Terry Hoy

REGULATION OF CLINICAL CHEMORESISTANCE BY bcl-2 AND BAX ONCOPROTEINS IN B-CELL CHRONIC LYMPHOCYTIC LEUKAEMIA

The bcl‐2 gene was first shown to be dysregulated in the majority of follicular lymphomas in which a t(14;18) chromosomal translocation is present, but is also over‐expressed in the absence of gene rearrangements in most cases of B‐cell chronic lymphocytic leukaemia (B‐CLL). The bcl‐2 oncoprotein is a regulator of apoptosis and the activity of this protein is opposed by bax, a homologous protein that accelerates the rate of cell death. B‐lymphocyte bcl‐2 and bax protein levels were found to be significantly altered in B‐CLL patients when compared to those of a normal control group. Increased bcl‐2/bax ratios were observed in both the treated and untreated patients when compared to those of normal controls. These alterations were particularly pronounced in those treated patients found to be clinically unresponsive to chemotherapy.

Elevated Bcl-2/Bax are a consistent feature of apoptosis resistance in B-cell chronic lymphocytic leukaemia and are correlated with in vivo chemoresistance.

We investigated the relationship between drug resistance and Bcl-2/Bax in B-cell chronic lymphocytic leukaemia (B-CLL). Apoptosis was induced in vitro with chlorambucil and cell death was monitored by dual-labelled FACS analysis using Annexin V and propidium iodide. Bcl-2 and Bax protein expression was quantified using FACS and a correlation between drug-induced apoptosis and Bcl-2/Bax was established. Cells were then sorted into viable and nonviable populations according to their forward and side-scatter characteristics and re-analysed for Bcl-2/Bax. The most resistant cells had elevated Bcl-2 levels and low Bax expression. Furthermore, those cells which were undergoing apoptosis showed only a marginal reduction in Bcl-2 expression, but significantly elevated Bax expression following exposure to chlorambucil. The Bcl-2/Bax was significantly greater in the cell fractions resistant to chlorambucil-induced apoptosis. This observation further supports the suggestion that Bax is the pivotal protein in determining the fate of cells following apoptotic signals.

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.

A novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry.

The deep red fluorescing agent (DRAQ5) is a synthetic anthraquinone with a high affinity for DNA and a high capacity to rapidly enter living cells or stain fixed cells. DRAQ5 is optimally excited by red-light emitting sources and yields a deep red emission spectrum which extends into the low infra-red. DRAQ5 shows excitation at sub-optimal wavelengths including the 488 nm line and the multi-line UV wavelengths emitted by argon-ion lasers. Single beam (488 nm) flow cytometry has been used to demonstrate the utility of DRAQ5-nuclear DNA fluorescence as a discriminating parameter for human leucocytes and lymphoma cells, in combination with fluorochrome-labelled antibodies for the detection of surface antigens and subpopulation recognition. DRAQ5 fluorescence was found to reflect cellular DNA content as evidenced by cell cycle distribution profiles for asynchronous and cell cycle-perturbed populations. Importantly, DRAQ5 can be used in combination with FITC and RPE-labelled antibodies, without the need for fluorescence compensation.

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.

The measurement of the total volume of red cells in man: a non-radioactive approach using biotin

Present methods for measuring red cell volume are based on the dilution of radioactively labelled cells. This precludes the investigation in neonates and pregnant women. We present a simple method for labelling red cells with biotin. These cells may be injected intravenously and subsequently detected using streptavidin‐FITC and flow‐cytometry. A comparison of the red cell volume estimated using both 51Cr and biotin labelled cells in 19 patients showed no consistent clinically significant difference between the two. This novel label appears to allow red volume to be reliably estimated without using radioactivity.

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.

Expression of the multiple drug resistance gene (mdr‐1) and epitope masking in chronic lymphatic leukaemia

Summary Resistance to cytotoxic agents is a common clinical problem in the treatment of chronic lymphatic leukaemia (CLL). The multidrug resistant (MDR) phenotype characterized by increased levels of a specific cell membrane p‐glycoprotein. confers cross resistance to a wide range of structurally dissimilar antineoplastic drugs. We have studied the expression of this p‐glycoprotein in chronic lymphatic leukaemia measured by immunofluorescence using a monoclonal antibody MRK 16 by flow cytometry.