Biochemical and Biophysical Research Communications
Drm/Gremlin transcriptionally activates p21Cip1 via a novel mechanism and inhibits neoplastic transformation
Section snippets
Materials and methods
Cell Culture. The cerebellar primitive neural ectodermal tumor (PNET-MB) cell line Daoy (HTB186) and the osteosarcoma-derived Saos-2 (HTB-85) were acquired from the American type culture collection. Cells were routinely maintained in DMEM supplemented with 10% fetal bovine serum (Life Technologies) and antibiotics.
Plasmid construction and transfections. An inducible Drm expression construct was generated using the ecdysone-inducible vector system obtained from Invitrogen. The HindIII/XbaI
Drm overexpression inhibits anchorage-independent growth in vitro and tumorigenesis in vivo
We generated ecdysone-inducible Daoy cells by both cotransfection and sequential transfection of the pVgRXR and pIND/Drm constructs, and control cells containing an empty pIND vector were generated in the same way. Western blot analysis showed that both glycosylated and non-glycosylated Drm protein expression [4] were inducible by ponasterone A in a concentration- and time-dependent manner, while no protein was detected in uninduced Daoy cells. Drm was detectable within 4 h of induction and the
Discussion
Regulators of the cell cycle frequently are directly involved in tumorigenesis and the genes encoding these regulators, including tumor suppressor genes, oncogenes, and DNA repair genes, perform functions that are often altered during oncogenesis [22]. In this report, we demonstrate that elevating Drm expression is sufficient to inhibit neoplastic transformation of Saos-2 and Daoy cells, and that this elevation correlates with increased levels of p21Cip1 protein, together with a decreased level
Acknowledgements
We thank Dr. Burt Vogelstein for providing the p21Cip1 promoter reporter construct, Dr. Draginia Djurickovic, and Louise Cromwell, SAIC-Frederick, for performing the animal experiments, Dr. Narayan Bhat and the SAIC Molecular Technology Group for performing the real-time PCR experiments, and Karen Cannon for preparation of the manuscript. This publication has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No.
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