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DNA damage has been implicated in the stimulation of the type 1 interferon (T1IFN) response. Here, we show that downregulation of the DNA repair protein, polynucleotide kinase/phosphatase (PNKP), in a variety of cell lines causes robust phosphorylation of STAT1, upregulation of interferon-stimulated genes and persistent accumulation of cytosolic DNA, all of which are indicators for the activation of the T1IFN response. Furthermore, this did not require damage induction by ionizing radiation. Instead, our data revealed that production of reactive oxygen species (ROS) synergises with PNKP loss to potentiate the T1IFN response, and that loss of PNKP significantly compromises mitochondrial DNA (mtDNA) integrity. Depletion of mtDNA or treatment of PNKP-depleted cells with ROS scavengers abrogated the T1IFN response, implicating mtDNA as a significant source of the cytosolic DNA required to potentiate the T1IFN response. The STING signalling pathway is responsible for the observed increase in the pro-inflammatory gene signature in PNKP-depleted cells. While the response was dependent on ZBP1, cGAS only contributed to the response in some cell lines. Our data have implications for cancer therapy, since PNKP inhibitors would have the potential to stimulate the immune response, and also to the neurological disorders associated with PNKP mutation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5'-DNA kinase/3'-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.

XRCC1 plays a central role in mammalian single-strand break repair. Although it has no enzymatic activity of its own, it stimulates the activities of polynucleotide kinase/phosphatase (PNKP), and this function is enhanced by protein kinase CK2 mediated phosphorylation of XRCC1. We have previously shown that non-phosphorylated XRCC1 stimulates the kinase activity of PNKP by increasing the turnover of PNKP. Here we extend our analysis of the XRCC1-PNKP interaction taking into account the phosphorylation of XRCC1. We demonstrate that phosphorylated and non-phosphorylated XRCC1 interact with different regions of PNKP. Phosphorylated XRCC1 binds with high affinity (K(d) = 3.5 nM and 1 : 1 stoichiometry) to the forkhead associated (FHA) domain, while non-phosphorylated XRCC1 binds to the catalytic domain of PNKP with lower affinity (K(d) = 43.0 nM and 1 : 1 stoichiometry). Under conditions of limited enzyme concentration both forms of XRCC1 enhance the activities of PNKP, but the effect is more pronounced with phosphorylated XRCC1, particularly for the kinase activity of PNKP. The stimulatory effect of phosphorylated XRCC1 on PNKP can be totally inhibited by the presence of excess FHA domain polypeptide, but non-phosphorylated XRCC1 is not susceptible to competition by the FHA domain. Thus, XRCC1 can stimulate PNKP by two independent mechanisms.

Background: Neither oral contraceptives (COC) nor metformin are an optimal modality for the long-term treatment of polycystic ovary syndrome (PCOS). The aim of this study was to evaluate whether a combination of both is beneficial over COC monotherapy., Methods: Altogether, 30 women were included in the study and 28 finished the protocol. The patients were randomly assigned to two groups treated with either COC (COC group) or COC and metformin (1500 mg/day) (METOC group) for 6 months. Anthropometric parameters, androgens, lipids, fasting insulin, glucose and sex hormone binding globulin (SHBG) concentrations were measured before and at the end of the sixth cycle of treatment. The insulin sensitivity index was evaluated using the euglycaemic clamp., Results: There were no significant changes in anthropometric parameters, fasting glucose or insulin sensitivity in either group. Total testosterone, free androgen index, androstenedione and dehydroepiandrosterone decreased and SHBG increased significantly in both groups. When comparing the effect of both treatments, only a more pronounced decrease in free androgen index was found in the METOC group., Conclusions: Adding metformin slightly modified the treatment effect of COC, causing a more significant decrease in the free androgen index but having no additional positive impact on lipids, insulin sensitivity, SHBG or testosterone. The available data do not offer enough evidence to advocate the standard use of combined treatment in PCOS. Whether the combination might be beneficial for specific subgroups of patients is of further interest.

The aim of our study was to compare insulin sensitivity in lean and obese European polycystic ovary syndrome (PCOS) women with lean healthy women. We performed the euglycemic hyperinsulinemic clamp in 83 women with PCOS [53 lean with body mass index (BMI) of 21.5 +/- 1.8 kg/m2 and 30 obese with BMI of 29.6 +/- 3.7 kg/m2] and in 15 healthy women with BMI of 21.6 +/- 1.8 kg/m2 to determine glucose disposal (M) and the insulin sensitivity index (ISI). Statistical evaluation was done using Kruskal-Wallis ANOVA followed by Kruskal-Wallis multiple-comparison z-value test. The basal blood glucose was significantly higher in lean and obese PCOS women than in controls (P < 0.02). Fasting insulin was significantly higher in both lean and obese PCOS women than in controls (P < 0.000001). Obese PCOS women were more insulin resistant than controls (P < 0.02 for M and P < 0.0008 for ISI); lean PCOS women did not differ from controls in M or ISI. Posthepatic insulin delivery was significantly higher in both lean and obese PCOS women compared with controls (P < 0.000008). We conclude that lean PCOS women are not more insulin resistant than healthy controls. Insulin hypersecretion, on the other hand, is present even in lean PCOS women.

Background: The aim of this study was to compare the pregnancy outcome, especially the prevalence of gestational diabetes mellitus (GDM), in a group of patients with polycystic ovary syndrome (PCOS), with a group of healthy weight-matched women., Methods: Retrospectively, we evaluated the pregnancies of 66 women with PCOS, who had been treated for infertility and who delivered at our department. These were compared with a group of 66 age- and weight-matched controls., Results: We did not find any significant differences in the prevalence of pregnancy complications such as gestational diabetes mellitus, pregnancy-induced hypertension (PIH) and premature deliveries between the group of PCOS patients and the controls., Conclusion: When differences in age and weight between PCOS patients and controls are negligible, PCOS is not associated with a higher risk of pregnancy complications.

Unlabelled: Insulin resistance is a frequent (although not constant) abnormality in both obese and nonobese women with polycystic ovary syndrome (PCOS). It plays a key role in the predisposition to type 2 diabetes, which is the most important health consequence of the syndrome. Identification of patients with insulin resistance is significant both for follow-up and for therapeutic reasons. The aim of the study was to evaluate the relationships between insulin sensitivity, measured by euglycemic clamp, and both endocrine and metabolic indices and to identify the best model for predicting insulin sensitivity. A total of 41 nonobese women fulfilling the diagnostic criteria for PCOS were enrolled in the study. None of the androgens correlated with the insulin sensitivity index. All clamp parameters correlated with SHBG, triglycerides, and body mass index, although no correlation was found with waist to hip ratio or waist circumference. The close relationship between insulin sensitivity and SHBG was documented by factor analysis and by its presence in all prediction models as the most significant (or even the single) predictor of the insulin sensitivity index., In Conclusion: 1) a decreased level of SHBG can be used as a single reliable parameter in the prediction of insulin sensitivity in nonobese women with PCOS; and 2) waist to hip ratio, waist circumference, and androgen concentrations have no predictive value.

Background: Combined oral contraceptives (COC) effectively suppress hyperandrogenism in women with polycystic ovary syndrome (PCOS), though deterioration of insulin sensitivity during treatment is assumed. The study aim was to investigate insulin action and androgen production during treatment with COC containing low-androgenic progestin., Methods: A total of 13 PCOS women and nine controls was enrolled into the study. Only non-obese women with a body mass index (BMI)

Polycystic ovarian syndrome (PCOS) is an obvious indication for long-term treatment. Combined oral contraceptives (COC) remain the first choice for the treatment of hyperandrogenism in most patients. However, differences in endocrine and metabolic parameters between obese and lean patients have been postulated. This is the first study evaluating the effect of COC treatment in obese versus non-obese PCOS patients. In total, 28 lean [body mass index (BMI) <25 kg/m(2))] and 15 obese (BMI >30 kg/m(2)) women patients were enrolled in the study. The concentrations of androgens, sex hormone-binding globulin (SHBG) and lipids were measured before and after 6 months of treatment with COC containing low-androgenic progestins. Clinical androgenic symptoms were monitored. There was a lower concentration of SHBG in obese patients, but there were no differences in androgen concentrations between both groups before the study. Highly significant changes in concentrations of testosterone (P < 0.001), androstenedione (P < 0.0001), SHBG (P < 0.001) and LH (P = 0.01) were demonstrated in lean patients, with only less significant changes in SHBG (P < 0.01) and testosterone (P < 0.05) in obese patients during the study. Clinical androgenic symptoms improved significantly (P = 0.05) only in the group of lean women. No reduction in low-density lipoprotein-cholesterol/high-density lipoprotein-cholesterol ratio was observed in either group. In conclusion, the positive effect of COC treatment on androgen production, serum androgen binding capacity, and clinical androgenic symptoms was negatively influenced by an increased BMI.

Background: Although many arguments have been put forth supporting the role of androgens in the aetiology of acne, their part in determining the severity of the disease is not well established., Objectives: The aim of our study was to evaluate the relationship between acne severity and the clinical and laboratory markers of androgenicity in a large group of patients., Methods: Ninety women over 17 years of age with acne were enrolled into the study. The levels of testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulphate and sex hormone binding globulin (SHBG) were measured. Menstrual cycle regularity, hirsutism score, acne severity and ultrasound evaluation of polycystic ovaries were recorded. One-way analysis of variance, chi(2)-test and correlation analysis were used for data processing., Results: Hirsutism was documented in 19 (21%) subjects, elevated levels of at least one androgen in 73 (81%) subjects, an irregular cycle was reported by 43 (48%) women, and polycystic ovaries were found in 45 (50%) women. The patients were divided into three groups according to acne severity. Acne was graded using the Leeds technique as minor in 43 (48%) cases, mild in 27 (30%) and moderate in 20 (22%). We did not demonstrate a positive correlation between the grade of acne severity and any of the clinical or laboratory markers of androgenicity assessed. On the contrary, women with a higher grade of acne severity showed lower values of the index of free testosterone, a lower hirsutism score and higher SHBG levels., Conclusions: Our study suggests that the severity of acne manifestation in adult women is not determined by androgen production.

The aim of the study was to determine the prevalence of non-insulin dependent diabetes mellitus (NIDDM), arterial hypertension, coronary artery disease and the risk factors for these diseases in perimenopausal women with a history of polycystic ovary syndrome (PCOS) treatment. A group of 28 women was selected from a large group of patients who had undergone wedge ovarian resection. A total of 752 controls was selected by age (45-59 years) from a random female population sample. There was no difference between the two groups in body mass index, waist circumference or waist-hip ratio. Both groups were found to have identical family histories of NIDDM, hypertension, and coronary artery disease and identical smoking habits. We did not find a difference between the mean concentrations of lipids and fasting glucose. The two groups did not differ in the proportions of women with elevated lipid concentrations. The prevalence of NIDDM and coronary artery disease was significantly higher in PCOS women. In conclusion, women in the general population have the same level of risk factors at perimenopausal age as PCOS women. Patients with markedly expressed clinical symptoms of PCOS made up a subgroup in the general population at high risk for developing NIDDM and coronary artery disease.