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 (Deltapsi(m)), 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 Deltapsi(m), 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.

Expression and function of TRAF-3 splice-variant isoforms in human lymphoma cell lines.

TRAF-3 gene products are signaling adaptor molecules required for lymphocytes to mediate T-dependent antibody responses in vivo. Previous work identified 8 splice-variant TRAF-3 mRNA species by RT-PCR that have the potential to encode novel isoforms, seven of which induce NF-kappaB activation when over-expressed in 293 cells. Here, their expression was characterized by RNAse protection assay, which showed the T cell line Jurkat D1.1 and the B cell lines BJAB, Daudi, and Raji each expressed mRNA encoding TRAF-3 splice-variants in approximately the same rank order (from highest to lowest); TRAF-3 Delta103aa, Delta83aa, full-length, Delta25aa, Delta52aa, Delta56aa, Delta27aa, and Delta221aa mRNA. The TRAF-3 Delta130aa mRNA was not detectable in any of the cell lines examined. The functional effect of over-expressing each TRAF-3 splice-variant on NF-kappaB activation was studied in the TRAF-5-responsive B cell line, BJAB. Of the seven TRAF-3 splice-variant isoforms that induce NF-kappaB activation in 293 cells, only TRAF-3 Delta27aa, Delta103aa, or Delta130aa induce NF-kappaB activation in BJAB cells. Together, these data indicate that a number of TRAF-3 splice-variant mRNAs are expressed and function in B and T lymphoma lines, which suggests that certain TRAF-3 splice-variant isoforms may participate in mediating the known functions of the TRAF-3 gene in lymphocytes.

Fas ligand is constitutively secreted by prostate cancer cells in vitro.

LNCaP, DU145, and PC3 prostate carcinoma cells secrete the 27-kDa soluble Fas ligand (sFasL) into their local environment. sFasL arises from the 40-kDa membrane-bound form (mFasL), which can be found on the cell surface in the LNCaP line, as demonstrated by monoclonal antibody staining. mFasL was also found in extracts of all three cell lines, as demonstrated by Western blotting. FasL mRNA was detected not only in the cell lines, but in the normal prostate as well. sFasL protein could also be detected immunohistochemically in prostate secretions and in human semen. Cleavage of mFasL to sFasL could be inhibited by several matrix metalloprotease inhibitors without a change in the cellular levels of FasL. Prostate-derived sFasL is biologically active, as demonstrated by its induction of apoptosis in Fas-positive Ramos cells, which was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Mitoxantrone induces cellular apoptosis in all three prostate cancer cell lines. Mitoxantrone treatment and doxorubicin treatment also cause up-regulation of Fas, the cell surface receptor for FasL, in LNCaP cells, but not in DU145 or PC3 cells. Furthermore, the up-regulation of Fas expression by mitoxantrone at a high concentration was potentiated by hydrocortisone. When FasL interacts with its Fas, the Fas-bearing cell undergoes apoptosis. When LNCaP cells were treated with mitoxantrone and incubated with an anti-FasL monoclonal antibody, apoptosis was partially blocked. This not only further suggests that the sFasL is biologically active, but that the up-regulation of Fas in the presence of sFasL accounts, in part, for the cytotoxicity of mitoxantrone.

Thymic nurse cell rescue of early CD4+CD8+ thymocytes from apoptosis.

We have now developed temperature sensitive lines of thymic nurse cells (TNCs), using the SV40 viral mutant tsA58, that maintain the ability to selectively internalize a subpopulation of alpha beta TCR+CD4+CD8+ thymocytes in vitro. One line, tsTNC-1, was shown to be able to rescue a subset of CD4+CD8+ thymocytes from programmed cell death at 32 degrees C, the temperature at which binding and internalization were detected. Rescue was significantly diminished at 38 degrees C, the temperature at which thymocyte binding was not observed. The rescued population of thymocytes showed a reduced level of apoptosis as measured by the DNA fragmentation assay. TNC rescue resulted in a shift of CD4+CD8+ thymocytes from immature TCRlow PNArhigh cells to the more mature TCRint PNArlow phenotype but no changes in cell surface levels of HSA nor CD69 were detected. The rescue activity of tsTNC-1 cells at 32 degrees C was significantly reduced with the addition of antibodies to either class I or class II MHC antigens. These results suggest that we have established TNC lines, using the SV40 viral mutant tsA58, that have the ability to rescue a subset of the TNC interactive thymocyte population from programmed cell death. The thymocyte population rescued by TNCs matures to a phenotype within the double positive stage of development that is indicative of positive selection.

The binding, internalization, and release of thymocytes by thymic nurse cells.

Recent studies in our laboratory have described the development of the SV40-transformed thymic nurse cell (TNC) line SVT-II2, that maintains the ability to internalize thymocytes in vitro. SVT-II2 cells were shown to bind and internalize a subset of the alpha beta TCR+, CD4+CD8+ thymocyte population exclusively. Also, SVT-II2 cells express cell surface class I and class II MHC antigens. These data are consistent with reports that suggest that TNCs may have a role in thymic education. In this report, we used scanning electron microscopy, transmission electron microscopy, and long-term video microscopy to study binding, internalization, and release of thymocytes by TNCs. The results of these experiments showed the internalization event to be selective and dynamic. The process appears to involve programmed cooperation between the two cell types that terminates with the release of selected thymocytes. Although no changes in thymocyte cell surface phenotype were detected as a result of their interaction with TNCs in vitro, over 90% remained viable after a 48-hr incubation period.