Correlation between the levels of survivin and survivin promoter-driven gene expression in cancer and non-cancer cells.
Survivin, a member of the inhibitor of apoptosis (IAP) protein family, is associated with malignant transformation and is over-expressed in most human tumors. Using lipoplex-mediated transfection, we evaluated the activity of the reporter enzyme, luciferase, expressed from plasmids encoding the enzyme under the control of either the cytomegalovirus (CMV) or survivin promoters, in tumor- and non-tumor-derived human and murine cells. We also examined whether there is a correlation between the survivin promoter-driven expression of luciferase and the level of endogenous survivin. Human cancer cells (HeLa, KB, HSC-3, H357, H376, H413), oral keratinocytes, GMSM-K, and chemically immortalized human mammary cells, 184A-1, were transfected with Metafectene at 2 microl/1 microg DNA. Murine squamous cell carcinoma cells, SCCVII, mouse embryonic fibroblasts, NIH-3T3, and murine immortalized mammary cells, NMuMG, were transfected with Metafectene PRO at 2 microl/1 microg DNA. The expression of luciferase was driven by the CMV promoter (pCMV.Luc), the human survivin promoter (pSRVN.Luc-1430), or the murine survivin promoters (pSRVN.Luc-1342 and pSRVN.Luc-194). Luciferase activity was measured, using the Luciferase Assay System and expressed as relative light units (RLU) per ml of cell lysate or per mg of protein. The level of survivin in the lysates of human cells was determined by ELISA and expressed as ng survivin/mg protein. In all cell lines, significantly higher luciferase activity was driven by the CMV promoter than by survivin promoters. The expression of luciferase driven by the CMV and survivin promoters in murine cells was much higher than that in human cells. The cells displayed very different susceptibilities to transfection; nevertheless, high CMV-driven luciferase activity appeared to correlate with high survivin-promoter driven luciferase expression. The survivin concentration in lysates of cancer cells ranged from 5.8 +/- 2.3 to 24.3 +/- 2.9 ng/mg protein (mean, 13.7 ng/mg). Surprisingly, elevated survivin protein was determined in lysates of non-tumor-derived cells. Survivin levels for GMSM-K and 184A-1 cells, were 16.7 +/- 8.7 and 13.5 +/- 6.2 ng/mg protein, respectively. The expression of endogenous survivin did not correlate with the level of survivin promoter-driven transgene activity in the same cells. The expression of survivin by non-tumorigenic, transformed cell lines may be necessary for their proliferative activity. The level of survivin promoter-driven gene expression achieved via liposomal vectors in OSCC cells was too low to be useful in cancer-cell specific gene therapy.
Department of Microbiology, University of the Pacific, Arthur A. Dugoni School of Dentistry, San Francisco, CA 94115, USA. kkonopka@pacific.edu
Krystyna
K
Konopka
Christopher
C
Spain
Allison
A
Yen
Nathan
N
Overlid
Senait
S
Gebremedhin
Nejat
N
Düzgüneş
D000818:Animals; D016923:Cell Death; D045744:Cell Line, Tumor; D003587:Cytomegalovirus / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D000077022:Survivin; D014162:Transfection; D019076:Transgenes
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D000077022:Survivin; D008156:Luciferases