Anal Biochem 1999 Aug 1;272(2):171-81
Abstract
Transient transfection of recombinant genes into cells is a commonly used
approach for analyzing cell-cycle- and/or apoptotic-related activities of
cell-cycle control proteins. In this approach, information regarding the
functional consequence of expressing a recombinant protein transiently is
garnered by comparing against results obtained from cells which are
transfected with either a control expression plasmid and/or with mutant
expression plasmids. In general however, little attention is paid to whether
the transfection procedure itself influences these experiments. Using the
calcium phosphate transfection method, we show that the introduction of DNA
into cells induces signaling of the cell-cycle control machinery. In Hela
cells, a transient increase in G0/G1 cells is observed 8 h after
transfection. Furthermore, the introduction of DNA into several cell lines
induces apoptosis. Transfection-mediated apoptosis can be elicited through a
p53-independent mechanism, suggesting the possible extrapolation to many
tumor cell lines. Last, we show that due to a likely cell-cycle-specific
entry of marker genes into the nucleus, a highly biased cell-cycle
distribution is observed in successfully transfected cells at early times
following transfection. The importance of these issues in the interpretation
as well as the design of transient transfection-based cell-cycle experiments
is discussed. Copyright 1999 Academic Press.