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Driver of DNA repair pathway

Posted in Curation, Developmental biology, DNA repair, tagged endogenous DNA repair, self renewal of iPCs on January 13, 2016| Leave a Comment »

Driver of DNA repair pathway

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Molecule Driving DNA Repair Disorder Revealed

GEN News http://www.genengnews.com/gen-news-highlights/molecule-driving-dna-repair-disorder-revealed/81252230/

http://www.genengnews.com/Media/images/GENHighlight/thumb_106235_web1881582438.jpg

This image shows a teratoma formed by induced pluripotent stem cells with defective Fanconi anemia DNA repair. Even with defective FA DNA repair, the induced stem cells were able to start the process of forming specific tissues until the DNA repair defect killed them off. Teratomas are benign tumors containing a haphazard array of cells and tissues of different organ systems. [Cincinnati Children’s Hospital Medical Center]

The importance of maintaining genomic integrity becomes readily apparent when determining the underlying causes of various genetic diseases with mutations residing within DNA repair pathways. Researchers at the Cincinnati Children’s Hospital Medical Center believe they have identified a molecular target and experimental treatment strategy for DNA repair defects behind Fanconi anemia—a multifaceted genetic disorder responsible for birth anomalies, organ damage, anemia, and cancer.

“This study provides an experimental platform to test new therapies that could prevent pre- and post-natal Fanconi anemia (FA) conditions, which have no cure and limited treatment options,” explained senior study author Susanne Wells, Ph.D., director of the epithelial carcinogenesis and stem cell program at the Cincinnati Children’s Hospital Medical Center. “Our findings also raise a number of important questions, so there is a lot more to be done.”

The findings from this study were published recently in Stem Cell Reports through an article entitled “Overcoming Pluripotent Stem Cell Dependence on the Repair of Endogenous DNA Damage.”

In the current study, the investigators utilized induced pluripotent stem cells (iPSCs), which have the ability to be reprogrammed into any cell type in the body. The induced stem cells were donated by FA patients and contained the defective DAN repair pathway. The research team carefully studied the stem cells in laboratory cultures and cells injected into humanized mouse models, monitoring their genetic, molecular, and developmental progression.

Interestingly, even with defective FA DNA repair, the stem cells were still able to transform into different tissues. However, the researchers said the DNA repair defect eventually started to kill off the pluripotent stem cells by blocking cell division and causing programmed cell death.

“To determine the effect of failed repair of endogenous DNA lesions on PSC biology, we generated iPSCs harboring a conditional FA pathway,” the authors wrote. “Upon FA pathway loss, iPSCs maintained pluripotency but underwent profound G2 arrest and apoptosis, whereas parental fibroblasts grew normally.”

Upon subsequent examination, the researchers noticed an enzyme that serves as a DNA regulatory checkpoint during cell division (CHK1) showed a dramatic increase in activity within the stem cells—speeding up their deaths. Using existing pharmacologic inhibitors of CHK1 to block the hyperactive enzyme at a critical stage of the stem cell cycle, the investigators were able to override what usually are unfixable errors in the FA repair pathway.

“Our findings establish that the FA pathway is essential for proliferation and survival of iPSCs and implicates CHK1 as a crucial factor in their extreme sensitivity to accrued DNA damage,” the authors stated.

Surprisingly, after targeted treatment, FA-pathway-deficient pluripotent stem cells resumed dividing and expanding normally. Moreover, the scientists were amazed that the resumption of cell growth occurred without what they had expected to be massive chromosome abnormalities, leading the ranchers to postulate that a compensating DNA repair process is engaged in the reinvigorated cells.

“A key question for us is what type of DNA repair kicks in under these conditions—and is it error free or error prone?” noted Dr. Wells. “A novel mode of emergency DNA repair might indeed be discovered in the iPSC cells. We believe some type of compensatory DNA repair must be driven by CHK1 inhibition when cells have FA pathway loss. Otherwise, the cells would have died off very quickly.”

The scientists plan to follow up this study with additional testing in humanized and genetic mouse models, attempting to improve embryonic development and post-birth fitness in FA-pathway deficient mice with a uniform application of the CHK1 inhibitor. The researchers hope that their findings and approach may lead to treatments for all of the clinical manifestations of the disease.

Overcoming Pluripotent Stem Cell Dependence on the Repair of Endogenous DNA Damage

Timothy M. Chlon, Sonya Ruiz-Torres, Logan Maag, …., James M. Wells, Susanne I. Wells correspondence

Stem Cell Reports Jan 2016; 6(1), 44–54, 12 http://dx.doi.org/10.1016/j.stemcr.2015.12.001

http://www.cell.com/cms/attachment/2043257303/2055826965/fx1.jpg

Highlights

•Self-renewal but not pluripotency of iPSCs depends on FA pathway function
•Hyperactive CHK1 limits self-renewal in a conditional FA-deficient iPSC model
•CHK1 inhibition rescues long-term growth of FA-deficient iPSCs

Summary

Pluripotent stem cells (PSCs) maintain a low mutation frequency compared with somatic cell types at least in part by preferentially utilizing error-free homologous recombination (HR) for DNA repair. Many endogenous metabolites cause DNA interstrand crosslinks, which are repaired by the Fanconi anemia (FA) pathway using HR. To determine the effect of failed repair of endogenous DNA lesions on PSC biology, we generated iPSCs harboring a conditional FA pathway. Upon FA pathway loss, iPSCs maintained pluripotency but underwent profound G2 arrest and apoptosis, whereas parental fibroblasts grew normally. Mechanistic studies revealed that G2-phase FA-deficient iPSCs possess large γH2AX-RAD51 foci indicative of accrued DNA damage, which correlated with activated DNA-damage signaling through CHK1. CHK1 inhibition specifically rescued the growth of FA-deficient iPSCs for prolonged culture periods, surprisingly without stimulating excessive karyotypic abnormalities. These studies reveal that PSCs possess hyperactive CHK1 signaling that restricts their self-renewal in the absence of error-free DNA repair.