Dependence of granzyme B-mediated cell death on a pathway regulated by Bcl-2 or its viral homolog, BHRF1

Abstract

The molecular pathways responsible for apoptosis in response to granzyme B have remained unresolved. Here we present data supporting the notion that granzyme Bmediated cell death is largely dependent on a pathway that is inhibitable by Bcl-2 or its viral analog BHRF1. We used a panel of stably transfected FDC-P1 mouse myeloid cell lines to show that overexpression of functional, wild-type Bcl-2 or BHRF1 rescued cells from granzyme B-mediated apoptosis, whereas mutated (Gly 145 ?Glu) Bcl-2, or wild-type Bcl-2 directed to the plasma membrane conferred no protection. Overexpression of Bcl-2 resulted in inhibition of multiple parameters of apoptosis in response to purified perforin and granzyme B, including DNA fragmentation, changes in light scatter profile indicating cell shrinkage and increased refractivity, loss of mitochondrial membrane potential and inhibited colony formation in clonogenic assays. Nevertheless, when exposed to cytotoxic lymphocytes, FDC-P1 and YAC-1 cells overexpressing Bcl-2 remained susceptible to death imparted by cytolytic granules, irrespective of whether the granules contained granzyme B. Thus, alternative granzyme Bindependent pathways can be activated by intact lymphocytes to overcome Bcl-2-like inhibitors of apoptosis, enabling CTLs to overcome potential viral blocks to granzyme B-mediated cell death. Cell Death and Differentiation (2000) 7, 973 ± 983.

A Central Role for Bid in Granzyme B-induced Apoptosis

“…On this basis, it was proposed that mitochondria are more appropriately seen as an amplification loop in the pathway to granzyme B-mediated apoptosis. These data challenge the hypothesis that Bid is required for granzyme B-induced cell death (16,17) and is inconsistent with studies that have shown a role for mitochondria in this pathway (1)(2)(3).…”

“…These studies cumulatively suggested that neither Bid nor MOMP is required for granzyme B-mediated cell death and proposed that MOMP acts as an amplification loop that may be dispensable during granzyme B-induced apoptosis. Although these studies propose to provide insight into the molecular mechanism of granzyme B-induced cell death, they are inconsistent with a number of studies showing that cells overexpressing Bcl-2 are protected against granzyme B-induced apoptosis (1-3) and maintain their proliferative potential following exposure to cytotoxic concentrations of granzyme B (2,3).…”

“…Bcl-2 blocks granzyme B-induced cell death (1-3) and maintains the proliferative potential of the treated cells (2,3), suggesting that mitochondria are critical targets for a granzyme B substrate. Consistent with this hypothesis, granzyme B has been shown to cleave and activate the pro-apoptotic Bcl-2 family member Bid (1, 10, 11, 24), which facilitates caspase activation by releasing proteins from the mitochondrial intermembrane space (23).…”

“…Overexpression of the oncogene Bcl-2 renders cells resistant to granzyme B-induced apoptosis (1,2), and the cells maintain their ability to proliferate (2,3). Bcl-2 is one of a large family of proteins that regulate mitochondrial outer membrane permeabilization (MOMP) during apoptosis (4).…”

BH3 domains define selective inhibitory interactions with BHRF-1 and KSHV BCL-2

“…EBV encodes a viral homolog of BCL-2, anti-apoptotic BHRF-1, 10 which has conserved BH1 and BH2 domains homologous to BCL-2. 11 BHRF-1 can inhibit apoptosis induced by a number of death insults including serum depletion, 12 death induced by tumor necrosis factor a and anti-Fas antibody, 13,14 g irradiation, chemotherapeutic drugs, 15,16 deregulated c-myc, 17 granzyme B, 18 Sindbis virus infection 19 and the tumor suppressor protein p53. 20 BHRF-1 is expressed during the productive/lytic replication cycle.…”