AT-101 induced cell death of CLL lymphocytes in both suspension culture as well as stromal coculture. (A,B) Dose and time response to AT-101. CLL lymphocytes in suspension culture were incubated with AT-101 at different concentrations (1, 3, 10, 15, 20, 30 μM; 1A; patients 2 ♦, 18 ○, 4 ▵, and 19 ▿) for 24 hours or with 20 μM AT-101 at 4, 8 and 24 hours; 1B; (patients 2 ♦, 16 ○, 19 ▿, 14 □, and 15 ▵) and the induction of apoptosis was measured by annexin-binding assay. (C,D) AT-101–induced apoptosis is independent of Rai stage or other prognostic factors. CLL lymphocytes in suspension culture were incubated with 20 μM AT-101 for 24 hours and assayed for annexin positivity (“% Apoptosis”). p53 indicates 17p deletion; ATM, 11q deletion; ZAP-70, > 20% ZAP-70 positivity; B2M, β2 microglobulin level higher than 2; and VH, IgV_H gene mutation based on less than 96% nucleic acid sequence homology. (E) AT-101–induced apoptosis in CLL B cells was not abolished by stromal cells. CLL lymphocytes from patients (n = 12) were cultured either in suspension medium (C) in suspension medium with 20 μM AT-101 (C + AT), or with stromal cells in the absence (C + S) or presence of AT-101 (C + S + AT) and the apoptosis was measured after 24 hours (72 hours for samples from patients 31 and 32) by annexin-binding assay. The numbers below the abscissa indicate patient numbers, which coincide with the numbers given in Table S1. (F) Fludarabine-induced apoptosis in CLL B cells was reduced by stromal cell cocultures. CLL lymphocytes from patients (n = 6) were either cultured in suspension medium (C), in suspension medium with 10 μM fludarabine (C + FL) or with stromal cells in the absence (C + S) or presence (C + S + FL) of fludarabine, and the apoptosis was measured after 24 hours (72 hours for samples from patients 31 and 32) by annexin-binding assay. Numbers below the abscissa are patient identification numbers, which coincide with the numbers given in Table S1.