Oncogenic functions of hMDMX in in vitro transformation of primary human fibroblasts and embryonic retinoblasts.

BACKGROUND: In around 50% of all human cancers the tumor suppressor p53 is mutated. It is generally assumed that in the remaining tumors the wild-type p53 protein is functionally impaired. The two main inhibitors of p53, hMDM2 (MDM2) and hMDMX (MDMX/MDM4) are frequently overexpressed in wild-type p53 tumors. Whereas the main activity of hMDM2 is to degrade p53 protein, its close homolog hMDMX does not degrade p53, but it represses its transcriptional activity. Here we study the role of hMDMX in the neoplastic transformation of human fibroblasts and embryonic retinoblasts, since a high number of retinoblastomas contain elevated hMDMX levels. METHODS: We made use of an in vitro transformation model using a retroviral system of RNA interference and gene overexpression in primary human fibroblasts and embryonic retinoblasts. Consecutive knockdown of RB and p53, overexpression of SV40-small t, oncogenic HRasV12 and HA-hMDMX resulted in a number of stable cell lines representing different stages of the transformation process, enabling a comparison between loss of p53 and hMDMX overexpression. The cell lines were tested in various assays to assess their oncogenic potential. RESULTS: Both p53-knockdown and hMDMX overexpression accelerated proliferation and prevented growth suppression induced by introduction of oncogenic Ras, which was required for anchorage-independent growth and the ability to form tumors in vivo. Furthermore, we found that hMDMX overexpression represses basal p53 activity to some extent. Transformed fibroblasts with very high levels of hMDMX became largely resistant to the p53 reactivating drug Nutlin-3. The Nutlin-3 response of hMDMX transformed retinoblasts was intact and resembled that of retinoblastoma cell lines. CONCLUSIONS: Our studies show that hMDMX has the essential properties of an oncogene. Its constitutive expression contributes to the oncogenic phenotype of transformed human cells. Its main function appears to be p53 inactivation. Therefore, developing new drugs targeting hMDMX is a valid approach to obtain new treatments for a subset of human tumors expressing wild-type p53.

Array comparative genomic hybridization with cyanin cis-platinum-labeled DNAs.

Fluorescent cis-platinum compounds that react with the N7 atom of guanine are useful for labeling nucleic acids influorescence hybridization applications. Here we report that cyanin (CyN) cis-platinum labeling of DNA samples for array comparative genomic hybridizations (arrayCGH) can be achieved reproducibly and reliably. We demonstrate that degrees of labeling of approximately 1% of all nucleotides in test and reference DNA samples with CyN3- and CyN5-cis-platinum produces arrayCGH signal-to-background ratios ranging from 30 to 40. The arrayCGH results achieved during analyses of mouse and human tumor samples were comparable to those achieved using enzymatic labeling. Thus, we conclude that Cy-cis-platinum labeling is an alternative to enzymatic labeling for arrayCGH.