The atypical protein kinase ATM is activated by double-stranded DNA breaks (DSBs), phosphorylating proteins involved in DNA repair and cell cycle checkpoint activation. The MRN complex is required for ATM activation in response to DSBs. We have studied ATM activation in the Xenopus egg extracts in response to linear DNA molecules. Based on our results we propose that MRN/ATM complexes bind at DSB ends, initiating ATM activation. However, full activation of ATM requires its binding to sequences flanking the DSB, with multiple ATM molecules being activated at each DSB through a cooperative proximity mechanism, which may require nucleosome/chromatin on the flanking DNA. This can account for the massive degree of ATM activity amplification achieved in response to a few DNA breaks in the cell. In addition, we have found that downstream of ATM, CtIP, a transcriptional regulator that is also implicated in the DNA damage checkpoint, plays a critical role in the transition from initial DSB sensing to DNA end resection, which generates the ssDNA regions at the DSB that lead to ATR activation.

Noninvasive breast lesions, such as ductal carcinoma in situ (DCIS), display a disruption in the orderly positioning of luminal epithelial cells that line the ducts and lobules of the mammary gland. To investigate this loss of epithelial architecture we have used spinning-disk microscopy to carry out real-time imaging of H2B-GFP MCF-10A mammary epithelial cells grown in a 3D culture system, using a 4-hydroxytamoxifen-inducible form of Raf-1 (Raf:ER) to activate ERK MAPK signaling. Raf:ER activation disrupts the hollow spherical acinar architecture generated by MCF-10A cells; cells become motile, migrating on the basement membrane and also into the lumen moving on other epithelial cells, but fail to invade the basement membrane surrounding the acinus. Motility requires myosin 2 activity, and MLC 2 phosphorylation, which activates myosin ATPase, and is blocked by combined inhibition of MLCK and ROCK, which phosphorylate MLC2. Increased actomyosin-based cell motility signaling and reduced expression of E- and N-cadherin cell-cell adhesion proteins define changes in epithelial character that promote this noninvasive motility. Our results suggest that motility and invasiveness are distinct phases of tumorigenesis.