Molecular characterization of apoptosis in B-cell chronic lymphocytic leukemia

Abstract

B-cell chronic lymphocytic leukemia (B-CLL), characterized by an accumulation of monclonal B cells, is the most common adult leukemia in the Western world. Defective apoptosis is considered to contribute to cell accumulation, disease progression and resistance to therapy in BCLL. In this thesis regulation of apoptotic pathways in B-CLL cells were studied in relation to disease progression and chemotherapy responses.

TRAIL potently induces apoptosis in many tumor cells but exerts minimal cytotoxicity toward normal human cells. In the first study the TRAIL-apoptosis pathway was studied in BCLL. BCLL cells were relatively resistant to in vitro induction of apoptosis by recombinant TRAIL, although they expressed TRAIL-death receptors. Actinomycin D increased B-CLL susceptibility to TRAIL-induced apoptosis, which was not associated with the modulation of TRAILreceptors. Down-regulation of the expression of FLIPL and FLIPs was correlated with the sensitization of B-CLL to TRAIL-induced apoptosis by actinomycin D. FLIP protein was also found to be expressed at higher levels in B-CLL cells as compared to normal tonsil B cells. Our results suggest the involvement of FLIP in the regulation of TRAIL resistance in B-CLL cells.

In the second study the proapoptotic BH3-only protein, Bmf was studied in B-CLL cells. Two new splice variants, named bmf-II and bmf-III were described. They lacked the BH3 domain and, in agreement with this, also lacked the proapoptotic function of the previously described form of Bmf, but instead could promote survival, when overexpressed in HeLa cells. Expression of the isoforms was detected in B-CLL and normal B cells. In B-CLL undergoing serum deprivationinduced apoptosis, the pro-apoptotic form of Bmf was up-regulated while Bmf-III was down regulated. Taken together, we show that alternative splicing is used to switch between the apoptotic/non-apoptotic function of the Bmf protein and suggest that the relative levels of Bmf isoforms may have a role in regulating growth and survival in B cells and leukemic B-CLL cells.

In the third study the expression profile of apoptosis-regulating genes in B-CLL was investigated, in relation chemoresistance and disease progression. We found higher expression of the anti-apoptotic Bcl-2-like proteins, Bfl-1, Mcl-1 and Bcl-2 in apoptosis-resistant B-CLL cells as compared to sensitive B-CLL. Bfl-1 was the most clearly discriminating gene between sensitive and resistant B-CLL cells. Investigation of the modulation of gene expression during serum deprivation-induced apoptosis was undertaken. A number of pro-apoptotic genes were induced and bfl-1 mRNA was down-regulated in B-CLL cells.

In the fourth study the bfl-1 mRNA expression level was determined in a larger amount of patients and found to be significantly higher in patients failing to respond to chemotherapy compared to patients who responded to therapy and untreated patients. Bfl-1 expression was inversely correlated with in vitro fludarabine-induced apoptosis but its levels did not correlate with progression. RNA interference, that down-regulated bfl-1 expression in apoptosis-resistant B-CLL cells with high expression of bfl-1, led to induction of apoptosis in these cells.

Taken together this indicates that bfl-1 might contribute to the development of apoptosis resistant phenotype in chemotherapy refractory BCLL and could thus represent a future potential therapeutic target in B-CLL.