Rituximab Sensitizes B-CLL Cells with Inactivated p53 to Fludarabine.

Background: Defects in the p53 gene predispose B-CLL patients for an inferior outcome, particularly a resistance to treatment by conventional chemotherapeutics. Very little data exist, however, about the efficacy of monoclonal antibody rituximab on B-CLL cells bearing p53 abnormalities. One reason for that might be a methodological rituximab alone does not have virtually any effect on the viability of B-CLL cells when cultivated in vitro, unless an active human plasma is added. After that, however, the cells are quickly lysed by complement, what is a process independent on p53. Aims: We used an in vitro system (not containing the active human plasma) to monitor a rituximab activity on B-CLL cells with p53 inactivation in relation to subsequently used nucleoside analog fludarabine, which demonstrably acts through the p53 in B-CLL cells. Methods: The p53 abnormalities in blood samples of B-CLL patients were detected by FISH and by functional yeast analysis coupled to sequencing, as described previously (Trbusek et al., Leukemia 2006, 20: 1159-1161). Vitally frozen samples were used in all cases, after 24h pre-cultivation. Mononuclear cells were cultivated for 72h with or without 20µg/ml of rituximab and subsequently for another 48h with four different concentrations of fludarabine (40µg/ml-0,625µg/ml). The cell viability was determined by a WST-1 assay. A sensitization effect of rituximab pretreatment was determined by an ANOVA analysis, with the value p=0,05 being a threshold for a statistical significance. To monitor an apoptosis (a suppossed mechanism of fludarabine action), the Western blotting was used for the caspase-3 cleavage, which was proved previously to occur in drug-treated B-CLL cells. Results: In the subgroup of eleven p53-wt samples the three cases manifested sensitization by rituximab for fludarabine activity, one case showed an oposite (antagonistic) effect, while there was no significant difference for another seven samples. Among ten p53-mutated samples there was just one case exhibiting no influence of rituximab pretreatment (with the p53 alleles being deletion / 281 Asp→Glu), one sample manifested with antagonistic effect (del / 220 Tyr→Cys), while the remaining eight cases showed a statistically significant sensitization by rituximab (del / truncated protein aa 314, del / no protein, del/ wt protein, del / 248 Arg→Gln, del / 249 Arg→Gly, del / del aa 252, del / del aa 252-254, and a composed mutant 281Asp→Asn / 254 Ile→Thr). We noticed a statistically significant potentiation also in three out of four ATM-deleted samples (ATM is the p53-regulatory kinase). An apoptosis occured after fludarabine addition both in pretreated and control cells, as evidenced by the caspase-3 cleavage in some (but not all) samples. Conclusions: We show, to our konwledge for the first time, that rituximab can significantly sensitize the B-CLL cells bearing different types of p53 mutations to fludarabine. This result warrants further investigation of the mechanism behind. Supported by grant NR8445-3/2005 provided by IGA of the Ministry of Health of the Czech Republic. E-mail: mtrbusek@fnbrno.cz.