研習報告與討論
Bcl-2 Family Gene Cloning and Its Role in Apoptosis
姓名： 劉 昆 瑋
學號： 891610
研習地點：Dr. Yi-Te Hsu’s lab,
Dept. of Biochemistry & Molecular Biology, Medical University of South Carolina, SC , USA
1. Abstract
Bcl-2 and Bcl-XL are pro-survival members of the Bcl-2 family and have been widely shown to antagonize the death promoting activity of Bax. However, it was recently reported by Kitanaka et al. that in a transient expression system, overexpression of Bcl-2 was unable to block Bax-induced cell death [1]. In stably expressed Bcl-2 and Bcl-XL cell lines, our lab found that Bax translocation to mitochondria was strongly inhibited. On the other hand, in transient transfection studies, we found that Bcl-2 was unable to inhibit Bax-mediated cell death. However, this lack of Bax inhibition is not due to the failure of Bcl-2 in blocking Bax localization to mitochondria but rather due to the intrinsic toxicity associated with Bcl-2 overexpression.
Furthermore, we intend to examine the effects in mice and rats, thus my work was focused on the cloning of mouse Bcl-XL , Rat Bcl-2 and Bcl-XL. And with the immunofluorescence technique, I found that rBcl-2 toxicity in transient expression system may involve translocation of Bcl-2 to endoplasmic reticulum, which will be furthered confirmed in following series of experiments.
2. Introduction
Apoptosis, or programmed cell death is an essential physiological process that plays a critical role in development and tissue homeostasis. Members of the Bcl-2 family (including Bcl-2, Bcl-XL , Bax ) play key roles in the regulation of apoptosis. They are membrane-associated and have been proposed to regulate apoptosis through both homodimerization and heterodimerization [2]. So far, over two dozen homologs of Bcl-2 have been identified [3]. It was shown that overexpression of Bcl-2 can promote cell survival against various cytotoxic insults [4]. Bcl-XL, like Bcl-2, inhibits apoptosis induced by a number of stimuli [5].
In addition, Bcl-2, Bcl-XL, and Bax have a hydrophobic segment at their carboxyl terminal ends. For Bcl-2 and Bcl-XL, these hydrophobic segments are responsible for the localization of these proteins to nuclear envelopes, endoplasmic reticulum and mitochondrial outer membranes [6-7]. For Bax, despite the presence of this hydrophobic segment, is predominantly a soluble protein in healthy living cells. Upon the induction of apoptosis, Bax is believed to undergo a conformational change, leading to the exposure of this hydrophobic segment and the translocation of Bax from the cytosol to mitochondria [8]. Bax subsequently oligomerizes and forms pores on mitochondrial outer membrane surface to cause the release of cytochrome c [9-10]. Cytochrome c then activates caspases to induce cell death [11].
Bcl-2 and Bcl-XL have been shown to antagonize the pro-apoptotic activities of Bax [2,12]. The primary mechanism of this antagonism appears to be their inhibition of Bax translocation from the cytosol to mitochondria. Most of the Bcl-2/Bax coexpression studies were carried out using stable cell lines expressing Bcl-2 and Bcl-XL. However, it was recently shown that the transient expression of Bcl-2 failed to block Bax-mediated cell death [1].
3. Materials and Methods
Generation of Bcl-XL and Bcl-2 expression constructs –The cDNAs encoding human and mouse Bcl-2 and human Bcl-XL were kind gifts of Drs. Stanley Korsmeyer, Carlos Croce, and Craig Thompson. Full-length rat Bcl-2 and Bcl-XL were PCR-amplified and cloned into the EcoRI restriction site of pcDNA 3 expression vector.
Cell culture, transfection of cell lines –Cos-7, HeLa, and H9c2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplements with 2 mM glutamine and 10 % fetal bovine serum and maintained at 37 °C in the presence of 5% CO2. The day prior to transfection, Cos-7, HeLa, and H9c2 cells were plated onto 6-well plates to approximately 70-80 % confluency. For Cos-7 and HeLa cells, FuGENE (Roche) was used for the transfection using the protocol described by the manufacturer (typically 3 μl FuGENE reagent per well). For co-transfection studies with C3-EGFP or C3-EGFPBax plasmids, a 1:3 ratio (0.25 μg: 0.75 μg/well) of GFP reporter plasmid to Bcl-2 or Bcl-XL plasmid constructs was used. For some studies, 25 μM z-VAD-fmk was added at 6 hours post transfection to prevent the rounding up of cells resulted from cell death due to the overexpression of Bcl-2 or Bax.
Immunofluorescence microscopy – Cos-7 cells were plated onto 6-well plates and transfected with 0.5 μg/well of either C3-EGFP-Bax, C3-EGFP-Bcl-2, or C3-EGFP- Bcl-XL plasmid. At 6 hours post-transfection, 25 μM z-VAD-fmk was added. At 14 hours post-transfection, cells were washed with PBS, fixed in 4% paraformaldehyde for 10 min and permeabilized in 0.02 % saponin in PBS for 15 min. The cells were then blocked for 30 min in the blocking buffer containing PBS, 5 % fetal bovine serum, and 0.02 % saponin. The cells were subsequently incubated in 2 μg/ml monoclonal antibody diluted in the blocking buffer for 3 hours. After washing with PBS/0.02 % saponin, the cells were incubated with 7.5 μg/ml rhodamine (TRITC) labeled goat anti-mouse IgG for one hour. The cells were then washed with PBS and treated with the antifade reagent. Monoclonal antibodies for Bcl-2, Bcl-XL ,Bax were previously generated by Dr. Hsu’s research group colleagues. Secondary antibody is conjugated with FITC (green) or TRITC (red) in fluorescence microscopy.
SDS-PAGE and ECL Immunoblotting (Western Blotting) – For western blotting analysis of the transfected cells, cells from each well of the 6-well plate were washed with phosphate buffered saline (PBS) and solubilized in 150 μl 10 mM Hepes pH 7.4, 1 % Triton X-100, 0.1 % sodium dodecyl sulphate (SDS), 150 mM NaCl, and 25 μg/ml phenylmethylsulfonyl fluoride. The lysate was spun at 16,000xg in a microfuge and the supernatant was mixed 2:1 with the SDS gel loading buffer. The samples were then analyzed by SDS-PAGE and ECL western blotting.
Confocal Microscopy – Detecting further protein localizations by examining several single thin layer confocal pictures inside the cells and, then overlapping them to have a three-dimensional view.
RNA purification and two-step RT-PCR – Total RNAs were isolated from KB cell line cultured in 3.5cm-diameter dish by the TRIZOL ® Reagent provided by GIBCO BRL®. cDNA was synthesized by M-MLV Reverse Transcriptase provided by Invitrogen TM and then underwent PCR reaction.
Generation of the anti-Bcl-2 monoclonal antibody – A new anti-Bcl-2 monoclonal antibody uBcl-2 3B3 was generated by immunizing mice with keyhole limpet hemocyanin conjugated to a peptide corresponding to amino acids 5- 17 of human Bcl-2 (CGRTGYDNREIVMK). Splenocytes taken from the immunoreactive mouse were fused to NS-1 myeloma cells by PEG 4000 and the resulting hybridomas were selected with a hypoxanthine/aminopterine/thymidine medium [12, 13].
4. Results and Discussion
Transient overexpression of Bcl-2 leads to cell death – In order to determine whether the morphological changes associated with the transient expression of GFP-Bax and Bcl-2 is caused by Bax or Bcl-2 or a combination of both, Cos-7 cells were co-transfected with the GFP reporter in the presence of either pcDNA 3 vector control, human Bax, or human Bcl-2. As shown in Fig 1., the GFP/vector controltransfected cells displayed a normal cellular morphology. On the other hand, approximately a third of the cells transfected with GFP and human Bax were rounded-up and some had blebbed membranes, consistent with the previous report that Bax overexpression leads to cell death [1]. Surprisingly, over half of the cells transfected with GFP and human Bcl-2 were also rounded-up with blebbed membranes. And the toxicity of transiently expressed Bcl-2 might involve the E.R. participation, which will be further confirmed.
Subcellular Localization of rat Bcl-2, Bcl-XL within the cells– Rat Bcl-2, Bcl-XL were cloned and transfected into cell lines as previously mentioned. And fluorescence microscopy showed that both proteins were mitochondria-bound when transiently expressed. (Fig 2.)
Rat Bcl-2, Bcl-XL co-transfection – Rat Bcl-2 , Bcl-XL were co-transfected by FuGENE reagent as previously mentioned. And the result is shown (Fig 3.)
Bcl-2 has been widely shown to be capable of antagonizing the pro-apoptotic function of Bax. These studies were mainly carried out in stably expressed Bcl-2 cell lines. The primary mechanism by which the pro-survival family members such
as Bcl-2 and Bcl-XL block Bax's function appears to be their inhibition of Bax
translocation to mitochondria [15,16]. Further analysis indicates that there is an intrinsic toxicity associated with Bcl-2. In fact, we found that transient expression of Bcl-2 is even more toxic than that of Bax. This toxicity of Bcl-2 has thus masked its anti-Bax activity and this appears to be the primary reason why transient expression of Bcl-2 fails block Bax-mediated cell death.
Recent reports showed that transient expression of human Bcl-2 is cytotoxic[17-18]. This suggests that Bcl-2's cytotoxic nature must have somehow been suppressed or eliminated in stably expressing cells.
Finally, Bcl-2's pro-survival function has made this protein an ideal candidate in gene therapy-based intervention of unwanted cell death. The viral-based gene delivery systems, however, are quite similar in principle to transient expression of Bcl-2 and they could potentially result in the promotion of cell death rather than cell survival. This particular issue would thus need to be taken into consideration for Bcl-2-based gene therapy approaches.
GFP + pcDNA 3 GFP + hBax GFP + hBcl-2
Fig 1. Transient expression of Bcl-2 and Bcl-XL blocks Bax translocation to mitochondria but transient expression Bcl-2 alters Cos-7 cell morphology.
3B3 , rBcl-2 10C4 , rBcl-2
7D9 , rBcl-xl 2H12 , rBcl-xl
Fig 2. Subcellular Localization of rat Bcl-2, Bcl-XL within the cell.
Fig 3. Rat Bcl-2, Bcl-XL co-transfection and underwent ECL western blotting by monoclonal antibodies uBcl-XL 2H12 and mBcl-2 10C4 simultaneously.
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