P. Bentley

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.

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.

Alterations of the retinoic acid receptor α(RARα) gene in myeloid and lymphoid malignancies

The retinoic acid receptor α (RARα) protein plays a central role in myeloid differentiation, and chromosomal translocations disrupting the RARα gene are implicated in the development of acute myeloid leukaemia (AML). To identify haemopoietic malignant disorders which may also be linked to RARα abnormalities, Southern blot analysis was performed in DNA from 153 patients with haematological malignancies other than AML FAB type M3 using RARα cDNA probes. Alterations of RARα were detected in 1/42 myelodysplastic syndromes (MDS), 2/24 AML, 3/47 B‐chronic lymphocytic leukaemias (B‐CLL), 0/40 lymphomas and 0/60 normal individuals. These data strongly suggest that alterations of RARα might predispose for myeloid and lymphoid disorders.