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Updated: Aug 5, 2026

Analysis of Cell Cycle Position in Mammalian Cells
Published on: January 21, 2012
Cell cycle regulation of the G0/G1 transition in 5-fluorouracil-sensitive and -resistant human colon cancer cell
C J McGinn1, B C Pestalozzi, J C Drake
1Department of Radiation Oncology, University of Michigan, Ann Arbor, USA.
Purpose:
Resistance to 5-fluorouracil (5-FU) has been associated with thymidylate synthase (TS) gene amplification and increased TS protein levels. Increased TS protein expression has also been found to be a significant independent prognostic factor for disease-free survival and overall survival in patients treated with adjuvant 5-FU-based chemotherapy. In these studies and in our prior preclinical studies, TS has been considered a marker of proliferative capacity. The purpose of the current study was to further evaluate the association between TS levels and cell cycle regulation, by investigating cell cycle kinetics in a 5-FU-resistant cell line with constitutive overexpression of TS. The influence of increased TS levels on cell cycle progression may provide insight into methods to overcome 5-FU resistance.
Materials:
5-FU-sensitive NCI H630(WT) and 5-FU-resistant NCI H630(R1) (with 15- to 20-fold higher TS protein levels) were utilized in this investigation to determine the influence of constitutive overexpression of TS on cell cycle kinetics.
Results:
There was no apparent influence of increased TS levels on cell cycle distribution during asynchronous growth, and both cell lines reach plateau growth phase in 120 hours, arresting in G0/G1 as determined by flow cytometry. In the H630(WT) cells, this G0/ G1 arrest was associated with a 14- to 17-fold reduction in TS activity and protein levels (using the TS-106 monoclonal antibody), whereas in the H630(R1) cells, only a two- to fivefold reduction was noted. Flow cytometry analysis utilizing Ki-67 indicated that there was no evidence of a G0 population in the confluent H630(R1), whereas 26% +/- 7% of confluent H630(WT) cells were Ki-67 negative (G0) and the remainder had low Ki-67 signal intensity. Analysis of pRb phosphorylation and p16 and p21 expression suggested that the arrest point for both cell lines was before the point at which Rb phosphorylation takes place, yet the confluent H630(R1) cells had threefold higher p21 than confluent H630(WT) cells.
Discussion:
These data suggest that the 5-FU-resistant H630(R1) cell lines arrest at a later point in G0/G1 and have a potentially greater capacity for proliferation.
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