Abstract
Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine.
Highlights
Cells respond to damage to their DNA or interference with their DNA replication by activating genome surveillance pathways such as cell cycle checkpoints
We evaluated the effects of daunorubicin and rebeccamycin on tight junction (TJ) integrity, and investigated the association between DNA damage signaling and the components of TJs in human intestinal cells
Because epithelial TJ barrier function is controlled by the various TJ components, we examined the effect of daunorubicin and rebeccamycin on the transcription of TJ components. Quantitative reverse transcriptase-PCR (qRT-PCR) analysis showed that daunorubicin mainly increased the expression of claudin-5, -8, and -11, whereas rebeccamycin significantly increased the expression of claudin-5 and -10 (Fig 3A)
Summary
Cells respond to damage to their DNA or interference with their DNA replication by activating genome surveillance pathways such as cell cycle checkpoints. Chk Activation Enhances Intestinal TJ Barrier telangiectasia mutated and Rad3-related protein (ATR). When ATM and ATR are recruited to sites of DNA damage, they activate checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2), respectively, which regulate the function of downstream effector proteins such as p21, Cdc25A, and cyclin-dependent kinases [1]. Depending on the extent of the DNA damage, cells either go into cell cycle arrest to allow time for repair or go into apoptosis [3]. This orderly, accurate response to DNA damage is essential to maintain the genetic stability of cells
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