Longchuan Chen

Cell Cycle-dependent Complex Formation of BRCA1·CtIP·MRN Is Important for DNA Double-strand Break Repair

BRCA1 plays an important role in the homologous recombination (HR)-mediated DNA double-strand break (DSB) repair, but the mechanism is not clear. Here we describe that BRCA1 forms a complex with CtIP and MRN (Mre11/Rad50/Nbs1) in a cell cycle-dependent manner. Significantly, the complex formation, especially the ionizing radiation-enhanced association of BRCA1 with MRN, requires cyclin-dependent kinase activity. CtIP directly interacts with Nbs1. The in vivo association of BRCA1 with MRN is largely dependent on the association of CtIP with the BRCT domains at the C terminus of BRCA1, whereas the N terminus of BRCA1 also contributes to its association with MRN. CtIP, as well as the interaction of BRCA1 with CtIP and MRN, is critical for IR-induced single-stranded DNA formation and cellular resistance to radiation. Consistently, CtIP itself is required for efficient HR-mediated DSB repair, like BRCA1 and MRN. These studies suggest that the complex formation of BRCA1·CtIP·MRN is important for facilitating DSB resection to generate single-stranded DNA that is needed for HR-mediated DSB repair. Because cyclin-dependent kinase is important for establishing IR-enhanced interaction of MRN with BRCA1, we propose that the cell cycle-dependent complex formation of BRCA1, CtIP, and MRN contributes to the activation of HR-mediated DSB repair in the S and G2 phases of the cell cycle.

The Interaction of CtIP and Nbs1 Connects CDK and ATM to Regulate HR–Mediated Double-Strand Break Repair

CtIP plays an important role in homologous recombination (HR)–mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.

The Mre11-Rad50-Nbs1 Complex Acts Both Upstream and Downstream of Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR) to Regulate the S-phase Checkpoint following UV Treatment

The Mre11-Rad50-Nbs1 (MRN) complex is required for mediating the S-phase checkpoint following UV treatment, but the underlying mechanism is not clear. Here we demonstrate that at least two mechanisms are involved in regulating the S-phase checkpoint in an MRN-dependent manner following UV treatment. First, when replication forks are stalled, MRN is required upstream of ataxia telangiectasia mutated and Rad3related protein (ATR) to facilitate ATR activation in a substrate and dosage-dependent manner. In particular, MRN is required for ATR-directed phosphorylation of RPA2, a critical event in mediating the S-phase checkpoint following UV treatment. Second, MRN is a downstream substrate of ATR. Nbs1 is phosphorylated by ATR at Ser-343 when replication forks are stalled, and this phosphorylation event is also important for down-regulating DNA replication following UV treatment. Moreover, we demonstrate that MRN and ATR/ATR-interacting protein (TRIP) interact with each other, and the forkhead-associated/breast cancer C-terminal domains (FHA/BRCT) of Nbs1 play a significant role in mediating this interaction. Mutations in the FHA/BRCT domains do not prevent ATR activation but specifically impair ATR-mediated Nbs1 phosphorylation at Ser-343, which results in a defect in the S-phase checkpoint. These data suggest that MRN plays critical roles both upstream and downstream of ATR to regulate the S-phase checkpoint when replication forks are stalled.

The Mre11 Complex Mediates the S-Phase Checkpoint through an Interaction with Replication Protein A

ABSTRACT The Mre11/Rad50/Nbs1 complex (MRN) plays an essential role in the S-phase checkpoint. Cells derived from patients with Nijmegen breakage syndrome and ataxia telangiectasia-like disorder undergo radioresistant DNA synthesis (RDS), failing to suppress DNA replication in response to ionizing radiation (IR). How MRN affects DNA replication to control the S-phase checkpoint, however, remains unclear. We demonstrate that MRN directly interacts with replication protein A (RPA) in unperturbed cells and that the interaction is regulated by cyclin-dependent kinases. We also show that this interaction is needed for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with RPA leads to pronounced RDS without affecting phosphorylation of Nbs1 or SMC1 following IR. Moreover, MRN is recruited to sites at or adjacent to replication origins by RPA and acts there to inhibit new origin firing upon IR. These studies suggest a direct role of MRN at origin-proximal sites to control DNA replication initiation in response to DNA damage, thereby providing an important mechanism underlying the intra-S-phase checkpoint in mammalian cells.

Neutralizing Anti-Vascular Endothelial Growth Factor (VEGF) Antibody Reduces Severity of Experimental Ulcerative Colitis in Rats: Direct Evidence for the Pathogenic Role of VEGF

In ulcerative colitis (UC), an increased expression of vascular endothelial growth factor (VEGF) correlates with disease activity, but a causal relationship is unknown. We tested the hypothesis that VEGF plays a mechanistic role in the pathogenesis of experimental UC and that VEGF neutralization may exert therapeutic effect. UC was induced in Sprague-Dawley rats by 6% iodoacetamide given intracolonically. Neutralizing anti-VEGF antibody (50 μg/rat), nonspecific IgG, or saline (0.1 ml/rat) was injected intramuscularly on the 3rd and 5th days after iodoacetamide enema. Rats were euthanized on the 7th day. We examined the extent of macroscopic, histologic, and clinical features of colitis and colonic vascular permeability. Colonic VEGF mRNA and protein expressions increased as early as 0.5 h after iodoacetamide enema and remained elevated in the active phase of colitis. Treatment with anti-VEGF antibody markedly improved the clinical and morphologic features of UC. Colonic lesion area was significantly reduced from 370 ± 140 or 311 ± 170 mm2 in saline- or IgG-treated groups to 122 ± 57 mm2 in the anti-VEGF-group (p < 0.05). Increased colonic vascular permeability was decreased by the anti-VEGF antibody (p < 0.05) and the Src inhibitor PP1 [pyrazolopyrimidine, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine] (p < 0.01). The number of acute and chronic inflammatory cells in the lesion area was significantly reduced in anti-VEGF-treated rats. In the anti-VEGF-treated group, mucosal levels of VEGF, platelet-derived growth factor, and basic fibroblast growth factor were also reduced. In conclusion: 1) Neutralizing anti-VEGF antibody significantly ameliorates experimental UC in rats in part by reducing excessive vascular permeability and decreasing inflammatory cells infiltration; and 2) VEGF seems to mediate increased colonic vascular permeability in experimental UC via the Src-dependent mechanism.

Mesalamine Restores Angiogenic Balance in Experimental Ulcerative Colitis by Reducing Expression of Endostatin and Angiostatin: Novel Molecular Mechanism for Therapeutic Action of Mesalamine

Mesalamine (5-aminosalicylate acid, 5-ASA) is an effective treatment for ulcerative colitis (UC). The mechanisms of its actions are not fully understood. Because angiogenesis is critical for healing UC, we examined whether 5-ASA alters the angiogenic balance between angiogenic factors [e.g., vascular endothelial growth factor (VEGF)] and antiangiogenic factors (e.g., endostatin and angiostatin) in the colon in experimental UC. Rats were treated with saline or 5-ASA (100 mg/kg) twice daily and euthanized 3 or 7 days after iodoacetamide-induced UC. Clinical signs (e.g., lethargy, diarrhea) and UC lesions were measured. Expression of VEGF, endostatin, angiostatin, tissue necrosis factor α (TNF-α), and matrix metalloproteinases (MMPs) 2 and 9 was determined by Western blots, enzyme-linked immunosorbent assay, and zymography in the distal colon. 5-ASA treatment reduced lethargy and diarrhea and significantly decreased colonic lesions (by ∼50%) compared with saline treatment in UC (both, P < 0.05). 5-ASA did not reverse the increased levels of VEGF, but it significantly reduced expression of endostatin and angiostatin in UC compared with vehicle treatment (both, P < 0.05). Furthermore, 5-ASA treatment significantly diminished increased activity of TNF-α and MMP9 in UC. This is the first demonstration that 5-ASA treatment reverses an imbalance between the angiogenic factor VEGF and antiangiogenic factors endostatin and angiostatin in experimental UC. The effect of 5-ASA in UC may be caused by the down-regulation of expression of endostatin and angiostatin by modulation of MMP2 and MMP9 via inhibition of TNFα. The inhibition of antiangiogenic factors may represent a novel molecular mechanism of the therapeutic action of 5-ASA.

Dbf4 Is Direct Downstream Target of Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia and Rad3-related (ATR) Protein to Regulate Intra-S-phase Checkpoint

Background: S-phase checkpoint is important for maintaining genome stability upon DNA damage in S-phase. Results: A replication essential protein Dbf4 is phosphorylated by checkpoint kinases when DNA is damaged. Conclusion: Dbf4 is a downstream target of the S-phase checkpoint to mediate DNA damage responses. Significance: These studies help understand how the genome is protected from DNA damage to prevent tumorigenesis. Dbf4/Cdc7 (Dbf4-dependent kinase (DDK)) is activated at the onset of S-phase, and its kinase activity is required for DNA replication initiation from each origin. We showed that DDK is an important target for the S-phase checkpoint in mammalian cells to suppress replication initiation and to protect replication forks. We demonstrated that ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins directly phosphorylate Dbf4 in response to ionizing radiation and replication stress. We identified novel ATM/ATR phosphorylation sites on Dbf4 and showed that ATM/ATR-mediated phosphorylation of Dbf4 is critical for the intra-S-phase checkpoint to inhibit DNA replication. The kinase activity of DDK, which is not suppressed upon DNA damage, is required for fork protection under replication stress. We further demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for preventing DNA rereplication upon loss of replication licensing through the activation of the S-phase checkpoint. These studies indicate that DDK is a direct substrate of ATM and ATR to mediate the intra-S-phase checkpoint in mammalian cells.

New molecular mechanisms of duodenal ulceration.

Abstract:  Stress is a major etiologic factor in the pathogenesis of gastric and duodenal ulceration, as first described in rats by Hans Selye. In patients with “peptic ulcers” duodenal ulcers are more frequent than gastric ulcers (except in Japan). Thus, our research during the last three decades focused on the molecular mechanisms of duodenal ulcer in rodent models of chemically induced duodenal ulceration, and here we review our three recent findings: Endothelins (ET‐1), the immediate early gene egr‐1 and imbalance of angiogenic/antiangiogenic molecules. Namely, we found an enhanced expression and release of ET‐1 within 15–30 min after the administration of duodenal ulcerogen cysteamine, resulting in local ischemia that triggers the expression of hypoxia‐inducible factors (HIF‐1α). Our gene expression studies also revealed an early (0.5–2 h) increase in the expression of egr‐1 that is followed (12–24 h) by upregulation of angiogenic growth factors (e.g., VEGF, bFGF, PDGF). Surprisingly, this event is also associated with an enhanced production of angiostatin and endostatin that probably counteract the beneficial effect of angiogenic molecules. Thus, the initial injury to endothelial and epithelial cells in duodenal ulceration seems to be aggravated (and not initiated) by HCl and proteolytic enzymes. The resulting mucosal necrosis does not rapidly heal because of the imbalance of VEGF and angiostatin/endostatin, hence duodenal ulcers develop. The experimental ulcers Selye described morphologically are now characterized at the molecular and genome level, involving unexpected mediators like ET‐1, egr‐1 and angiogenesis‐related molecules.

New mechanistic explanation for the localization of ulcers in the rat duodenum: Role of iron and selective uptake of cysteamine

Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [(14)C]cysteamine uptake. [(14)C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [(14)C]cysteamine by IEC-6 was Na(+)-independent, saturable, inhibited by structural analogs, H(2)-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [(14)C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis.

Role of anti-angiogenic factor endostatin in the pathogenesis of experimental ulcerative colitis

AIMS Vascular endothelial growth factor (VEGF) and pathologic angiogenesis have been demonstrated to play a pathogenic role in the development and progression of inflammatory bowel disease. Thus, we hypothesized that the potent anti-angiogenic factor endostatin might play a beneficial role in experimental ulcerative colitis (UC). MAIN METHODS We used three animal models of UC: (1) induced by 6% iodoacetamide (IA) in rats, or (2) by 3% dextran sulfate sodium (DSS) in matrix metalloproteinase-9 (MMP-9) knockout (KO) and wild-type mice, and (3) interleukin-10 (IL-10) KO mice. Groups of MMP-9 KO mice with DSS-induced UC were treated with endostatin or water for 5days. KEY FINDINGS We found concomitant upregulation of VEGF, PDGF, MMP-9 and endostatin in both rat and mouse models of UC. A positive correlation between the levels of endostatin or VEGF and the sizes of colonic lesions was seen in IA-induced UC. The levels and activities of MMP-9 were also significantly increased during UC induced by IA and IL-10 KO. Deletion of MMP-9 decreased the levels of endostatin in both water- and DSS-treated MMP-9 KO mice. Treatment with endostatin significantly improved DSS-induced UC in MMP-9 KO mice. SIGNIFICANCE 1) Concomitantly increased endostatin is a defensive response to the increased VEGF in UC, 2) MMP-9 is a key enzyme to generate endostatin which may modulate the balance between VEGF and endostatin during experimental UC, and 3) endostatin treatment plays a beneficial role in UC. Thus, anti-angiogenesis seems to be a new therapeutic option for UC.