G1 and S phase gene expression cannot be analyzed in mammalian cells synchronized by inhibition.

Sequence analyses of mRNA from cells arrested with either a G1 phase DNA content or an S phase DNA content were interpreted as indicating that these two cell populations differentially expressed particular transcripts (Earle-Hughes et al. Genome Sci Technol 1, 89-128, 1996). Approximately 13% of the total transcript population appeared to be differentially expressed between the G1 and S phases of the cell cycle. The question arises if it is possible, using inhibition methods (double-thymidine block to produce cells with an S phase DNA content and inhibition with dibutyryl adenosine 3'5'-cyclic monophosphate and theophylline to produce cells with a G1 phase DNA content), to truly synchronize cells and obtain cell populations representative of specific phases of the division cycle. Here it is argued that this is not possible. Cells may have specific amounts of DNA (i.e., G1 phase or S phase DNA content) yet not be representative of these particular phases. It is proposed that the differential gene expression observed may be due to the effects of the inhibitors and does not necessarily reflect the actual transcription pattern in unperturbed cells in specific cell cycle phases. This proposal has a wide applicability and should not be confined to the specific article under discussion.