Figure 1 . Transient transfection methods differentially induce hsp 72. Immunofluorescent staining for hsp 72 was determined by flow cytometric analysis and data presented as percent expression over non-transfected controls. ( A ) The expression of hsp 72 was determined 24 h following calcium phosphate and DEAE-dextran methods of transfection in HeLa cells. Percent expression of hsp 72 was determined in HUT 78 cells following electroporation. ( B ) Hsp 72 expression was determined following liposome-mediated transfection of varied times in serum-free conditions. * +- s.e.m., n = 3 (liposome-mediated, calcium phosphate, DEAE-dextran).

HeLa cells were transfected by liposome-mediated transfer using a commercially available product (Transfectam®, Promega, Madison, WI). Hela cells were transfected for 3, 6 or 24 h following the recommended assay protocol for media without serum by the manufacturer. Following transfections, media was removed and cells returned to regular culture conditions (Eagles's minimal essential medium supplemented with 10% FCS, 1.2 mM glutamine, 60 U/ml penicillin and 60 [mu]g/ml streptomycin) and hsp 72 expression determined 24 h later. In HeLa cells the serum-free conditions of liposome-mediated transfection resulted in an increased expression of hsp 72 which correlated with the amount of time the cells were cultured in serum-free media (Fig. 1 B). Serum-free conditions for 3 h did not result in hsp 72 expression as determined by flow cytometry. Cells left in contact with serum-free medium for 6 and 24 h had a mean expression (16 and 37%) of hsp 72 respectively (Fig. 1 B).

Hela cells were transfected by calcium phosphate mediated transfer (5 Prime -> 3 Prime, Inc., Boulder, CO) or DEAE-dextran-mediated transfer (Promega, Madison, WI) using the recommended assay protocol by the manufacturer. Calcium phosphate-mediated transfections did not elicit increased expression of hsp 72 in HeLa cells (Fig. 1 A). DEAE-dextran-mediated transfection resulted in a mean expression of 16% of hsp 72 in transfected cells as compared to non-transfected controls (Fig. 1 A).

While many chemical or physical agents are known to induce the stress response, the mechanisms in which they mediate the response with subsequent enhancement of hsp gene expression is complex and not completely understood ( 2 ). Electroporation mediates entry of plasmid DNA into cells by generating an electric field which creates cellular pores through which the DNA diffuses. However, the high-voltage electrical pulse can result in membrane disruption and alteration of protein structure (i.e. denaturation) ( 6 ) which are known to induce the stress response ( 7 - 9 ).

Serum-free conditions greatly enhance transfection efficiency when using liposome-mediated DNA delivery system ( 10 ). Liposome-mediated transfections in serum-free conditions are often performed overnight, however we found that the degree of expression of hsp 72 correlated with the amount of time the cells remain in serum-free conditions before returning to normal culture condition (i.e. serum-stimulation). Serum-stimulation following serum-free conditions is a potent inducer of the stress response ( 11 ). Therefore, when analyzing stress responsive genes, long transfection times with this method may inhibit or mask a stress-mediated response.

The liposome-mediated DNA delivery system resulted in the greatest transfection efficiency in HeLa cells as compared to calcium phosphate and DEAE-dextran (data not shown). This was determined by comparing reporter gene activity following transfection with an SV40 luciferase reporter plasmid (pGL2-Control, Promega, Madison, WI). Because of the pronounced induction of hsp 72 in HUT 78 cells following electroporation, we excluded this method from promoter analysis studies. A 3 h transfection time with liposome-mediated transfer (i.e. serum-free conditions) did not result in increased expression of hsp 72 by flow cytometric analysis (Fig. 1 B), therefore we chose this transfection protocol for subsequent kinetic experiments.

Even though no increase in expression of hsp 72, as compared to controls, was found with a 3 h transfection time, we wanted to establish if the cells still required a recovery period before stress induction. To test the influence of an optimized transfection method on promoters known to be responsive to cellular stress, we examined both the human T-cell lymphotropic virus type 1 long terminal repeat (HTLV-1-LTR) ( 12 ) and human cytomegalovirus immediate early transcriptional unit (CMV-IE) ( 13 ). The CMV-IE luciferase plasmid contains the immediate early gene cloned into the Xho I- Sal I site of a luciferase reporter plasmid (pGL2-Basic). The stress response was induced in HeLa cells with 640 [mu]M of Na arsenite as previously described ( 12 ) at 6 or 24 h following transfection. Luciferase activity was measured by liquid scintillation counting following the manufacturer's recommendations (Promega) in cell extracts collected at 3, 6, 12 and 24 h post-stress and compared to non-stressed extracts. Both promoter constructs were non-responsive to cell stress when only allowed a 6 h recovery prior to induction of the cellular stress response by Na arsenite (Fig. 2 A and B). However,when the cells remained in normal culture conditions for 24 h before induction of stress, an increase in promoter activity was observed at 3, 6, 12 and 24 h following transfection (Fig. 2 C and D).Our data indicates that maximal stress responsive gene activity with the HTLV-1-LTR and CMV-IE promoters is dependent on the cells being maintained in normal culture conditions for 24 h before induction of the cellular stress response. The lack of promoter responsiveness with only a 6 h recovery period may be related to multiple variables,which includes phase of cell cycle (e.g. G ₁ /S block) or development of thermotolerance initiated by the serum-free to serum-stimulation conditions of the liposome-mediated transfections ( 14 ). Thus, the cells must be allowed to recover >6 h before maximal induction of the stress response can be elicited.

Figure 2 . Maximal stress induced increase in promoter activity was detected after HeLa cells rested for 24 h following liposome-mediated transfection. The stress response was induced at 6 h ( A and B ) or 24 h ( C and D ) following transfection with an HTLV-1-LTR reporter construct and a CMV-IE reporter construct. Reporter gene activity was determined at 3, 6, 12 and 24 h following stress induction. Luciferase activity of extracts from non-stressed transfected cells was assigned a value of 1.0. The data is presented as the fold increase in luciferase activity.

In conclusion, we found that certain transfection techniques do result in induction of the cellular stress response. Therefore, when analyzing stress-mediated changes in gene expression by transient transfection methods, the technique itself may mask any observed changes. Additionally, we demonstrate that the maximal stress-mediated enhancement of transcription with both promoters did not occur until the cells recovered for 24 h following transfection. In this study we used two viral promoters as a model system to study stress-mediated enhancement, however our data have important implications for investigations of any cellular stress responsive genes employing these commonly used transfection methods.

ACKNOWLEDGEMENT

This work was supported by the National Institutes of Health (National Institute of Allergy and Infectious Diseases grant K11 AI01190).

REFERENCES

1 Tjian, R. and Maniatis,T. (1994) Cell 77, 5-8. MEDLINE Abstract

2 Nover, L. (1991) Inducers of hsp synthesis: heat shock and chemical stressors, in Nover, L. (ed.), Heat Shock Response. CRC Press, Inc. Baca Raton, Florida, pp. 6-40.

3 Welch, W. J., Kang, H. S., Beckmann, R. P. and Mizzen, L. A. (1991) Response of mammalian cells to metabolic stress; changes in cell physiology and structure/function of stress proteins, in Kaufmann,S.H.E. (ed.) Current Topics in Microbiology and Immunology, vol. 167. Springer-Verlag Berlin, Heidelberg, pp. 31-55.

4 Gazdar, A. F., Carney, D. N., Bunn, P. A., Russell, E. K., Jaffe, E. S., Schechter, G. P. and Guccion, J. G. (1980) Blood 55, 409-417.

5 Andrews, J. M., Oglesbee, M. J., Trevino, A. V., Guyot, D. J., Newbound, G. C. and Lairmore, M. D. (1995) Virology 208, 816-820.

6 Neumann, E., Schaefer-Ridder, M., Wang, Y. and Hofschneider, P. H. (1982) EMBO J. 1, 841-845. MEDLINE Abstract

7 Edington, B. V., Whelan, S. A. and Hightower, L. E. (1989) J. Cell. Physiol. 139, 219-228.

8 Thomas, B. P. and Mathews, M. B. (1984) Mol. Cell. Biol. 4, 1063-1072.

9 Lee, K.-J. and Hahn, B. M. (1988) J. Cell. Physiol. 136, 411-420. MEDLINE Abstract

10 Barthel, F., Remy, J. S., Loeffler, J. D. and Behr, J. P. (1993) DNA Cell Biol. 12, 553-560. MEDLINE Abstract

11 Wu, B. J. and Morimoto, R. I. (1985) Proc. Natl. Acad. Sci. USA 82, 6070-6074.

12 Andrews, J. M., Newbound, G. C., Oglesbee, M. J., Brady, J. N. and Lairmore, M. D. (1997) J. Virol. 71, 741-745.

13 Geelen, J. L. M. C., Boom, R., Klaver, G. P. M., Minnaar, R. P., Feltkamp, M. C. W., Van Milligen, F. J., Sol, C. J. A. and Van der Noordaa, J. (1987) J. Gen. Virol. 68, 2925-2931.

14 Missen, La. A. and Welch, W. J. (1988) J. Cell Biol. 106, 1105-1116.

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*To whom correspondence should be addressed at present address: Department of Microbiology, Pathology and Parasitology, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA. Tel: +1 919 515 7406; Fax: +1 919 515 4237; Email: jan_andrews@ncsu.edu
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