In vitro models for tumor protein d52 function in cancer cells
© BioMed Central 2005
Published: 17 June 2005
Tumor protein D52 (TPD52), a tumor-associated antigen, is overexpressed in the majority of breast, prostate and ovarian cancers, where it is also amplified in a proportion of cases. Comparing TPD52 (or D52) protein expression with clinical parameters indicates that increased D52 expression is an early event in the development of prostate cancer and possibly other cancers. The D52 protein is a member of a family that includes the related proteins D53 and D54. Studies to date indicate that while D52-like proteins share common molecular functions as putative adaptor proteins, D52-like genes are not equally overexpressed or targeted by gene amplification in cancer. As a first step in allowing the specific targeting of D52 overexpression in cancer, it is therefore imperative to determine the effects of increasing or reducing the expression of D52 and related proteins in mammalian cells.
Expression vectors encoding human D52, two human isoforms of both D53 and D54, and mouse D52 have been constructed in the pCDNA3.1 vector. Similarly, expression constructs have been derived that encode pEGFP-tagged forms of human D52, and two human D53 isoforms. Transient and stable DNA transfections were carried out using Lipofectamine 2000 reagent into the MDA-MB-231 human breast carcinoma cell line, and the Balb/c 3T3 fibroblastic cell line. In addition, an Ambion pSilencer system is being developed to reduce D52 expression in MCF-7 breast carcinoma cells. Protein expression in transfected cell populations is assessed using western blotting and indirect immunofluorescence. Cell proliferation rates are assessed using MTT assays, and anchorage-independent growth is assessed by quantitating colony formation in soft agar after a 2-week assay period.
We have previously studied the effects of expressing D52 or D53 in MDA-MB-231 breast carcinoma cells, which express relatively low levels of both proteins. While stably transfected MDA-MB-231 cell lines could be derived when D52 or D53 were expressed from the β-actin promoter, these could not be obtained when pEGFP-tagged D52 and D53 or untagged D53 were expressed from the CMV promoter. Transient transfections revealed that expression of these proteins commonly produced high proportions of multinucleated cells (40–44% by 24 hours post transfection), compared with vector controls. Expressing mouse D52 under the control of the CMV promoter increased the cellular proliferation rate in pooled stable 3T3 transfectants, relative to the vector alone, as measured using MTT assays. We are currently carrying out similar transfections with additional D52-like expression constructs in 3T3 cells, and knocking down D52 expression in MCF7 breast carcinoma cells, which express high levels of D52 and D53 proteins.
These studies indicate that the exogenous expression of D52-like proteins produces different phenotypes in different cell types. Expressing multiple D52-like proteins in MDA-MB-231 cells adversely affects their ability to complete mitosis, whereas increased proliferation rates in mouse D52-expressing 3T3 cells support previous results implicating D52 as a regulator of cell proliferation in human leukemic and chick neuroretinal cells. Further studies are now required to determine whether reducing D52 expression negatively impacts upon the growth of breast cancer cells.