Ludo M. Evers

Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation

We investigated the apoptosis gene expression profile of chronic lymphocytic leukemia (CLL) cells in relation to (1) normal peripheral and tonsillar B-cell subsets, (2) IgVH mutation status, and (3) effects of cytotoxic drugs. In accord with their noncycling, antiapoptotic status in vivo, CLL cells displayed high constitutive expression of Bcl-2 and Flip mRNA, while Survivin, Bid and Bik were absent. Paradoxically, along with these antiapoptotic genes CLL cells had high-level expression of proapoptotic BH3-only proteins Bmf and Noxa. Treatment of CLL cells with fludarabine induced only the proapoptotic genes Bax and Puma in a p53-dependent manner. Interestingly, the degree of Puma induction was more pronounced in cells with mutated IgVH genes. Thus, disturbed apoptosis in CLL is the net result of both protective and sensitizing aberrations. This delicate balance can be tipped via induction of Puma in a p53-dependent matter, the level of which may vary between groups of patients with a different tendency for disease progression.

CD20-induced B cell death can bypass mitochondria and caspase activation

The apoptotic pathway activated by chimeric anti-CD20 monoclonal antibodies (rituximab, IDEC.C2B8) was analyzed using the Burkitt lymphoma cell line Ramos. Crosslinking of CD20 (CD20XL) induced apoptosis in Ramos cells, which involved loss of mitochondrial membrane potential (Δψm), the release of cytochrome-c (cyt-c), and activation of caspases-9 and -3. Nevertheless, several lines of evidence showed that the apoptotic outcome did not depend on these events. First, under circumstances where Ramos cells display resistance to either CD95- or B cell receptor (BCR)-induced apoptosis, CD20XL-induced apoptosis was not affected, pointing to a distinct pathway. Second, the broad-spectrum caspase inhibitor zVAD-fmk prevented processing of caspase-9, -3 and PARP as well as DNA fragmentation, but did not block apoptosis as measured by annexin V staining, cell size and membrane integrity. Lastly, Bcl-2 overexpression blocked cyt-c release and the decrease in Δψm, and completely prevented CD95- or BCR-mediated apoptosis; however, it did not affect CD20XL-induced cell death. We conclude that although CD20XL can initiate the mitochondrial apoptosis pathway, CD20-induced apoptosis does not necessarily require active caspases and cannot be blocked by Bcl-2. Since most chemotherapeutic drugs require the activation of caspases to exert their cytotoxicity, these findings provide an important rationale for the use of CD20 mAbs in chemoresistant malignancies.

Apoptosis via the B cell antigen receptor requires Bax translocation and involves mitochondrial depolarization, cytochrome C release, and caspase‐9 activation

Various routes to apoptosis can be active during B cell development. In a model system of mature B cells, differences in caspase‐3 processing have suggested that antigen receptor (BCR)‐mediated apoptosis may involve a zVAD‐insensitive initiator protease(s). In search of the events leading to caspase‐3 activation, we now establish that both CD95‐ and BCR‐mediated apoptosis depend on Bax activation and cytochrome C (cytC) release. Nevertheless, the timing and caspase‐dependence of mitochondrial membrane depolarization differed considerably after CD95‐ or BCR‐triggering. To delineate events subsequent to cytC release, we compared apoptosis induced via BCR triggering and via direct mitochondrial depolarization by CCCP. In both cases, partial processing of caspase‐3 was observed in the presence of zVAD. By expression in 293 cells we addressed the potential of candidate initiator caspases to function in the presence of zVAD, and found that caspase‐9 efficiently processed caspase‐3, while caspase‐2 or –8 were inactive. Finally, retroviral expression of dominant‐negative caspase‐9 inhibited both CD95‐ and BCR‐mediated apoptosis. In conclusion, we obtained no evidence for involvement of a BCR‐specific protease. Instead, our data show for the first time that the BCR‐signal causes Bax translocation, followed by mitochondrial depolarization, and cytC release. Subsequent caspase‐9 activation can solely account for events further downstream.

Treatment of AIDS-related non-Hodgkin's lymphoma with chemotherapy (CNOP) and r-hu-G-CSF: Clinical outcome and effect on HIV-1 viral load

PURPOSE The optimal treatment of AIDS-related NHL (ARL) has yet to be defined. The purpose of this study was 1) to evaluate the efficacy and toxicity of the CNOP-regimen (cyclophosphamide, mitoxantrone, vincristine, and prednison) in combination with G-CSF; and 2) to study the effect of this regimen on HIV-1 viral replication. PATIENTS AND METHODS A phase II study was performed in 21 previously untreated patients with ARL. RESULTS Based on intention to treat, the response rate was 43%: four complete and five partial remissions. Median survival was only five months. Only one patient had an opportunistic infection during treatment; three patients had localized infections and one episode of septicaemia was seen. Remarkably, during treatment, in 94% of cases p24 antigen levels either remained undetectable or showed a substantial decrease, even though antiretroviral therapy had been discontinued just prior to the first cycle of chemotherapy in all patients. HIV-1 RNA load decreased or remained unchanged in 82% of patients and increased in three patients. CONCLUSIONS Our data demonstrate, 1) that the CNOP-regimen in combination with G-CSF, although associated with a low risk of both opportunistic and bacterial infections, can not be recommended in the treatment of ARL; but 2) that G-CSF can be used safely to sustain haematopoiesis in patients with ARL treated with chemotherapy.

Addition of granulocyte colony‐stimulating factor to chemotherapy in patients with AIDS‐related lymphoma: effects on neutrophil Fcγ receptor expression and soluble FcγRIII plasma levels

AIDS‐related neutropenia and neutrophil dysfunction can (partly) be reversed by granulocyte‐colony stimulating factor (G‐CSF). We studied the effect of G‐CSF on neutrophil increment and levels of soluble Fcγ receptor type III in 15 patients with AIDS‐related lymphoma (ARL) undergoing chemotherapy. In six of these patients we performed a detailed kinetic analysis of the membrane expression of the functionally important Fcγ‐receptors type I, II and III. In all these patients G‐CSF induced FcγRI positive neutrophils with a decreased expression of the FcγRIII receptor. These changes were similar to those seen both in healthy volunteers and in non‐HIV‐infected individuals treated with chemotherapy. Interestingly, the mean neutrophil and sFcγRIII increment were significantly lower and more patients had a nadir granulocyte count < 0.5 × 109/l after the first cycle than after the second cycle of chemotherapy. This may be related to a therapy‐associated decrease in HIV‐1 viral load.