Human Molecular Genetics, 2001, Vol. 10, No. 21 2329-2339
© 2001 Oxford University Press
Bcl2-L-10, a novel anti-apoptotic member of the Bcl-2 family, blocks apoptosis in the mitochondria death pathway but not in the death receptor pathway
University Women’s Hospital Basel, Schanzenstrasse 46, CH-4031 Basel, Switzerland
Received April 11, 2001; Revised and Accepted July 23, 2001.
DDBJ/EMBL/GenBank accession no. AF285092.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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By GenBank database searches and PCR, we have identified a novel human Bcl2-like gene, Bcl2-L-10, which contains conserved BH4, BH1 and BH2 domains but lacks BH3 domain. The Bcl2-L-10 gene has been assigned to chromosome 15q21.2. Transfection experiments demonstrated that Bcl2-L-10 can block apoptosis induced by interleukin-3 withdrawal and Bax expression, by prevention of cytochrome C release, caspase-3 activation and mitochondrial membrane potential collapse. Bcl2-L-10 cannot block TNF
-induced apoptosis. Furthermore, both the BH4 domain and the transmembrane domain of Bcl2-L-10 are necessary for its suppressive action on cell death. Our results demonstrated that Bcl2-L-10 is a newly detected anti-apoptotic member of the Bcl-2 family and that it blocks apoptosis in the mitochondrial death pathway but not in the death receptor pathway. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Programmed cell death or apoptosis plays an essential role in the development of multicellular organisms and in pathological processes (for reviews see 1,2). Dysregulation of apoptosis is involved in the development of many diseases such as cancer, autoimmunity and various degenerative diseases.
Proteins of the Bcl-2 gene family, which are evolutionarily conserved from the Sponges to man (3), are the most prominent regulators of apoptosis and are of considerable importance for clinical medicine. The Bcl-2 family consists of a growing number of proteins, which contain four conserved Bcl-2 homology domains (BH1, BH2, BH3 and BH4), together with a transmembrane domain, all being identified as crucial for the regulation of apoptosis.
Based on functional studies and the retention of BH domains, the Bcl-2 family can be divided into three subgroups (1). The Bcl-2 subgroup includes all anti-apoptotic proteins, such as Bcl-2, Bcl-xL, A1/Bfl-1 and Mcl-1. The Bax subgroup consists of pro-apoptosis members, such as Bax, Bak and Bad. Both groups contain more than one BH domain. The third subgroup contains BH3-only proteins, such as Bid and Bim, which can interact with either anti-apoptotic proteins or pro-apoptosis members and promote apoptosis (4). The observation that supressors and promoters of cell death interact with each other by forming homodimers or heterodimers suggests that apoptosis is regulated, at least in part, by protein–protein interaction (5,6).
Despite the clear cut functions of the distinctive Bcl-2 family proteins either in the promotion or in the suppression of apoptosis, they play non-redundant roles in normal embryonic development, in tissue homeostasis and in a multitude of pathological processes. Both immunohistochemical and mRNA analysis have shown consistently that the expression patterns of the Bcl-2 gene family proteins are distinctive, albeit with certain overlappings, suggesting that the Bcl-2 gene family proteins regulate cell death at specific stages of cell differentiation through tissue-specific control of their expression (7–10). Gene knockout studies further demonstrated that the Bcl-2 gene family proteins play different roles in embryogenesis (11–20).
Therefore, identification of all Bcl2-like proteins will permit us to understand the structure, the function and the mutual functional relationships of the various members of the Bcl2 family, which control apoptosis in vivo.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Bcl2-L-10 is a novel human Bcl-2 related gene
In order to isolate novel Bcl-2 related genes, TBLASTN was used to screen public GenBank database with the amino acid sequences of all known Bcl-2 family members from various species (see Materials and Methods). Two short sequences (GenBank accession nos AA005293 and AA098865), which were identical and shared significant amino acid sequence similarities with the BH1 domain of Bcl-2 family members, were identified from the GenBank database expressed sequence tags (ESTs). Subsequently, the nucleotide sequence of the ESTs was used as a probe to query search the Human Genome Project DataBase, which revealed a matching genome sequence. Conceptually translation analysis revealed that the deduced amino acid sequence contains an open reading frame (ORF) of 204 amino acids, which we termed Bcl2-L-10 and considered as a Bcl2-like-10 protein. Accordingly, the 5' cDNA sequence was then obtained by PCR using the human placenta cDNA as a template (Materials and Methods). The full cDNA sequence contains a 5'-untranslated region (5'-UTR) of 80 bp and a 3'-UTR of 581 bp, including a poly(A)+ signal (Fig. 1A). The deduced amino acid sequence corresponded to a 23 kDa polypeptide. The coding sequence thus depicts an N-terminal region of 13 amino acids (residues 17–29) with homology to a BH4 domain, BH1 and BH2 domains and a C-terminal region (residues 183–199) being a putative membrane anchor sequence present in many anti-apoptotic members (1,21) (Fig. 1C). The Bcl2-L-10 amino acid sequence also contains a potential PEST sequence, a domain rich in proline, glutamate, serine, threonine and aspartate residues (22), and an amino acid stretch (LAAS), which has not been found in any other Bcl2-like proteins (Fig. 1C).
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Comparison of the primary structure of Bcl2-L-10 and other Bcl-2 related proteins (23) revealed that the amino acid sequence of Bcl2-L-10 showed 49% identity with mouse Boo/Diva (24,25) and 37% identity with avian NR-13 (26). We view Bcl2-L-10 as an evolutionarily distinctive gene but related to other members (Fig. 1B). We analyzed the predicted secondary structure of Bcl2-L-10 by NNPREDICT, a protein secondary structure prediction program (27) and compared it with Bcl-xL, Bcl-2 and Bax, of which the three dimensional structures have been determined (28–30). We found that Bcl2-L-10 also has eight predicted
-helices similar to Bcl-xL, Bcl-2 and Bax (Fig. 1C).
The Bcl2-L-10 gene is localized at chromosomal 15q21
Comparison of the cloned cDNA sequence with the genome sequence deposited in the Human Genomic Database revealed the genomic structure and the chromosomal localization of the Bcl2-L-10 gene. As shown in Figure 1A, the gene consists of two exons. The intron/exon junctions followed the GT/AG rule and were flanked by conserved sequences (data not shown). This gene is located on human chromosome 15q21.2 within human genomic bacterial artificial chromosome (BAC) clone CTD-2184D3 (GenBank accession no. AC023906) by using the Human Genome Browser program.
BCl2-L-10 mRNA is differentially expressed in human tissues
To compare the tissue distribution of Bcl2-L-10 mRNA expression with other Bcl-2 related genes, we have used a semiquantitive PCR-based system, the Rapid-Scan gene expression panel, to profile Bcl2-L-10 mRNA expression in 24 different human organ tissues. In contrast to the relatively restricted expression of Boo/Diva mRNA in the ovary (24,25), Bcl2-L-10 mRNA appeared to be more widely expressed. While Bcl2-L-10 expression could be detected in the brain, heart, kidney, spleen, liver, colon lung, small intestine, muscle, stomach, testis and placenta, the highest levels were detected in the bone marrow, whereas high expression levels were also found in all glands (Fig. 2A). Surprisingly, the expression in ovary, uterus and prostate was relatively low, since they could only be detected after more rounds of PCR amplification (Fig. 2B). Inspection of Bcl2-L-10 at the UniGene collection at the NCBI revealed several human ESTs derived from cDNA libraries prepared from pregnant uterus, fetal liver and spleen, infant brain, kidney as well as kidney tumor cells. These data suggest that the Bcl2-L-10 mRNA is almost ubiquitously expressed.
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Bcl2-L-10 is associated with cellular membranes
Like many other bcl-2 family members, Bcl2-L-10 contains a possible transmembrane stretch (‘TM’) in its C-terminus (Fig. 1C), which has been shown previously to anchor proteins, such as Bcl-2 and Bcl-xL, to the outer membranes of mitochondria, the endoplasmic reticulum (ER) and the nuclear envelope (for a review see 2). Since it was found that the His-tagged Bcl-L-10 was located diffusely throughout the transiently transfected REF52 cells, a rat fibroblast cell (not shown), we then studied the subcellular distribution of Bcl2-L-10 in cellular fractions of stably transfected FL5.12 cells. As shown in Figure 2D, the majority of Bcl2-L-10 was located in the membrane fraction (92%), as quantitated by densitometry, and Bcl2-L-10 was also found in the cytosol fraction (8%), indicating that Bcl2-L-10 is associated with cellular membranes.
Bcl2-L-10 is an anti-apoptotic protein which can block apoptosis induced by interleukin-3 (IL-3) withdrawal and Bax expression but cannot block TNF-induced apoptosis
Based on its function and the retention of different BH domains, the Bcl-2 family is divided into three classes: the anti-apoptotic members, the pro-apoptotic members and the BH3-only members (2). To assess the function of Bcl2-L-10 on apoptosis, we ectopically expressed Bcl2-L-10 in various cell lines.
FL5.12 cells is a mouse pro-B cell line, which undergoes apoptosis after IL-3 deprivation (31,32). The cell was transfected with pcDNA3/His Bcl2-L-10 expression vectors and two clones were selected. As shown in Figure 3A, FL5.12 neo cells and FL5.12 Bax cells lost their viability after IL-3 deprivation. Both Bcl2-L-10 expressing clones displayed resistance to apoptosis compared with that of the Bcl-2 clone. Similar results were obtained in the presence of the protein synthesis inhibitor, cycloheximide (CHX) (data not shown). The degree of resistence may be due to variable protein expression levels, as the expression of Bcl2-L-10 was slightly different in the two clones (Fig. 3B).
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Bcl2-L-10 expression vectors were also studied in REF52 cells, which undergo apoptosis upon serum deprivation (21) and Bax expression (33). The Bcl2-L-10 constructs were transfected into REF52 rat fibroblasts together with a vector (pEGFP-N1) encoding the green fluorescent protein (GFP) and pcDNA3 Bax (33). GFP served as marker for successfully transfected cells, whereas expression of Bax and Bcl-xL as positive controls. At 24 h after transfection, the number of apoptotic REF52 cells were counted (33). Figure 4A shows that only
5% of cells transfected with the control plasmid (pcDNA3) were apoptotic, whereas the expression of Bax increased the number of apoptosis by 40%. Approximately the same percentage of the Bax transfected cells could be rescued by Bcl-xL and Bcl2-L-10 expression. These results indicate that Bcl2-L-10 is an anti-apoptotic protein.
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Two major pathways leading to caspase activation during apoptosis have been documented previously (34,35). While one pathway is mediated by the mitochondrial death pathway, the other is initiated by death receptors. However, the exact function of the anti-apoptotic proteins in the death receptor pathway remains controversial (36,37). To examine this issue, we tested the response of FL5.12 Bcl2-L-10 cell to TNF
in the presence of the protein synthesis inhibitor CHX, since it has been shown that TNF can only trigger programmed cell death of FL5.12 with CHX and not alone (38). As shown in Figure 3C, 36 % of the FL5.12 cells transfected with a control vector (Neo) were viable, as determined by trypan blue exclusion after an 18 h incubation with 10 ng/ml TNF and 2.5 µg/ml CHX. When FL5.12 cells overexpressing His-Bcl2-L-10 were incubated with TNF and CHX, there was no effect on viability, which was found to approximate 40%. To investigate whether the failure of Bcl2-L-10 to prevent apoptosis induced by TNF relates to rapid degradation of Bcl2-L-10 in the presence of CHX, we examined the expression level of Bcl2-L-10 in the presence of CHX. As shown in Figure 3D, the protein level of Bcl2-L-10 did not significantly alter during a period of 36 h. These results indicate that CHX did not affect the stability of Bcl2-L-10 but rather enhanced the FL5.12 cells’ sensitivity to TNF by other mechanisms. Therefore, the fact that His-Bcl2-L-10 cells die to the same extent as wild-type cells implies that the addition of the His-Bcl2-L-10 does not confer any selective advantages for cell survival. The inability of His-Bcl2-L-10 to block TNF-induced apoptosis was intrinsic to the signal and not to the cell, because these molecules were sufficient to block IL-3 withdrawal-induced cell death in the same cells (Fig. 3A and C), regardless of CHX. Moreover, caspase-3 (casp-3) activation in His-Bcl2-L-10 cells was inhibited even after IL-3 deprivation but not in the TNF and CHX treatment (Fig. 3E).
The BH4 domain and the transmembrane domain of Bcl2-L-10 are necessary for its suppression of cell death
Bcl2-L-10 has an N-terminus with a BH4 domain which has been shown to be essential for the anti-apoptotic activity of Bcl-2/Bcl-xL (39–42). Some of the conserved residues of the BH4 domain, such as L8 of Bcl-xL, were reported to be crucial for the anti-apoptotic activity of Bcl-2/Bcl-xL (43). A potential PEST sequence, which may affect protein stability (22), has been found in the amino acids 32–50 (Fig. 1C). To explore the structure–function relationship of Bcl2-L-10, we deleted the BH4 domain (Materials and Methods). The N-terminal truncated Bcl2-L-10 construct (Bcl2-L-10-BH4), in which the first BH 4 domain has been deleted, was then transfected together with pEGFP-N1 and Bax vectors in the REF52 cells apoptosis assay. As shown in Figure 4A, Bcl2-L-10-BH4 lost its death suppression activity compared with the plasmid carrying a full-length cDNA (Bcl2-L-10), indicating that the N-terminal region is necessary for its anti-apoptotic function.
The C-terminal truncated construct, Bcl2-L-10-TM, which lacks a putative transmembrane domain, also lost its anti-apoptosis activity (Fig. 4A). These results are consistent with the previous observations that mutants of the Bcl-2 related proteins lacking the transmembrane domain in the C-terminus are functionally ineffective. The loss of function of the C-terminal mutant indicated that the localization of Bcl2-L-10 is important for its function. Thus, these results provided evidence of intrinsic functions of the N-terminal sequence with the BH4 domain and the C-terminal sequence with transmembrane domain of Bcl2-L-10 for this anti-apoptotic protein.
It has been demonstrated that the BH4 domain of Bcl2 contains a caspase cleavage site and that deletion of BH4 domain converted Bcl2 into a Bax-like death effector (44). We tested the function of Bcl2-L-10-BH4 along with GFP but without Bax. The transfections revealed that the molecule with the deleted BH4 domain became pro-apoptotic, whereas the Bcl2-L-10-TM showed neither protecting nor killing activity (Fig. 4B).
Bcl2-L-10 prevents cytochrome C (cyt. c) release, casp-3 activation and mitochondrial membrane potential (Mmp) collapse
As demonstrated above, Bc12-L-10 can only block the mitochondrial death pathway. We therefore tested the possibility that Bcl2-L-10 mediates apoptosis by preventing cyt. c release (45), casp-3 activation (46) and Mmp collapse (47). We first fractionated FL5.12 cells into cytosolic and membrane-bound fractions to detect cyt. c by immunoblotting. The membrane-bound fraction contained mitochondria (48). The results are presented in Figure 5A. In control cells (Fig. 5A, ‘Neo/+IL-3’), cyt. c was present in mitochondria. After 24 h of IL-3 deprivation, cyt. c in the mitochondrial fraction was decreased, and this was accompanied by the presence of cyt. c in the cytosol (Fig. 5A, ‘Neo/–IL-3’). Release of mitochondrial cyt. c was attenuated by overexpression of Bcl2-L-10 (Fig. 5A, ‘B-L-10/–IL-3’).
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We then determined caspase activation in the cytosolic fractions. As shown in Figure 5B, casp-3 activation was stimulated by IL-3 deprivation (Fig. 5B, ‘Neo/–IL-3’compared with ‘Neo/+IL-3’) (32). Significantly, casp-3 activation was prevented after IL-3 deprivation in the cells overexpressing Bcl2-L-10 (Fig. 5B, ‘Bcl2-L-10’) as well as in Bcl-2 expressing cells (Fig. 5B, ‘Bcl-2’) and by the synthetic peptide inhibitors (Fig. 5B, ‘Neo/–IL-3+DEVDfmk’). However, casp-3 activation was not inhibited in the cells overexpressing Bcl2-L-10 treated with TNF
(Fig. 3D). The loss of Mmp is an early physiological change during apoptosis (47). Cells were incubated with 3,3’-dihexyloxacarbocyanine iodide (DiOC6), followed by the flow cytometric analysis of red fluorescence (32). The ‘M1’ cell population in Figure 5B indicates the percentage of apoptotic cells. When FL5.12 control cells were deprived of IL-3, the Mmp dropped markedly in a large number of cells within 12 h (Fig. 5B, ‘Neo/–IL3’ compared with ‘Neo/+IL-3’). FL5.12 Bcl2-L-10 overexpressing cells showed no sign of changes in Mmp (Fig. 5B, ‘B-L-10/–IL3’ compared with ‘B-L-10/+IL-3’). No Mmp change has been found in FL5.12 Bcl-2 cells (‘Bcl-2/–IL3’ compared with ‘Bcl-2/+IL-3’). These studies indicate that Bcl2-L-10 inhibits apoptosis through prevention of cyt. c release, casp-3 activation and Mmp collapse.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In the present study, we described a novel member of the Bcl-2 gene family, Bcl2-L-10. The deduced protein sequence comprises conserved BH1, BH2 and BH4 domains but lacks the BH3 domain. We have mapped the Bcl-L-10 gene to the human chromosome 15q21.2. The amino acid sequence of Bcl-L-10 has 49% identity to mouse Diva/Boo. Diva/Boo is localized on chromosome 9 (25). Our comparative mapping studies in human and mouse further revealed that the locus of mouse Diva/Boo is tightly linked to D9Mit263 (H.Zhang, unpublished data). Both human Bcl2-L-10 and mouse Diva/Boo are located closely with gene MAPK6 and MYO5A. We therefore consider mouse Diva/Boo as a human Bcl2-L-10 ortholog. However, they may play different roles as refracted by several differences in the expression pattern and protein structure.
Bcl2-L-10 mRNA is widely expressed in human tissues, in contrast to mouse Boo/Diva, which is expressed only in few mouse tissues (24,25). Although the 3'-UTR of human Bcl2-L-10 and mouse Boo/Diva shared 56% identity (H.Zhang, unpublished data), an ATTTA motif is present in the mouse sequence but not in the human sequence. The cis-acting ATTTA motifs are thought to be destabilizing elements associated with rapid mRNA degradation (reviewed in 49). Interestingly, the ATTTA motif was also found in mouse Bok, a pro-apoptotic member of the Bcl-2 family gene, which is expressed exclusively in ovary (50). In contrast to mouse Bok, the human Bok gene contains no ATTTA motif and is expressed much more widely (51 and H.Zhang, unpublished data). It is possible that the ATTTA motif regulates the mRNA degradation of both Diva/Boo and Bok, so that the expression of both genes can only be detected in tissues with the highest expression.
Structurally, although the conserved BH3 amino acids are not present in the Bcl2-L-10 protein, an -helix ( 2) structure can still be predicted by secondary structure prediction analysis (Fig. 1C). Thus the overall folding of the protein should be similar to that of Bcl-xL (28), Bcl-2 (29) and Bax (30). However, a long amino acid stretch (comprising 20 amino acids) was found to be present in Bcl2-L10, forming a long loop between the BH1 and BH2 domain (5–6 helices), which has not been observed in any other Bcl-2 family members. Our secondary structure prediction and transmembrane domain analysis revealed that amino acids 93–116 and 155–179 also have transmembrane potentials (Materials and Methods). BH1 and BH2 domains have an essential function for dimerization with other members and for channel formation. The 5–6 helices are believed to be inserted into the membrane bilayer, perpendicular to the membrane surface, with the loop connecting 5 and 6 presumably protruding from the other side of the membrane (52). The long loop in Bcl2-L10 may determine the selective binding activity of BH3 domains of the Bcl2 members. Therefore, it would be interesting to further determine whether the long amino acid stectch may alter conformation of the hydrophobic cleft composed by BH1, 2 and 3 domains.
One important difference could be found between human Bcl2-L-10 and mouse Diva/Boo. The amino acid residues aligned to positions 95 (Gly) and 156 (TRP) of Bcl2-L-10 are conserved as in other Bcl2 members, which are two of the essential residues for their function. Substitution of Gly 145 in BH1 domain or Trp 188 in BH2 domain completely abrogated Bcl-2’s death-repressor activity and disrupted its heterodimerization with Bax (53,54). However, both residues are substituted by Serine 88 and Arginine 144 in Diva/Boo, respectively (Fig. 1B). The functional importance of these substitutions needs to be found by future experiments.
Bcl2-L-10 contains a conserved BH4 domain followed by a PEST sequence, a domain rich in proline, glutamate, serine, threonine and aspartate residues (22). Our data demonstrated that deletion of the BH4 domain of Bcl2-L-10 abrogated its anti-apoptotic ability and created a molecule which displayed pro-apoptotic activity, indicating that BH4 is crucial for the apoptotic suppressing activity. These results were consistent with previous observations that deletion of BH4 from Bcl-2/Bcl-xL leads to loss of their anti-apoptotic ability (43,44). The BH4 domain has been reported to bind with other proteins regulating apoptosis, including calcineurin (55), Raf-1 (56) and Ced-4 (42). The BH4-deletion results also shed light on the function of the PEST sequence in the Bcl2-L-10. PEST sequences prevail in a number of proteins rapidly degraded by a non-ubiquitin-mediated process (22). This raises the possibility that Bcl2-L-10 may be cleaved near the PEST sequence and that such a cleavage might be a mechanism in regulating apoptosis. Mouse Boo/Diva contains a PEST sequence in the same position as that of Bcl2-L-10 (H.Zhang, unpublished data). The exact function of mouse Boo/Diva remains controversial, because it was considered as a negative regulator of cell death (25), whereas at the same time it has been shown to induce apoptosis (24). This discrepancy may be explained by the PEST sequence proteolysis, which may be active differently according to the cell type.
In summary, we have demonstrated that Bcl2-L-10 is an anti-apoptosis protein and that the BH4 domain, together with the transmembrane domain of Bcl2-L-10, is necessary for its suppression of apoptosis. In addition, we demonstrated that Bcl2-L-10 blocks apoptosis in the mitochondrial death pathway by preventing cyt. c release, casp-3 activation and Mmp collapse, but does not block apoptosis in the death receptor pathway (Fig. 6).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Molecular cloning and chromosomal location of Bcl2-L-10
The conserved amino acid sequences of BH1 and BH2 from all known Bcl-2 family members were subjected to homology searches using the BLASTN algorithm (57) from the National Center for Biotechnology Information web server against the human EST database (dbEST). Two overlapping short sequences (GenBank accession nos AA005293 and AA098865) were identified; both clones were essentially identical containing amino acid sequences with significant similarities to the BH1 domain. The clones were obtained from the IMAGE consortium (58) through Research Genetics Inc. and sequenced using insert-flanking vector primers. Subsequently, the nucleotide sequence of the ESTs was used as a probe to query search the Human Genome Project Database. The search revealed that a BAC sequence (accession no. AC023906) matched 100%. A genomic sequence was identified with conceptually translation analysis which contained an ORF of 204 amino acids, and was termed BCl2-L-10. Accordingly, the further 5' sequences were obtained by PCR using the human placenta cDNA (Clontech) as a template with primers 5'-GGAATTCCACGGCAGCCCTACCCTGG-3' and 5'-ATTTGCGGCCGCCAGTTCTTGTTCTCACACATCTGTCA-3'. The full length cDNA sequence then corresponded to that combined from EST cDNA clones and the PCR clone. The GenBank accession no. for Bcl2-L-10 is AF285092. This gene was located on human chromosome 15q21.2 by using the Human Genome Browser program created by Jim Kent at University of California at Santa Cruz, CA.
Structure analysis
Multiple alignment was performed by using the multiple alignment program (Multalin) (59). Potential PEST sequence was predicted using PESTfind at the EMBnet Austria server. Protein secondary structure analysis was performed by the NNPREDICT program at the University of California at San Francisco (UCSF). Hydrophobicity study was performed using the TMpred program (60), which revealed that amino acids 93–116, 155–179 and 183–199 have transmembrane potentials.
Plasmid constructions
The original PCR products amplified from the human placenta cDNA were cloned in the EcoRI/NotI site of vector pZero-1 (Invitrogen) and sequenced. The full length cDNAs containing the coding sequence were amplified with 5'-GGAATTCATGGTTGACCAGTTGCGGGA-3' and 5'-ATTTGCGGCCGCCAGTTCTTGTTCTCACACATCTGTCA-3' and subcloned in-frame into the mammalian expression vector pcDNA3.1/His (Invitrogen). To construct the deletion mutants, the full length cDNAs were used as a template for PCR. Since the nucleotide sequences around the PEST motif are very G/C rich, we then created the Bcl2-L-10 BH4 deletion mutant, Bcl2-L-10-BH4, by cutting the sequence encoding the BH4 domain. Forward and reverse primers were 5'-GGAATTCTACCTGGGGTACTGCGCCCGGGAA-3' and 5'-ATTTGCGGCCGCCAGTTCTTGTTCTCACACATCTGTCA-3'. To create the Bcl2-L-10 transmembrane deletion mutant, Bcl2-L-10-TM, forward and reverse primers were 5'-GGAATTCATGGTTGACCAGTTGCGGGA-3' and 5'-ATTTGCGGCCGCCAGTTCTTGTTCTCACACATCTGTCA-3'. All fragments generated by PCR were cloned in-frame into the EcoRI/NotI sites of pcDNA3.1/His (Invitrogen) and confirmed by sequencing. Other Bcl2-related gene constructs were described previously (21).
Tissue expression pattern analysis by RT–PCR
In order to determine the expression patterns of Bcl2-L-10 mRNA in different human tissues, PCR was performed using the primers 5'-GCAAATGGCTCTTCCTTGAG-3' and 5'-AGCAGCACATGAAGTTGTGG-3' on a panel of first-strand cDNAs from various human tissues (Origene). The PCR conditions were as described in the manufacturer’s protocol with 30 cycles performed on the ß-actin control employing Taq polymerase. Low expression was detected by amplification for 35 cycles. Semiquantitation of ß-actin controls was performed using human ß-actin primers provided by the manufacturer.
Cell lines, cell culture and transfection
Mouse pro-B cell line FL5.12 wild-type, FL5.12 Bcl-2 and FL5.12 Bax CL16 cells were obtained from Dr S.Korsmeyer (Washington University School of Medicine, St Louis, MO) (31). The cells were grown in full medium [RPMI 1640 (Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL) and 10% WEHI-3B conditional medium as a source of IL-3] in 5% CO2 at 37°C. Transfection of FL5.12 cells was as described previously (32). Briefly, 5 x 106 cells/ml resuspended in 400 µl of ice-cold PBS buffer (Gibco BRL) were co-electroporated with 15 µg of DNA at 400 V, 1000 mF in a Bio-Rad electroporation cuvette (Bio-Rad), followed by 2 days of recovery. The clones were selected by G418.
REF52 rat fibroblasts were obtained from the American Type Culture Collection and cultured as described previously (21). For transfection, REF52 cells were seeded at 10–30% confluency (5 x 104 cells) in 12-well cell culture plates and grown for 24 h. Transfections were performed successfully with either Lipofect AMINE Reagent (Gibco BRL) or Superfect (Qiagen). Cells were transfected with 1 µg of plasmids encoding the GFP (pEGFP-N1, Clontech) together with 1 µg of pcDNA3, carrying the genes to be tested. The pcDNA3 plasmid served as control.
Cellular fractionation and western blotting
FL5.12 cells (107) were grown with or without IL-3 for the time periods as indicated. Cells were pelleted and suspended in 100 µl of an enzymatic reaction buffer (32), supplemented with protease inhibitors, lysed by sonication for 15 s in an ice bath. Subsequently, the lysate was centrifugated for 10 min at 1000 g for removal of unlysed cells and nuclei. The supernatant was then centrifugated for 1 h at 100 000 g (Kontron Instruments). The final supernatant served as a cytosolic fraction, the pellet as a crude membrane fraction which was resuspended in one volume of the lysis buffer with 10% Triton X-100. The protein concentration of both cytosolic and membrane fractions was determined by a Bio-Rad protein assay. Fractionated cell lysates were boiled for 10 min. Twenty micrograms of samples were separated on 15% SDS–PAGE, electrotransferred to an Immobilon-P membrane (Millipore), immunoblotted with a rabbit polyclonal anti-His antibody (Milan Analytica AG/CH) or anti-cyt. c antibody (Pharmingen), and developed with NBT/BCIP (Sigma). The total density of proteins present on the blotted membrane was quantified by computerized scanning densitometry using ImageQuan (Molecular dynamics).
Cell viability assay
REF52 cells were transfected with 1 µg of plasmids encoding GFP (pEGFP-N1, Clontech) together with 1 µg of pcDNA3, carrying genes to be tested. Green fluorescent cells were monitored, and normal flat and round apopotic cells were counted by microscopic examination after 24 h of transfection. At least 100 cells from each individual culture were analyzed. Cell survival is expressed as percentage of surviving cells per total number of cells, given with the standard deviation of the assay (21). For IL-3 deprivation experiments, transfected FL5.12 cells were washed three times in serum-free medium to remove IL-3 and cultured at 105 cells per ml in triplicate. At various time points at least 100 cells from each individual culture were analyzed by trypan blue exclusion staining. Viability was determined at various time points by trypan blue exclusion, counting at least 100 cells from each individual culture. The percentage of cell survival was calculated as the number of surviving cells per total cell count (32). Statistical differences (P < 0.05) between mean values were analyzed by the Kruskal–Wallis test.
Measurement of casp-3 activity and assessment of mitochondrial potential
ApoAlert CPP32 assay kits (Clontech) were used to measure casp-3 activity, following the manufacturer’s instructions. Enzyme reactions contained 20 µM DEVD-pNA (Clontech) and 10 µg of cellular extracts with or without 1 µM of the inhibitor DEVD-fmk (Clontech). The spectrophotometric reading was performed at 405 nm using a 96-well plate reader. Statistical differences (P < 0.05) between mean values were analyzed by the Kruskal–Wallis test.
To evaluate changes of mitochondrial potential, 106 cells/ml in PBS were incubated with DiOC6 (400 nM, Molecular Probes) for 15 min at 37°C, followed by FACS analysis of 105 cells measuring red fluorescence described previously (32). Control experiments were performed in the presence of 5 µM mC1CCP (Molecular Probes) for 15 min at 37°C.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr S.Korsmeyer for providing us with the FL5.12 clones employed in this study: wild-type, Neo, Bcl-2 overexpressing and Bax overexpressing. The study is supported by The Basler Cancer League (Krebsliga Beider Basel, grant 8/99 to H.Z.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +41 61 3259354; Fax: +41 61 3259060; Email: hzhang@uhbs.ch
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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