Alun Thomas

Antisense-mediated suppression of Bcl-2 highlights its pivotal role in failed apoptosis in B-cell chronic lymphocytic leukaemia.

Although advances have been made in the development of more effective treatment modalities, B‐cell chronic lymphocytic leukaemia (B‐CLL) remains incurable due to the development of drug resistance. Defective programmed cell death mechanisms rather than dysregulation of cell cycle appears to predominate in B‐CLL and it is likely that a failure to initiate apoptosis contributes to chemoresistance. Most B‐CLL cells contain high levels of the anti‐apoptotic protein Bcl‐2 and high Bcl‐2/Bax ratios have been associated with in vitro resistance to cytotoxic agents. In this study we evaluated the cellular responses to a Bcl‐2 antisense oligonucleotide in terms of Bcl‐2 mRNA and protein expression and the induction of apoptosis. The antisense molecule induced a specific reduction in Bcl‐2 mRNA and protein expression over the 48 h culture period and was associated with increased apoptosis. The study indicates that Bcl‐2 protein is central to the mediation of resistance to apoptosis in B‐CLL. Therefore Bcl‐2 antisense oligonucleotides might be useful in the treatment of B‐CLL.

Flow cytometric assessment of three different methods for the measurement of in vitro apoptosis

Chlorambucil-induced apoptosis was assessed by three different flow cytometric methods in B-cell chronic lymphocytic leukaemia (B-CLL) cells cultured in vitro and the results were compared with those derived from the morphological assessment of the same samples. Spontaneous apoptosis was consistently observed in the control cultures in the absence of drug but this accounted for less than 12% of all cells in every case. The methods under investigation were the Annexin V labelling assay, the terminal deoxynucleotidyl transferase (TdT) end-labelling assay and the labelling of a 38 kDa mitochondrial membrane protein (7A6 antigen) which is exposed on cells undergoing apoptotic cell death (Apo2.7 assay). The Annexin V assay consistently stained a higher percentage of cells and with a greater separation between the positive and negative cell populations. We conclude that the phosphatidyl serine translocation to the outer leaflet of the cell membrane following an apoptotic signal, as labelled by Annexin V, probably occurs before the development of the DNA strand breaks or the exposure of 7A6 antigen in those cells triggered to die by apoptosis.

Flavopiridol circumvents Bcl‐2 family mediated inhibition of apoptosis and drug resistance in B‐cell chronic lymphocytic leukaemia

Flavopiridol, a synthetic flavone, is currently under clinical investigation for the treatment of B‐cell chronic lymphocytic leukaemia (B‐CLL). In this study, we examined the in vitro effects of flavopiridol and fludarabine on B‐CLL cells from 64 patients (36 treated and 28 untreated) in terms of apoptosis induction and Bcl‐2 family expression. Both flavopiridol and fludarabine induced apoptosis in all the samples tested with mean LD50 values (± SD) of 59·7 nmol/l (± 36·5) and 6·2 μmol/l (± 7·5) respectively. Mean flavopiridol LD50 values were not significantly different between the treated and untreated patient groups (P = 0·35), whereas the fludarabine LD50 values were significantly higher in the previously treated patient group (P = 0·01). Bcl‐2 and Mcl‐1 expression were downregulated in both flavopiridol and fludarabine‐induced apoptotic cells, but the increase in Bax expression that accompanied fludarabine‐induced apoptosis was not evident in flavopiridol‐treated cells. In addition, Bcl‐2:Bax ratios were not predictive of flavopiridol cytotoxicity (P = 0·82), whereas they were highly predictive of in vitro responsiveness to fludarabine (P = 0·001). Overall, these findings suggest that flavopiridol exerts its cytotoxic effect through a novel cell‐death pathway that is not subject to the Bcl‐2 family mediated resistance mechanisms that reduce the efficacy of many conventional chemotherapeutic drugs.

Chlorambucil resistance in B‐cell chronic lymphocytic leukaemia is mediated through failed Bax induction and selection of high Bcl‐2‐expressing subclones

Our previous data have shown that high Bcl‐2/Bax ratios in chronic lymphocytic leukaemia (B‐CLL) correlate with in vitro apoptosis and clinical resistance. We have now monitored the in vitro viability of B‐CLL cells in relation to Bcl‐2 and Bax expression over a 48 h time course following exposure to chlorambucil. The results showed that Bax up‐regulation was essential for chlorambucil‐induced apoptosis in B‐CLL cells and a 3‐fold increase in expression within 4 h of exposure to drug was typically observed in sensitive cells; resistant cells failed to up‐regulate Bax at all. In contrast, the constitutively high levels of Bcl‐2 found in B‐CLL cells were found to be down‐regulated in apoptotic cells but the mean Bcl‐2 expression in viable cells was increased, probably as a result of the loss of lower Bcl‐2‐expressing cells into the apoptotic compartment. Taken together, these data add further weight to the suggestion that Bcl‐2/Bax ratios may be pivotal in determining the fate of B‐CLL cells. Furthermore, the Bcl‐2/Bax ratios found in apoptotic B lymphocytes were remarkably similar in the treated, untreated and normal control cells, which suggests that there is a universal Bcl‐2/Bax ratio threshold for cell survival and cell death.

The P2X7 receptor gene polymorphism 1513 A→C has no effect on clinical prognostic markers, in vitro sensitivity to fludarabine, Bcl‐2 family protein expression or survival in B‐cell chronic lymphocytic leukaemia

Summary. A cohort of 121 patients with B‐cell chronic lymphocytic leukaemia (B‐CLL) was investigated for a single nucleotide polymorphism in the P2X7 receptor gene (1513 A→C), and the findings were correlated with clinical prognostic markers, in vitro sensitivity to fludarabine, expression of Bcl‐2 family proteins and overall survival. The frequency of the polymorphism in B‐CLL samples was not significantly different from that found in normal healthy controls (P = 0·27; Fishers exact test). Furthermore, when the B‐CLL patients were analysed according to P2X7 genotype (1513 A/A versus 1513 A/C), there was no significant difference in age at diagnosis, stage at diagnosis, lymphocyte doubling time, time to first treatment, progression‐free survival and overall survival, and neither was there any evidence of bias in terms of VH gene mutational status, CD38 expression, in vitro sensitivity to fludarabine or expression of Bcl‐2, Bax or Mcl‐1 between the two groups. These results indicate that the 1513 A→C polymorphism of the P2X7 gene is unlikely to play a significant role in the pathogenesis or disease progression of B‐CLL.

Bcl-2 Antisense Oligonucleotides Enhance the Cytotoxicity of Chlorambucil in B-Cell Chronic Lymphocytic Leukaemia Cells

We have previously shown that the Bcl-2 antisense oligonucleotide ODN 2009 can induce apoptosis in B-cell chronic lymphocytic leukaemia (B-CLL) cells. In this study we evaluated whether ODN 2009 could increase the sensitivity of B-CLL cells to Chlorambucil-induced cell death in vitro in order to establish whether the notion of antisense-mediated chemosensitisation could be applied to B-CLL. Bcl-2 antisense in combination with Chlorambucil resulted in a more marked reduction in Bcl-2 protein expression (p = 0.003), enhanced Bax expression (p < 0.0001) and increased apoptosis when compared to cells incubated with Chlorambucil alone (p = 0.03). This increased in vitro cytotoxicity demonstrates a proof of the concept that a combination of Bcl-2 antisense Oligonucleotides with conventional chemotherapeutic drugs may elicit an enhanced therapeutic effect in B-CLL and should therefore be considered for further investigation in the form of a clinical trial.

Bcl-2 and Bax expression and chlorambucil-induced apoptosis in the T-cells and leukaemic B-cells of untreated B-cell chronic lymphocytic leukaemia patients

Chlorambucil and other cytotoxic drugs kill cells, non-selectively, by inducing apoptosis. In this study, we measured the apoptotic response to chlorambucil in T- and B-cells from untreated B-CLL patients and T-cells, from normal control subjects. We found increased chemosensitivity in the T-cells of B-CLL patients compared to the controls (P=0.0002). The chlorambucil ID(50) values for T-cells from B-CLL patients showed a direct correlation with Bcl-2 expression (P=0.002) and an inverse correlation with CD3 cell count (P<0.0001), suggesting a trend of increasing chemosensitivity and decreasing Bcl-2 expression with an elevated T-cell count. There was no differential expression of Bcl-2 or Bax between the CD4(+) and CD8(+) cells of B-CLL patients, isolated by immunomagnetic separation. We found correlations in the leukaemic B-cells between chlorambucil ID(50) values and both Bcl-2 expression (P=0.006), and Bcl-2/Bax ratios (P=0.002), suggesting a role for the Bcl-2/Bax ratio in predicting the response of untreated CLL patients to cytotoxic treatment. Chlorambucil produced almost identical changes in Bcl-2 and Bax expression in normal T-cells and leukaemic B-cells triggered to die by apoptosis, which together with the correlation between Bcl-2 and chemosensitivity confirms a pivotal role for Bcl-2 in regulating a distal step in the apoptotic pathway following cytotoxic cellular damage.

Flavopiridol Induces Apoptosis in B-cell Chronic Lymphocytic Leukaemia Cells Through a p38 and ERK MAP Kinase-dependent Mechanism

Flavopiridol, a synthetic flavone, has been previously shown to induce apoptosis in B-cell chronic lymphocytic leukaemia (B-CLL) cells in vitro. The apoptosis was associated with a concomitant activation of caspase-3 without evidence of dependence on functional p53 or Bcl-2 family modulation. In this study, we examined flavopiridol-induced apoptosis in terms of upstream caspase activity, cell cycle distribution and signal transduction, in order to elucidate the mechanism of action of this potent cytotoxic agent. Flavopiridol-induced apoptosis was significantly abrogated by the caspase-9 inhibitor Z-LEHD-FMK (p =0.002; paired t -test) but was not altered by the caspase-8 inhibitor Z-IETD-FMK (p =0.37; paired t -test). There was a concentration-dependent increase in a sub G 0 /G 1 peak indicative of apoptotic cells but if these cells were excluded by gating no other cell cycle perturbations were observed suggesting that flavopiridol is capable of inducing apoptosis in cells in all phases of the cell cycle. Significantly, apoptosis was associated with activation of p38 MAP kinase and suppression of ERK activity (p =0.0036 and p =0.0048, respectively; paired t -test). These results show for the first time that flavopiridol modulates specific cellular signal transduction pathways in B-CLL cells thereby altering the balance between survival and cell death signals and providing a rationale for the p53-independent nature of flavopiridol-induced apoptosis. Further work is required to identify whether combinations of conventional chemotherapeutic drugs and novel agents like flavopiridol can be used to improve patient outcomes in the treatment of B-CLL.

Pleiotropic drug resistance in B-cell chronic lymphocytic leukaemia — the role of Bcl-2 family dysregulation

B-cell chronic lymphocytic leukaemia (B-CLL) is an incurable clonal disease which shows initial responsiveness to a number of chemotherapeutic drugs. However, most treated patients become resistant to treatment and this represents a major problem in the successful management of the condition. Experimental evidence points to the fact that most chemotherapeutic drugs ultimately exert their cell killing effect through the process of apoptosis. In this study we compared the apoptotic responses of B-CLL cells in vitro following exposure to several chemotherapeutic drugs. We found that there was a correlation between ID50 values for all the drugs under investigation; particularly between Chlorambucil and Fludarabine (P = 0.0002). In addition, we analysed the expression of Bcl-2 and Bax, two proteins pivotal to the regulation of apoptosis, both immediately ex vivo and in viable and apoptotic sub-populations following exposure to drug. Our data suggest that high Bcl-2/Bax ratios may be predictive of a drug resistant phenotype in B-CLL cells and that modulation of these proteins is essential for the induction of cell death. Furthermore, it seems likely that the superior potency that has been ascribed to Fludarabine is due to it being administered in a more optimised dose. A recently reported clinical trial of Fludarabine against high-dose Chlorambucil supports this view since it showed that both treatment modalities were comparable in terms of response rate and survival times.

Leukemic and Non-Leukemic Lymphocytes from Patients with Li Fraumeni Syndrome Demonstrate Loss of p53 Function, Bcl-2 Family Dysregulation and Intrinsic Resistance to Conventional Chemotherapeutic Drugs But Not Flavopiridol

Li Fraumeni syndrome (LFS) is characterised by a predisposition to the early onset of certain tumors and is associated with germline mutation of the anti-oncogene p53. In this study we analysed the in vitro responses of lymphocytes from two LFS patients to chemotherapeutic drugs in terms of apoptosis induction and the expression of key intracellular proteins that regulate this process. One of the LFS patients also suffered from B-cell chronic lymphocytic leukemia (B-CLL) and hence presented with a light-chain restricted B-cell lymphocytosis while the other patient had entirely normal blood counts. The B-lymphocytes from both LFS patients showed a marked degree of resistance to chlorambucil and fludarabine when compared to age-matched controls but were remarkably sensitive to the novel flavone, flavopiridol. Loss of function of p53 was demonstrated by a failure to induce Bax and p21 protein expression. In addition, altered basal expression patterns of Bcl-2 and Bax, two key regulators of apoptosis, were found in the LFS lymphocytes when compared with controls. These results suggest that LFS lymphocytes carrying a p53 mutation show intrinsic resistance to conventional chemotherapeutic drugs and this is associated with dysregulation of Bcl-2 family proteins. Furthermore, The innate resistance profile was similar in leukemic and non-leukemic lymphocytes and was therefore independent of genetic changes acquired during malignant transformation. Novel agents that induce p53-independent cell killing may be useful not only in the treatment of LFS-associated tumors but also drug resistant tumors in general where p53 and/or Bcl-2 family dysregulation is a feature.