Cell cycle regulation of the G(0)/G(1) transition in 5-fluorouracil-sensitive and -resistant human colon cancer cell lines

PURPOSE Resistance to 5-fluorouracil (5-FU) has been associated with thymid ylate synthase (TS) gene amplification and increased TS protein levels. Inc reased TS protein expression has also been found to be a significant indepe ndent prognostic factor for disease-free survival and overall survival in p atients treated with adjuvant 5-FU-based chemotherapy. In these studies and in our prior preclinical studies, TS has been considered a marker of proli ferative capacity. The purpose of the current study was to further evaluate the association between TS levels and cell cycle regulation, by investigat ing cell cycle kinetics in a 5-FU-resistant cell line with constitutive ove rexpression of TS. The influence of increased TS levels on cell cycle progr ession may provide insight into methods to overcome 5-FU resistance. MATERIALS 5-FU-sensitive NCI H630(WT) and 5-FU-resistant NCl H630(R1) (with 15- to 20-fold higher TS protein levels) were utilized in this investigati on 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 cycl e distribution during asynchronous growth, and both cell lines reach platea u growth phase in 120 hours, arresting in G(0)/G(1), as determined by flow cytometry, In the H630(WT) cells, this G(0)/G(2) 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 fi vefold reduction was noted. Flow cytometry analysis utilizing Ki-67 indicat ed that there was no evidence of a G(0) population in the confluent H630(R1 ), whereas 26% +/- 7% of confluent H630(WT) cells were Ki-67 negative (G(0) ) and the remainder had low Ki-67 signal intensity. Analysis of pRb phospho rylation and p16 and p21 expression suggested that the arrest point for bot h cell lines was before the point at which Rb phosphorylation takes place, yet the confluent H630(R1) cells had threefold higher p21 than confluent H6 30(WT) cells. DISCUSSION These data suggest that the 5-N-resistant H630(R1) cell lines ar rest at a later point in G(0)/G(1) and have a potentially greater capacity for proliferation.