5-fluorouracil (5FU) resistance has been studied extensively. Many mechanisms have been implicated, such as pharmacokinetic resistance, decreased accumulation of activated metabolites, and altered effects on thymidylate synthase (TS; 1,2). Cell cycle disturbance and apoptosis induction also result in 5FU resistance (3–7). TS protein or mRNA expression predictive value was tested clinically in many tumoral pathologies (8–11). However, although the prognostic value of TS was accepted in colorectal tumors, its significance remains debated (9, 12–14). In head and neck pathologies, TS expression was unrelated to 5FU treatment outcome (15, 16). Other parameters were tested to predict 5FU response, but none were universally used or accepted (15, 17). More recently, cell cycle parameters (18) and apoptosis-related factors were implicated in the 5FU response (19, 20).
The study of human tumor proliferation has been facilitated by the development of monoclonal antibodies (mAbs) that recognize halogenated pyrimidines, such as iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU), followed by cytometric analysis. Pyrimidine analogs are incorporated into DNA during S-phase. The cell kinetic information generated by this approach is more informative than that obtained from in vitro incubation with DNA precursors such as tritiated thymidine or by single-parameter DNA analysis (21). The most widely used proliferation parameters are labeling index (LI) and potential doubling time (Tpot). Tpot is defined as the time that would be required by a tumor to double in cell number in the absence of cell loss (22). It can be calculated in vivo by flow cytometric analysis of a tissue biopsy obtained after a single infusion of a thymidine analog, such as IdU or BrdU, which can be detected using a specific mAb, avoiding the use of radioactive isotopes (22, 23). Proliferation index (S-phase fraction or LI) and DNA ploidy cytometric analysis are of clinical interest for making treatment decisions or for predicting response and survival (24–28). As we previously described in vitro, basal LI values are related to 5FU cytotoxicity (18). The present study was designed to compare cell cycle cytometric analysis using monovariate propidium iodide (PI) or bivariate BrdU versus PI labelling, either before or after 5FU exposure. In vitro LI and G1/S subpopulation evaluated using BrdU incorporation are related to 5FU sensitivity, both before and after 5FU treatment.
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- LITERATURE CITED
With regards to clinical response to 5FU-based therapy, only TS expression was evaluated as a prognostic factor (8–11). Although TS expression analysis has demonstrated potential prognostic significance in patients with advanced colorectal cancer (12, 31), its clinical usefulness remains in question. Prospective studies are still required to confirm its importance in adjuvant chemotherapy. TS enzymatic activity can be altered by genomic polymorphism or mutation, phosphorylation level, and subcellular localization. These do not necessarily modify TS protein level but can affect TS prognostic value (32–35). TS is regulated throughout the cell cycle phase (36, 37).
Recent studies demonstrate that cell cycle kinetic studies have clinical value in predicting 5FU sensitivity. S-phase fraction has clinical utility for patients with breast cancer (28) and Tpot can predict patients with a low probability of achieving long-term local control with conventional fractionation of radiotherapy in head and neck carcinoma (26, 28, 38–40). However, standardization and quality control of these parameters have to be improved before Tpot can be implemented for routine use in community settings (28, 41, 42).
Cell cycle distribution or kinetics parameters can also be used to predict 5FU sensitivity because fluoropyrimidine treatment leads to cell cycle arrest (3, 43). In our panel of cell lines, 5FU equitoxic treatment results in G0/G1 accumulation as demonstrated by PI analysis (Table 1, Fig. 2). Cell cycle phase subpopulation was not statistically correlated to 5FU sensitivity. Conversely, significant G0/G1 accumulation induced after 5FU treatment was only observed for the most 5FU-sensitive cell lines (Table 1). However, BrdU analysis showed that a cell cycle delay took place specifically at the G1/S interface as previously reported (44, 45; Table 2, Fig. 3).
As BrdU is a thymidine analog that can be used by cells for DNA synthesis, it can lead to progression from late G1 to early S-phase. Therefore, the influence of BrdU incubation during or after 5FU exposure was assessed both by PI and BrdU analysis (Table 3). Using either PI or bivariate BrdU analysis, no BrdU influence on cell cycle distribution was seen. Therefore, the G1/S subpopulation cannot be considered to be derived from late G1 during BrdU incubation. In fact, inhibition of TS by 5FU was almost irreversible and BrdU could only rescue new synthesized TS molecules. A 20-min BrdU incubation was not enough long to initiate new DNA synthesis.
The LI was statistically correlated to 5FU sensitivity as we have previously described (18), both before and after 5FU exposure (Fig. 5A1,A2). The G1/S subpopulation was also strikingly correlated to 5FU sensitivity (Fig. 5B1,B2). In addition, considering the G1/S interface (Fig. 1), cytometric analysis with bivariate staining analysis (BrdU versus PI) compared with the single-parameter analysis of DNA content (PI) did not provide the same information. Consequently, these two methods will not have the same usefulness for the determination of prognostic indicators. In fact, BrdU analysis showed that G1 cells, as determined by PI analysis, was composed of BrdU-positive and BrdU-negative cell subpopulations (Figs. 1, 3). These subpopulations cannot be distinguished using PI analysis alone. This was the consequence of the cell fraction that was able to incorporate BrdU and maintain its G1 DNA content. This fraction was considered to be in G1 by PI analysis, whereas it was included in the labeled subpopulation by BrdU analysis. Thus, cells performing repair synthesis of DNA, with no net DNA increase, would be included in the LI score, but could still have a DNA content that was indistinguishable from G1 cells. The number of BrdU-positive cells within this peak increased with duration of 5FU exposure and was related to p21 mRNA expression induction (data not shown). On the other hand, differences in molecular mechanisms gave rise to the existence of these two subpopulations. For example, cyclin D is required for progression through early to mid-G1. Following this, expression and activity of cyclin E increase at the G1/S phase border and result in S-phase progression. Cyclin D and E cooperate temporally by successive pRb phosphorylation (46). Synchronization of cells, caused by a delay in G1/S, may be related to programmed cell death initiation. Induced delay in G1/S was representative of apoptosis involvement and of 5FU treatment sensibility. Actually, chromatin condensation and endonuclease-mediated DNA fragmentation were detected using fluorescent microscopy in the most 5FU-sensitive cells (data not shown). The amount of cells displaying DNA fragmentation was also related to 5FU sensitivity (data not shown). 5FU exposure leads to an LI increase. However, LI modifications were only related to G1/S phase accumulation, and not to a real enhanced S-phase fraction. In conclusion, cell cycle parameters using BrdU analysis could be used as predictive markers for 5FU response to complement the predictive value of TS.