No abstract

This study further investigated the mechanisms that control apoptosis in leukaemic CD5+ B cells, and focused on the Bcl-2 gene family. The pattern of expression of Bcl-2, Bcl-xL, Bcl-xS and Bax genes, selected because of their interrelated role in the control of apoptosis, was analysed in a series of CD5+ B-cell chronic lymphoid leukaemias. Cells from 34 patients with chronic lymphoid leukaemia of B-cell type (23 B-chronic lymphocytic leukaemia (B-CLL) and 11 mantle cell lymphoma (MCL) in leukaemic phase) were investigated. High levels of Bcl-2 mRNA were observed by Northern blot and high levels of Bcl-2 protein were detected by cytofluorograph analysis with a specific monoclonal antibody (MAb) in all cases. Strong Bax expression was detected by RT-PCR in 20/23 B-CLL cases; Bax was also observed in 8/11 MCL in leukaemic phase with variable degree of intensity. In both B-CLL and MCL samples the presence of Bax protein was confirmed by cytofluorograph analysis. RT-PCR detected high levels of Bcl-xL in 16/23 B-CLL and in 8/11 MCL in leukaemic phase, whereas Bcl-xS was detectable in low to trace amounts respectively in 13/23 B-CLL and in 6/11 MCL in leukaemic phase. According to the functional role of Bcl-2, Bcl-xL, Bcl-xS and Bax, these data indicate that the pattern of Bcl-2 family genes expression in leukaemic CD5+ B cells is skewed toward prevention of apoptosis and may thus favour the relentless accumulation of CD5+ leukaemic B cells.

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

CD5+ B‐chronic lymphocytic leukaemia (B‐CLL) and mantle cell lymphoma (MCL) in leukaemic phase are characterized by defects in cell death induction that primarily involves the Bcl‐2 family of genes. Fludarabine (9‐β‐D‐arabinofuranosyl‐2‐fluoradenine, F‐ara‐A) is a potent inducer of apoptosis in CLL cells. This study aimed to determine whether F‐ara‐A‐induced apoptosis might be related to Bcl‐2 modifications and to evaluate in vitro/in vivo correlations.  Peripheral blood lymphocytes from eight B‐CLL and four leukaemic MCL were cultured in the presence of different concentrations of F‐ara‐A ±methylprednisolone (MP). F‐ara‐A down‐regulated the expression of Bcl‐2 in 5/12 cases. mRNA down‐regulation was maximal at 48 h; protein down‐regulation was prominent after 48 h. Both events were dose‐dependent. The amount of apoptosis was significantly higher in the samples treated with F‐ara‐A than in those exposed to MP alone. In the seven remaining cases, no Bcl‐2 down‐regulation was observed after exposure to F‐ara‐A and the degree of F‐ara‐A‐induced apoptosis overlapped that induced by MP.  The in vivo outcome after treatment with three to six courses of F‐ara‐A was evaluable in 10 patients: 4/5 cases, whose cells had shown in vitro Bcl‐2 down‐regulation and prominent apoptosis after exposure to F‐ara‐A, had a complete response (CR) and a partial response (PR) was observed in the remaining patient. Of the five patients whose cells had shown no in vitro Bcl‐2 modulation after exposure to F‐ara‐A, two had a PR, but the other three did not show any in vivo clinical response.

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.