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.

[Research Progress on Expression Regulation, Function and Clinical Significance of CASP8AP2 Gene].

We systematically reviewed the results of the studies on expression regulation, biological functions, and clinical prognostic significance of CASP8AP2 gene. At present, the studies showed that the expression of CASP8AP2 gene was regulated by Homeobox proteins and DNA methylation, and could be silenced by miRNA-210. This protein was involved in apoptosis mediated by FAS and TNFα, NF-κB activation mediated by TNFα, regulation of gene expression induced by glucocorticoid and mineralocorticoid receptor, comprising Cajal body and histone locus body, transcription of replication-dependent histone, 3' end processing of histone, regulation of S phase progression, in addition to functioning as coactivator of transcription factors c-Myb and p73 to activating many genes' expression. On the other hand, low expression of CASP8AP2 gene was associated with relapse in childhood ALL. The deletion of this gene was related to the poor prognosis of children with T-ALL and T lymphoblastic lymphoma. Furthermore, 3 SNPs in this gene were possibly correlated with genesis of diffuse large B cell lymphoma and childhood leukemia. In conclusions, CASP8AP2 was a multifunctional protein. It could function to regulate cell proliferation, apoptosis, and gene expression. In childhood hematological malignancies, CASP8AP2 was a promising molecular marker with prognostic significance. Some SNPs were possibly correlated with leukemo- and lymphomogenesis.

Low expressions of ARS2 and CASP8AP2 predict relapse and poor prognosis in pediatric acute lymphoblastic leukemia patients treated on China CCLG-ALL 2008 protocol.

ARS2 protein is important to early development and cell proliferation, in which ARS2-CASP8AP2 interaction is implicated. However, the predictive significance of ARS2 in childhood acute lymphoblastic leukemia (ALL) is unknown. Here we evaluate the predictive values of ARS2 expression and combined ARS2 and CASP8AP2 expression in relapse. We showed that ARS2 expression in ALL bone marrow samples at initial diagnosis was markedly lower than that in complete remission (CR). Likewise, the levels of ARS2 expression in the patients suffering from relapse were significantly lower than that of patients in continuous CR. Furthermore, low expression of ARS2 was closely correlated to poor treatment response including poor prednisone response and high minimal residual disease (MRD), and the patients with high MRD (≥10(-4)) and low ARS2 were more subject to relapse. The multivariate analyses for relapse free survival and event free survival revealed that ARS2 expression remained an independent prognostic factor after adjusting other risk factors. In addition, combined assessment of ARS2 and CASP8AP2 expression was more accurate to predict relapse, based on which an algorithm composed of ARS2 and CASP8AP2 expression, prednisone response and MRD (day 78) was proposed. Together, ARS2 and CASP8AP2 expressions can precisely predict high-risk of relapse and ALL prognosis.

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.

Effect of microRNA-210 on prognosis and response to chemotherapeutic drugs in pediatric acute lymphoblastic leukemia.

Many studies have demonstrated that microRNA-210 (miR-210) expression is intensively upregulated in hypoxic states and differentially regulated in most types of cancer cells. However, the clinical significance of miR-210 and its effects on the response of leukemic cells to chemotherapeutic drugs in childhood acute lymphoblastic leukemia (ALL) remain unknown. In the current study, using real-time qRT-PCR to detect miR-210 expression in bone marrow samples from 114 children at initial diagnosis of ALL, we investigated the prognostic significance of miR-210 and determined its associations with common clinical characteristics and treatment outcome. We further examined its effect on the response to chemotherapeutic drugs in the Reh and RS4;11 cell lines. Results showed that miR-210 expression was significantly lower in patients suffering from relapse and induction failure than in other patients (P < 0.001). Using the receiver operating characteristic curve, 3.8243 was selected as the cut-off value of miR-210 expression in our test cohort (38 cases). A significantly poorer treatment outcome (P < 0.05) was found in the low-expression group and verified in the validation cohort (76 cases, P < 0.05). Patients with low expression of miR-210 and positive minimal residual disease at the end of induction had a much higher rate of relapse or induction failure (P = 0.001). Increasing/decreasing miR-210 expression using agomir/antagomir could enhance or reduce the response of Reh cells and RS4;11 cells to daunorubicin/dexamethasone/L-asparaginase and daunorubicin/dexamethasone/vincristine, respectively. In conclusion, miR-210 may be a good prognostic factor and a useful predictor of drug sensitivity, and is a potential therapeutic target for pediatric ALL.

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.

Hypermethylation of two CpG sites upstream of CASP8AP2 promoter influences gene expression and treatment outcome in childhood acute lymphoblastic leukemia.

DNA hypermethylation of Caspase 8 associated protein 2 (CASP8AP2) and its role in childhood acute lymphoblastic leukemia (ALL) is unclear. We analyzed methylation status of CpG sites upstream of CASP8AP2 gene in 86 children with ALL by bisulfite sequencing and quantitative PCR. Methylation percentage of two CpG sites at positions of -1189 and -1176 was inversely correlated with mRNA expression (Spearman correlation: -0.333, P=0.002). High methylation was associated with the existence of minimal residual disease (MRD) at day 78 (P=0.035), The patients in high methylation group had a poor treatment outcome. The combination of methylation level and MRD at day 33 might improve current risk stratification.

microRNA-7 suppresses the invasive potential of breast cancer cells and sensitizes cells to DNA damages by targeting histone methyltransferase SET8.

SET8 (SET domain containing 8) is a histone H4 lysine 20 (H4K20)-specific monomethyltransferase in higher eukaryotes that exerts diverse functions in transcription regulation, DNA repair, tumor metastasis, and genome integrity. The activity of SET8 is tightly controlled during cell cycle through post-translational modifications, including ubiquitination, phosphorylation, and sumoylation. However, how the expression of SET8 is regulated is not fully understood. Here, we report that microRNA-7 is a negative regulator of SET8. We demonstrated that microRNA-7 inhibits H4K20 monomethylation and suppresses epithelial-mesenchymal transition and the invasive potential of breast cancer cells. We showed that microRNA-7 promotes spontaneous DNA damages and sensitizes cells to induced DNA damages. Our experiments provide a molecular mechanism for the regulation of SET8 and extend the biological function of microRNA-7 to DNA damage response, supporting the pursuit of microRNA-7 as a potential target for breast cancer intervention.

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.

The tyrosine kinase c-Src directly mediates growth factor-induced Notch-1 and Furin interaction and Notch-1 activation in pancreatic cancer cells.

The proteolytic activity of Furin responsible for processing full length Notch-1 (p300) plays a critical role in Notch signaling. The amplitude and duration of Notch activity can be regulated at various points in the pathway, but there has been no report regarding regulation of the Notch-1-Furin interaction, despite its importance. In the present study, we found that the Notch-1-Furin interaction is regulated by the non-receptor tyrosine kinase, c-Src. c-Src and Notch-1 are physically associated, and this association is responsible for Notch-1 processing and activation. We also found that growth factor TGF-α, an EGFR ligand, and PDGF-BB, a PDGFR ligand, induce the Notch-1-Furin interaction mediated by c-Src. Our results support three new and provocative conclusions: (1) The association between Notch-1 and Furin is a well-regulated process; (2) Extracellular growth factor signals regulate this interaction, which is mediated by c-Src; (3) There is cross-talk between the plasma growth factor receptor-c-Src and Notch pathways. Co-localization of Notch-1 and c-Src was confirmed in xenograft tumor tissues and in the tissues of pancreatic cancer patients. Our findings have implications for the mechanism by which the Notch and growth factor receptor-c-Src signaling pathways regulate carcinogenesis and cancer cell growth.