ABSTRACT
The G(2) checkpoint is an indispensable pathway for cancers lacking p53 function, for delaying cell cycle progression, and for completing DNA repair. Therefore, disruption of this pathway is expected to offer selective therapy for these highly prevalent cancers. The aim of this study was to identify an inhibitor of the G(2) checkpoint including the ataxia-telangiectasia-mutated and Rad3-related checkpoint kinase 1 pathway that selectively suppresses the growth of p53-deficient cells. To obtain molecules with a novel mechanism of action, we constructed a high-throughput screening system that detected abrogation of the G(2) checkpoint in X-irradiated HT-29 cells. The screening resulted in identification of a guanidine analog, CBP-93872 that dose dependently inhibited the G(2) checkpoint induced by DNA damage. Interestingly, CBP-93872 directly suppressed the growth of p53-mutated cancer cell lines with wild-type CDKN2A by eliciting G(1) arrest, but not CDKN2A-deleted and/or wild-type p53 lines. CBP-93872 decreased phospho-cdc2 Y15 by inhibiting phosphorylation of Chk1, but did not suppress phospho-Chk2 or the kinase activities of either Chk1 or Chk2 in cellular or cell-free assays. These results suggest that a checkpoint modulator through suppression of Chk1 phosphorylation provides synthetic lethality to p53-deficient cells.
Subject(s)
Aniline Compounds/pharmacology , Antineoplastic Agents/pharmacology , G2 Phase/drug effects , High-Throughput Screening Assays/methods , Propanolamines/pharmacology , Tumor Suppressor Protein p53/genetics , CDC2 Protein Kinase , Camptothecin/pharmacology , Cell Proliferation , Checkpoint Kinase 1 , Cyclin B/metabolism , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinases , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , G1 Phase/drug effects , G1 Phase/genetics , HT29 Cells/drug effects , HT29 Cells/radiation effects , Humans , Mutation , Phosphorylation/drug effects , Protein Kinases/metabolism , Reproducibility of ResultsABSTRACT
In order to study osteoblast differentiation we subcloned a cell derived from a mouse a bone marrow stromal cell line, Kusa O, and obtained a number of clones representative of three different phenotypes. One that neither differentiated into osteoblasts nor into adipocytes, a second that differentiated into osteoblasts but not adipocytes, and a third that differentiated into both osteoblasts and adipocytes. Four subclones were selected for further characterization according to their ability to mineralize and/or differentiate into adipocytes. The non-mineralizing clone had no detectable alkaline phosphatase activity although some alkaline phosphatase mRNA was detected after 21 days in osteoblast differentiating medium. Alkaline phosphatase activity and mRNA in the three mineralizing clones were comparable with the parent clones. Osteocalcin mRNA and protein levels in the non-mineralizing clone were low and non-detectable, respectively, while both were elevated in the parent cells and mineralizing subclones after 21 days in differentiating medium. PTH receptor mRNA and activity increased in the four subclones and parent cells with differentiation. mRNA for two other osteoblast phenotypic markers, osteopontin and bone sialoprotein, were similarly expressed in the parent cells and subclones while mRNAs for the transcription factors, Runx2 and osterix, were detectable in both parent and subclone cells. Runx2 was unchanged with differentiation while osterix was increased. Interestingly, PPARgamma mRNA expression did not correlate with cell line potential to differentiate into adipocytes. Indian hedgehog mRNA and its receptor (patched) mRNA levels both increased with differentiation while mRNA levels of the Wnt pathway components beta-catenin and dickkopf also increased with differentiation. Although we have focussed on characterizing these clones from the osteoblast perspective it is clear that they may be useful for studying both osteoblast and adipocyte differentiation as well as their transdifferentiation.
Subject(s)
Bone Marrow Cells/cytology , Cell Differentiation/physiology , Stromal Cells/physiology , Zebrafish Proteins , Adipocytes/physiology , Animals , Bone Marrow Cells/physiology , Cell Line , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , DNA Primers , Hedgehog Proteins , Mice , Osteoblasts/cytology , Osteoblasts/physiology , Phenotype , Polymerase Chain Reaction , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA, Messenger/metabolism , Stromal Cells/cytology , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Wnt Proteins , beta CateninABSTRACT
Dorsoventral patterning depends on the local concentrations of the morphogens. Twisted gastrulation (TSG) regulates the extracellular availability of a mesoderm inducer, bone morphogenetic protein 4 (BMP-4). However, TSG function in vivo is still unclear. We isolated a TSG cDNA as a secreted molecule from the mouse aorta-gonad-mesonephros region. Here we show that TSG-deficient mice were born healthy, but more than half of the neonatal pups showed severe growth retardation shortly after birth and displayed dwarfism with delayed endochondral ossification and lymphopenia, followed by death within a month. TSG-deficient thymus was atrophic, and phosphorylation of SMAD1 was augmented in the thymocytes, suggesting enhanced BMP-4 signaling in the thymus. Since BMP-4 promotes skeletogenesis and inhibits thymus development, our findings suggest that TSG acts as both a BMP-4 agonist in skeletogenesis and a BMP-4 antagonist in T-cell development. Although lymphopenia in TSG-deficient mice would partly be ascribed to systemic effects of runtiness and wasting, our findings may also provide a clue for understanding the pathogenesis of human dwarfism with combined immunodeficiency.
