A recombinant immunotoxin, BL22 has shown clinical efficacy against hairy cell leukemia and other B-cell non-Hodgkin’s lymphomas. BL22 contains an antibody-derived domain that recognizes CD22, and PE38, a truncated Pseudomonas exotoxin A domain, for inhibition of protein synthesis. Previous studies have shown that Pseudomonas exotoxin A-based immunotoxins exert their cytotoxicity by apoptosis and additional, yet uncharacterized mechanisms. We investigated the cytotoxicity of BL22 in two mantle cell lymphoma lines, NCEB-1 and Granta-519, and in the diffuse, large-cell lymphoma line SU-DHL-4. Presence of the CD22 target was confirmed by flow cytometry. Treatment of NCEB-1 cells to 1 μg/ml BL22 for 24-32h reduced the numbers of viable cells in comparison to cells exposed to a non-binding immunotoxin. Induction of apoptosis, determined by activation of caspase-3 and exposure of phosphatidylserine, was not detected before 48h of exposure. Incorporation of bromo-deoxyuridine into DNA was, however, almost completely inhibited after 24h. Correspondingly, the numbers of G1 phase cells were increased upon treatment with BL22 as determined by flow cytometry. G1-arrest resulted from decreased enzyme activities of cyclin-dependent kinases (cdk)-2 and 4 as assessed in vitro with respective substrates, histone H1 and the retinoblastoma susceptibility (RB) protein. Immunoblotting revealed markedly reduced amounts of cyclins A, E, D1 and D3 whereas cdk2 and cdk4 protein expression levels remained unchanged. Expression of the RB protein was decreased, and the protein was hypophosphorylated in immunotoxin-treated cells. Pulse-labeling with [35S]-labeled amino acids, followed by immunoprecitation of cyclins E and D1 confirmed that BL22 inhibited synthesis of these cyclins. Thus, immunotoxin-induced cell cycle arrest results from insufficient synthesis of regulatory cyclins required for progression through G1 into S-phase. Similar results were obtained in Granta-519 cells. SU-DHL-4 diffuse, large-cell lymphoma cells were highly sensitive to BL22, and G1 arrest was observed as early as after 8–24h in these cells. Expression of cyclins A, D3 and E was decreased while cyclin D1 was not expressed in SU-DHL-4 cells. Interestingly, cell cycle arrest did not prevent from subsequent induction of apoptosis in lymphoma cells continuously exposed to the immunotoxin. In conclusion, we have characterized cell cycle arrest as a previously unknown mechanism that contributes to the cytotoxicity of Pseudomonas exotoxin-based immunotoxins.

Author notes

Corresponding author

Sign in via your Institution