miR-21-mediated Radioresistance Occurs via Promoting Repair of DNA Double Strand Breaks.

miR-21, as an oncogene that overexpresses in most human tumors, is involved in radioresistance; however, the mechanism remains unclear. Here, we demonstrate that miR-21-mediated radioresistance occurs through promoting repair of DNA double strand breaks, which includes facilitating both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR). The miR-21-promoted NHEJ occurs through targeting GSK3B (a novel target of miR-21), which affects the CRY2/PP5 pathway and in turn increases DNA-PKcs activity. The miR-21-promoted HRR occurs through targeting both GSK3B and CDC25A (a known target of miR-21), which neutralizes the effects of targeting GSK3B-induced CDC25A increase because GSK3B promotes degradation of both CDC25A and cyclin D1, but CDC25A and cyclin D1 have an opposite effect on HRR. A negative correlation of expression levels between miR-21 and GSK3ß exists in a subset of human tumors. Our results not only elucidate miR-21-mediated radioresistance, but also provide potential new targets for improving radiotherapy.

Identification of a tumor-suppressive human-specific microRNA within the FHIT tumor-suppressor gene.

Loss or attenuated expression of the tumor-suppressor gene FHIT is associated paradoxically with poor progression of human tumors. Fhit promotes apoptosis and regulates reactive oxygen species; however, the mechanism by which Fhit inhibits tumor growth in animals remains unclear. In this study, we used a multidisciplinary approach based on bioinformatics, small RNA library screening, human tissue analysis, and a xenograft mouse model to identify a novel member of the miR-548 family in the fourth intron of the human FHIT gene. Characterization of this human-specific microRNA illustrates the importance of this class of microRNAs in tumor suppression and may influence interpretation of Fhit action in human cancer.

MicroRNA-376a sensitizes cells following DNA damage by downregulating MEPE expression.

MicroRNAs (miRNAs) are a class of endogenous molecules that post-transcriptionally regulate target gene expression and play an important role in many developmental processes. Matrix extracellular phosphoglycoprotein (MEPE) is related to bone metabolism. We recently reported that MEPE protects cells from DNA damage-induced killing. The purpose of this study is to investigate whether miRNAs targeting MEPE play an important role in DNA damage response. We report in this study that miR-376a directly targets MEPE, and overexpression of miR-376a reduces the G2 arrest of the cells and sensitizes the cells to DNA damage-induced killing. These results indicate an association of MEPE gene inactivation with decreased survival after DNA damage and also provide useful information for miRNA-based drug development: a new target for sensitizing human tumor cells to radiotherapy or chemotherapy.

microRNA-143 protects cells from DNA damage-induced killing by downregulating FHIT expression.

MicroRNAs (miRNAs) are posttranscriptional modulators of gene expression and play an important role in many developmental processes. Recent studies suggest roles of miRNAs in carcinogenesis. Fragile histidine triad (FHIT) gene deletion, methylation, and reduced Fhit protein expression occur in about 70% of human epithelial tumors and are clearly associated with tumor progression. Although it has been previously reported that Fhit(-/-)cells exhibit more resistance to multi-DNA damage inducers, including ionizing radiation, it remains unclear how miRNAs targeting FHIT in DNA damage response play the role. This study reports that miR-143 directly targets FHIT and that overexpression of miR-143 results in significant G2-phase arrest and protects cells from DNA damage-induced killing. These results indicate an association of FHIT gene inactivation with increased survival after DNA damage and also provide useful information for miRNA-based drug development in two directions: protect cells from DNA damage-induced killing and sensitize cells to radiation therapy.

A small peptide mimicking the key domain of MEPE/OF45 interacting with CHK1 protects human cells from radiation-induced killing.

Checkpoint activation benefits DNA homologous recombination repair and therefore protects cells from ionizing radiation (IR)-induced killing. CHK1 is one of the most important checkpoint regulators in mammalian cells. We recently reported that matrix extracellular phosphoglycoprotein/osteoblast factor 45 (MEPE/OF45) stabilizes CHK1 through interacting with CHK1, thus protecting cells from IR-induced killing. The purpose of this study is to investigate whether a small peptide that mimics the key domain of MEPE/OF45 could interact with CHK1 and protect cells from IR-induced killing. We showed here that the synthesized peptide with 18 amino acids (aa) could enter human transformed lymphoblasts when it is linked to fatty acid CH3(CH2)8CO. After the 18 aa peptide entered the human cells, it interacted with CHK1, increased the CHK1 level and induces stronger G2 arrest in the cells following IR. More importantly, the 18 aa peptide could protect the cells from IR-induced killing. Our data indicate that the 18 aa peptide, similar to MEPE/OF45, reduces CHK1 degradation and protects cells from IR-induced killing. We believe that these results provide useful information for drug development in two directions: protect cells from IR induced damage and sensitize cells to radiation therapy.

MEPE/OF45 protects cells from DNA damage induced killing via stabilizing CHK1.

Matrix extracellular phosphoglycoprotein/osteoblast factor 45 (MEPE/OF45) was cloned in 2000 with functions related to bone metabolism. We identified MEPE/OF45 for the first time as a new co-factor of CHK1 in mammalian cells to protect cells from DNA damage induced killing. We demonstrate here that MEPE/OF45 directly interacts with CHK1. Knocking down MEPE/OF45 decreases CHK1 levels and sensitizes the cells to DNA damage inducers such as ionizing radiation (IR) or camptothicin (CPT)-induced killing. Over-expressing wild-type MEPE/OF45, but not the mutant MEPE/OF45 (depleted the key domain to interact with CHK1) increases CHK1 levels in the cells and increases the resistance of the cells to IR or CPT. MEPE/OF45, interacting with CHK1, increases CHK1 half-life and decreases CHK1 degradation through the ubiquitine-mediated pathway. In addition, the interaction of MEPE/OF45 with CHK1 decreases CHK1 levels in the ubiquitin E3 ligases (Cul1 and Cul4A) complex, which suggests that MEPE/OF45 competes with the ubiquitin E3 ligases binding to CHK1 and thus decreases CHK1 from ubiquitin-mediated proteolysis. These findings reveal an important role of MEPE/OF45 in protecting cells from DNA damage induced killing through stabilizing CHK1, which would provide MEPE/OF45 as a new target for sensitizing tumor cells to radiotherapy or chemotherapy.

Low dose radiation-induced adaptive response preventing HPRT mutation is Fhit independent.

PURPOSE: To study whether fragile histidine triad (Fhit) prevents IR-induced hypoxanthineguanine phosphoribosyltransferase (HPRT) mutation and whether Fhit plays any role in preventing HPRT mutation through low dose-induced adaptive response. MATERIALS AND METHODS: Establishing human cell lines with or without Fhit expression by making constructs expressing hemagglutinin (HA) alone or HA-Fhit fusion protein and transfecting the vector to HeLa cells. The effects of Fhit on ionising radiation (IR)-induced mutation were examined by observing HPRT mutation rates in the established cell lines following different doses of IR. The role of Fhit on low dose IR-induced adaptive response were examined by observing HPRT mutation rates in the established cell lines that were exposed to 0.1 Gy and followed with high dose IR or ultraviolet (UV) exposure. RESULTS: Low dose (0.1 Gy) does not affect HPRT mutation rates in these cell lines. Fhit prevents high dose IR (> or = 2 Gy)-induced mutation as it prevents UV-induced mutation. However, low dose of IR (0.1 Gy)-induced adaptive response prevents both high doses of IR and UV-induced mutation in both the cells with and without Fhit expression. CONCLUSIONS: Fhit prevents IR-induced HPRT mutation and preventing mutation through low dose of IR-induced adaptive response is Fhit independent.

Fhit and CHK1 have opposing effects on homologous recombination repair.

Fragile histidine triad (FHIT) gene deletion or promoter methylation and reduced Fhit protein expression occur in approximately 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified. We previously reported that compared with Fhit+/+ cells, Fhit-/- cells with an overactivated ATR/CHK1 pathway show increased mutation frequency and resistance to DNA damage-induced killing, indicating that Fhit and the CHK1 pathway have opposing roles in cells responding to DNA damage. In this study, we show that cells, with or without Fhit expression, have similar DNA double-strand break induction levels and similar rejoining rates following ionizing radiation, indicating that the effect of Fhit on cell radiosensitivity is independent of nonhomologous end-joining. By combining I-SceI-induced-DNA double-strand break system and small interfering RNA approach, we also show that knocking down Fhit increases the efficiency of homologous recombination repair of cells, but knocking down Chk1 decreases the efficiency of homologous recombination repair, associated with the sensitivity to ionizing radiation-induced killing. Taken together, the results show that the role of Fhit in affecting the sensitivity of cells to ionizing radiation-induced killing is through the CHK1 pathway linked to homologous recombination repair. These results also illustrate the importance of balanced checkpoint activation in genomic stability and suggest a connection between the radioresistance and mutagenesis, carcinogenesis, as well as tumor progression in Fhit-deficient cells or tissue.

CHK1 affecting cell radiosensitivity is independent of non-homologous end joining.

CHK1 is one of the most important checkpoint proteins in mammalian cells for responding to DNA damage. Cells defective in CHK1 are sensitive to ionizing radiation (IR). The mechanism by which CHK1 protects cells from IR-induced killing remains unclear. DNA double strand breaks (DSBs) induced by IR are critical lesions for cell survival. Two major complementary DNA DSBs repair pathways exist in mammalian cells, homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). By using CHK1 kinase dead human cell lines established in our laboratory, we show here that although these human cell lines have different CHK1 activities with different sensitivities to IR-induced killing and G(2) accumulation, all these cell lines show similar inductions and rejoining rates of DNA DSBs. These results indicate that the different radiosensitivities and G(2) checkpoint responses in these cell lines are independent of NHEJ, suggesting that CHK1-regulated checkpoint facilitates HRR and therefore protects cells from IR-induced killing.

Involvement of the Fhit gene in the ionizing radiation-activated ATR/CHK1 pathway.

Fragile Histidine Triad (Fhit) gene deletion, methylation, and reduced Fhit protein expression occur in about 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified, although it has been shown that Fhit overexpression leads to apoptosis in many cancer cell lines. We report in this study that Fhit-/- cells derived from gene knockout mice show much stronger S and G2 checkpoint responses than their wild type counterparts. The strong checkpoint responses are regulated by the ATR/CHK1 pathway, which contributes to the radioresistance of Fhit-/- cells. These results indicate an association of Fhit gene inactivation with increased survival after DNA damage, which is related to the over-active checkpoints regulated by the ATR/CHK1 pathway. These results also suggest the potential effects of Fhit-dependent DNA damage response on tumor progression.

Involvement of Hus1 in the chain elongation step of DNA replication after exposure to camptothecin or ionizing radiation.

DNA damage-induced S phase (S) checkpoint includes inhibition of both replicon initiation and chain elongation. The precise mechanism for controlling the two processes remains unclear. In this study, we showed that Hus1-deficient mouse cells had an impaired S checkpoint after exposure to DNA strand break-inducing agents such as camptothecin (CPT) (>or=1.0 micro M), or ionizing radiation (IR) (>or=15 Gy). The Hus1-dependent S checkpoint contributes to cell resistance to CPT. This impaired S checkpoint induced by CPT or IR in Hus1-deficient cells reflected mainly the chain elongation step of DNA replication and was correlated with the reduction of dissociation of PCNA from DNA replication foci. Although Hus1 is required for Rad9 phosphorylation following exposure of cells to CPT or IR, Hus1-deficient cells showed normal activation of ATR/CHK1 and ATM kinases at doses where the checkpoint defects were manifested, suggesting that Hus1 is not a component of the sensor system for activating these pathways in S checkpoint induced by CPT or IR.

An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells.

Induction of checkpoint responses in G1, S, and G2 phases of the cell cycle after exposure of cells to ionizing radiation (IR) is essential for maintaining genomic integrity. Ataxia telangiectasia mutated (ATM) plays a key role in initiating this response in all three phases of the cell cycle. However, cells lacking functional ATM exhibit a prolonged G2 arrest after IR, suggesting regulation by an ATM-independent checkpoint response. The mechanism for this ataxia telangiectasia (AT)-independent G2-checkpoint response remains unknown. We report here that the G2 checkpoint in irradiated human AT cells derives from an overactivation of the ATR/CHK1 pathway. Chk1 small interfering RNA abolishes the IR-induced prolonged G2 checkpoint and radiosensitizes AT cells to killing. These results link the activation of ATR/CHK1 with the prolonged G2 arrest in AT cells and show that activation of this G2 checkpoint contributes to the survival of AT cells.

An ATM-independent S-phase checkpoint response involves CHK1 pathway.

After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.