Novel role for MyoD as a sensor of DNA damage

Aim 1: To assess the activation of the kinases involved in the DNA damage checkpoint pathway (ATM, chk1, cdc25) as a consequence of culture in DM or etoposide.

Skeletal myoblasts will be cultured in GM or DM for 4 or 8 hours or in GM with or without etopside as described (2).  Lysates will be prepared and Western analysis will be performed using antibodies to assess the level of activated, phosphorylated ATM, phosphorylated Chk1 (indicative of ATM activation) and phosphorylated cdc25 (indicative of Chk1 activation).  To confirm that any observed differences are a consequence of increased activation/phosphorylation and not a consequence of increased expression of total ATM, Chk1, or cdc25, Western analysis will also be performed using antibodies to detect total ATM, Chk1, or cdc25.  To control for equal sample loading and electrophoretic transfer, Western analysis will also be performed for actin. All experiments will be performed in triplicate.

 

Aim  2: To assess the contribution of signaling by checkpoint kinases on the MyoD-mediated increased expression of PUMA in response to culture in DM or treatment with etoposide.  

We will begin our analysis using pharmacological inhibitors and will follow up with genetic analysis as appropriate.  KU55933 and AZD7762 are inhibitors of ATM and Chk1, respectively.  23A2 myoblasts will be treated with varying concentration of inhibitors or vehicle for 4 or 8 hours in DM or GM supplemented with etoposide.  Inhibition of kinase activity will be assessed by Western analysis with appropriate antibodies as described in Aim 1.  The appropriate concentration of inhibitor will then be used to assess the contribution of each respective kinase to the induction of PUMA in myoblasts after 3 hours of culture in DM or after treatment with etoposide. All experiments will be performed in triplicate.