ZFP36 loss-mediated BARX1 stabilization promotes malignant phenotypes by transactivating master oncogenes in NSCLC.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, with high morbidity and mortality worldwide. Although the dysregulation of BARX1 expression has been shown to be associated with malignant cancers, including NSCLC, the underlying mechanism remains elusive. In this study, we identified BARX1 as a common differentially expressed gene in lung squamous cell carcinoma and adenocarcinoma. Importantly, we uncovered a novel mechanism behind the regulation of BARX1, in which ZFP36 interacted with 3'UTR of BARX1 mRNA to mediate its destabilization. Loss of ZFP36 led to the upregulation of BARX1, which further promoted the proliferation, migration and invasion of NSCLC cells. In addition, the knockdown of BARX1 inhibited tumorigenicity in mouse xenograft. We demonstrated that BARX1 promoted the malignant phenotypes by transactivating a set of master oncogenes involved in the cell cycle, DNA synthesis and metastasis. Overall, our study provides insights into the mechanism of BARX1 actions in NSCLC and aids a better understanding of NSCLC pathogenesis.

The m6A methyltransferase METTL3 modifies PGC-1α mRNA promoting mitochondrial dysfunction and oxLDL-induced inflammation in monocytes.

Mitochondrial biogenesis and energy metabolism are essential for regulating the inflammatory state of monocytes. This state is partially controlled by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a coactivator that regulates mitochondrial biogenesis and energy metabolism. Disruption of these processes can also contribute to the initiation of chronic inflammatory diseases, such as pulmonary fibrosis, atherosclerosis, and rheumatoid arthritis. Methyltransferase-like 3 (METTL3)-dependent N6-methyladenosine (m6A) methylation has recently been shown to regulate a variety of inflammatory processes. However, the role of m6A mRNA methylation in affecting mitochondrial metabolism in monocytes under inflammation is unclear, nor is there an established relationship between m6A methylation and PGC-1α. In this study, we identified a novel mechanism by which METTL3 acts during oxidized low-density lipoprotein (oxLDL)-induced monocyte inflammation, where METTL3 and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) cooperatively modify PGC-1α mRNA, mediating its degradation, decreasing PGC-1α protein levels, and thereby enhancing the inflammatory response. METTL3 coordinated with YTHDF2 to suppress the expression of PGC-1α, as well as that of cytochrome c (CYCS) and NADH:ubiquinone oxidoreductase subunit C2 (NDUFC2) and reduced ATP production and oxygen consumption rate (OCR). This subsequently increased the accumulation of cellular and mitochondrial reactive oxygen species (ROS) and the levels of proinflammatory cytokines in inflammatory monocytes. These data may provide new insights into the role of METTL3-dependent m6A modification of PGC-1α mRNA in the monocyte inflammation response. These data also contribute to a more comprehensive understanding of the pathogenesis of monocyte-macrophage inflammation-associated diseases, such as pulmonary fibrosis, atherosclerosis, and rheumatoid arthritis.

A Systematic Analysis of Dysregulated Long Non-Coding RNAs/microRNAs/mRNAs in Lung Squamous Cell Carcinoma.

BACKGROUND: Lung squamous cell carcinoma (LUSC) accounts up for approximately 30% of all lung cancers with a high mortality. The study was aimed at finding genes critical in the diagnosis and prognosis of LUSC. MATERIALS AND METHODS: The differentially expressed (DE) genes (DEGs) and DE lncRNAs (DELs) from 501 LUSC and 49 normal lung tissues, and DE miRNAs (DEMs) from 478 LUSC and 45 normal lung tissues were respectively obtained via the TCGA database. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and co-expression network analyses were performed. Survival analysis and receiver operating characteristic curve of hub mRNAs were also analyzed. Competitive endogenous RNA networks of lncRNAs, miRNAs and mRNAs were constructed. RESULTS: A total of 5747 DEGs, 378 DEMs and 3141 DELs in LUSC were identified in LUSC. The DEGs including AUARK, CDK1, KIF11 and EXO1 were proven to be significant metastatic indicators in LUSC, and 2 DEGs were significantly associated with the survival in LUSC patients. Some genes might have connections with many other gene nodes through a co-expression network. Four lncRNAs, 2 mRNAs and 2 miRNAs were identified as the candidates for the competitive miRNA-mRNA-lncRNA network and might serve as prognostic markers in LUSC. CONCLUSIONS: We identified the differentially expressed lncRNAs, miRNAs and mRNAs in LUSC, providing further insights into the molecular mechanism of LUSC tumorigenesis and the potential prognostic biomarkers or therapeutic targets for LUSC.

MiR-509-3-5p-NONHSAT112228.2 Axis Regulates p21 and Suppresses Proliferation and Migration of Lung Cancer Cells.

BACKGROUND: Although the involvement of individual microRNA and lncRNA in the regulation of p21 expression has largely been evidenced, less is known about the roles of functional interactions between miRNAs and lncRNAs in p21 expression. Our previous work demonstrated that miR-509- 3-5p could block cancer cell growth. METHODS: To gain an insight into the role of miR-509-3-5p in the regulation of p21 expression, we performed in silico prediction and showed that miR-509-3-5p might target the NONHSAT112228.2, a sense-overlapping lncRNA transcribed by a non-code gene overlapping with p21 gene. Mutation and luciferase report analysis suggested that miR-509-3-5p could target NONHSAT112228.2, thereby blocking its expression. Consistently, NONHSAT112228.2 expression was inversely correlated with both miR-509-3-5p and p21 expression in cancer cells. Ectopic expression of miR-509-3-5p and knockdown of NONHSAT112228.2 both promoted proliferation and migration of cancer cells. RESULTS: Interestingly, high-expression of NONHSAT112228.2 accompanied by low-expression of p21 was observed in lung cancer tissues and associated with lower overall survival. CONCLUSION: Taken together, our study found a new regulatory pathway of p21, in which MiR-509-3-5p functionally interacts with NONHSAT112228.2 to release p21 expression. MiR-509-3-5p- NONHSAT112228.2 regulatory axis can inhibit the proliferation and migration of lung cancer cells.

DNA Damage-Response Pathway Heterogeneity of Human Lung Cancer A549 and H1299 Cells Determines Sensitivity to 8-Chloro-Adenosine.

Human lung cancer H1299 (p53-null) cells often display enhanced susceptibility to chemotherapeutics comparing to A549 (p53-wt) cells. However, little is known regarding to the association of DNA damage-response (DDR) pathway heterogeneity with drug sensitivity in these two cells. We investigated the DDR pathway differences between A549 and H1299 cells exposed to 8-chloro-adenosine (8-Cl-Ado), a potential anticancer drug that can induce DNA double-strand breaks (DSBs), and found that the hypersensitivity of H1299 cells to 8-Cl-Ado is associated with its DSB overaccumulation. The major causes of excessive DSBs in H1299 cells are as follows: First, defect of p53-p21 signal and phosphorylation of SMC1 increase S phase cells, where replication of DNA containing single-strand DNA break (SSB) produces more DSBs in H1299 cells. Second, p53 defect and no available induction of DNA repair protein p53R2 impair DNA repair activity in H1299 cells more severely than A549 cells. Third, cleavage of PARP-1 inhibits topoisomerase I and/or topoisomerase I-like activity of PARP-1, aggravates DNA DSBs and DNA repair mechanism impairment in H1299 cells. Together, DDR pathway heterogeneity of cancer cells is linked to cancer susceptibility to DNA damage-based chemotherapeutics, which may provide aid in design of chemotherapy strategy to improve treatment outcomes.

MiR-509-3-5p causes aberrant mitosis and anti-proliferative effect by suppression of PLK1 in human lung cancer A549 cells.

MicroRNAs (miRNAs) are potent post-transcriptional regulators of gene expression and play roles in DNA damage response (DDR). PLK1 is identified as a modulator of DNA damage checkpoint. Although down-regulation of PLK1 by certain microRNAs has been reported, little is known about the interplay between PLK1 and miR-509-3-5p in DDR. Here we have demonstrated that miR-509-3-5p repressed PLK1 expression by targeting PLK1 3'-UTR, thereby causing mitotic aberration and growth arrest of human lung cancer A549 cells. Repression of PLK1 by miR-509-3-5p was further evidenced by over-expression of miR-509-3-5p in A549, HepG2 and HCT116p53(-/-) cancer cells, in which PLK1 protein was suppressed. Consistently, miR-509-3-5p was stimulated, while PLK1 protein was down-regulated in A549 cells exposed to CIS and ADR, suggesting that suppression of PLK1 by miR-509-3-5p is a component of CIS/ADR-induced DDR pathway. Flow cytometry and immunofluorescence labeling showed that over-expression of miR-509-3-5p in A549 induced G2/M arrest and aberrant mitosis characterized by abnormal bipolar mitotic spindles, condensed chromosomes, lagging DNA and chromosome bridges. In addition, over-expression of miR-509-3-5p markedly blocked A549 cell proliferation and sensitized the cells to CIS and ADR treatment. Taken together, miR-509-3-5p is a feasible suppressor for cancer by targeting PLK1. Our data may provide aid in potential design of combined chemotherapy and in our better understanding of the roles of microRNAs in response to DNA damage.

E2F1 regulates p53R2 gene expression in p53-deficient cells.

The p53R2 gene encoding a small subunit of the ribonucleotide reductase has been identified as a p53-inducible gene. Although this gene is discovered as a target for p53 family proteins, the mechanism underlying p53R2 induction by DNA damage in p53-defiencient cells remains to be elucidated. In this study, we demonstrate that transcription factor E2F1 regulates the p53R2 gene expression in p53-deficient cells. We found that p53R2 was a target for E2F1 in DNA damage response (DDR), because ectopic expression of E2F1 in HCT116-p53(-/-) cells resulted in the increase of p53R2 mRNA and protein expression, and silencing E2F1 diminished its basic expression. Combination of luciferase reporter assay with overexpression or knockdown of E2F1 revealed that E2F1 directly activates the p53R2 gene. Chromatin immunoprecipitation (ChIP) assay showed E2F1 directly bound to the site (TTTGGCGG) at position -684 to -677 of the promoter under E2F1 overexpression or adriamycin (ADR) exposure. Moreover, silencing p53R2 could enhance apoptotic cell death in both HCT116-p53(-/-) and HCT116-p53(+/+) compared to ADR exposure, indicating that p53R2 may protect cancer cell from ADR-induced apoptosis. Together, we have identified a new role of E2F1 in the regulation of p53R2 expression in DDR, and silencing p53R2 may sensitize cancer cells to ADR-induced apoptosis. Our data support the notion that p53R2 is a potential target for cancer therapy. The involvement of E2F1-dependent p53R2 activation in DDR will provide further insight into the induction of p53R2 in p53-deficient cells. These data also give us a deeper understanding of E2F1 role in DDR.

E2F1-regulated DROSHA promotes miR-630 biosynthesis in cisplatin-exposed cancer cells.

DNA damage may regulate microRNA (miRNA) biosynthesis at the levels of miRNA transcription, processing and maturation. Although involvement of E2F1 in the regulation of miRNA gene activation in response to DNA damage has been documented, little is known about the role of E2F1 in miRNA processing. In this study we demonstrate that E2F1 enhances miR-630 biosynthesis under cisplatin (CIS) exposure through promoting DROSHA-mediated pri-miR-630 processing. Northern blot and RT-qPCR revealed that CIS exposure caused not only an increase in pri-miR-630 but also much more increase in pre-miR-630 and mature miR-630. The increases in pri-miR-630 and pre-miR-630 expression in unmatched proportion indicated that primary transcript processing was involved in CIS-stimulated miR-630 biosynthesis. Furthermore, combination of reporter enzyme assay with mutation and over-expression of E2F1 showed that induction of DROSHA promoted miR-630 expression, in which CIS-induced E2F1 activated DROSHA gene expression by recognizing and binding two E2F1 sites at the positions -214/-207 and -167/-160 of the DROSHA promoter. The increased binding of E2F1 to the DROSHA promoter in CIS-exposed cells was further evidenced by chromatin immunoprecipitation assay. Together, E2F1-regulated DROSHA promotes pri-miR-630 processing, thereby, contributes to CIS-stimulated miR-630 expression. The involvement of E2F1-dependent DROSHA activation in pri-miRNA processing under DNA damage stress will provide further insight into the regulation of miRNA biosynthesis. These data also give us a deeper understanding of E2F1 role in response to DNA damage.

ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage.

The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein-protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage.

p53 Suppresses E2F1-dependent PLK1 expression upon DNA damage by forming p53-E2F1-DNA complex.

E2F1 is implicated in transcriptional activation of polo-like kinase-1 (PLK1), but yet the mechanism is not fully understood. PLK1 suppression plays an important checkpoint role in response to DNA damage. Suppression of the PLK1 gene by binding of p53 to upstream p53RE2 element in the promoter has been recently revealed. Here we report another mechanism, in which p53 interacts with E2F1 to form p53-E2F1-DNA complex repressing E2F1-dependent PLK1 expression. PLK1 was downregulated in cisplatin exposed HCT116p53(+/+) but not HCT116p53(-/-) cells, indicating p53-suppressed PLK1 upon DNA damage. Co-transfection and reporter enzyme assays revealed that p53 suppressed but E2F1 promoted PLK1 gene activation. 5'-Deletion and substitution mutations showed multiple positive cis-elements residing in the PLK1 promoter, of which at least two E2F1 sites at positions -75/-68 and -40/-32 were required for the full activity of the promoter. Combination of 5'-deletion and substitution mutations with over-expression of p53 showed that suppression of the PLK1 gene by p53 was E2F1-dependent: mutation of the E2F1 site at position -75/-68 partially abrogated suppression activity of p53; mutation of E2F1 site at position -40/-32 released from p53 suppression of PLK1 gene completely. Co-immunoprecipitation and electrophoretic mobility shift assay showed that DNA damage promoted p53-E2F1 interaction, thereby creating a p53-E2F1 complex assembly on the PLK1 promoter in vitro. The in vivo formation of p53-E2F1-PLK1 promoter complex upon DNA damage was further evidenced by chromatin immunoprecipitation (ChIP) and re-ChIP. In addition, we showed that suppression of PLK1 by p53 promoted apoptosis. Our data suggest that p53 may interact with E2F1 to form p53-E2F1-DNA complex suppressing E2F1-dependent PLK1 expression. The model of p53 action on E2F1-activated PLK1 gene may explain at least partly how p53 as a suppressor regulates the downstream effects of E2F1 in cellular stresses including DNA damage stress.

E2F1-mediated DNA damage is implicated in 8-Cl-adenosine-induced chromosome missegregation and apoptosis in human lung cancer H1299 cells.

Although E2F1-mediated DNA double-stranded breaks (DSBs) and tetraploid have been extensively studied, the role of E2F1 in mitotic catastrophe is still unknown. We have previously shown that 8-chloro-adenosine (8-Cl-Ado) induces DNA DSBs and aberrant mitosis in human lung cancer cells, followed by delayed apoptosis. Here, we demonstrate that E2F1-mediated DNA damage is implicated in 8-Cl-Ado-induced chromosome missegregation and apoptosis in lung cancer H1299 cells. We showed that E2F1 was accumulated upon 8-Cl-Ado-induced DNA DSBs. Induction of E2F1 by 8-Cl-Ado caused DNA damage in cycling cells including M cells. In contrast, silencing of E2F1 expression decreased 8-Cl-Ado-induced DNA DSBs, particularly eliminated E2F1-mediated mitotic DNA damage. Over-expression of E2F1 and/or 8-Cl-Ado exposure resulted in aberrant mitotic spindles and chromosome segregation errors. Furthermore, over-expression of E2F1 expression enhanced 8-Cl-Ado-induced apoptosis. Together, our data indicate that E2F1-mediated DNA damage, in particular mitotic DNA damage, is an important fraction of 8-Cl-Ado-induced DNA damage, which is implicated in 8-Cl-Ado-induced mitotic catastrophe and delayed apoptosis. Induction of E2F1 by 8-Cl-Ado may contribute at least partly to the drug-inhibited proliferation of cancer cells.

Effects of myogenin on expression of late muscle genes through MyoD-dependent chromatin remodeling ability of myogenin.

MyoD and myogenin (Myog) recognize sets of distinct but overlapping target genes and play different roles in skeletal muscle differentiation. MyoD is sufficient for near-full expression of early targets, while Myog can only partially enhance expression of MyoD-initiated late muscle genes. However, the way in which Myog enhances the expression of MyoD-initiated late muscle genes remains unclear. Here, we examine the effects of Myog on chromatin remodeling at late muscle gene promoters and their activation within chromatin environment. Chromatin immunoprecipitation (ChIP) assay showed that Myog selectively bound to the regulatory sequences of late muscle genes. Overexpression of Myog was found to overcome sodium butyrateinhibited chromatin at late muscle genes in differentiating C2C12 myoblasts, shifting the transcriptional activation of these genes to an earlier time period. Furthermore, overexpression of Myog led to increased hyperacetylation of core histone H4 in differentiating C2C12 myoblasts but not NIH3T3 fibroblasts, and hyperacetylated H4 was associated directly with the late muscle genes in differentiating C2C12, indicating that Myog can induce chromatin remodeling in the presence of MyoD. In addition, co-immunoprecipitation (CoIP) revealed that Myog was associated with the nuclear protein Brd4 in differentiating C2C12 myoblasts. Together, these results suggest that Myog enhances the expression of MyoD-initiated late muscle genes through MyoD-dependent ability of Myog to induce chromatin remodeling, in which Myog-Brd4 interaction may be involved.

E2F1 enhances 8-chloro-adenosine-induced G2/M arrest and apoptosis in A549 and H1299 lung cancer cells.

The E2F1 transcription factor is a well known regulator of cell proliferation and apoptosis, but its role in response to DNA damage is less clear. 8-Chloro-adenosine (8-Cl-Ado), a nucleoside analog, can inhibit proliferation in a variety of human tumor cells. However, it is still elusive how the agent acts on tumors. Here we show that A549 and H1299 cells formed DNA double-strand breaks after 8-Cl-Ado exposure, accompanied by E2F1 upregulation at protein level. Overexpressed wild-type (E2F1-wt) colocalized with double-strand break marker γ-H2AX and promoted G2/M arrest in 8-Cl-Ado-exposed A549 and H1299, while expressed S31A mutant of E2F1 (E2F1-mu) significantly reduced ability to accumulate at sites of DNA damage and G2/M arrest, suggesting that E2F1 is required for activating G2/M checkpoint pathway upon DNA damage. Transfection of either E2F1-wt or E2F1-mu plasmid promoted apoptosis in 8-Cl-Ado-exposed cells, indicating that 8-Cl-Ado may induce apoptosis in E2F1-dependent and E2F1-independent ways. These findings demonstrate that E2F1 plays a crucial role in 8-Cl-Ado-induced G2/M arrest but is dispensable for 8-Cl-Ado-induced apoptosis. These data also suggest that the mechanism of 8-Cl-Ado action is complicated.

Calcium signal-initiated early activation of NF-kappaB in neurons is a neuroprotective event in response to kainic acid-induced excitotoxicity.

We demonstrate that activation of nuclear factor kappaB (NF-kappaB) in neurons is neuroprotective in response to kainic acid (KA)-induced excitotoxicity. Combination of Western blotting, immunocytochemistry, and electrophoresis mobility shift assay showed that KA exposure induced a fast but transient nuclear translocation of the NF-kappaB p65 subunit and increased DNA-binding activity of NF-kappaB in primary cultured cortical neurons. The transient NF-kappaB activity was associated with upregulation of antiapoptotic Bcl-xL and XIAP gene products revealed by real-time PCR. Knockdown of p65 decreased neuronal viability and antiapoptotic gene expression. In addition, we showed that KA-stimulated DNA-binding activity of NF-kappaB was associated with reactive oxygen species and calcium signals, using AMPA/KA receptor antagonist, calcium chelator, and antioxidant. These results suggest that the fast and transient activation of NF-kappaB initiated by calcium signals is one of the important proximal events in response to KA-induced excitotoxicity, which has neuroprotective effect against KA-induced apoptosis.

p14ARF interacts with E2F factors to form p14ARF-E2F/partner-DNA complexes repressing E2F-dependent transcription.

Primarily, E2F factors such as E2F1, -2, and -3 stimulate cell-cycle progression, while ARF tumor suppressor mediates growth suppression. The ARF gene can be induced by oncogenic signal through activating E2F-dependent transcription. In turn, ARF may target E2F for its degradation via a p53-dependent mechanism. However, it remains unclear how the cell keeps the balance between the functional opposites of E2F and ARF. In this study, we demonstrate that p14ARF interacts with E2F1-3 factors to directly repress their transcriptional activities through forming p14ARF-E2F/partner-DNA super complexes, regardless of E2F protein degradation. The inhibition of E2F transcriptional activities by p14ARF in this manner occurs commonly in a variety of cell types, including p53-deficient and p53-wild type cells. Thus, E2F-mediated activation of the ARF gene and ARF-mediated functional inhibition of E2F compose a feedback loop, by which the two opposites act in concert to regulate cell proliferation and apoptosis, depending on the cellular context and the environment.

Stabilization of mitochondrial function by tetramethylpyrazine protects against kainate-induced oxidative lesions in the rat hippocampus.

Mitochondria are critical regulators of cell death, a key feature of neurodegeneration. Reactive oxygen species (ROS) are crucial to Ca(2+)-mediated effects of glutamate receptor activation leading to neuronal degeneration. Tetramethylpyrazine (TMP) is a principal ingredient of Ligusticum wallichi Franchat (a Chinese herb), used for treatment of cardiovascular and cerebrovascular ischemic diseases. However, its protection against oxidative brain injury associated with excessive activation of glutamate receptors is unknown. In this study, we demonstrate TMP neuroprotection against kainate-induced excitotoxicity in vitro and in vivo. We found that TMP could partly alleviate kainate-induced status epilepticus in rats and prevented and rescued neuronal loss in the hippocampal CA3 but not the CA1 region. The partial prevention and rescue of neuronal loss by TMP were attributable to the preservation of the structural and functional integrity of mitochondria, evidenced by maintaining the mitochondrial membrane potential, ATP production, and complex I and III activities. Stabilization of mitochondrial function was linked to the observation that TMP could function as a reductant/antioxidant to quench ROS, block lipid peroxidation, and protect enzymatic antioxidants such as glutathione peroxidase and glutathione reductase. These results suggest that TMP may protect against oxidative brain injury by stabilization of mitochondrial function through quenching of ROS.

Synergistic up-regulation of muscle LIM protein expression in C2C12 and NIH3T3 cells by myogenin and MEF2C.

Although the role of muscle LIM protein (MLP, also known as CRP3), a LIM-only protein of LIM domain-containing protein family, is well-characterized, the mechanism by which the MLP gene expresses remains unclear. Herein, we demonstrate that myogenin and myocyte enhancer factor 2C (MEF2C) cooperate in activating the MLP gene in myogenesis. RT-PCR, real-time PCR and Western blotting showed that overexpression of myogenin or myogenin plus MEF2C led to induction of the MLP gene in differentiating C2C12 and NIH3T3 fibroblasts. By contrary, knocking-down of myogenin by RNA interference (RNAi) suppressed MLP expression in differentiating C2C12. Deletion and reporter enzyme assay revealed that the promoter activity was determined largely by the region extending from -260 to -173, which containing three E-box (CANNTG motif) candidates. Site-directed mutagenesis demonstrated that the E-box at position -186 to -180 was crucial for activating the promoter by myogenin. Furthermore, MEF2C could enhance myogenin-mediated activation of the promoter. In addition, chromatin immunoprecipitation (ChIP) and re-ChIP showed that myogenin and MEF2C were associated with the activated MLP promoter. Together, these results suggest that myogenin and MEF2C cooperate in the MLP gene activation. The linking of the MLP gene activation with myogenin and MEF2C may facilitate myogenin-mediated differentiation of striated muscle.

Inhibition of topoisomerase II by 8-chloro-adenosine triphosphate induces DNA double-stranded breaks in 8-chloro-adenosine-exposed human myelocytic leukemia K562 cells.

8-Chloro-cAMP and 8-chloro-adenosine (8-Cl-Ado) are known to inhibit proliferation of cancer cells by converting 8-Cl-Ado into an ATP analog, 8-chloro-ATP (8-Cl-ATP). Because type II topoisomerases (Topo II) are ATP-dependent, we infer that 8-Cl-Ado exposure might interfere with Topo II activities and DNA metabolism in cells. We found that 8-Cl-Ado exposure inhibited Topo II-catalytic activities in K562 cells, as revealed by decreased relaxation of the supercoiled pUC19 DNA and inhibited decatenation of the kinetoplast DNA (kDNA). In vitro assays showed that 8-Cl-ATP, but not 8-Cl-Ado, could directly inhibit Topo IIalpha-catalyzed relaxation and decatenation of substrate DNA. Furthermore, 8-Cl-ATP inhibited Topo II-catalyzed ATP hydrolysis and increased salt-stabilized closed clamp. In addition, 8-Cl-Ado exposure decreased bromo-deoxyuridine (BrdU) incorporation into DNA and led to enhanced DNA double-stranded breaks (DSBs) and to increased formation of gamma-H2AX nuclear foci in exposed K562 cells. Together, 8-Cl-Ado/8-Cl-ATP can inhibit Topo II activities in cells, thereby inhibiting DNA synthesis and inducing DNA DSBs, which may contribute to 8-Cl-Ado-inhibited proliferation of cancers.

Inhibition of CHK1 kinase by Gö6976 converts 8-chloro-adenosine-induced G2/M arrest into S arrest in human myelocytic leukemia K562 cells.

8-Chloro-cAMP (8-Cl-cAMP) and its metabolite 8-chloro-adenosine (8-Cl-Ado) inhibit cell growth by 8-Cl-Ado-converted 8-Cl-ATP that targets cell-cycle control and RNA metabolism. However, the cell-cycle checkpoint pathways remain to be identified. Recent studies have shown that 8-Cl-cAMP administration and 8-Cl-Ado exposure may damage chromosomal DNA in vivo and in vitro. In this study, we demonstrate that 8-Cl-Ado-induced DNA damage activates G2/M phase checkpoint, which is associated with ATM-activated CHK1-CDC25C-CDC2 pathway joined by BRCA1-CHK1 branch in apoptosis-resistant human myelocytic leukemia K562 (p53-null) cells. Inhibition of CHK1 kinase by Gö6976, an inhibitor of CHK1 activity, can promote DNA damage and lead to the activation of CHK2, converting G2/M checkpoint into intra-S-phase checkpoint in which two parallel branches, the ATM-CHK2-CDC25A-CDK2 and the ATM-NBS1/SMC1 cascades, are involved. These observations may provide aid in better understanding of the mechanisms of 8-Cl-cAMP and 8-Cl-Ado actions and in potential design of the combined therapy.

Exposure of human lung cancer cells to 8-chloro-adenosine induces G2/M arrest and mitotic catastrophe.

8-Chloro-adenosine (8-Cl-Ado) is a potent chemotherapeutic agent whose cytotoxicity in a variety of tumor cell lines has been widely investigated. However, the molecular mechanisms are uncertain. In this study, we found that exposure of human lung cancer cell lines A549 (p53-wt) and H1299 (p53-depleted) to 8-Cl-Ado induced cell arrest in the G2/M phase, which was accompanied by accumulation of binucleated and polymorphonucleated cells resulting from aberrant mitosis and failed cytokinesis. Western blotting showed the loss of phosphorylated forms of Cdc2 and Cdc25C that allowed progression into mitosis. Furthermore, the increase in Ser10-phosphorylated histone H3-positive cells revealed by fluorescence-activated cell sorting suggested that the agent-targeted cells were able to exit the G2 phase and enter the M phase. Immunocytochemistry showed that microtubule and microfilament arrays were changed in exposed cells, indicating that the dynamic instability of microtubules and microfilaments was lost, which may correlate with mitotic dividing failure. Aberrant mitosis resulted in mitotic catastrophe followed by varying degrees of apoptosis, depending on the cell lines. Thus, 8-Cl-Ado appears to exert its cytotoxicity toward cells in culture by inducing mitotic catastrophe.