DNA-damage response, survival and differentiation in vitro of a human neural stem cell line in relation to ATM expression.
Ataxia-telangiectasia (A-T) is a neurodegenerative disorder caused by defects in the ATM kinase, a component of the DNA-damage response (DDR). Here, we employed an immortalized human neural stem-cell line (ihNSC) capable of differentiating in vitro into neurons, oligodendrocytes and astrocytes to assess the ATM-dependent response and outcome of ATM ablation. The time-dependent differentiation of ihNSC was accompanied by an upregulation of ATM and DNA-PK, sharp downregulation of ATR and Chk1, transient induction of p53 and by the onset of apoptosis in a fraction of cells. The response to ionizing radiation (IR)-induced DNA lesions was normal, as attested by the phosphorylation of ATM and some of its substrates (e.g., Nbs1, Smc1, Chk2 and p53), and by the kinetics of gamma-H2AX nuclear foci formation. Depletion in these cells of ATM by shRNA interference (shATM) attenuated the differentiation-associated apoptosis and response to IR, but left unaffected the growth, self-renewal and genomic stability. shATM cells generated a normal number of MAP2/beta-tubulin III+ neurons, but a reduced number of GalC+ oligodendrocytes, which were nevertheless more susceptible to oxidative stress. Altogether, these findings highlight the potential of ihNSCs as an in vitro model system to thoroughly assess, besides ATM, the role of DDR genes in neurogenesis and/or neurodegeneration.
Carlessi
L
L
Department of Experimental Oncology, Fondazione IRCSS Istituto Nazionale Tumori, Milan, Italy.
De Filippis
L
L
Lecis
D
D
Vescovi
A
A
Delia
D
D
D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D002135:Calcium-Binding Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D002470:Cell Survival; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006657:Histones / Q000378:metabolism; D006801:Humans; D009457:Neuroglia / Q000166:cytology*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D009836:Oligodendroglia / Q000166:cytology / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000378:metabolism*; D034622:RNA Interference; D011839:Radiation, Ionizing; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000172:deficiency / Q000378:metabolism*
C109198:CIB1 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases