3416400803

© 2002 Wilcy-Liss, Inc.

Cytometry 48:6-13(2002)

G_a/S but not Go/Gj Celi Fraction Is Related to

5-Fluorouracil Cytotoxicity

J.-F. Młrjolet, C. Didelot, M. Barberl-Heyob,* andJf.-L. Merlln

Centre Alexis Yautrin, Laboratoire de Recherche en Oncologie, Vandoeuvre-les-Nancy, France

Rccełvcd 21 Scptcmbcr 2000; Rcvision Rcccivcd 4 Scptcmbcr 2001; Acccptcd 20 Scptcmbcr 2001

Background: Bromodeoxyuridine (BrdU) celi cycle analysis using łlow cytometry is of clinical intercst for making treatment decisions or for predicting response and sur-vival, through prolifcration ratę (labeling index or S-phase fraction) assessment or T_pot calculation. Thymidylate syn-thase expression was tested in vitro, in vivo, and clinically as a prognosdc factor for 5-fluorouracil (5FU) sensitivity. Howevcr, results were still controversial. Moreover, we had reported that 5FU sensitivity was related to the labeling index of untreated celi cultures.

Methods: We used six human cancer celi lines that ex-hibited a wide rangę of 5FU sensitivity. Celi cycle analysis was performed using flow cytometry monovariate pro-pidium iodide (PO analysis and bivariate distributions of BrdU incorporation versus DNA content. 5FU sensitivity was assayed using a 3<4,5-dimethylthiazol-2-yI>2,5-diphe-nyl-2H-tetrazolium bromide (MTO colorimetric assay.

Results: In all celi lines, 5FU exposure resultcd in a statistically signifkant G_x/S accumulation. No statistically significant relationship was seen between Gq/G, delay determined by monovariatc analysis and 5FU sensitivity. However, 5FU sensitivity was statistically correlated to the labeling index and G,/S subpopulation assessed with bi-variate analysis using BrdU incorporation versus DNA content.

Concluslons: Cellular proliferation parameters using BrdU incorporation are morę informative than PI for in vitro 5FU sensitivity. Because BrdU incorporation could be assessed clinically, it could also be informative for 5FU clinical response prediction. Cytometry 48:6-13, 2002. © 2002 Wilcy-Liss, Inc.

Key terms: 5FU sensitivity; celi cycle; G,/S delay; labeling index; bromodeoxyuridine; flow cytometry

5-fluorouracil (5FU) resistance has been studied exten-sively. Many mechanisms have been implicated, such as pharmacokinetic resistance, decreased accumulation of activated metabolites, and altered effeets on thymidylate synthase (TS; 1,2). Celi 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 outeome (15,16). Other parameters were tested to predict 5FIJ response, but nonę were universally used or accepted (15,17). Morę rccently, celi cycle parameters (18) and apoptosis-related factors were implicated in the 5FU respoase (19,20).

The study of human tumor proliferation has been facil-itated by the development of monoclonal antibodies (mAbs) that recognize halogenatcd pyrimidines, such as iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU), followed by cytometric analysis. Pyrimidine analogs are incorporated into DNA during S-phase. The celi kinetic informadon generated by this approach is morę infomia-tive than that obtained from in vitro ineubadon with DNA precursors such as tritiated thymidine or by single-param-eter DNA analysis (21). The most widely used proliferation parameters are labeling index (LI) and potential doubling time (T^t). T^, is defined as the time that would be required by a tumor to double in celi number in the absence of celi 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). Proliferadon index (S-phase fraction or U) and DNA ploidy cytometric analysis are of clinical interest for making treatment decisions or for prediedng 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

Grant sponsor: Liguc contrc Ic Canccr; Póle Kuropćcn de Santć: Region l.orrainc; Communautć Urbainc du Grand Nancy; Alcxis Vautrin Canccr Center privatc rcsearch funds.

•Corrcspondcncc to: Muriel Barl>cri-Heyob, PhD, laboratoire dc Re-chcrche en Oncologie, Ccntrc Alcxis Vautrin, Avcnuc dc Bourgognc, P-54511 Vandocuvrc-lcs-Nancy Ccdcx, France.

E-mall: [e-mail]

Publishod onlinc in Wilcy InterScicncc (www.intcrscicnccAvilcy.com).

DOI: 10.1002/cyto. 10087