Intracellular reduction and oxidation pathways regulate protein functionality through both reversible and irreversible mechanisms. The Cdc25 phosphatases, which control cell cycle progression, are potential subjects of oxidative regulation. Many of the more potent Cdc25 phosphatase inhibitors reported to date are quinones, which are capable of redox cycling. Therefore, we used the previously characterized quinolinedione Cdc25 inhibitor DA3003-1 [NSC 663284 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] and a newly synthesized congener JUN1111 [7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] to test the hypothesis that quinone inhibitors of Cdc25 regulate phosphatase activity through redox mechanisms. Like DA3003-1, JUN1111 selectively inhibited Cdc25 phosphatases in vitro in an irreversible, time-dependent manner and arrested cells in the G1 and G2/M phases of the cell cycle. It is noteworthy that both DA3003-1 and JUN1111 directly inhibited Cdc25B activity in cells. Depletion of glutathione increased cellular sensitivity to DA3003-1 and JUN1111, and in vitro Cdc25B inhibition by these compounds was sensitive to pH, catalase, and reductants (dithiothreitol and glutathione), consistent with oxidative inactivation. In addition, both DA3003-1 and JUN1111 rapidly generated intracellular reactive oxygen species. Analysis of Cdc25B by mass spectrometry revealed sulfonic acid formation on the catalytic cysteine of Cdc25B after in vitro treatment with DA3003-1. These results indicate that irreversible oxidation of the catalytic cysteine of Cdc25B is indeed a mechanism by which these quinolinediones inactivate this protein phosphatase.

Cdc25 phosphatases propel cell cycle progression by activating cyclin-dependent kinases (Cdk). DNA damage is generally thought to inhibit Cdc25 functionality by inducing proteasomal degradation of Cdc25A and phosphorylation-mediated sequestration of Cdc25B and Cdc25C to the cytoplasm. More recently, a critical role for Cdc25B in the resumption of cell cycle progression through mitosis after DNA damage has been identified. In this study, the fate of Cdc25B after mechanistically distinct DNA-damaging agents (etoposide, cisplatin, bleomycin, ionizing irradiation, or UV irradiation) was examined, and surprisingly a rapid increase in cellular Cdc25B levels was observed after DNA damage. Using UV irradiation as the prototypic damaging agent, we found that the increase in Cdc25B levels was checkpoint dependent and was controlled by a p53-independent mechanism. Cdc25B levels controlled the number of cells progressing into mitosis after UV, but they did not affect G 2 -M checkpoint engagement immediately after DNA damage. Increased Cdc25B reduced the time required for cell cycle resumption. These data support a model in which Cdc25B accumulation is an important anticipatory event for cell cycle resumption after DNA damage.

The tumor suppressor p53 exerts its activity by preventing DNA-damaged cells from dividing until either the chromosomal repair is effected or the cell undergoes apoptosis. Reactive oxygen species (ROS) are enhanced through the action of p53-mediated transcription of apoptosis-promoting genes; however, p53 also can promote the expression of many antioxidant genes that prevent apoptosis. New research indicates that in low cellular stress, low concentrations of p53 induce the expression of antioxidant genes, whereas in severe cellular stress, high concentrations of p53 promote the expression of genes that contribute to ROS formation and p53-mediated apoptosis. p53-depleted cells injected into athymic mice increased significantly in tumor volume, whereas injected p53(+/+) cells did not grow to the same degree. Interestingly, administration of the antioxidant compound N-acetylcysteine (NAC) inhibited the growth of tumor volume in p53-depleted injected cells, and NAC supplementation of p53(-/-) mice from birth greatly decreased the number of karyotype abnormalities and tumors formed in these mice by six months of age. Thus, under normal (or low stress) conditions, p53 appears to have an antioxidant role that protects cells from oxidative DNA damage and although this effect might depend on the concentration of p53, other cellular factors likely participate in a cell's final fate.

For prevention of a recurrent preterm birth (PTB), intramuscular 17-α-hydroxy progesterone caproate (IM 17 OHPC) weekly is recommended. Vaginal progesterone is preferred for women at risk for PTB due to a short cervical length, but may be useful in women with a prior PTB. However, there is no consensus about the optimal vaginal formulation or its efficacy as compared to 17 OHPC to prevent recurrent PTB. We randomised 100 women with a singleton pregnancy between 16 and 24 weeks of gestation and ≥ one prior spontaneous PTB, of a singleton (>16 to <37 weeks of gestation) to receive the 200 mg vaginal progesterone effervescent tablet daily (Group A) or IM 17-OHPC, 250 mg weekly (Group B) till 37 weeks of gestation or delivery. The spontaneous PTB rate of <37 weeks was similar (20% in Group A and 20.8% in Group B, p =  .918). The PTB rate of <34 weeks or <28 weeks were also comparable. The mean birth weight and other neonatal outcomes were similar in the two groups. Two neonates in Group A and four neonates in Group B required NICU admission, one of whom (Group B) died due to prematurity. Twenty percent of women in Group A and 29.2% in Group B reported adverse effects from their respective study medications (p =  .408, NS). Thus, there did not appear to be a difference between vaginal progesterone and 17-OHPC when used for the prevention of a recurrent PTB. Impact statement What is already known on this subject? Progesterone administration is useful for prevention of a recurrent preterm birth (PTB) and these women are prescribed the intramuscular 17-α-hydroxy progesterone caproate (IM 17 OHPC), 250 mg, weekly. Some studies found that vaginal progesterone (once daily) is also beneficial in these women, but there is no consensus regarding its efficacy when compared to 17 OHPC, or its optimal formulation and dose. What do the results of this study add? In the present study, 100 women with a singleton pregnancy between 16 and 24 weeks of gestation and ≥ one prior spontaneous singleton PTB or mid-trimester abortion were randomised to receive 200 mg of vaginal progesterone effervescent tablet daily (Group A) or 250 mg IM 17-OHPC weekly (Group B) till 37 weeks of gestation or delivery. The spontaneous PTB rate <37 weeks was similar in the two groups (20% in Group A and 20.8% in Group B, p = .918). The PTB rate <34 weeks or <28 weeks were also comparable. The mean birth weight and other neonatal outcomes were similar. Twenty percent of women in Group A and 29.2% of women in Group B reported adverse effects from their respective study medications (p = .408, NS). Thus, there did not appear to be a difference between the vaginal progesterone effervescent tablet and 17-OHPC when used for the prevention of a recurrent PTB. What are the implications of these findings for clinical practice and/or further research? The vaginal progesterone effervescent tablet may be a suitable alternative to IM 17 OHPC to prevent recurrent PTB. Future studies should identify the most appropriate route (IM or vaginal) and vaginal progesterone formulat...