VARIABLE GENE EXPRESSION IN DIFFERENT HUMAN ORAL CANCER CELLS
DRIVEN BY THE SURVIVIN OR CYTOMEGALOVIRUS PROMOTERS
Allison Yen1*, Nathan Overlid2, Fenghzi Li3, Nejat Düzgüneş2 and Krystyna Konopka2
1
Doctor of Dental Surgery Program, 2Department of Microbiology, University of the Pacific,
Arthur A. Dugoni School of Dentistry, San Francisco, CA 3Department of Pharmacology and
Therapeutics, Roswell Park Cancer Institute, Buffalo, NY
OBJECTIVES: Survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, is
associated with malignant transformation and is over-expressed in oral squamous cell carcinoma
(OSCC), but is undetectable in most normal adult tissues. This property may be exploited for the
expression of therapeutic genes specifically in cancer cells. We examined the activity of the
reporter gene, luciferase, expressed from plasmids encoding the enzyme under the control of
either the cytomegalovirus promoter (pCMV.Luc) or the human survivin promoter (pSRVN.Luc)
in human OSCC and non-tumor-derived cells.
METHODS: KB cells derived from epidermoid carcinoma of the nasopharynx, HSC-3, H357,
and H413 cells derived from SCCs of the tongue, and non-tumor-derived human oral
keratinocytes, GMSM-K, were seeded in 48-well culture plates one day before transfection, and
used at ~80% confluence. The cells were transfected with a polycationic liposome, Metafectene
(Biontex Laboratories GmbH, Munich) at a ratio of 2 µl Metafectene:1 µg DNA. Transfection
efficacy was evaluated by measuring luciferase activity, using the Luciferase Assay System
obtained from Promega and a Turner Designs TD-20/20 luminometer. The data were expressed
as relative light units (RLU) per ml of cell lysate, or per mg of protein (determined by the BioRad BCA assay).
RESULTS: In all five cell lines, significantly higher level of luciferase activity was driven by
the CMV promoter when compared to luciferase expression under the control of the human
survivin promoter. Although in HSC-3 cells the luciferase activity driven by pSRVN.Luc was
relatively high (1,700 ± 890 RLU/ml), the other three tumor cell lines did not display notably
greater expression than that in the non-tumor GMSM-K cells. The four OSCC cell lines
displayed very different susceptibilities to transfection by pCMV.Luc. When compared to KB
and HSC-3 cells (63,000 ± 15,000 and 33,000 ± 3,000 RLU/ml, respectively) the expression of
luciferase in H357 and H413 cells was much lower (1200 ± 580 and 3400 ± 680 RLU/ml,
respectively).
CONCLUSIONS: The inability of the survivin promoter to drive high-level gene expression
appears to preclude its potential use in gene therapy. The rationale for the different susceptibility
to transfection observed with different cells may include differences in binding, endocytosis and
intracellular transport of liposome-DNA complexes, as well as transcription activity. Future
studies to elucidate the nature of these factors and their effects on the transfection process may
enable us to develop improved gene transfer vectors for eventual gene therapy applications.
This work was supported by Research Pilot Project Award 03-Activity 040 from the Arthur A.
Dugoni School of Dentistry, and an AADR Research Fellowship (A. Yen).