Replication protein A (RPA) is the major single-stranded DNA (ssDNA) binding protein that is essential for DNA replication and processing of DNA double-strand breaks (DSBs) by homology-directed ...repair pathways. Recently, small molecule inhibitors have been developed targeting the RPA70 subunit and preventing RPA interactions with ssDNA and various DNA repair proteins. The rationale of this development is the potential utility of such compounds as cancer therapeutics, owing to their ability to inhibit DNA replication that sustains tumor growth. Among these compounds, (1Z)-1-(2-hydroxyanilino) methylidene naphthalen-2-one (HAMNO) has been more extensively studied and its efficacy against tumor growth was shown to arise from the associated DNA replication stress. Here, we study the effects of HAMNO on cells exposed to ionizing radiation (IR), focusing on the effects on the DNA damage response and the processing of DSBs and explore its potential as a radiosensitizer. We show that HAMNO by itself slows down the progression of cells through the cell cycle by dramatically decreasing DNA synthesis. Notably, HAMNO also attenuates the progression of G2-phase cells into mitosis by a mechanism that remains to be elucidated. Furthermore, HAMNO increases the fraction of chromatin-bound RPA in S-phase but not in G2-phase cells and suppresses DSB repair by homologous recombination. Despite these marked effects on the cell cycle and the DNA damage response, radiosensitization could neither be detected in exponentially growing cultures, nor in cultures enriched in G2-phase cells. Our results complement existing data on RPA inhibitors, specifically HAMNO, and suggest that their antitumor activity by replication stress induction may not extend to radiosensitization. However, it may render cells more vulnerable to other forms of DNA damaging agents through synthetically lethal interactions, which requires further investigation.
We have recently reported that in G2-phase cells (but not S-phase cells) sustaining low loads of DNA double-strand break (DSBs), ATM and ATR regulate the G2-checkpoint epistatically, with ATR at the ...output-node, interfacing with the cell cycle through Chk1. However, although inhibition of ATR nearly completely abrogated the checkpoint, inhibition of Chk1 using UCN-01 generated only partial responses. This suggested that additional kinases downstream of ATR were involved in the transmission of the signal to the cell cycle engine. Additionally, the broad spectrum of kinases inhibited by UCN-01 pointed to uncertainties in the interpretation that warranted further investigations. Here, we show that more specific Chk1 inhibitors exert an even weaker effect on G2-checkpoint, as compared to ATR inhibitors and UCN-01, and identify the MAPK p38α and its downstream target MK2 as checkpoint effectors operating as backup to Chk1. These observations further expand the spectrum of p38/MK2 signaling to G2-checkpoint activation, extend similar studies in cells exposed to other DNA damaging agents and consolidate a role of p38/MK2 as a backup kinase module, adding to similar backup functions exerted in p53 deficient cells. The results extend the spectrum of actionable strategies and targets in current efforts to enhance the radiosensitivity in tumor cells.
The aim of the present study was to investigate the efficacy of recombinant human endostatin (ES) (rh-ES) combined with radiation on rat cardiomyocyte apoptosis and the regulatory mechanism of ...transforming growth factor beta1 (TGF-β1)/Sma and Mad-related protein 3 (Smad3)/connective tissue growth factor (CTGF) signaling.
The primary cardiomyocytes were isolated from neonatal Sprague-Dawley rats for culture in vitro and divided into blank control group (without treatment), 10 Gy radiation + siTGF-β1 siRNA (gene silencing) group, ES + siTGF-β1 siRNA group, and 10 Gy radiation + ES + siTGF-β1 siRNA group. Methyl thiazolyl tetrazolium assay was used to calculate the half-maximal inhibitory concentration (IC
) of rh-ES on cardiomyocytes. Adenoviral vector was constructed for virus packaging to silence TGF-β1 expression in cardiomyocytes. Quantitative real-time polymerase chain reaction and Western blot were carried out to analyze TGF-β1, Smad2, Smad3 and CTGF expression at both gene and protein levels. Flow cytometry and electron microscope were used to examine cell apoptosis.
ES had a dose-dependent inhibitory effect on the proliferation of primary rat cardiomyocytes. ES combined with radiotherapy significantly inhibited cardiomyocyte proliferation and promoted cell apoptosis (P < 0.01). The gene and protein expression of TGF-β1, Smad2, Smad3 and CTGF were significantly up-regulated in primary cardiomyocytes transfected with TGF-β1 gene (P < 0.05).
The combination therapy with rh-ES and radiation can promote cardiomyocyte apoptosis and aggravate myocardial cell damage via TGF-β1/Smad3/CTGF signaling pathway.
We have recently reported that in G
-phase cells (but not S-phase cells) sustaining low loads of DNA double-strand break (DSBs), ATM and ATR regulate the G
-checkpoint epistatically, with ATR at the ...output-node, interfacing with the cell cycle through Chk1. However, although inhibition of ATR nearly completely abrogated the checkpoint, inhibition of Chk1 using UCN-01 generated only partial responses. This suggested that additional kinases downstream of ATR were involved in the transmission of the signal to the cell cycle engine. Additionally, the broad spectrum of kinases inhibited by UCN-01 pointed to uncertainties in the interpretation that warranted further investigations. Here, we show that more specific Chk1 inhibitors exert an even weaker effect on G
-checkpoint, as compared to ATR inhibitors and UCN-01, and identify the MAPK p38α and its downstream target MK2 as checkpoint effectors operating as backup to Chk1. These observations further expand the spectrum of p38/MK2 signaling to G
-checkpoint activation, extend similar studies in cells exposed to other DNA damaging agents and consolidate a role of p38/MK2 as a backup kinase module, adding to similar backup functions exerted in p53 deficient cells. The results extend the spectrum of actionable strategies and targets in current efforts to enhance the radiosensitivity in tumor cells.
Among the pleotropic roles of transforming growth factor-β (TGFβ) signaling in cancer, its impact on genomic stability is least understood. Inhibition of TGFβ signaling increases use of alternative ...end joining (alt-EJ), an error-prone DNA repair process that typically functions as a "backup" pathway if double-strand break repair by homologous recombination or nonhomologous end joining is compromised. However, the consequences of this functional relationship on therapeutic vulnerability in human cancer remain unknown. Here, we show that TGFβ broadly controls the DNA damage response and suppresses alt-EJ genes that are associated with genomic instability. Mechanistically based TGFβ and alt-EJ gene expression signatures were anticorrelated in glioblastoma, squamous cell lung cancer, and serous ovarian cancer. Consistent with error-prone repair, more of the genome was altered in tumors classified as low TGFβ and high alt-EJ, and the corresponding patients had better outcomes. Pan-cancer analysis of solid neoplasms revealed that alt-EJ genes were coordinately expressed and anticorrelated with TGFβ competency in 16 of 17 cancer types tested. Moreover, regardless of cancer type, tumors classified as low TGFβ and high alt-EJ were characterized by an insertion-deletion mutation signature containing short microhomologies and were more sensitive to genotoxic therapy. Collectively, experimental studies revealed that loss or inhibition of TGFβ signaling compromises the DNA damage response, resulting in ineffective repair by alt-EJ. Translation of this mechanistic relationship into gene expression signatures identified a robust anticorrelation that predicts response to genotoxic therapies, thereby expanding the potential therapeutic scope of TGFβ biology.
We have recently reported that in Gsub.2-phase cells (but not S-phase cells) sustaining low loads of DNA double-strand break (DSBs), ATM and ATR regulate the Gsub.2-checkpoint epistatically, with ATR ...at the output-node, interfacing with the cell cycle through Chk1. However, although inhibition of ATR nearly completely abrogated the checkpoint, inhibition of Chk1 using UCN-01 generated only partial responses. This suggested that additional kinases downstream of ATR were involved in the transmission of the signal to the cell cycle engine. Additionally, the broad spectrum of kinases inhibited by UCN-01 pointed to uncertainties in the interpretation that warranted further investigations. Here, we show that more specific Chk1 inhibitors exert an even weaker effect on Gsub.2-checkpoint, as compared to ATR inhibitors and UCN-01, and identify the MAPK p38α and its downstream target MK2 as checkpoint effectors operating as backup to Chk1. These observations further expand the spectrum of p38/MK2 signaling to Gsub.2-checkpoint activation, extend similar studies in cells exposed to other DNA damaging agents and consolidate a role of p38/MK2 as a backup kinase module, adding to similar backup functions exerted in p53 deficient cells. The results extend the spectrum of actionable strategies and targets in current efforts to enhance the radiosensitivity in tumor cells.
Purpose The aim of the present study was to investigate the efficacy of recombinant human endostatin (ES) (rh-ES) combined with radiation on rat cardiomyocyte apoptosis and the regulatory mechanism ...of transforming growth factor beta1 (TGF-beta1)/Sma and Mad-related protein 3 (Smad3)/connective tissue growth factor (CTGF) signaling. Method The primary cardiomyocytes were isolated from neonatal Sprague-Dawley rats for culture in vitro and divided into blank control group (without treatment), 10 Gy radiation + siTGF-beta1 siRNA (gene silencing) group, ES + siTGF-beta1 siRNA group, and 10 Gy radiation + ES + siTGF-beta1 siRNA group. Methyl thiazolyl tetrazolium assay was used to calculate the half-maximal inhibitory concentration (IC.sub.50) of rh-ES on cardiomyocytes. Adenoviral vector was constructed for virus packaging to silence TGF-beta1 expression in cardiomyocytes. Quantitative real-time polymerase chain reaction and Western blot were carried out to analyze TGF-beta1, Smad2, Smad3 and CTGF expression at both gene and protein levels. Flow cytometry and electron microscope were used to examine cell apoptosis. Results ES had a dose-dependent inhibitory effect on the proliferation of primary rat cardiomyocytes. ES combined with radiotherapy significantly inhibited cardiomyocyte proliferation and promoted cell apoptosis (P < 0.01). The gene and protein expression of TGF-beta1, Smad2, Smad3 and CTGF were significantly up-regulated in primary cardiomyocytes transfected with TGF-beta1 gene (P < 0.05). Conclusion The combination therapy with rh-ES and radiation can promote cardiomyocyte apoptosis and aggravate myocardial cell damage via TGF-beta1/Smad3/CTGF signaling pathway. Keywords: Recombinant human endostatin, Radiotherapy, Signaling pathway, Apoptosis, Lung cancer
Purpose: Gefitinib plays an important role in non‐small‐cell lung cancer (NSCLC) treatment; however, progression of the disease occurs in most patients even after an initial response. The role of ...erlotinib after gefitinib failure has been investigated but continues to be debated, especially in heavily treated patients with poor performance status (PS). Therefore, a retrospective matched‐pair case–control study was carried out to evaluate the role of erlotinib after gefitinib failure in advanced NSCLC patients.
Methods: A total of 58 patients were identified. The two groups were balanced with demographic and baseline clinical characteristics. All patients had PS ≥2 and most of them (89.7%) had received more than two systemic therapies before erlotinib or best supportive care (BSC). The epidermal growth factor receptor (EGFR) and KRAS genotypes were analyzed in 36 (62.1%) patients, 19 of them were in the erlotinib group.
Results: Median overall survival (OS) for all patients was 6 months. Median OS for patients who received erlotinib and BSC was 10 and 3 months, respectively (P= 0.001). Disease control rate (DCR) and objective response rate (ORR) were 51.7% and 10.3% in patients receiving erlotinib, respectively, while median time to progression (TTP) was 3 months. Among the 19 patients in the erlotinib group with biomarker results available, those with EGFR mutation achieved longer median TTP (P= 0.016) and better DCR (P= 0.177) than those with wild‐type EGFR.
Conclusions: A switch to erlotinib after gefitinib failure may represent a better therapeutic option for advanced NSCLC patients with poor PS, and an EGFR mutation seemed to be associated with better survival rate.
Hydroacoustics is a non-invasive fish stock assessment sampling technique that plays an important role in fishery science and management. However, non-standard hydroacoustic surveys could lead to ...biased results, and the factor of the sampling period (e.g., season and diel cycle) is extremely critical as it can greatly affect hydroacoustic results. Efforts to improve the accuracy and credibility of the hydroacoustic survey results are getting more and more attention. Thus, we conducted two diel hydroacoustic surveys
in situ
in summer and winter to detect whether there were diel and seasonal differences in density, Target Strength (
T
S
) and vertical distribution of fish. The results indicated that nighttime had significantly higher fish mean density than daytime in summer and winter. No significant difference between summer and winter daytime, however, significant difference between summer and winter nighttime, but this bias could be accepted from the fisheries management perspective; The mean
T
S
of the summer daytime was significantly higher than that of summer nighttime, winter daytime and winter nighttime, but there were no significant differences among summer nighttime, winter daytime, and winter nighttime, mainly due to mean
T
S
may be overestimated from fish schooling behavior during summer daytime; The fish vertical distribution had significant seasonal correlations and was more dispersed in different water layers during the nighttime, proving that the assessment was better at nighttime than during the daytime. Consequently, the hydroacoustic surveys in Yudong Reservoir and other similar plateau deep water reservoirs should be performed at nighttime, which will yield relatively accurate density and
T
S
, and dispersed vertical distribution of fish.
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