Type of serum influences the rituximab dependent cytotoxicity and apoptosis of chronic lymphocytic leukemia cells in vitro

Abstract

The aim of the study was to compare the influence of types of serum on the in vitro viability and on either spontaneous or rituximab (RIT)-induced apoptosis of chronic lymphocytic leukemia (CLL) cells. The influence of fetal calf serum (FCS), patients' autologous serum (AS) and human AB-serum (ABS), used alone and in combinations consisting of two of them (v/v-1:1), on RIT-dependent cytotoxicity, apoptosis, detection of active forms of caspases-3,-9,-8 and disruption of mitochondrial membrane potential (ΔΨm) were assessed by flow cytometry. RIT was used at the concentration of 10 µg/ml. The spontaneous apoptosis was assessed in culture without RIT. AS revealed the protective action on CLL cells, however this serum added in vitro to the culture either alone or in combination with FCS was the only one to allow RIT to exert its cytotoxic action against CLL cells. RIT-induced apoptosis involved changes in ΔΨm and activation of caspases-3,-8,-9 when AS+FCS was applicated. Drug induced apoptosis (DIA) was 6.02 and 0.34, when FCS+AS and FCS alone were used, respectively (p<0.01). The RIT-dependent cytotoxic effect decreased when FCS+AS or FCS+ABS were used, as compared to effect of AS used separately. The cytotoxic effect of RIT did not depend on drug concentration, but on the type of serum added to the culture. The strongest cytotoxic effect of RIT in the presence of AS suggests that this drug activity towards CLL cells is enhanced by known cytotoxic mechanisms, caspase-dependent apoptotic pathway and possible influence of other extracellular factors present in the patients' sera.