Role of caspases and possible involvement of retinoblastoma protein during TGFβ-mediated apoptosis of human B lymphocytes

Abstract

In this study, we investigated the involvement of caspases in TGFb-induced apoptosis in human B cells. Our results show that TGFb-mediated nuclear fragmentation, observed in the Epstein ± Barr virus-negative Burkitt's Lymphoma cell line BL41, was abolished in the presence of the tripeptide caspase inhibitor ZVAD-fmk or the speci®c caspase-3 inhibitor DEVD-fmk. Other apoptotic manifestations such as cell shrinkage, surface phosphatidylserine expression and chromatin condensation were strongly inhibited by ZVAD-fmk but only partially by DEVD-fmk. This suggests that other caspases in addition to caspase-3 control these apoptotis-associated features. Speci®c activation of caspase-3 during TGFbinduced apoptosis was demonstrated by the DEVD-fmksensitive expression of the active p17 subunit of caspase-3 and by in vivo cleavage of PARP. In addition, TGFb treatment of BL41 promoted the expression of both dephosphorylated and truncated forms of the retinoblastoma protein. Inhibition of caspase-3 activity abolished both nuclear fragmentation and expression of the truncated retinoblastoma protein, without modifying the G1 cell cycle arrest induced by TGFb. Our data thus demonstrate that TGFb-induced apoptosis of lymphoma B lymphocytes is dependent on caspase activation and involves caspase-dependent cleavage of the retinoblastoma protein.

Apoptosis Induced by TGF-β1 in Burkitt’s Lymphoma Cells Is Caspase 8 Dependent But Is Death Receptor Independent

“…The cleavage of caspase 8 into its p43, p40, and p18 active forms was detected following 12-24 h of TGF-␤1 treatment. We could only detect the active p20 and p19 cleavage products of caspase 3 after 24 h of TGF-␤1 treatment consistent with other findings (24,25). The necessity for long exposures of autoradiogrammes indicated that a small percentage of the total zymogen pool of caspases 3, 8, and 9 is cleaved into their active subunits.…”

“…Schrantz et al (25) have also recently suggested that pRb cleavage may be important in TGF-␤1-mediated apoptosis in BL41 cells. Western blotting analysis with a pRbspecific mAb revealed the appearance of a faster migrating form of pRb following 24 h of TGF-␤1 treatment and a general reduction in the steady-state levels of all forms of pRb by 48 h of TGF-␤1 treatment (Fig.…”

“…Concomitant with our studies, others reported that this process involves activation of caspase 3 (24,25) and may involve down-regulation of the Bcl-2 family member Bcl-X L (24) and cleavage of the retinoblastoma protein (pRb). In this study, we show that these aforementioned events are late in TGF-␤1-mediated apoptosis and are preceded by activation of caspases 2, 7, 8, and 9 in a caspase 8-dependent, but caspase 3-independent manner.…”

“…We (26) and others (20,(22)(23)(24)(25) have previously shown that TGF-␤1 can induce apoptosis in the BL41, Ramos, and L3055 EBV-negative BL cell lines. We have also recently shown that this is the case for the majority of EBV-negative and Group I BL cell lines (26).…”

“…TGF-␤1 pretreatment of primary B cells has been demonstrated to block activation signal-induced proliferation (11)(12)(13)(14) and to inhibit Ig secretion (15)(16)(17), and may promote class switching to an IgA phenotype (18). It has also been suggested that TGF-␤1 can induce apoptosis in human primary B lymphocytes (19 -21) and BL cell lines (20,(22)(23)(24)(25)(26). The induction of apoptosis by TGF-␤1 has also been observed in myeloid leukemia cells (27), gastric carcinoma cells (28), primary endometrial cells (29), primary hepatocytes and hepatoma cells (30), human cervical carcinoma cell lines (31), and human lung epithelial cell lines (32).…”