Dual Inhibition of H3K9me2 and H3K27me3 Promotes Tumor Cell Senescence without Triggering the Secretion of SASP

Chemotherapy remains the most common cancer treatment. Although chemotherapeutic drugs induce tumor cell senescence, they are often associated with post-therapy tumor recurrence by inducing the senescence-associated secretory phenotype (SASP). Therefore, it is important to identify effective strategies to induce tumor cell senescence without triggering SASP. In this study, we used the small molecule inhibitors, UNC0642 (G9a inhibitor) and UNC1999 (EZH2 inhibitor) alone or in combination, to inhibit H3K9 and H3K27 methylation in different cancer cells. Dual inhibition of H3K9me2 and H3K27me3 in highly metastatic tumor cells had a stronger pro-senescence effect than either inhibitor alone and did not trigger SASP in tumor cells. Dual inhibition of H3K9me2 and H3K27me3 suppressed the formation of cytosolic chromatin fragments, which inhibited the cGAS-STING-SASP pathway. Collectively, these data suggested that dual inhibition of H3K9 and H3K27 methylation induced senescence of highly metastatic tumor cells without triggering SASP by inhibiting the cGAS-STING-SASP pathway, providing a new mechanism for the epigenetics-based therapy targeting H3K9 and H3K27 methylation.     

Regulation of Epigenetic Modifications in the Placenta during Preeclampsia: PPARγ Influences H3K4me3 and H3K9ac in Extravillous Trophoblast Cells

The aim of this study was to analyze the expression of peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RxRα), a binding heterodimer playing a pivotal role in the successful trophoblast invasion, in the placental tissue of preeclamptic patients. Furthermore, we aimed to characterize a possible interaction between PPARγ and H3K4me3 (trimethylated lysine 4 of the histone H3), respectively H3K9ac (acetylated lysine 9 of the histone H3), to illuminate the role of histone modifications in a defective trophoblast invasion in preeclampsia (PE). Therefore, the expression of PPARγ and RxRα was analyzed in 26 PE and 25 control placentas by immunohistochemical peroxidase staining, as well as the co-expression with H3K4me3 and H3K9ac by double immunofluorescence staining. Further, the effect of a specific PPARγ-agonist (Ciglitazone) and PPARγ-antagonist (T0070907) on the histone modifications H3K9ac and H3K4me3 was analyzed in vitro. In PE placentas, we found a reduced expression of PPARγ and RxRα and a reduced co-expression with H3K4me3 and H3K9ac in the extravillous trophoblast (EVT). Furthermore, with the PPARγ-antagonist treated human villous trophoblast (HVT) cells and primary isolated EVT cells showed higher levels of the histone modification proteins whereas treatment with the PPARγ-agonist reduced respective histone modifications. Our results show that the stimulation of PPARγ-activity leads to a reduction of H3K4me3 and H3K9ac in trophoblast cells, but paradoxically decreases the nuclear PPARγ expression. As the importance of PPARγ, being involved in a successful trophoblast invasion has already been investigated, our results reveal a pathophysiologic connection between PPARγ and the epigenetic modulation via H3K4me3 and H3K9ac in PE.     

Autoimmune Diseases: Enzymatic cross Recognition and Hydrolysis of H2B Histone,Myelin Basic Protein,and DNA by IgGs against These Antigens

As shown in many studies, one of the earliest statistically significant indicators of the development of many autoimmune diseases (ADs) is the appearance in the blood of antibodies with catalytic activities (abzymes) hydrolyzing different autoantigens. Antibodies-abzymes having different enzymatic activities are a specific and essential feature of some ADs. Most abzymes are harmful to humans. Free histones in the blood are damage-associated proteins, and their administration to animals drives systemic inflammatory and toxic effects. Myelin basic protein (MBP) is the most critical component of the axon myelin-proteolipid sheath. Hydrolysis of MBP by abzymes leads to the disruption of nerve impulses. Here, we analyzed the possible pathways for the formation of unusual antibodies and abzymes that exhibit polyspecificity in recognition during complex formation with partially related antigens and possess the ability to catalyze several different reactions for the first time. Using IgGs of HIV-infected and multiple sclerosis patients against five individual histones (H1–H4), MBP, and DNA, it was first shown that abzymes against each of these antigens effectively recognize and hydrolyze all three antigens: histones, MBP, and DNA. The data obtained indicate that the formation of such polyspecific abzymes, whose single active center can recognize different substrates and catalyze several reactions, can occur in two main ways. They can be antibodies against DNA–protein complex hybrid antigenic determinants containing proteins and nucleic sequences. Their formation may also be associated with the previously described phenomenon of IgG extensive LH half-molecule (containing one L-light and one H-heavy chains) exchange leading to H₂L₂ molecules containing HL halves with variable fragments recognizing different antigens.     

Evaluation of Calyculin A Effect on γH2AX/53BP1 Focus Formation and Apoptosis in Human Umbilical Cord Blood Lymphocytes

Dephosphorylation inhibitor calyculin A (cal A) has been reported to inhibit the disappearance of radiation-induced γH2AX DNA repair foci in human lymphocytes. However, other studies reported no change in the kinetics of γH2AX focus induction and loss in irradiated cells. While apoptosis might interplay with the kinetics of focus formation, it was not followed in irradiated cells along with DNA repair foci. Thus, to validate plausible explanations for significant variability in outputs of these studies, we evaluated the effect of cal A (1 and 10 nM) on γH2AX/53BP1 DNA repair foci and apoptosis in irradiated (1, 5, 10, and 100 cGy) human umbilical cord blood lymphocytes (UCBL) using automated fluorescence microscopy and annexin V-FITC/propidium iodide assay/γH2AX pan-staining, respectively. No effect of cal A on γH2AX and colocalized γH2AX/53BP1 foci induced by low doses (≤10 cGy) of γ-rays was observed. Moreover, 10 nM cal A treatment decreased the number of all types of DNA repair foci induced by 100 cGy irradiation. 10 nM cal A treatment induced apoptosis already at 2 h of treatment, independently from the delivered dose. Apoptosis was also detected in UCBL treated with lower cal A concentration, 1 nM, at longer cell incubation, 20 and 44 h. Our data suggest that apoptosis triggered by cal A in UCBL may underlie the failure of cal A to maintain radiation-induced γH2AX foci. All DSB molecular markers used in this study responded linearly to low-dose irradiation. Therefore, their combination may represent a strong biodosimetry tool for estimation of radiation response to low doses. Assessment of colocalized γH2AX/53BP1 improved the threshold of low dose detection.     

3-苯基-2-（4-叔丁基苯氧基）-3＇-氧环己烷并噻吩并[2，3-d]嘧啶-4（3H）-酮的合成及其与BSA的相互作用

通过Aza-Wittig反应。碳二亚胺与酚类化合物在碱性催化条件下成环,合成了3-苯基-2-（4-叔丁基苯氧基）-3’-氧环己烷并噻吩并[2,3-胡嘧啶-4（3H）-酮（PTP）。在生理条件下。采用紫外吸收光谱法及荧光光谱法研究了PTP与牛血清白蛋白（BSA）的相互作用。并采用同步荧光法考察了PTP的加入对BSA构象的影响。结果表明,PTP对BSA的荧光猝灭机理为静态猝灭过程。PTP与BSA的结合反应为自发进行,反应中主要作用力是氢键和范德华力,两者的结合位点数为1、结合距离为1．78nm．PTP的加入对BSA构象没有明显影响。