphate (IP1) formed were conducted by using rat tracheal rings and slices. Hyperosmolarity solutions of 350, 450 and 600 mOsm were made with dissolving glucose in Krebs-Henseleit (K-H) solution. Hyperosmolarity attenuated dose-dependently CCh-induced contraction of rat trachea (1.86 ± 0.13 g at 300 mOsm, 1.85 ± 0.16 g at 350 mOsm, 1.37 ± 0.07 g at 450 mOsm and 0.50 ± 0.04 g at 600 mOsm, respectively), and also attenuated CCh- induced IP1 accumulation (5.77 ± 0.33 Bq at 300 mOsm, 3.38 ± 0.26 Bq at 350 mOsm, 2.08 ± 0.30 Bq at 450 mOsm and 1.71 ± 0.40 Bq at 600 mOsm, respectively), and AF-induced IP1 accumulation (3.93 ± 0.22 Bq at 300 mOsm, 1.63 ± 0.14 Bq at 450 mOsm and 1.02 ± 0.14 Bq at 600 mOsm, respectively). The results suggest that hyperosmolarity would inhibit G-protein-coupled phospholipase C, resulting in attenuation of CCh-induced airway smooth muscle contraction

UBC13-Mediated Ubiquitin Signaling Promotes Removal of Blocking Adducts from DNA Double-Strand Breaks

Chemical modifications and adducts at DNA double-strand break (DSB) ends must be cleaned before re-joining by non-homologous end-joining (NHEJ). MRE11 nuclease is essential for efficient removal of Topoisomerase II (TOP2)-DNA adducts from TOP2 poison-induced DSBs. However, mechanisms in MRE11 recruitment to DSB sites in G1 phase remain poorly understood. Here, we report that TOP2-DNA adducts are expeditiously removed through UBC13-mediated polyubiquitination, which promotes DSB resection in G2 phase. We found that this ubiquitin signaling is required for efficient recruitment of MRE11 onto DSB sites in G1 by facilitating localization of RAP80 and BRCA1 to DSB sites and complex formation between BRCA1 and MRE11 at DSB sites. UBC13 and MRE11 are dispensable for restriction-enzyme-induced "clean" DSBs repair but responsible for over 50% and 70% of NHEJ-dependent repair of γ-ray-induced "dirty" DSBs, respectively. In conclusion, ubiquitin signaling promotes nucleolytic removal of DSB blocking adducts by MRE11 before NHEJ

Comparison of proportions and prognostic impact of pathological complete response between evaluations of representative specimen and total specimen in primary breast cancer after neoadjuvant chemoradiotherapy: an ancillary study of JCOG0306

Background In JCOG0306 trial, a phase II study to examine the efficacy of neoadjuvant chemotherapy followed by radiation therapy (NAC-RT) to primary breast cancer, pathological complete response (pCR) was evaluated from specimens of the representative cross-section including the tumor center that had been accurately marked [representative specimen (RS) method]. In this ancillary study, we examined if the RS method was comparable to the conventional total specimen (TS) method, which is widely employed in Japan, to identify the pCR group showing excellent prognosis. Methods We obtained long-term follow-up data of 103 patients enrolled in JCOG0306 trial. As histological therapeutic effect, pCR (ypT0 and ypT0/is) and quasi-pCR [QpCR, ypT0/is plus Grade 2b (only a few remaining invasive cancer cells)] were evaluated with RS and TS methods. Concordance of pCR between these two methods and associations of the pCR with prognosis were examined. Results ypT0, ypT0/is, and QpCR were observed in 28 (27.2%), 39 (37.9%), and 45 (43.7%) patients with RS method, whereas these were 20 (19.4%), 25 (24.3%) and 40 (38.9%) with TS method, respectively. Between RS and TS methods, concordance proportions of ypT0 and ypTis were 92.2% and 86.4%, respectively. Risk of recurrence of ypT0/is group was lower than that of non-ypT0/is group (HR 0.408, 95% CI [0.175–0.946], P = 0.037) and risk of death of ypT0/is group was lower than that of non-ypT0/is group (HR 0.251, 95% CI [0.073–0.857], P = 0.027). The ypT0 and ypT0/is groups with RS method showed excellent prognosis similarly with those with TS method, and RS method was able to differentiate the OS and RFS between pCR and non-pCR than TS method significantly even if pCR was classified ypT0 or ypT0/is. With TS method, QpCR criteria stratified patients into the better and worse prognosis groupsmore clearly than pCR criteria of ypT0 or ypT0/is. Conclusions RS method was comparable to TS method for the evaluation of pCR in the patients who received NAC-RT to primary breast cancer provided the tumor center was accurately marked. As pCR criteria with RS method, ypT0/is appeared more appropriate than ypT0