Upregulation of bfl-1 is a potential mechanism of chemoresistance in B-cell chronic lymphocytic leukaemia

Abstract

B-cell chronic lymphocytic leukaemia (B-CLL) is characterised by the progressive accumulation of monoclonal CD5(+) B cells. In a previous study, we have analysed the expression profile of apoptosis-regulating genes using a cDNA-based microarray and found overexpression of the antiapoptotic bcl-2 family member, bfl-1, in B-CLL cells with an apoptosis-resistant phenotype. In this study, bfl-1 mRNA levels have been determined by competitive PCR in an extended population of B-CLL patients to characterise its role in disease progression and development of chemoresistance. bfl-1 levels were significantly higher in patients with no response (NR) to last chemotherapy than in patients responding (partial response (PR)) to last chemotherapy (P<0.05) and in patients who had not required treatment (P<0.05). We found no correlation between bfl-1 mRNA levels and disease progression, IGHV mutational status or other clinical parameters. In addition, bfl-1 mRNA levels were inversely correlated with apoptotic response to in vitro fludarabine treatment of B-CLL cells. Specific downregulation of bfl-1 using siRNA induced apoptosis in resistant cells. Our data suggest that bfl-1 contributes to chemoresistance and might be a therapeutic target in B-CLL.

Figure 1

Correlation between bfl-1 and bcl-2 expression and therapy requirement and outcome bfl-1 (A) and blc-2 (B) mRNA expression was determined using competitive PCR. Results are shown as mRNA expression relative to the median of the sample population. Boxes show intraquartile range. Whiskers correspond to the 10th and 90th percentile, and horizontal line within boxes represents the median value. Data correspond to 17 untreated patients, 13 patients with no response (NR) to treatment and 7 with partial response (PR). bfl-1 comparison NR vs PR, P<0.05 and NR vs untreated, P<0.05. bcl-2 comparison NR vs PR, P<0.05 and PR vs untreated, P<0.01. (C) Comparison of bfl-1 and bcl-2 mRNA expression, as determined by competitive PCR, and therapy outcome. Data are represented as fold increase/decrease mRNA expression relative to the median of the sample population.

Figure 2

Correlation between bfl-1 expression and in vitro fludarabine-induced apoptosis. Isolated B cells (0.5 × 10⁶) from B-CLL patients (all 37 patients included in the study, Table 1) were cultured in 96-well plates in medium alone (non-supplemented with FBS) or with fludarabine (5 μM). Apoptosis was measured after 48 h of culture using AnnexinV staining and fludarabine-specific apoptosis was calculated as described in Materials and Methods section. Cells were considered resistant if specific fludarabine-induced apoptosis was less than 25% (resistant, n=11; sensitive, n=26). bfl-1 mRNA expression was determined before culture using competitive PCR. Results are shown as mRNA expression relative to the median of the sample population. Boxes show intraquartile range. Whiskers correspond to the 10th and 90th percentile, and horizontal line within boxes represents the median value. Comparison: resistant vs sensitive, P<0.01.

Figure 3

Correlation between bfl-1 and bcl-2 expression and chromosomal aberrations. The prognostically relevant chromosomal aberrations del(13q), trisomy 12, del(11q) and del(17p) were determined by FISH analysis in 28 B-CLL patients included in the study (Table 1). Patients were grouped in good prognosis del(13q)/‘normal’ group (n=14) and intermediate/bad prognosis trisomy 12/del(11q)/del(17p) group (n=13). bfl-1 and blc-2 mRNA expression was determined using competitive PCR and results are shown as mRNA expression relative to the median of the sample population. Boxes show intraquartile range. Whiskers correspond to the 10th and 90th percentile, and horizontal line within boxes represents the median value. Comparison del(13q)/‘normal’ vs trisomy 12/del(11q)/del(17p) for bfl-1, P<0.01, and for bcl-2 P<0.02.

Figure 4

siRNA-mediated targeting of bfl-1 in B-CLL cells. B-CLL cells from four in vitro fludarabine-resistant and bfl-1 high-expressing cases (CLL-24, CLL-32, CLL-27 and CLL-33) were transfected with bfl-1-specific siRNA, control siRNA oligo or were not transfected. At 24 h cells were harvested, mRNA was isolated and analysed for bfl-1 mRNA expression by RT–PCR. (A) At the same time apoptosis was quantified using AnnexinV staining to quantify apoptosis. (B) Data show one representative experiment out of two.