Percentage Of γH2AX Positive Cells Research Articles

Abstract 410: ASXL3 is a potential biomarker of response of SCLC to targeted therapies

Abstract Small Cell Lung Cancer (SCLC) has been designated a recalcitrant cancer and a priority by National Cancer Institute. Despite recent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential, these advances have yet to be fully translated to effective regimens for the treatment of SCLC. Previously, we identified Additional Sex Combs Like-3 (ASXL3) as a novel mediator of pluripotency in human respiratory epithelia and potential therapeutic target in SCLC. ASXL3 is one of three members of the ASXL family, whereas the functions of ASXL1 and ASXL2 as components of the PR-DUB complex have been extensively studied, the functions and significance of ASXL3 during malignant transformation remain elusive. To further investigate the relevance of ASXL3 activation in SCLC, we initially studied ASXL3 expression levels in various SCLC subtypes by qRT-PCR, digital RT-PCR, RNA-seq, and western blotting analysis. These studies demonstrated that ASCL (A) and ASCL and NeuroD1 (A+N) subtypes of SCLC cells contain thousand-folds more ASXL3 (ASXL3high) than Pou2f3 (P) and YAP1 (Y) SCLC subtypes (ASXL3low). ASXL3 levels were elevated when cancer cells were cultured under stemness-enriched conditions. Under normal culture conditions, ASXL3high SCLC cells showed higher intrinsic DNA damage, evidenced by a high percentage of γ-H2AX positive cells compared to ASXL3low SCLC cells. Additionally, ASXL3high SCLC cells displayed 38% γ-H2AX positive metaphase cells, and 42% of abnormal chromosome segregations during mitosis, including lagging chromosomes, DNA bridges, and unequal chromosome segregation indicative of profound genome instability. One reason for this extensive genomic instability is that ATM pathway activation in response to DNA replication stress is impaired in ASXL3high SCLC cells. Following exposures to 1mM hydroxyurea (HU) for 4, 8, and 24 hours, ATM autophosphorylation levels in ASXL3high SCLC cells were much lower than in ASXL3low SCLC cells. This insufficiency significantly affected the activation of downstream cascade targets including CHK2, KAP-1 and EZH2. Consistent with these findings, ASXL3high SCLC cells showed reduced sensitivities to ATM inhibitors. However, ASXL3high SCLC cells exhibited higher activity of PLK1 and were more sensitive to hydroxyurea. Importantly, low dose HU reduced the IC50 values of the PLK1 inhibitors, BI2536 or NMS-P937 by 80-100-fold in ASXL3high but not ASXL3low SCLC cells, suggesting a strong synergy between these two drugs in ASXL3high SCLC cells. Collectively, these findings suggest that ASXL3 functions to protect cells from lethality induced by genomic instability through monitoring ATM activation. Experiments are underway using SCLC PDX models to demonstrate the efficacy of this combined treatment in-vivo as a prelude to possible evaluation of this drug combination in SCLC patients. Citation Format: Ruihong Wang, Haitao Wang, Xinwei Wu, Keerti Mishra, Stephanie Shiffka, Frank Hernandez, Mary Zhang, Umay Tuana Tolunay, Sudheer Gara, Sichuan Xi, Shamus R. Carr, Choung D. Hoang, David S. Schrump. ASXL3 is a potential biomarker of response of SCLC to targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 410.

Combined inhibition of Wee1 and Chk1 as a therapeutic strategy in multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative stress. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways. In this study, we reported that high CHK1 and WEE1 expression is associated with poor outcome in independent cohorts of MM patients treated with high dose melphalan chemotherapy or anti-CD38 immunotherapy. Combined targeting of Chk1 and Wee1 demonstrates synergistic toxicities on MM cells and was associated with higher DNA double-strand break induction, as evidenced by an increased percentage of γH2AX positive cells subsequently leading to apoptosis. The therapeutic interest of Chk1/Wee1 inhibitors' combination was validated on primary MM cells of patients. The toxicity was specific of MM cells since normal bone marrow cells were not significantly affected. Using deconvolution approach, MM patients with high CHK1 expression exhibited a significant lower percentage of NK cells whereas patients with high WEE1 expression displayed a significant higher percentage of regulatory T cells in the bone marrow. These data emphasize that MM cell adaptation to replicative stress through Wee1 and Chk1 upregulation may decrease the activation of the cell-intrinsic innate immune response. Our study suggests that association of Chk1 and Wee1 inhibitors may represent a promising therapeutic approach in high-risk MM patients characterized by high CHK1 and WEE1 expression.

In vitro assessment of the DNA damage and senility of CD117+ stem cells collected from diabetic mice.

Angiogenesis impairment is a common feature of diabetes mellitus (DM), whereas CD117+ bone marrow cells (BMCs) injury might be responsible for such complication. In this study, we studiedtheeffectofhyperglycemia on the DNA damage and senility of CD117+ bone marrow cells. We isolated CD117+ BMCs from the Streptozotocin(STZ) induceddiabetes and healthy control mice. Oxidative stress was detected by flow cytometric analysis. γ-H2AX, which is the DNA damage mark, was detected by using Western blotting and immunofluorescence histochemistry. We also detected the expression of γ-H2AX and p16 by using Western blotting. Compared with the control mice, the level of reactive oxygen species (ROS) was increased significantly in the CD117+ BMCs collected from the diabetic mice (p<0.05), and the percentage of γ-H2AX positive cells was higher significantly (p<0.01). The expression of γ-H2AX and p16 was increased significantly in the CD117+ BMCs from the diabetic mice. Our experiments demonstrated the oxidative stress in CD117+ BMCs under DM conditions, while accelerating the DNA damage and senility in CD117+ BMCs as well.

Aging and Senescence in Canine Testes.

Senescent cells accumulate with age but tissue-based studies of senescent cells are limited to selected organs from humans, mice, and primates. Cell culture and xenograft studies have indicated that senescent cells in the microenvironment may play a role in tumor proliferation via paracrine activities. Dogs develop age-related conditions, including in the testis, but cellular senescence has not been confirmed. We hypothesized that senescent cells accumulate with age in canine testes and in the microenvironment of testicular tumors. We tested the expression of the established senescence markers γH2AX and p21 on normal formalin-fixed, paraffin-embedded testes from 15 young dogs (<18 months of age) and 15 old dogs (7-15 years of age) and correlated the findings with age-dependent morphological changes. A statistically significant age-dependent increase in the percentage of p21-expressing cells was observed for testicular fibroblasts (4-fold) and Leydig cells (8-fold). However, p21-expressing cells were still a rare event. In contrast, the percentage of γH2AX-positive cells did not increase with age. P21- and γH2AX-expressing cells were rare in the microenvironments of tumors. Age-dependent morphological changes included an increased mean number of Leydig cells per intertubular triangle (2.95-fold) and a decreased spermatogenesis score. To our surprise, no age-related changes were recorded for interstitial collagen content, mean tubular diameter, and epithelial area. Opposed to our expectations based on previous in vitro data, we did not identify evidence of a correlation between age-associated accumulation of senescent cells and testicular tumor development. Understanding the role of the microenvironment in senescence obviously remains a challenging task.

Abstract 1376: Effect of gold nanoparticle on radiation induced DNA damage in MCF7 breast cancer cells

Abstract Nanoparticles (NPs) are 1-100 nm constructs explored for their application in cancer diagnosis and treatment. Exposure of cells to ionizing irradiation leads to DNA damage, with DNA double strand breaks (DSBs) being the most toxic that can lead to apoptosis. The presence of gold in NPs enhances radiation damage and DSBs, the latter measured by the levels of phosphorylated DNA histone protein H2AX (γH2AX). The goal of this study was to decipher biological mechanisms of NP radio-sensitization. We analyzed the effects of two different sizes of gold nanoparticles (AuNP) on DSBs in MCF-7 breast cancer cells by assessing H2AX phosphorylation at three photon energies. Cells were incubated with either 4 nm or 14 nm AuNP and irradiated with 2, 4 or 8 Gy using 2.5 FFF MV (60 MU/min), 6 MV (600 MU/min) or 10 FFF MV (2400 MU/min) X-rays. Percent of cells positive for γH2AX was determined by flow cytometry. Live cells (100000) were gated using forward (FSC; cell size) versus side scatter (SSC; complexity) characteristics (gate P1). Cells positive for γH2AX were further gated to identify subpopulations exhibiting brighter (gate P2) or dimmer (gate P3) fluorescence intensity. Treatment with 4 nm AuNP resulted in significantly more γH2AX positive cells after irradiation at all three energies, compared to their respective controls. When treated at 6 MV energy, increases in the percentage of cells positive for H2AX phosphorylation was detected at all three doses (2, 4 and 8 Gy). Increased phosphorylation was accompanied by an increase of cells in the P2 gate accompanied by an increase in their FCS and SSC characteristics, consistent with activation. In cells treated with 10 MV the effect was most pronounced at 4Gy dose, while 2 and 8 Gy resulted in a slight increase in the percentage of γH2AX positive cells. Nevertheless, these cells still exhibited prominent increases within the P2 gate and FCS vs SSC characteristics, compared to their irradiated controls. In cells treated with 2.5 MV energy similar results were observed using 4 and 8 Gy, while no effect was detected with 2 Gy. Data from the experiments using 14 nm size AuNP were less clear and require further investigation. The difference may be due to the localization of the smaller NPs within the nucleus and therefore cause more DNA damage thereby greater H2AX phosphorylation. We hypothesize that the NP radio-sensitization mechanism involves directing cells towards apoptosis by enhancing DNA damage and interfering with DNA repair. Hence, γH2AX positive cells exhibiting bright fluorescence (P2) and an increase in FCS and SSC identified even in the conditions without significant increase in the percent of total γH2AX positive cells, may reflect cells primed for apoptosis. Future studies are planned to elucidate the exact intracellular mechanisms of NP biological radio-sensitization effect. Citation Format: Branislava Janic, Fangchao Liu, Kevin Bobbitt, Stephen Brown, Guangzhao Mao, Indrin J. Chetty, Benjamin Movsas, Ning Winston Wen. Effect of gold nanoparticle on radiation induced DNA damage in MCF7 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1376.

Research of radiation resistance delivery through paracrine effects on non-small cell lung cancer

Objective To investigate the role and possible mechanisms of paracrine in radiation tolerance of non-small cell lung cancer (NSCLC) by observing the effects of radiation-resistant NSCLC H460R cell secretion on the radiosensitivity of parental H460 cells. Methods H460 cells were inoculated and cultured, and then divided into control group, conditional culturing group, irradiation group and irradiation combined conditional culturing group. One-time irradiation with 137Cs γ-rays was conducted with a dose rate of 1.02 Gy/min. Effect of H460R conditioned media on the radiation sensitivity of H460 cells was observed by clonogenic assay. The percentage of phospho-histone histone H2AX (γH2AX) positive cells was detected by immunofluorescence staining. The cyclical changes of the cells were detected by flow cytometry. The expression of DNA damage-associated proteins γH2AX and Rad51 were detected by Western Blot. Results The results showed that compared with the single γ-rays irradiation, the number of cell clones was significantly increased in the H460R cells after γ-rays irradiation combined with conditional culturing treatment at different doses of 2, 4, and 6 Gy, and the differences were statistically significant (2,4 Gy: all P<0.05; 6 Gy: P<0.01). The results of immunofluorescence staining showed that the percentage of γH2AX positive cells in the conditional culturing group was higher than that in the control group [(39.40±2.51)% vs. (25.21±2.05)%], and the difference was statistically significant (P<0.01). This value in the irradiation combined conditional culturing group was lower than that in irradiated group [(60.48±2.79)% vs. (80.65±2.05)%], and the difference was statistically significant (P<0.001). The flow cytometry analysis showed that the percentage of cells in G2/M phase for the irradiation (4 Gy) combined conditional culturing group was higher than that of irradiated group [(26.83±1.42)% vs. (15.73±1.29)%], and the difference was statistically significant (P<0.001). The Western Blot results showed that γH2AX and Rad51 protein expression in the irradiation (4 Gy) combined conditional culturing group respectively was lower and higher than that in irradiated group. Conclusion In the tumor microenvironment, radiation-resistant H460R cells can enhance the radioresistance of H460 cells by paracrine, which may be related to the promotion of DNA repair ability after irradiation Key words: Non-small cell lung cancer; Paracrine; Radioresistance; DNA repair

Effect of DNA damage marker γH2AX in the process of oxygen-induced retinal neovascularization

Background Changes in the concentration of oxygen induced retinal neovascularization and DNA damage repair response.Overexpression of angiogenic factors is the main reason of angiogenesis.Whether the DNA damage related to retinal neovascularization is unclear. Objective This study was to study the role of DNA damage marker γH2AX in the process of retinal neovascularization of mouse. Methods Seventy-two 17-day-old C57BL/6J mice were randomly classified into normal control group, oxygen induced retinopathy (OIR) model group, OIR positive control group and OIR negative control group, 18 for each group.The retinal tissue were obtained from the 4 groups, the retinal patch immunofluorescence was used to observe and compare the area of retinal neovascularization and non-perfusion region and γH2AX expression of the four groups.The human umbilical vein endothelial cells (HUVECs) were classified into normal control group, hypoxia model control group, positive interference group and negative interference group.The cells from the 4 groups were obtained 12 hours after treatment, the expression of the γH2AX from different HUVECs groups were compared by immunofluorescence.Western blot was performed to detect the expressions of the γH2AX from different HUVECs groups. Results The retinal patch immunofluorescence showed that the OIR model was successfully established.The area of retinal neovascularization and the area of non-perfusion region among the 4 groups had statistical significances (F=437.62, 93.05, both at P<0.01). The area of retinal neovascularization and non-perfusion region in OIR model group and OIR negative control group was larger than that in the normal control group.The non-perfusion region was smaller in the OIR positive control group than that in the OIR model group and OIR negative control group (both at P<0.01). The appearance of the retinal γH2AX focus congestion was consistent with the area of neovascularization and non-perfusion in the 17-day-old mouse.The difference of γH2AX positive percentage in the four groups of HUVECs was statistically significant (F=64.97, P<0.01). The percentages of γH2AX positive cells in the hypoxia model control group and negative interference group were significantly higher than that in the normal control group (both at P<0.01). The percentage of γH2AX positive cells in the positive interference group was lower than that in the hypoxia model control group and negative interference group (both at P<0.01). Conclusions γH2AX is abundant in OIR neovascularization.Inhibiting the formation of γH2AX may reduce the OIR neovascularization. Key words: Retina; Neovascularization; Human umbilical vein endothelial cells; γH2AX

ANKHD1 Silencing Delays S Phase Progression in Multiple Myeloma Cells Via Activation of ATM/ATR -CDC25a Pathway

Ankyrin repeat and KH domain-containing protein 1, ANKHD1, is highly expressed in myeloma cells and plays an important role in multiple myeloma (MM) progression and growth. ANKHD1 is found to be overexpressed in S phase of cell cycle in MM cells and silencing of ANKHD1 expression leads to accumulation of cells in S phase, suggesting a role in S phase progression (1). Earlier studies by our group reported that ANKHD1 silencing downregulates all replication dependent histones and that this downregulation may be associated with replication stress and DNA damage (2). We observed increased expression of γH2AX protein (phosphorylated histone H2A variant, H2AX, at Serine 139), a marker for DNA double strand breaks (DSBs) and an early sign of DNA damage induced by replication stress, in ANKHD1 silenced MM cells. In the present study we further sought to investigate the mechanisms underlying the induction of DNA damage on ANKHD1 silencing. We first confirmed the increased expression of γH2AX by flow cytometry analysis and observed that both the mean fluorescence intensity as well as percentage of γH2AX positive cells were higher in ANKHD1 silenced MM cells as compared to control cells. Phosphorylation of histone 2AX requires activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated)andATR (ATM-Rad3-related) that serves as central components of the signaling cascade initiated by DSBs. Hence, we checked for the expression of these kinases and observed increased phosphorylation of both ATM and ATR kinases in ANKHD1 silenced MM cells. There was no difference in the expressions of DNA-PKcs in control and ANKHD1 silenced cells by western blot. We next checked for the expression of CHK1 (checkpoint kinase 1) and CHK2 (checkpoint kinase 2), essential serine threonine kinases downstream of ATM and ATR. We observed a decrease in pCHK2 (phosphorylated CHK2 at Thr 68), with no change in expression of pCHK1 (phosphorylated CHK1 at Ser 345) total CHK1 or total CHK2. We also checked for expression of CDC25a (a member of the CDC25 family of dual-specificity phosphatases), that is specifically degraded in response to DNA damage (DSBs) and delays S phase progression via activation of ATM /ATR-CHK2 signaling pathway. Expression of CDC25a was significantly decreased in ANKHD1 silencing cells, confirming the induction of DSBs, and probably accounting for S phase delay on ANKHD1 silencing. Since there was decrease in active CHK2 (pCHK2) and no change in CHK1 required for degradation of CDC25a, we assume that decrease in CDC25a in ANKHD1 silenced MM cells may be via activation of ATM/ ATR pathway independent of CHK2/CHK1. Expression of several other downstream factors of DSBs induced DNA damage response and repair such as BRCA1, PTEN, DNMT1, SP1, HDAC2 were also found to be modulated in ANKHD1 silenced MM cells.In conclusion, ANKHD1 silencing in MM cells leads to DNA damage and modulates expression of several genes implicated in DNA damage and repair. DNA damage induced after ANKHD1 silencing in MM cells activates ATM/ ATR-CDC25a pathway which may lead to the activation of S phase checkpoint in MM cells. Results however are preliminary and further studies are required to understand the role of ANKHD1 in intra S phase check point.

The Flaxseed-Derived Lignan Phenolic Secoisolariciresinol Diglucoside (SDG) Protects Non-Malignant Lung Cells from Radiation Damage.

Plant phenolic compounds are common dietary antioxidants that possess antioxidant and anti-inflammatory properties. Flaxseed (FS) has been reported to be radioprotective in murine models of oxidative lung damage. Flaxseed’s protective properties are attributed to its main biphenolic lignan, secoisolariciresinol diglucoside (SDG). SDG is a free radical scavenger, shown in cell free systems to protect DNA from radiation-induced damage. The objective of this study was to investigate the in vitro radioprotective efficacy of SDG in murine lung cells. Protection against irradiation (IR)-induced DNA double and single strand breaks was assessed by γ-H2AX labeling and alkaline comet assay, respectively. The role of SDG in modulating the levels of cytoprotective enzymes was evaluated by qPCR and confirmed by Western blotting. Additionally, effects of SDG on clonogenic survival of irradiated cells were evaluated. SDG protected cells from IR-induced death and ameliorated DNA damage by reducing mean comet tail length and percentage of γ-H2AX positive cells. Importantly, SDG significantly increased gene and protein levels of antioxidant HO-1, GSTM1 and NQO1. Our results identify the potent radioprotective properties of the synthetic biphenolic SDG, preventing DNA damage and enhancing the antioxidant capacity of normal lung cells; thus, rendering SDG a potential radioprotector against radiation exposure.