An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.

PIG3 downregulation enhances the radiosensitivity of NSCLC cells by promoting G_(2)/M cell cycle arrest and apoptosis

Objective:To investigate the mechanism of p53-induced gene 3(PIG3)-regulation of radioresistance in human non-small cell lung cancer(NSCLC)cells,in order to explore new biomarkers and therapeutic targets to combat radioresistance and improve the 5-year survival rate.Methods:The PIG3 gene was knocked down in A549 cells using siRNA,and was overexpressed in H1299 cells using a PIG3 expression plasmid.After confirming PIG3 knockdown and overexpression through the Western blot analysis,the radiosensitivity,DNA damage,cell cycle distribution,and apoptosis in these cells were analyzed using colony formation assay,immunofluorescence staining forγH2AX,and flow cytometry,respectively.Results:PIG3 silencing markedly increased the radiosensitivity of NSCLC cells,with radiosensitization ratios of 1.12 and 1.25.Compared with the corresponding negative control,PIG3 knockdown significantly enhanced G2/M phase arrest(siNC:26.12±2.50,siPIG3#1:34.98±4.19,siPIG3#2:37.79±3.53,P<0.05),promoted radiation-induced apoptosis(siNC:14.61±1.85,siPIG3#1:17.26±1.14,siPIG3#2:20.70±2.04,P<0.05),and reduced the number ofγ-H2AX foci 0.5,1,and 2 h after radiation(P<0.05).Conversely,PIG3 overexpression markedly decreased the radiosensitivity of NSCLC cells,as evidenced by the reduction of G2/M phase arrest(NC:33.18±2.11 vs.PIG3:24.21±3.09,P<0.05)and apoptosis(NC:15.49±0.56 vs.PIG3:12.79±0.29,P<0.05),and increased DNA damage(P<0.05).Conclusions:PIG3 downregulation increases the radiosensitivity of NSCLC cells,and PIG3-upregulation leads to the progression in radioresistance.Therefore,PIG3 is a potential target for radiotherapy for NSCLC.

Risk and mechanism of metabolic syndrome associated with radiation exposure

Obesity, hyperglycemia, hypertension, hypertriglyceridemia and low high-density lipoprotein cholesterol are the typical features of Metabolic syndrome (MetS). Exploring the risk factors would benefit for prevention control. Several studies have revealed an association between ionizing radiation (IR) exposure and MetS, likely attributable to production of reactive oxygen species (ROS), oxidative stress, DNA damage. Understanding the health effects of IR exposure, which have long been overlooked, would improve knowledge on MetS and help identify effective strategies for targeted prevention of MetS. In this review, we first highlight the importance of IR and MetS, providing information on the wide use of IR in the field, IR-induced damage, and the prevalence and burden of MetS. Then, we summarize the findings association between IR and various components of MetS addressing the dual effects of IR on MetS in a dose-response manner. Although there remain unresolved challenges, study on the association of radiation and MetS could open new perspectives in the future.

DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer

Radiotherapy is one of the most common countermeasures for treating a wide range of tumors.However,the radioresistance of cancer cells is still a major limitation for radiotherapy applications.Efforts are continuously ongoing to explore sensitizing targets and develop radiosensitizers for improving the outcomes of radiotherapy.DNA double-strand breaks are the most lethal lesions induced by ionizing radiation and can trigger a series of cellular DNA damage responses(DDRs),including those helping cells recover from radiation injuries,such as the activation of DNA damage sensing and early transduction pathways,cell cycle arrest,and DNA repair.Obviously,these protective DDRs confer tumor radioresistance.Targeting DDR signaling pathways has become an attractive strategy for overcoming tumor radioresistance,and some important advances and breakthroughs have already been achieved in recent years.On the basis of comprehensively reviewing the DDR signal pathways,we provide an update on the novel and promising druggable targets emerging from DDR pathways that can be exploited for radiosensitization.We further discuss recent advances identified from preclinical studies,current clinical trials,and clinical application of chemical inhibitors targeting key DDR proteins,including DNA-PKcs(DNA-dependent protein kinase,catalytic subunit),ATM/ATR(ataxia–telangiectasia mutated and Rad3-related),the MRN(MRE11-RAD50-NBS1)complex,the PARP(poly[ADP-ribose]polymerase)family,MDC1,Wee1,LIG4(ligase IV),CDK1,BRCA1(BRCA1 C terminal),CHK1,and HIF-1(hypoxia-inducible factor-1).Challenges for ionizing radiation-induced signal transduction and targeted therapy are also discussed based on recent achievements in the biological field of radiotherapy.

DNA damage repair:historical perspectives,mechanistic pathways and clinical translation for targeted cancer therapy

Genomic instability is the hallmark of various cancers with the increasing accumulation of DNA damage.The application of radiotherapy and chemotherapy in cancer treatment is typically based on this property of cancers.However,the adverse effects including normal tissues injury are also accompanied by the radiotherapy and chemotherapy.Targeted cancer therapy has the potential to suppress cancer cells'DNA damage response through tailoring therapy to cancer patients lacking specific DNA damage response functions.Obviously,understanding the broader role of DNA damage repair in cancers has became a basic and attractive strategy for targeted cancer therapy,in particular,raising novel hypothesis or theory in this field on the basis of previous scientists'findings would be important for future promising druggable emerging targets.In this review,we first illustrate the timeline steps for the understanding the roles of DNA damage repair in the promotion of cancer and cancer therapy developed,then we summarize the mechanisms regarding DNA damage repair associated with targeted cancer therapy,highlighting the specific proteins behind targeting DNA damage repair that initiate functioning abnormally duo to extrinsic harm by environmental DNA damage factors,also,the DNA damage baseline drift leads to the harmful intrinsic targeted cancer therapy.In addition,clinical therapeutic drugs for DNA damage and repair including therapeutic effects,as well as the strategy and scheme of relative clinical trials were intensive discussed.Based on this background,we suggest two hypotheses,namely"environmental gear selection”to describe DNA damage repair pathway evolution,and"DNA damage baseline drift”,which may play a magnified role in mediating repair during cancer treatment.This two new hypothesis would shed new light on targeted cancer therapy,provide a much better or more comprehensive holistic view and also promote the development of new research direction and new overcoming strategies for patients.

HIF-1 signaling: A key orchestrator of cancer radioresistance

Hypoxia is a big roadblock for cancer radiotherapy,in which the hypoxia-inducible factor(HIF-1)creates a microenvironment and cancer cells’intrinsic signaling networks conferring radioresistance to cancers.HIF-1 is a heterodimeric transcription factor HIF-1α/HIF-1β,that regulates the transcription of a broad range of downstream genes possessing an E-box-like hypoxia response element(HRE).The expression of HIF-1αis oxygensensitive while HIF-1βis constitutively expressed.In addition to hypoxia,ionizing radiation can also induce the expression of HIF-1α.The HIF-1 modulates a set of signaling pathways to cause profound effects on the response of cancer to radiotherapy,including radiation-induced DNA damage response(DDR),vasculogenesis and glucose metabolism reprograming,epithelial mesenchymal transition(EMT),etc.In this review,our aim is to summarize the current knowledge about the role and the related signal pathways of HIF-1 in association with the resistance of cancers to radiotherapy.Targeting HIF-1 and its signal pathways is a promising strategy for sensitization of cancers to radiotherapy.

Radiation-induced non-targeted effect of immunity provoked by mitochondrial DNA damage triggered cGAS/ AIM2 pathways

Non-targeted effect is an important complement to the classical target theory of radiation biology which takesnuclear genomic DNA as the core target. The principle of radiation target theory is to assume that an organism orcell has one or more sensitive points or targets, hit and inactivation of which directly by radiation leads toconsiderable damage and the death event. Recent findings indicate that not only cell nucleus but also othercellular parts can be considered as possible targets. Mitochondrion is considered as a critical organelle where thenon-targeted effect is initiated. A series of recent studies have provided substantial evidence and solid data whichprofoundly facilitate the understanding of radiation-induced non-targeted effects emitted from mitochondrion inthe irradiated cells, such the major apparent performances, signaling pathways and biological significance.Mitochondrial genome is more sensitive to genotoxic than nuclear genome. Ionizing radiation can induce mtDNAsdouble-strand breaks directly or indirectly via increased mitochondrial ROS. Under stress conditions, mitochondrial DNA (mtDNA) fragments are released into the cytoplasm. The cytosol mtDNAs are sensed by cGAS andAIM2 proteins and they activate the corresponding signaling pathways, generating relevant inflammatory andimmune responses. These newly developed mitochondrial DNA-initiating pathways may boost the development oftargeted therapies for preventing normal tissue toxicity as well as radio-immunotherapy, an emerging trend forcancer therapies. Here we focus and discuss the mechanisms and biological significance of mtDNA-triggeringcGAS/AIM2 signaling pathways of immune response from the aspect of non-targeted effect of radiation.

Genome-wide identification of DNA-PKcs-associated RNAs by RIP-Seq

Dear Editor,The DNA-dependent protein kinase catalytic subunit(DNA-PKcs)forms a serine/threonine protein kinase complex with the Ku heterodimer(Ku70/Ku80)and plays an important role in the DNA damage response(DDR)and maintenance of genomic stability through nonhomologous end joining(NHEJ),wherein the Ku heterodimer recognizes and binds broken DNA ends,facilitating the recruitment and activation of DNA-PKcs.1 Activated DNA-PKcs phosphorylates and alters the function of factors that mediate NHEJ,including DNA-PKcs itself.2 In addition,DDR-independent roles of DNA-PKcs have been demonstrated.3 Studies further identified DNA-PKcs as a modulator of cancer-associated pathways distinct from DNA repair,including hypoxia,metabolism,the inflammatory response,and transcriptional regulation.