Organization of p21waf1/cip1 with Cdk cyclin complexes leads to decreased Cdk task, which, consequently, inhibits the phosphorylation/ inactivation of certain endogenous substrates, such as retinoblastoma protein, which are required for development through the cell cycle. Moreover, selective FAAH inhibitor is a common Cdk inhibitor and causes cell cycle arrest at G1/S or G2/M period. Based on studies, we recommended that primary osteoblasts might activate when confronted with ATO the DNA repair apparatus and/or the cell cycle machinery. The aims of the present study were, consequently, to look at changes in cell cycle progression of primary osteoblasts during ATO treatment and to handle the problem of how these results of ATO cause changes of checkpoint and Cdk dependent phosphorylation. ATO was obtained from Sigma Aldrich Co. LLC.. The ATM chemical KU55933 was from TOCRIS Bioscience. Collagenase form II was from Worthington Biochemical Corporation. The protease inhibitor cocktail and RNase A were from Sigma?Aldrich Co. LLC.. Protein analysis reagents were from Bio Rad Laboratories. All other chemicals were of analytical grade and were obtained from Sigma?Aldrich Co. LLC.. Mouse monoclonal antibodies against rat/ human ATM, phospho Ser1981 ATM, p53, phospho Ser20 p53, p21, Cdc2, phospho Tyr15 Cdc2, Bax, caspase 3, or cytochrome c, rabbit polyclonal antibodies against rat/human Bcl XL, phospho Thr68 Chk2, ATR, phospho Ser428 ATR, Nbs1, ATRIP, Cdc25C, Wee1, or cyclin B1, and goat polyclonal antibodies against Immune system rat/human Chk1, phospho Ser345 Chk1, phospho Ser216 Cdc25C, or t actin were obtained from Santa Cruz Biotechnology Inc.. Phycoerythrin conjugated rabbit monoclonal antibody against rat phospho Ser139 H2AX was ordered from Cell Signaling Technology Inc.. Horseradish peroxidaseconjugated anti mouse, goat, or rabbit IgG antibodies were obtained from Santa Cruz Biotechnology Inc.. Osteosarcoma cell lines, MG63 originated from individual and UMR106 order PFI-1 originated from rat, were received from the Bioresource Collection and Research Center within the Food Industry Research and Development Institute and were cultured in Minimum Crucial Medium containing ten percent fetal bovine serum, 2 mM L glutamine, 100 units/ml of penicillin, and 100 mg/ml of streptomycin at 37 8C in a chamber with 5% CO2. The rat calvaria osteoblast were isolated and cultured as described previously. Quickly, calvaria from newborn rats of both sexes were cultured for 5 days and cut into small pieces on collagen gel prepared in Minimum Essential Medium containing ten percent fetal bovine serum. Pre osteoblasts produced from your calvarium were obtained by treatment of collagen gel cultures with collagenase and cultured for seven days in MEM containing 10% FBS, 50 mg/ml of ascorbic acid, and 10 mg/ml of b glycerophosphate to acquire adult osteoblasts revealing osteocalcin.