The use of ‘double-thymidine block’ was the first widely accepted method for inducing cell synchrony and remains one of the most effective and frequently used techniques for analyzing the cell cycle today. While thymidine is in itself an inhibitor of DNA replication, thymidine blocks are typically used to generate cell synchrony at the G1/S boundary. We have previously presented the first evidence that shows the growth imbalance and altered expression levels of cyclins A, B1, D3 and E in MOLT-4 cells synchronized in the cell cycle by thymidine. The major objective of the present study was to compare the levels of cyclins A, B1, D3 and E in G1 phase of the cell cycle between synchronized and unperturbed asynchronously growing human lymphocyte leukemia MOLT-4 cells. Here, we demonstrate that the sorted, asynchronously growing MOLT-4 cells had considerably lower levels of cyclins A, B1, D3 and E than their counterparts of the cells arrested in G1/S phase, as assessed by flow cytometry. In addition, we confirmed these results by using post-sorting Western blotting, a new method we recently developed for examining protein expression in specific phases of sorted, synchronized or asynchronously growing cells. Our findings revealed that the levels of cyclins D3 and E in the asynchronously growing MOLT-4 cells were significantly lower than those in synchronized cultures. Interestingly, protein expression levels of cyclins A and B1 in the asynchronously growing MOLT-4 cells were barely measurable, suggesting that these proteins were either not expressed or under detectable levels. These studies indicate that our synchronization protocol may have disturbed cell proliferation and metabolism as evidenced by significant differences in the expression of cyclins between asynchronously growing and synchronized cells, and further suggest that the levels of cyclins A, B1, D3 and E in synchronized cultures cannot represent those in unperturbed, asynchronously growing cells. Thus, it appears that thymidine-treated, synchronized cells may not be suitable experimental models for the study of normal cell cycle.

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