Overexpressed full-length human BCL2 extends the survival of baculovirus-infected Sf9 insect cells.

Abstract

Full-length and truncated human BCL2 lacking the entire C-terminal hydrophobic domain have been overexpressed in Spodoptera frugiperda insect cells with the baculovirus expression system. Immunoblot analysis with BCL2-specific antibodies revealed that both full-length and truncated BCL2 are expressed as multiple immunoreactive species, suggesting posttranslational modifications. The expression of the full-length but not the truncated BCL2 extended the survival of baculovirus-infected cells by preventing virusinduced DNA cleavage. This result is consistent with the reported protective effect of BCL2 against apoptosis in mammalian lymphocytes and suggests a conserved function in evolution. Subcellular fractionation and indirect immunofluorescence studies in intact cells demonstrated that the recombinant full-length and truncated BCL2 proteins were expressed predominantly as nuclear membrane-associated proteins. These results imply that BCL2 must utilize hydrophobic domains other than the deleted domain for its association with the subcellular membranes. Metabolic labeling of insect cells expressing the full-length and the truncated form of BCL2 with 32p, demonstrated that BCL2 is a phosphoprotein.The protooncogene BCL2 has been isolated and characterized as a result of its involvement with the t(14;18) chromosomal translocation in follicular lymphomas (1-5). The t(14;18) translocation places BCL2 under the transcriptional regulation of the immunoglobulin heavy-chain locus, causing the deregulation ofBCL2 expression (4, 5). The BCL2 protein is a 26-kDa membrane-associated cytoplasmic protein (6-8). It is different from other protooncogene products in that it seems to function in enhancing the survival of hematopoietic cells of T and B origins rather than promoting their growth (9-15). The ability of BCL2 to enhance the survival of these cells is related to its ability to block programmed cell death, or apoptosis, initiated by several factors. BCL2 overexpression in pre-B lymphocytes inhibits apoptosis following cytokine deprivation (8,9,14) or glucocorticoid treatment (16). Recently, two independent studies (17, 18) have also shown that expression of BCL2 in immature thymocytes of transgenic mice protects them from glucocorticoid-, radiation-, and anti-CD3-induced apoptosis. BCL2 upregulation also inhibits apoptosis of Epstein-Barr virus-infected B-cell lines (19). The mechanism by which BCL2 inhibits apoptosis is not known.To gain insight into the structure and mechanism of action of BCL2, we overexpressed full-length and truncated BCL2 in insect cells with the baculovirus expression system. Because of the low abundance of BCL2 in lymphocytes, overexpression of this protein in Sf9 insect cells will avail large quantities of protein for structural, functional, and biochemical analysis. MATERIALS AND METHODSCell Culture. Sf9 insect cells were grown in TNM-FH medium (20, 21). Cells were maintained and infected either as monolayers or in suspension in a rotary shaker. When cells were grown in suspension the me...

The c-Myc-interacting adaptor protein Bin1 activates a caspase-independent cell death program

“…In contrast, there was no di erence in the susceptibility of cells overexpressing Bcl-2 or the FADD dominant inhibitor to death induction by Bin1 (see Figure 7a). On a di erent line of work, we had observed that recombinant Bin1 baculoviruses increased the kinetics of cell death in Sf9 cells, and that Bcl-2 could not inhibit this e ect (see Figure 7b), despite the fact that Bcl-2 inhibits baculovirus-induced cell death in this (Alnemri et al, 1992). In support of these results, we also obtained a set of negative results from experiments aimed at determining whether Bin1 caused cytochrome c release or altered mitochondrial membrane potential (data not shown).…”

“…This report also indicated that AEBSF does not block apoptosis induced by Fas activation, the Increased kinetics of insect cell death elicited by recombinant Bin1 baculovirus are una ected by Bcl-2. Sf9 cells were infected as described (Alnemri et al, 1992;Elliott et al, 1999b) and the proportion of viable cells in the culture were determined at various times post-infection. Inset: Western analysis of cell extracts processed at 24 h after infection demonstrating expression of Bin1 and Bcl-2 cytotoxic T cell granule protein granzyme B, or a variety of cytotoxic drugs, nor did other kinds of serine protease inhibitors a ect death by c-Myc.…”

“…Adherent and¯oating cells were harvested 36 h after adenoviral infection or 24 ± 30 h after staurosporine treatment and processed for osmium tetroxide staining and electron microscopy using standard methods. Baculovirus experiments were performed in the insect cell line Sf9 using full-length recombinant viruses expressing Bin1, Bcl-2 or no insert, essentially as described (Alnemri et al, 1992;Elliott et al, 1999b). Viable cell counts were determined at the indicated times after virus infection by the trypan blue exclusion method.…”

Subcellular and submitochondrial mode of action of Bcl-2-like oncoproteins

“…This manipulation abrogates (Alnemri et al, 1992;Nguyen et al, 1994;Tanaka et al, 1993) or diminishes (Hockenbery et al, 1993; the death-inhibitory e ects of Bcl-2, depending on the experimental system ( (Zha et al, 1996a). Yeast cells with mutations in nuclear genes coding for the mitochondrial F 0 F 1 ATPase are resistant to the lethal e ect of Bax (Matsuyama et al, 1998), a ®nding which underlines the functional impact of Bax on mitochondria.…”

True

“…36 Later, it was discovered that addition of IL-3 to growth factordeprived murine myeloid cells promoted serine phosphorylation of Bcl2. 34 Importantly, Bcl2 phosphorylation was also correlated with increased cell survival since addition of IL-3 protected cells from apoptosis induced by the chemotherapeutic drug etoposide.…”