γH2AX Expression Research Articles

Centipeda minima extract sensitizes lung cancer cells to DNA-crosslinking agents via targeting Fanconi anemia pathway

BackgroundIntrinsic and acquired chemoresistance remains a critical challenge in lung cancer chemotherapy. Fanconi anemia (FA) pathway plays an important role in antagonizing the cytotoxic effects of chemotherapeutics by repairing DNA damage. We recently demonstrated that the traditional Chinese medicinal herb, Centipeda minima (C. minima), possessed anti-inflammatory and antioxidant properties. However, the potential anticancer application of C. minima and the underlying mechanisms remain unclear. PurposeWe aimed to investigate the combined anticancer effects of the ethanol extract of C. minima (ECM) and DNA-crosslinking agents on non-small cell lung cancer (NSCLC) and elucidate the underlying mechanisms. MethodsCell viability and flow cytometry assay were performed to determine the synergistic cytotoxicity of ECM and DNA-crosslinking agents, cisplatin (CDDP) or mitomycin C (MMC), in NSCLC cells. Western blotting and immunofluorescence were conducted to examine the effects of ECM on protein expression in DNA damage repair pathway. Comet assay was applied to evaluate DNA damage levels. Subcutaneous xenografts of NSCLC were established to evaluate the combined anticancer effects of ECM and CDDP. ResultsCombined treatments with ECM and DNA-crosslinking agents exhibited synergistic cytotoxic effects against A549 and H1299 cells. FANCD2 was highly expressed in NSCLC that correlates with poor prognosis of NSCLC patients, based on the online database analysis. ECM significantly inhibited DNA damage-induced monoubiquitination and nuclear foci formation of FANCD2, thereby sensitizing NSCLC to CDDP- or MMC-induced DNA damage and apoptosis, as evidenced by increased expression of γ-H2AX, increased cleavage of caspases-3 and PARP, and enhanced Annexin V-FITC/PI staining. Further, ECM can also decrease the protein level of FANCD2 that contributes to the chemosensitizing effects. Moreover, ECM significantly attenuated CDDP-mediated S-phase arrest by antagonizing the activation of ATR/Chk1 pathway in NSCLC cells. Animal experiments further demonstrated that ECM and CDDP combination treatment synergistically inhibited tumor growth by decreasing FANCD2 protein level in tumor tissues. ConclusionOur results demonstrated that ECM can inhibit DNA-crosslinking agents-induced activation of FA pathway by attenuating both the expression and monoubiquitination of FANCD2. ECM and CDDP combination therapy exhibited synergistic anticancer effects both in vitro and in vivo, indicating that ECM and its active components might serve as novel anticancer drugs in the combination chemotherapy.

ClbG in Avian Pathogenic Escherichia coli Contributes to Meningitis Development in a Mouse Model.

Colibactin is a complex secondary metabolite that leads to genotoxicity that interferes with the eukaryotic cell cycle. It plays an important role in many diseases, including neonatal mouse sepsis and meningitis. Avian pathogenic Escherichia coli (APEC) is responsible for several diseases in the poultry industry and may threaten human health due to its potential zoonosis. In this study, we confirmed that clbG was necessary for the APEC XM strain to produce colibactin. The deletion of clbG on APEC XM contributed to lowered γH2AX expression, no megalocytosis, and no cell cycle arrest in vitro. None of the 4-week Institute of Cancer Research mice infected with the APEC XM ΔclbG contracted meningitis or displayed weakened clinical symptoms. Fewer histopathological lesions were observed in the APEC XM ΔclbG group. The bacterial colonization of tissues and the relative expression of cytokines (IL-1β, IL-6, and TNF-α) in the brains decreased significantly in the APEC XM ΔclbG group compared to those in the APEC XM group. The tight junction proteins (claudin-5, occludin, and ZO-1) were not significantly destroyed in APEC XM ΔclbG group in vivo and in vitro. In conclusion, clbG is necessary for the synthesis of the genotoxin colibactin and affects the development of APEC meningitis in mice.

Analysis of Radiation-Induced Changes in Cell Cycle and DNA Damage of Murine Hematopoietic Stem Cells by Multi-Color Flow Cytometry.

Exposure of bone marrow to genotoxic stress such as ionizing radiation (IR) results in a rapid decline of peripheral blood cells and stimulates entry of the normally quiescent hematopoietic stem cells (HSCs) into the cell cycle to reconstitute the hematopoietic system. While several protocols have employed flow cytometry analysis of bone marrow cells to study changes in specific cell populations with respect to cell cycle proliferation and/or expression of γ-H2AX, a marker of DNA damage, these parameters were examined in separate panels. Here, we describe a flow cytometry-based method specifically designed to examine cell cycle distribution using Ki-67 and FXCycle violet in combination with γ-H2AX in HSCs and hematopoietic progenitor cells (HPCs) within the same sample. This method is very useful, particularly in studies involving genotoxic stresses such as IR, which substantially reduce the absolute numbers of HSCs and HPCs available for staining. Additionally, we describe several important considerations for the analysis of markers of HSCs in irradiated versus unirradiated samples. Examples include the use of fluorescence minus one (FMO) controls, the gating strategy for markers whose expression is typically impacted by IR such as Sca1, tips for staining of intracellular antigens like Ki67, and ensuring the detection of signal from at least 500 events in each gate to ensure robustness of the results. © 2021 Wiley Periodicals LLC. Basic Protocol: Immunostaining protocol for bone marrow mononuclear cells using a multi-fluorophore panel.

Combination of rapamycin and SAHA enhanced radiosensitization by inducing autophagy and acetylation in NSCLC.

Radiotherapy plays an essential role in the treatment of non-small-cell lung cancer (NSCLC). However, cancer cells' resistance to ionizing radiation (IR) is the primary reason for radiotherapy failure leading to tumor relapse and metastasis. DNA double-strand breaks (DSB) repair after IR is the primary mechanism of radiotherapy resistance. In this study, we investigated the effects of autophagy-inducing agent, Rapamycin (RAPA), combined with the histone deacetylase inhibitor (HDACi), Suberoylanilide Hydroxamic Acid (SAHA), on the radiosensitivity of A549 and SK-MES-1 cells, and examined the combination effects on DNA damage repair, and determined the level of autophagy and acetylation in A549 cells. We also investigated the combination treatment effect on the growth of A549 xenografts after radiotherapy, and the level of DNA damage, autophagy, and acetylation. Our results showed that RAPA combined with SAHA significantly increased the inhibitory effect of radiotherapy compared with the single treatment group. The combined treatment increased the expression of DNA damage protein γ-H2AX and decreased DNA damage repair protein expression. RAPA combined with SAHA was induced mainly by regulating acetylation levels and autophagy. The effect of combined treatment to increase radiotherapy sensitivity will be weakened by inhibiting the level of autophagy. Besides, the combined treatment also showed a significantly inhibited tumor growth in the A549 xenograft model. In conclusion, these results identify a potential therapeutic strategy of RAPA combined with SAHA as a radiosensitizer to decreased DSB repair and enhanced DNA damage by inducing acetylation levels and autophagy for NSCLC.

Human adipose-derived mesenchymal stromal cells from face and abdomen undergo replicative senescence and loss of genetic integrity after long-term culture

Body fat depots are heterogeneous concerning their embryonic origin, structure, exposure to environmental stressors, and availability. Thus, investigating adipose-derived mesenchymal stromal cells (ASCs) from different sources is essential to standardization for future therapies. In vitro amplification is also critical because it may predispose cell senescence and mutations, reducing regenerative properties and safety. Here, we evaluated long-term culture of human facial ASCs (fASCs) and abdominal ASCs (aASCs) and showed that both met the criteria for MSCs characterization but presented differences in their immunophenotypic profile, and differentiation and clonogenic potentials. The abdominal tissue yielded more ASCs, and these had higher proliferative potential, but facial cells displayed fewer mitotic errors at higher passages. However, both cell types reduced clonal efficiency over time and entered replicative senescence around P12, as evaluated by progressive morphological alterations, reduced proliferative capacity, and SA-β-galactosidase expression. Loss of genetic integrity was detected by a higher proportion of cells showing nuclear alterations and γ-H2AX expression. Our findings indicate that the source of ASCs can substantially influence their phenotype and therefore should be carefully considered in future cell therapies, avoiding, however, long-term culture to ensure genetic stability.

Characteristic differences in radiation-induced DNA damage response in human papillomavirus-negative and human papillomavirus-positive head and neck cancers with accumulation of fractional radiation dose.

Superior treatment responses by patients with human papillomavirus (HPV) positive head and neck squamous cell carcinoma (HNSCC), compared to patients with HNSCC from other causes, drive biomarker research to optimize treatment. Most HNSCC patients receive radiation therapy delivered as a fractionated course. Changing HPV status in HNSCC from a positive prognostic marker to a predictive one requires biomarkers that capture cellular radiation response to cumulative dose. Nuclear enlargement, γH2AX expression and micronuclei count, were studied in six HNSCC cell lines after 4 Gy fractionated X-irradiation. All HNSCC cell lines displayed altered cellular responses, indicating increasing inability to repair radiation damage with subsequent radiation fractions. Increases in nuclear area were significantly greater among HPV positive cell lines (207% and 67% for the HPV positive and HPV negative groups, respectively). A different character of DNA repair dysfunction in the HPV positive group suggests greater chromosomal translocation with accumulated radiation dose.

Abstract 1183: Targeting the DNA repair pathway with BOLD-100 in BRAF mutant colorectal cancer

Abstract Background: BRAF mutations (BRAFMT) occur in ∼10-15% of metastatic colorectal cancer (mCRC) and have a poor clinical outcome, in particular those with microsatellite stable (MSS) disease. Anti-BRAF/EGFR combinations have shown some increases in response rate, but this is associated with minor increases in overall survival. There is an unmet need to understand the biology of poor prognostic BRAFMT CRC. Using publicly available gene expression data from 155 CRC cell lines (GSE59857), we recently identified that unfolded protein response (UPR) and DNA repair are dominant pathways deregulated in the consensus molecular subgroup 1/BRAFMT subgroup. The aim of this study was to investigate the role of BOLD-100, an inhibitor of the UPR regulator GRP78, in regulating the survival of CRC cells. Methods: A panel of isogenic paired and non-isogenic V600E BRAFMT and BRAFWT cells were used. BOLD-100, a ruthenium-based small molecule inhibitor, was obtained from BOLD Therapeutics. Cell Titre Glo, Flow Cytometry, Western blotting, Caspase 8, 3/7 activity, RNAi assays were used. RNA seq and IPA bioinformatic analyses were performed on BOLD-100-treated BRAFMT/WT CRC cells. A compound library including small molecules approved by the FDA was used. Results: In vitro CellTitre-Glo® and Annexin V/PI sensitivity studies showed that the BRAFMT, MSS CRC cells were highly sensitive to BOLD-100 with IC50 values between 9.25-31μM. Treatment with BOLD-100 resulted in early decreases in GRP78 levels and increases in expression levels of the endoplasmic reticulum stress protein CHOP, and this was associated with caspase-8 dependent cell death in the BRAFMT CRC cells. Notably, silencing of CHOP did not abrogate BOLD-100-induced cell death in BRAFMT CRC cells, indicating that the UPR pathway played no role in the cell death following BOLD-100. RNA seq and IPA analysis showed that cell cycle regulation and DNA repair were the most significant deregulated pathways following BOLD-100 treatment. Further mechanistic studies revealed that BOLD-100 induced rapid and potent increases in pATRT1989, pChk1S345 and γH2AX expression levels in BRAFMT cells. Using a small molecule compound library, we found that the ATR inhibitors AZD6783 and M4344 resulted in strong synergy and apoptosis when combined with BOLD-100, in particular in BRAFMT CRC cells. Moreover, we found that the ROS scavenger NAC abrogated BOLD-100 induced CHOP, pATRT1989, pChk1S345 and γH2AX levels and rescued cell death following BOLD-100 treatment in BRAFMT CRC cells. Conclusions: Taken together, we have identified a role for BOLD-100 in regulating the survival of BRAFMT CRC cells. Our data support further studies with BOLD-100, in particular in combination with ATRi, for the treatment of BRAFMT CRC tumours. Citation Format: Robbie Carson, Shivaali Karelia, Deborah Lavin, Vijay Tiwari, Richard Kennedy, Kienan Savage, Adam Carie, Jim Pankovich, Mark Bazett, Sandra Van Schaeybroeck. Targeting the DNA repair pathway with BOLD-100 in BRAF mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1183.

Abstract 2029: The ER stress protein IRE1a regulates intracellular calcium, ROS, and the UV damage response through the IP3R inhibitor CIB1

Abstract UV induced DNA damage is the primary driver of human skin cancer. UV can also cause endoplasmic reticulum (ER) stress, but the role of unfolded protein response (UPR) in the cellular response to UV exposure is not clear. Inositol-requiring enzyme 1α (IRE1α) is one of the key regulators of the UPR but its function in the UV response has not been resolved. Here we show that IRE1α, is transiently dephosphorylated and its RNase activity downregulated by UVB resulting in reduced XBP1 splicing. Keratinocytes with either IRE1α knockout or knockdown have elevated cytosolic calcium and ROS resulting in increased apoptosis after UV. Pharmacological inhibition of the ER inositol triphosphate receptor (IP3R) or phospholipase C (PLC) restored normal cytosolic calcium and ROS levels to cells lacking IRE1α. While loss of IRE1α did not alter the frequency of cells with UV-induced cyclo-pyrimidine dimers (CPD), it significantly reduced the percent of γH2AX positive keratinocytes 6 hours after UVB irradiation. This percent of CPD positive cells and the level of γH2AX 6 hours after UV suggesting a defect in the DNA repair response. Pharmacological inhibition of IRE1α RNase or Xbp1 knockdown did not phenocopy IRE1α loss, but overexpression of IRE1α in IRE1α knockdown cells restored γH2AX expression and reduced keratinocyte apoptosis. IRE1α overexpression also enhanced the survival of keratinocytes deficient in p53 function after UV irradiation. Additionally, mice with an epidermal deletion of IRE1α showed more rapid loss of UV-induced CPD positive keratinocytes compared to wildtype mice, primarily through accelerated sloughing of UV damaged cells from the skin. Knockdown of Calcium and integrin binding protein 1 (CIB1), which binds and inhibits the IP3R, elevated cytosolic calcium in keratinocytes and suppressed UV-induced γH2AX. Knockdown of Apoptosis signal-regulating kinase 1 (ASK1), which can interact with IRE1α through TRAF2 as well as CIB1, restored normal levels of UV-induced γH2AX to IRE1α knockdown keratinocytes. Together these results show that IRE1α is essential for maintaining normal cellular calcium and ROS levels and the UV-induced repair response, and suggest a model where IRE1a activation state drives CIB1 binding either to the IP3R or ASK1 to regulate ER calcium efflux, ROS and DNA repair responses following UV. Citation Format: Jeongin Son, Saie Mogre, Adam Glick. The ER stress protein IRE1a regulates intracellular calcium, ROS, and the UV damage response through the IP3R inhibitor CIB1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2029.

Targeting WEE1 Kinase after G2/M Checkpoint Arrest Induces Apoptosis in Chronic Myeloid Leukemia Progenitors

BackgroundThe integrity of signaling pathways involved in cell cycle arrest, chromatin remodeling and DNA repair are critical for maintaining the fidelity of replicated DNA. Normal cells repair damaged DNA during G1 arrest; in contrast, leukemic cells often have a deficient G1/S checkpoint and depend on a functional G2/M checkpoint for DNA repair. Aurora Kinase A (AKA) is a key component of centrosome cycle and polar spindle assembly required for regulated progression from G2 to M and throughout M phase. It is frequently overexpressed in cancers, where it correlates with a poor prognosis. Notably, AKA overexpression is associated with genomic instability, one major trait of chronic myeloid leukemia (CML). Our hypothesis is that AKA, together with Polo like kinase 1 (Plk1), cooperates with the constitutive TK activity of the BCR-ABL1 fusion protein by increasing DNA damage, promoting the occurrence of additional genomic alterations and driving TKIs resistance and disease progression to blast crisis.AimsIn this study, the effects of AKA and Plk1 inhibition were evaluated in K562 cells sensitive (K562-S) and resistant (K562-R) to imatinib.MethodsProtein expression and activation was assessed by Western Blotting (WB). Danusertib and Volasertib were used to investigate the effects of AKA and Plk1 inhibition in K562-S and K562-R cells. Both cell lines were incubated with increasing concentrations of Danusertib and Volasertib (0.5-1 µM) or control medium for 24, 48, 72 and 96 hours to perform growth curve experiments and to identify the best sub-lethal drug doses in liquid medium. The percentage of apoptotic cells was quantified by annexin V/propidium iodide staining and flow cytometry. WB experiments were performed to evaluate AKA and Plk1 expression and activation before and after treatment with Danusertib and Volasertib alone or in combination with the Wee1 inhibitor MK1775. Further experiments were performed using polyclonal antibodies against cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 to confirm apoptosis activation. PARP cleavage and γH2AX expression and activation were also tested. The effects of Danusertib and Volasertib on cell cycle progression were evaluated by flow cytometry in K562-S and K562-R cells after incubation in medium containing 0.5 µM of each drugs for 24 h.ResultsBoth Danusertib and Volasertib (0.5 µM for 24h) were effective in cell growth inhibition in K562-S and K562-R by inducing cells to undergo cell cycle arrest and apoptosis. Moreover, they caused a dose- and time-dependent reduction of the G0/G1 cell fraction and an increase of the G2/M fraction. Cell cycle arrest was associated with increased levels of phospho (p)-Chk1 and p-Chk2, p-cyclin B1, p-cdc2 and p-Wee1. Using a Wee1 inhibitor (MK1775) after 24h treatment with Danusertib and Volasertib 0.5 µM, when cells were arrested in G2 phase and Wee1 was overexpressed and hyper-activated, resulted in a synergistic inhibition of cell viability in both K562-S and -R (Figure 1). MK1775 combined with either Danusertib or Volasertib caused a time-dependent increase of annexin-V-positive cells by activating the mitochondrial apoptotic pathway as reflected by an increment of Bax expression and induction of the cleavage of caspase-3, -8 and -9 and PARP. Moreover, both drug combinations induced a significant increase of the DNA double-strand break marker γH2AX, suggesting that Wee1 inhibition promotes mitosis and propagates genomic instability by forcing the cells through successive replication cycles, ultimately resulting in apoptosis from mitotic catastrophe.ConclusionsOur results provide support for MK1775 to be used in synergistic combination therapies to improve the outcomes of patients with CML in blast crisis or resistant to multiple lines of TKI therapy.Supported by ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. [Display omitted] DisclosuresCastagnetti:Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gugliotta:Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Rosti:Incyte: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Martinelli:Johnson&Johnson: Consultancy; Roche: Consultancy; Ariad/Incyte: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen: Consultancy. Soverini:Bristol-Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy.

Bone Marrow Stromal Cell Mediated Resistance to Mertk Inhibition in Acute Myeloid Leukemia

MERTK is a receptor tyrosine kinase of the TAM family (TYRO-3, AXL, MERTK) that is aberrantly expressed in 80-100% of pediatric and adult primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by small molecule tyrosine kinase inhibitors (TKIs), UNC2025 or MRX-2843, altered cell cycle progression and induced apoptosis in leukemia cell cultures. Moreover, treatment with these MERTK TKIs prolonged survival in mouse xenograft models of acute leukemia, but was not curative. In these models, treatment with the TKI effectively reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance.To evaluate the role of the bone marrow stromal niche in mediating resistance to MERTK inhibition by MRX-2843, AML cell lines were cultured in the presence of a fibroblast-like cell line (HS27) and induction of cell death and apoptosis was determined by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with the HS27 cell line significantly reduced cell death in Kasumi-1 AML cell cultures in response to treatment with 200nM MRX-2843 compared to leukemia cells alone (31.9% versus 61.2%, p<0.05). Similar results were observed in NOMO-1 and OCI-AML5 AML cell cultures. Additionally, cell cycle profiles were determined by flow cytometry in the presence or absence of stromal cells and MRX-2843. Treatment of Kasumi-1 cell cultures with 200nM MRX-2843 in the absence of stromal cells resulted in a significant accumulation of cells in G2 phase relative to vehicle-treated cultures (8.9% versus 14.8%, p<0.01) and was accompanied by an increase in γH2AX expression detected by immunoblot. In contrast, there was no change in cell cycle progression or γH2AX expression in cultures treated with MRX-2843 when co-cultured with the stromal cell line. NOMO-1 cells also accumulated in G2 phase in response to treatment with MRX-2843 and were protected from changes in cell cycle progression by co-culture with HS27 cells. Additionally, MERTK inhibition resulted in induction of polyploidy in NOMO-1 cell cultures, but this effect was abrogated in the presence of stromal cells. Collectively these results indicate abrogation of cell cycle progression in leukemia cells in response to treatment with MRX-2843 and demonstrate protection from this effect mediated by stromal cell interactions.The MERTK ligand, GAS6, mediates resistance to cytotoxic chemotherapy in leukemia cells and is a poor prognostic marker in AML. To evaluate the role of the MERTK ligands GAS6 and PROS1 in resistance to MERTK inhibition, their expression in stromal cells was determined by immunoblot. GAS6, but not PROS1, was expressed in HS27 cells; however, MRX-2843 retained its ability to inhibit MERTK phosphorylation in stromal cell co-cultures. We hypothesized that upregulation of the other TAM kinases, AXL and/or TYRO3, may mediate resistance as they share common ligands. To test this hypothesis, Kasumi-1 cells were treated with UNC4241, a small molecule pan-TAM inhibitor, in the presence of stromal cells. While co-culture protected Kasumi-1 cells from MRX-2843 induced apoptosis, treatment with UNC4241 induced apoptosis in the presence of stromal cells (32.7% versus 20.4% in vehicle control, p<0.01).To further evaluate the interactions between stromal cells and leukemia cells, Kasumi-1 cells were cultured in HS27-conditioned medium in the presence or absence of MRX-2843. Conditioned medium was not sufficient to provide protection from MRX-2843 induced apoptosis (74.0% vs. 69.8% in unconditioned medium, p>0.05). Similarly, addition of 500nM exogenous GAS6 or PROS1 did not protect leukemia cells from apoptosis in the presence of MRX-2843. Together, these results suggest that direct cell-cell contact between leukemia and stromal cells is necessary to provide protection.Together these data support a model whereby direct cell-cell contact with stromal cells in the bone marrow niche protects leukemia cells from MRX-2843 induced apoptosis and cell cycle alterations and suggest that bone marrow mobilizing agents may be effective to overcome resistance to MERTK inhibitors. DisclosuresWang:Meryx: Equity Ownership; University of North Carolina: Patents & Royalties. Frye:University of North Carolina: Patents & Royalties; Meryx: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Earp:Meryx: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. DeRyckere:Meryx: Equity Ownership. Graham:Meryx: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Inhibiting p21-activated kinase (PAK7) enhances radiosensitivity in hepatocellular carcinoma.

In light of the upregulation of p21-activated kinase (PAK7) in a variety of cancers, including hepatocellular carcinoma (HCC), we aimed to investigate the effect of PAK7 on the sensitivity of HCC cells to radiotherapy. PAK7 expression was determined in normal adult liver epithelial THLE-2 and human HCC cell lines. The effect of ionizing radiation (IR) on the HCC cell viability was evaluated by Sulforhodamine B (SRB) assay. HCC cell lines Mahlavu and Huh7 were chosen to assess the effect of PAK7 shRNAs on the viability, clone formation, apoptosis, cycle distribution and γ-H2AX expression after exposure to IR. As compared to THLE-2 cells, PAK7 was upregulated in poorly differentiated Mahlavu and SK-Hep-1 cells, but moderately or lowly expressed in well-differentiated Huh7 and HepG2 cells. HCC cells with moderate or low expression of PAK7 presented a decreased viability at 2 Gy IR, which had no significant effect on PAK7high HCC cells. Mahlavu and Huh7 cells transfected with PAK7 shRNAs showed increased inhibitory effect of IR on viability. In addition, PAK7 shRNAs reduced clone formation, facilitated the cell apoptosis, arrested cells at G2/M phase, and increased γ-H2AX expression. Moreover, changes above were more evident in the HCC cells co-treated with IR and PAK7 shRNAs. PAK7 downregulation could inhibit the viability, promote the apoptosis, arrest cells in G2/M phase, and induce the DNA damage in HCC cells, thereby enhancing the radiosensitivity in HCC.

1,2-Dichloropropane induces γ-H2AX expression in human cholangiocytes only in the presence of macrophages

Recent epidemiological studies reported cases of cholangiocarcinoma in workers exposed to 1,2-dichloropropane (1,2-DCP) in an offset proof printing factory in Japan. The present study investigated the effects of 1,2-DCP on the expression of histone family member X (H2AX) phosphorylated on Ser 139 (γ-H2AX), a marker of DNA double strand break, in human immortalized cholangiocytes MMNK-1 cells. Mono-cultures of MMNK-1 cells and co-cultures of MMNK-1 cells with THP-1 macrophages were exposed to 1,2-DCP at concentrations of 100 and 500 μM for 24 h. Expression of γ-H2AX was visualized by immunofluorescence staining. Exposure to 1,2-DCP had no effect on the expression of γ-H2AX in mono-cultured MMNK-1 cells, but significantly increased the number of nuclear foci stained by γ-H2AX in MMNK-1 cells co-cultured with THP-1 macrophages. Exposure to 1,2-DCP also significantly increased the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in co-cultured MMNK-1 cells. The results suggest that macrophages play a critical role by producing cytokines in 1,2-DCP-induced DNA double strand break in MMNK-1 cells.

A time-resolved experimental-mathematical model for predicting the response of glioma cells to single-dose radiation therapy.

To develop and validate a mechanism-based, mathematical model that characterizes 9L and C6 glioma cells' temporal response to single-dose radiation therapy in vitro by explicitly incorporating time-dependent biological interactions with radiation. We employed time-resolved microscopy to track the confluence of 9L and C6 glioma cells receiving radiation doses of 0, 2, 4, 6, 8, 10, 12, 14 or 16 Gy. DNA repair kinetics are measured by γH2AX expression via flow cytometry. The microscopy data (814 replicates for 9L, 540 replicates for C6 at various seeding densities receiving doses above) were divided into training (75%) and validation (25%) sets. A mechanistic model was developed, and model parameters were calibrated to the training data. The model was then used to predict the temporal dynamics of the validation set given the known initial confluences and doses. The predictions were compared to the corresponding dynamic microscopy data. For 9L, we obtained an average (± standard deviation, SD) Pearson correlation coefficient between the predicted and measured confluence of 0.87 ± 0.16, and an average (±SD) concordance correlation coefficient of 0.72 ± 0.28. For C6, we obtained an average (±SD) Pearson correlation coefficient of 0.90 ± 0.17, and an average (±SD) concordance correlation coefficient of 0.71 ± 0.24. The proposed model can effectively predict the temporal development of 9L and C6 glioma cells in response to a range of single-fraction radiation doses. By developing a mechanism-based, mathematical model that can be populated with time-resolved data, we provide an experimental-mathematical framework that allows for quantitative investigation of cells' temporal response to radiation. Our approach provides two key advances: (i) a time-resolved, dynamic death rate with a clear biological interpretation, and (ii) accurate predictions over a wide range of cell seeding densities and radiation doses.

The Combination of Trametinib, a MEK Inhibitor, and Temsirolimus, an mTOR Inhibitor, Radiosensitizes Lung Cancer Cells.

We evaluated the radiosensitizing effect of the combination treatment of trametinib, a MEK inhibitor, and temsirolimus, an mTOR inhibitor, on non-small-cell lung carcinoma (NSCLC) cells. The effects of combining trametinib and temsirolimus with radiation in NSCLC cell lines were evaluated using clonogenic survival and apoptosis assays. DNA double-strand breaks and cell cycle distribution were analyzed using flow cytometry. Tumor volume was measured to determine the radiosensitivity in lung cancer xenograft models. Exposure of lung cancer cells to a combination of trametinib and temsirolimus reduced clonogenic survival and promoted radiation-induced apoptosis. Combined inhibition of MEK and mTOR induced prolonged expression of γH2AX after irradiation and resulted in prolonged G2/M cell cycle arrest after irradiation in A549 cells. In vivo studies revealed that co-administration of the drugs sensitizes lung cancer xenografts to radiotherapy. The combination of trametinib and temsirolimus can enhance lung cancer radiosensitivity in vitro and in vivo.

A first-in-human phase I study of ATR inhibitor M1774 in patients with solid tumors.

TPS3153 Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase plays a critical role in the DNA damage response by sensing and responding to DNA replication stress, and by inducing cell cycle arrest to prevent aberrant replication and mitotic catastrophe. Based on extensive preclinical and limited clinical evidence, ATR inhibition is a promising treatment strategy as monotherapy for patients with advanced tumors harboring synthetically lethal conditions, such as alternative lengthening of telomeres (ALT) and inactivating mutations in ARID1A and ATM. M1774 is a potent, selective, orally administered ATR inhibitor that has been shown to exert antitumor activity in patient-derived xenograft tumors and acute myeloid leukemia xenograft tumors that express the ATR inhibition sensitizing mixed lineage leukemia fusion protein. This study (NCT04170153) aims to evaluate the safety and tolerability, maximum tolerated dose, recommended dose for expansion (RDE) and pharmacokinetics (PK) of M1774 (part A1), the effect of food on M1774 PK (part A2), and the efficacy of M1774 in patients with tumors harboring selected mutations (part A3). An additional objective is to assess the pharmacodynamics of M1774 by measuring relative changes in baseline p-CHK1 and γ-H2AX expression in paired tumor biopsies and serial blood samples. Methods: Patients aged ≥18 years, with an Eastern Cooperative Oncology Group performance status ≤1, adequate baseline hematological, renal and hepatic function, and with locally advanced or metastatic disease refractory to standard therapy are eligible. Patients with tumors bearing loss-of-function (LoF) mutations (determined by site testing or a central trial assay) in ARID1A, ATM, or ATRX and/or DAXX as ALT status surrogate markers; and measurable disease according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, will be enrolled in part A3. In the dose escalation phase (part A1 [open]), 18–24 patients are due to receive M1774 at a starting dose of 5 mg once daily. Dose escalation is determined by the safety monitoring committee and guided by a Bayesian 2-parameter logistic regression model. The preliminary food assessment (part A2) will follow a randomized two-sequence two-period crossover design in which ≤12 patients will be randomized (1:1) to receive a single dose of M1774 on Day –7 at the RDE (determined in part A1) in either a fed or fasted condition. After the food assessment, patients will subsequently receive M1774 according to the part A1 dosing schedule. In the preliminary efficacy study (part A3), patients (n = 20–24 for each of the three planned cohorts) with tumors harboring LoF mutations in the genes for ARID1A, ATM, ATRX and/or DAXX, will receive M1774 at the RDE. The primary efficacy endpoint is overall response (RECIST). The study is open and currently recruiting. Patients have been enrolled to seven cohorts in part A1 with no DLTs observed; dose escalation is ongoing. Clinical trial information: NCT04170153.

The functional significance of ATM phosphorylation of Bub3 on Serine 135 in sensitivity to DNA damaging agents.

e15048 Background: Identification of biomarkers to assess and modify the sensitivity of cancer cells to radiotherapy and chemotherapy is critical to improve cancer treatment outcome. Budding uninhibited by benzimidazoles 3 (Bub3) is a mitotic checkpoint protein, and it is frequently overexpressed in many cancers and associated with low survival rates. Bub3 is involved in the repair of DNA damage induced by radiotherapy and chemotherapy. We recently identified that the ATM kinase phosphorylated Bub3 on Serine 135 (S135) via the stable isotope labeled amino acid in cell culture -mass spectrometry analysis. This study aims to explore the mechanism of ATM-phosphorylated Bub3 in the DNA damage response (DDR) and its effect on tumor sensitivity to DNA damaging agents. Methods: The radiosensitivity of the cells was detected by clonal formation assay, the proliferation ability of the cells was detected by the MTS assay. The expression of DDR protein γ-H2AX in the nucleus was detected by immunofluorescence assay. Genomic instability was observed by multinuclear formation. Co-immunoprecipitation and Western Blot were used to explore the internal mechanism of ATM phosphorylation of Bub3 in DDR. Results: We showed that ionizing radiation (IR) could induce Bub3 S/TQ (the specific ATM consensus motif) phosphorylation in an ATM dependent manner. Mutation of Bub3 Serine 135 to alanine (S135A) led to a phosphorylation defect. Phenotypic experiments showed hypersensitivity to IR in cells expressing Bub3 S135A. Bub3 S135A prolonged existence of γ-H2AX foci and increased the proportion of cells containing micronuclei. Further, we found that Ku70 and Ku80 showed a significant increase after IR in their interactions with Bub3, while Bub3 S135A mutation significantly reduced the interaction, leading to impaired DNA repair. Conclusions: We demonstrate that Bub3 S135 phosphorylation mediated by ATM is essential for an optimal DDR and disruption of this pathway increases tumor sensitivity to DNA damaging agents.

LncRNA CCAT1 downregulation increases the radiosensitivity of non-small cell lung cancer cells.

This study aims to investigate if the radiosensitivity of non-small cell lung cancer (NSCLC) cells can be regulated by long noncoding RNA (lncRNA) colon cancer associated transcript1 (CCAT1). CCAT1 was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in NSCLC cells (A549, H1299, SK-MES1, H460, and H647) and human bronchial epithelial cells (16HBE). H460 and A549 cells were then selected for the determination of CCAT1 expression after exposure to radiation (0, 2, 4, 6Gy) at different time points (0, 6, 12, 24 h). Colony forming assay was performed to evaluate the effects of CCAT1 siRNA or pcDNA3.1-CCAT1 vector on the radiosensitivity of H460 and A549 cells. Then, flow cytometry, western blotting and qRT-PCR were also conducted. CCAT1 was increased in NSCLC cells when compared with 16HBE cells, which was declined in a time- and dosage-dependent manner after exposure to radiation. The H460 and A549 cell colonies were decreased and the γ-H2AX expression was elevated with the increase of radiation dosage, which was more obvious in those transfected with CCAT1 siRNA. CCAT1 downregulation arrested NSCLC cells at G2/M phase. Moreover, the enhanced apoptosis of radiotherapy-treated NSCLC cells with reductions of p-p38/p38, p-ERK/ERK, and p-JNK/JNK was promoted by siCCAT1, but it was reversed by pcDNA3.1-CCAT1 vector. Inhibiting CCAT1 regulated cell cycle, DNA damage and apoptosis of NSCLC cells, and affected MAPK pathway, eventually improving the radiosensitivity of NSCLC.

Centromere protein N may be a novel malignant prognostic biomarker for hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is one of the deadliest tumors. The majority of HCC is detected in the late stage, and the clinical results for HCC patients are poor. There is an urgent need to discover early diagnostic biomarkers and potential therapeutic targets for HCC.MethodsThe GSE87630 and GSE112790 datasets from the Gene Expression Omnibus (GEO) database were downloaded to analyze the differentially expressed genes (DEGs) between HCC and normal tissues. R packages were used for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses of the DEGs. A Search Tool for Retrieval of Interacting Genes (STRING) database was used to develop a protein-protein interaction (PPI) network, and also cytoHubba, Molecular Complex Detection (MCODE), EMBL-EBI, CCLE, Gene Expression Profiling Interactive Analysis (GEPIA), and Oncomine analyses were performed to identify hub genes. Gene expression was verified with a third GEO dataset, GSE25097. The Cancer Genome Atlas (TCGA) database was used to explore the correlations between the hub genes and clinical indexes of HCC patients. The functions of the hub genes were enriched by gene set enrichment analysis (GSEA), and the biological significance of the hub genes was explored by real-time polymerase chain reaction (qRT-PCR), western blot, immunofluorescence, CCK-8, colony formation, Transwell and flow cytometry assays with loss-of-function experiments in vitro.ResultsCentromere protein N (CENPN) was screened as a hub gene affecting HCC tumorigenesis. Evaluation by Cox regression showed that a high level of CENPN expression was an independent danger variable for poor prognosis of HCC. GSEA showed that high CENPN expression was linked to the following pathways: liver cancer subclass proliferation, cell cycle, p53 signaling pathway, Rb1 pathway, positive regulation of cell cycle G1/S phase transition, and DNA damage response signal transduction by p53 class moderators. Further cell experiments showed that knocking down CENPN expression decreased the proliferation and colony-forming abilities of HepG2 and Huh7 cells as well as Ki67 expression in these cell lines. The cell cycle was arrested in G1 phase, which is consistent with previous experiments on CENPN downregulation., but neither migration nor invasion were significantly affected. Western blot results revealed that the expression of p53, p27, p21, CDK4, cyclin D1, CDK2, cyclin E, pRb, E2F1 and c-myc decreased after CENPN knockdown, but there was no significant change in total Rb levels. In addition, CENPN-knockdown cells subjected to irradiation showed significantly enhanced of γ-H2AX expression and reduced colony formation.ConclusionCENPN functions as an oncogene in HCC and may be a therapeutic target and promising prognostic marker for HCC.

Synergistic Effect of Bazedoxifene and PARP Inhibitor in the Treatment of Ovarian Cancer Regardless of BRCA Mutation.

Poly (ADP-ribose) polymerase inhibitors (PARPis) are one of the targeted therapies proven to treat breast cancer gene (BRCA)-mutant ovarian cancer. Because most ovarian cancers are BRCA wild-type, it is necessary to extend the usage of PARPis. In the present study, we combined the PARPi, talazoparib, and the IL-6 inhibitor, bazedoxifene, for the treatment of human ovarian cancer cells. The human ovarian cancer cell lines, SKOV3, UWB1.289 (BRCA1-null) and OV75, were treated with talazoparib and bazedoxifene, as monotherapy or combination treatment. The effects of treatment on cell viability, migration, growth and colony formation were examined. Western blot was used to investigate pathways that may be involved in the antitumor effects of the two agents. The combination of talazoparib and bazedoxifene showed synergistic inhibition of cell viability, cell migration, cell growth, and cell colony formation on all the studied cell lines. The expression of p-AKT, c-myc, p-ERK, ERα was inhibited, and γ-H2AX expression was induced. Combined inhibition of PARP and IL-6 may be an efficacious treatment for ovarian cancer, independently of BRCA mutation status.

Adipose Derived Mesenchymal Stem Cells from a Hypoxic Culture Reduce Cartilage Damage.

The method to benifit tissue engineering of adipose-derived stem cells (ADSCs) to cartilage has been an objective of intense research in treating increasing cartilage-related disease. In this study, whether hypoxic expansion would enhance the proliferation and in vitro chondrogenic differentiation of ADSCs was studied, and then hypoxic expansion was applied to reduce cartilage damage in a rat model in vivo. Hypoxic expansion increased the proliferation and decreased the expression of aging-related genes, including p16, p21, and p53, of human ADSCs in comparison with normoxic expansion. In addition, the γH2AX expression was reduced in the hypoxic ADSCs. The chondrogenic markers were enhanced in the hypoxic ADSC differentiated chondrogenic pellets, including SOX9 on day 7 and gene expressions of COL 2 and COL 10 on day 21. To determine the in vitro chondrogenic differentiation potential of ADSCs, ADSC differentiated 21-day chondrogenic pellets were stained by Alcian blue staining and the immunostaining of COL 2 and COL 10, the results of which confirmed the enhancement of differentiation potential after the hypoxic expansion. Moreover, cartilage injury in a rat model was reduced by hypoxic ADSC treatment that was determined by histological and immunohistochemical staining detections. The effects of hypoxic expansion of ADSCs and bone marrow-derived stem cells (BMSCs) on chondrogenic differentiation potential were also compared. Smaller sizes were presented in the in vitro hypoxic BMSC differentiated chondrogenic pellets, whereas the chondrogenic marker expressions were significantly higher than those of the hypoxic ADSCs. However, there was no significant difference between the treatments of the hypoxic ADSCs and BMSCs in the cartilage injury in vivo. In conclusion, hypoxic expansion increases the chondrogenic differentiation potential of ADSCs and BMSCs in vitro and enhances them to reduce cartilage damage in vivo. Although the hypoxic BMSCs showed compact chondrogenic pellet formation and higher potential of chondrogenesis, the easy access and large resources of ADSCs still uplifted the application.