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In vitro 5-fluorouracil sensitivity and Bcl-2/Bax rałio 1385

ct al, I995), moreover p53 and mdm2 proteins constitute an autoregulatory feedback loop in which p53 activity is self-limited through mdm2 production (Kubbutat et al, 1997). Wild-type cel! lines (CAL5I and KB) showed a marked increase in mdm2 tran-script corresponding to mdm2-p53RE and no up-regulation was detccted when p53 gene was mutated or dcleted, except for FaDu. Mutant p53 was prcviously demonstrated to be able to bind mdm2-p53RE, according to the naturę and position of thc amino-acid substitution (Gorgoulis et al, I998). 0’Connor ct al (I993) also demonstrated induction of mdm2 mRNA for three mutant p53 lines, which cxhibitcd altcration of codon 248 as observed in FaDu celi linę. This mutation was classified as ‘contact mutant', sińce residue 248 is directly involved in p53 DNA-binding, and then rcduced the afflnity of p53 for its consensus sites by rcmoving criti-cal contact with DNA (Clio et al, 1994; Arrowsmith and Morin, 1996) without changing p53 confonnation (Ory et al, 1994). p21 mRNA basal expression was rcduced in p53 mutated celi lines in agreement with Elbendary et al (1996). Relalionship could be evidcnced between p2l basal expression and p53 status. p21 mRNA overexprcssion was still detected in all celi lines indepen-dently of p53 status, suggesling that p21 could be up-regulated by other pathways (Maclcod ct al, 1995; Loignon ct al, 1997; Wouters et al, 1999). In all celi lines after 5-FU cxposure p21 induction, mediated or not by p53, results in Gl arrest (el-Dciry ct al, 1993; 1994). Uowever, Gl accumulation was statistically significant only for CAL51, KB, and FaDu lines, which exhibited p53 tran-scriptional functionality leading to Gl arrest, which should be morę related to p53 functionality than p53 status.

Since Bax was identified as a p53 early-response gene (Sclvakumaran et al, 1994), and a uniquc p53-regulatcd gene which induced apoptosis (Zhan et al, 1994), and Bcl-2 as an apop-tosis antagonist (Oltvai et al, 1993; Reed, 1994), lcvcls of Bax and Bcl-2 were analysed in celi lines after cxposurc to 5-FU equitoxic concentrations. Bcl-2 Family proteins were demonstrated to be important apoptosis regulators after 5-FU treatment (Koshiji ct al, 1997; Nita ct al, I998a). I.ikcwisc, CAI.5I celi linę displayed a significant increase in Bax, as well as a significant dccrease in Bcl-2 protein cxprcssion. All p53 mutant ccii lines displayed either no moditication of Bax and Bcl-2 protein expression (CAL33 and FaDu), or a significant decrease in Bax as well as an increase in Bcl-2 protein expression (CAL27 and PANC3) after 5-FU cxpo-sure. Our results also showed that HPV 18 positive KB celi linę, in which p53 was not up-regulated, displayed no Bax protein up-rcgulation, but Bcl-2 down regulation. 5-FU-rcsistanl celi lines {CM21 ct PANC3) showed an increase in Bcl-2 protein cxprcs-sion reported to protect the cells against thymidylate synthase inhibitor (Fisher ct al. 1993), as well as other anticanccr drugs (Reed, 1994). Bcl-2 and Bax induction significantly correlates with 5-FU sensitivily and whatever p53 status, Bcl-2 to Bax rcla-tive expression ratio was also correlated with 5-FU sensitivity.

In vitro, 5-FU sensitivity was related to diffcrcnl mechanisms (Pincdo and Peters, 1988; Spears et al, 1988; Zhang et al, 1992; Peters ct al, 1995). Although each of the mechanisms have been well documcntcd, their rclative contribution to the development of clinical drug resistance remains incertain. However, therc is a growing body of evidence to suggest that sensitivity to the cyto-toxic effects of fluoropyrimidines may be mediated via TS and DPD process (Peters et al, 1995). Although TS and DPD have demonstrated potcntial prognostic significance, their prognostic values are still controversial (Beck et al, 1994; Nita et al, 1998/;; Eticnne ct al, 1999; Kirihara ct al. 1999). Morę recently, Bcl-2 family proteins were implicated in chemothcrapy-induced celi death (Simonian et al, 1997) and previous results suggest that some members of the Bcl-2 family of proteins, in human colon canccr celi lines, are modulated by 5-FU, and that the ratio of Bcl-X(L) to Bax may be related to chemoscnsitivity to 5-FU (Nita ct al, 1998a).

In conclusion, for celi cyclc control, p53 functionality appeared to be morę essential than mutational status. Moreover, whatever p53 status or functionality, 5-FU sensitivity was related to Bcl-2 family proteins expression and Bcl-2/Bax ratio could be a relevant marker to predict 5-FU treatment response.

ACKNOWLEDGEMENTS

This work was |>erformed within the framework of the ‘Póle Europeen de Santd Region Lorrainc Communautó Urbaine du Grand Nancy’ and was supported by the French ‘Liguc contrę Ic Cancer’.

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© 2000 Cancer Research Campaign

British Journal of Cancer (2000) 83(10), 1380-1386