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The historic distinction between academic- and industry-driven drug discovery, whereby investigators at universities worked to uncover the elusive principles of basic science and drug companies advanced the identification of drug targets and probe discovery, has been blurred by an academic high throughput chemical genomic revolution. It is now common for academic labs to use biochemical or cell-based high throughput screening (HTS) to investigate the effects of thousands or even hundreds of thousands of chemical probes on one or more targets over a period of days or weeks. To support the efforts of individual investigators, many universities have established core facilities where screening can be performed collaboratively with large chemical libraries managed by highly trained HTS personnel and guided by the experience of computational, medicinal and synthetic organic chemists. The identification of large numbers of promising hits from such screens has driven the need for independent labs to scale-down secondary in vitro assays in the hit to lead identification process. In this chapter we will describe the use of luminescent and quantitative reverse transcription real-time PCR (qRT-PCR) technologies that permit evaluation of the expression patterns of multiple Unfolded Protein Response (UPR) and apoptosis-related genes and simultaneously evaluate proliferation and cell death in 96 or 384 well format.
The ability of the Unfolded Protein Response (UPR) to modulate cell death, following an unsuccessful attempt to restore homeostatic protein folding in the ER lumen, remains an incomplete story. Recently some of the key molecular players have been identified and at the transcriptional level and it has become clear that multiple proteins interacting in the nucleus to coordinately shut off survival genes and activate pro-death genes is a common theme. The ATF4-mediated induction of CHOP, following PERK activation and eIF2α phosphorylation, is a key event in the switch, under stress, from adaptation toward death and has received the most attention in the literature. Initial clues implicating CHOP as a participant in the UPR-mediated cell death program came to light when it was reported that overexpression of CHOP could induce cell cycle arrest and apoptosis (Barone, Crozat et al. 1994; Matsumoto, Minami et al. 1996); and that Chop null mice were partially resistant to ER stress-mediated apoptosis (Zinszner, Kuroda et al. 1998; Oyadomari, Koizumi et al. 2002). Though it is clear that CHOP has an important role in ER stress-induced apoptosis a comprehensive analysis of its target (downstream of CHOP or DOC) genes has not revealed a smoking gun (Wang, Kuroda et al. 1998) (and our un-published observation), suggesting that this effect might be indirect.
Though CHOP target genes capable of directly inducing apoptosis have not been identified it can induce the expression of death receptor 5 (DR5) and tribbles-related protein 3 (TRB3) in a stress-dependent fashion to modulate the UPR death response. DR5 is a member of the TNFR family and can mediate cell death via the FADD signaling complex (Chaudhary, Eby et al. 1997). Thapsigargin enhanced DR5 expression was found to be mediated by CHOP in human cancer cell lines and sensitized them to TRAIL-induced cell death (Hetschko, Voss et al. 2008). Increased expression of DR5 enhanced ligand binding and led to the recruitment of adaptor proteins at the intracellular DR5 death domain and initiated a signaling cascasde that culminated in the cleavage and activation of caspase 8 similar to TNFR1, Fas and DR3 and DR4. The discovery that CHOP could modulate DR5 expression linked the UPR to the “extrinsic” death receptor-mediated apoptosis pathway which, following caspase 8 cleavage, culminates in the activation of executioner caspases 3 and 7 to target substrates in the nucleus such as a lamins and PARP immediately prior to DNA fragmentation. It should be noted that additional in vitro experiments revealed that siRNA knockdown of DR5 could interfere with the conformational change of Bax and caspase 3 activation required for apoptotic cell death following stress (Yamaguchi and Wang 2004). Tribbles-related protein 3 (TRB3) has also been identified as an ER stress-inducible target of CHOP/ATF4 signaling that can modulate UPR-dependent cell death induced by various ER stressors (Ohoka, Yoshii et al. 2005; Ord and Ord 2005; Ord, Meerits et al. 2007). Though there is a report that siRNA knockdown of TRB3 could reduce ER stress-dependent cell death in 293 cells (Ohoka, Yoshii et al. 2005) most studies have reported that TRB3 antagonizes the anti-proliferative and cytotoxic effects of the UPR by down-regulating ATF4 transcriptional activity thereby lowering the level of intracellular reactive oxygen species (ROS) (Ord, Meerits et al. 2007).
The UPR utilizes the BCL2 family during the cell death process via distinct and complementary mechanisms. CHOP induction can dramatically reduce cellular levels of BCL2 to directly potentiate the release of cytochrome c and initiate the mitochondrial or “intrinsic” cell death pathway (McCullough, Martindale et al. 2001). The subset of BCL2 family members that possess only the BCL2 homology domain 3 (BH3 domain), in stark contrast to BCL2, are all known to be pro-apoptotic. In general this small group of proteins have a similar modus operandi in the apoptotic push toward death which is characterized by their ability to interact with BCL2 impeding its ability to keep Bax and Bak in an inactive conformation. Activation of Bax or Bak precipitates the release of cytochrome c from mitochondria and Ca+2 from the ER, thus setting in motion the process of apoptosis. Though currently 9 members of the BH3-only protein family have been identified only NBK/BIK, BIM, NOXA and PUMA have been closely associated with the UPR-mediated cell death (Morishima, Nakanishi et al. 2004; Fribley, Evenchik et al. 2006; Kieran, Woods et al. 2007; Shimazu, Degenhardt et al. 2007; Zou, Cao et al. 2009).
A number of molecules in addition to CHOP, ATF4, Bax/Bak, and caspase 12 are known to be involved with UPR-mediated cell death. It has been known for over a decade that thapsigargin could activate the c-Jun NH(2)-terminal kinase cascade and apoptosis in an oxidative stress-dependent fashion (Srivastava, Sollott et al. 1999). Several years later it was reported that the activation of IRE1α led to the formation of a tripartite complex at the cell membrane with TRAF2 and ASK1 prior to the activation of the JNK cell death program (Urano, Wang et al. 2000; Matsuzawa, Nishitoh et al. 2002; Nishitoh, Matsuzawa et al. 2002). It is clear that JNK plays an important role in UPR-mediated cell death. Since we will not describe any large scale methods focused to identify the activation of JNK signaling, further discussion has been omitted. For thorough and recent reviews of stress mediated activation of JNK signaling:[(Nagai, Noguchi et al. 2007; Rincon and Davis 2009)
When cells undergo apoptosis many distinct biochemical changes occur that can be readily detected to identify early or late stages of the death process. Recent advances in fluorescent and luminescent technology has made it possible to detect these changes with very limited numbers of cells and reagents in a 96 or 384 well format in a relatively cost-effective fashion. Importantly, we will describe how to monitor cell viability/proliferation and caspase activation in parallel to clearly establish the kinetics of cell growth/death and caspase activation to determine the contribution of apoptosis in UPR-mediated cell death. Members of the caspase family of cysteine proteases are necessary for nearly all apoptotic responses. Importantly, caspase 3−/−, caspase 7−/− and caspase 9−/− murine embryonic fibroblast were found to be resistant to thapsigargin, tunicamycin, brefeldin A and calcium ionophore-induced ER stress suggesting an essential role for mitochondria-mediated or intrinsic cell death following unresolved protein folding defect (Masud, Mohapatra et al. 2007). Although this group demonstrated that caspase 8−/− MEF’s were not protected from these stresses it has been reported that in murine cells caspase 12 can activate caspase 8 following UPR activation (Morishima, Nakanishi et al. 2002; Rao, Castro-Obregon et al. 2002). The caspase 12 gene in humans is inactive due to a single nucleotide polymorphism (Saleh, Vaillancourt et al. 2004).
Luminescent assays for simultaneous detection of proliferation and caspase 3/7 activation
Special note on “edge effect”. Unequal distribution of attached cells in 96 or 384 well plates, incubator heat gradient fluctuations or levelness of incubators can lead to a phenomenon known as edge effect. Edge effect is characterized by a ring or crescent-shaped pattern of adherent cells at the periphery of a well and can have dramatic effects on inter-well reproducibility. Several solutions including not using the outermost wells of a plate have been proposed. Another simple technique has been described whereby plates are allowed to sit in the tissue culture hood at room temperature for 1 hour before placing cultures in an incubator (Lundholt, Scudder et al. 2003).
The use of 96 or 384 well thermocyclers for qRT-PCR has moved academic laboratories’ ability to analyze gene expression light years beyond the Northern blot; however, most protocols still rely on the use of cumbersome and time consuming phenol-based extractions from relatively large numbers of cells for RNA isolation. The recent introduction of Cells to CT from (Applied Biosystems/Ambion) has provided a phenol-free system that we have found can provide enough high quality RNA template for the production of cDNA from as few as 500 cells less in than 10 minutes. In this section we will describe a slight protocol modification that increases by 2-fold the number of cDNA reactions that can be performed from the manufacturer’s indication. We will then describe how the cDNA can be diluted and interrogated with TaqMan (Applied Biosystems) primer probe sets to evaluate the expression of UPR and apoptosis genes in stressed cells.
|Component||Per Rxn (μl)||96 Rxns‡ (μl)||384 Rxns‡ (μl)|
|2X RT Buffer||12.5||1320||5280|
|20X RT Enzyme Mix||1.25||132||528|
|Reverse transcription||1||1||37||60 min|
|RT inactivation||2||1||95||5 min|
The TaqMan Gene Expression Assay and Master Mix cocktail and cDNA templates are added to 96 or 384 well plates separately, as described:
|Component||Per Rxn (μl)|
|20X TaqMan Gene Expression Assay||0.25|
|2X TaqMan Gene Expression Master Mix||2.5|
|cDNA template (diluted 1:50)||2.25|
Calculate the volume of TaqMan Gene Expression Assay and Master Mix cocktail required to measure each cDNA in triplicate. For example: To measure the expression of 18S (housekeeping gene) in 8 cDNA samples would require TaqMan Gene Expression Assay and Master Mix cocktail for 24 wells; (Figure 2, gray shaded wells). Calculations include a 10% overage to account for errors in pipetting.
|TaqMan Gene Expression Assay/Master Mix cocktail for 8 samples (24 Rxns)|
|Component||Per Rxn (μl)||24 Rxns (μl)|
|20X TaqMan Gene Expression Assay||0.25||6.6|
|2X TaqMan Gene Expression Master Mix||2.5||66|
Each cDNA should initially be diluted 1:50 in nuclease-free water; calculate the volume of diluted cDNA needed for triplicate samples (Figure 2, black shaded wells). Calculations include a 10% overage to account for errors in pipetting.
|Diluted cDNA Template:|
|Component||Per Rxn (μl)||12 Rxns (μl)|
|cDNA Template (1:50)||2.25||29.7|
Methods of calculating changes in gene expression vary by investigator and instrument and are therefore, omitted.
For semi-quantitative PCR analysis of XBP1 unspliced (XBP1u) and spliced (XBP1s) with Cells to CT™-derived cDNA the Taq PCR Core Kit (Qiagen) is routinely utilized, according the manufacture’s protocol.
|Forward Primer||1 (10 pmol/μl)||200 nM|
|Reverse Primer||1||200 nM|
|dNTP mix||1||200 μM each dNTP|
|10X Buffer (w/15mM MgCl2)||5||1X|
|Taq DNA Polymerase||0.25||2.5 U|
|Final Volume Master Mix||45|
|Initial Denaturation||1||1||1||94||3 min|
|Final Extension||3||1||72||10 min|
Note: We have improved throughput of hXBP1 amplicon analysis with the QIAxcel System (Qiagen). For amplicon analysis with this machine, the appropriate cartridge is the QIAxcel DNA High Resolution Gel Cartridge (number); and the analysis can be performed using the OM500 method.
The hallmark of late-stage apoptosis has come to be the endonucleolytic cleavage (laddering) of DNA between nucleosomes into different sized fragments (Schwartzman and Cidlowski 1993).
DNA lysis buffer prepared by mixing:
Proteinase K (cat# P6656, Sigma)
Standard agarose gel electrophoresis equipment and reagents
Note: A laddered appearance of the DNA is indicative of apoptosis; a smeared appearance suggests the observed cell death is the result of some other form cell death such as necrosis.
The authors appreciate the critical review of this manuscript by Dr. Harmeet Malhi. Portions of this work were supported by NIH grants DK042394, HL052173, and HL057346, as well as a MH084182 and MH089782 (RJK). Additionally, AMF is supported by DE019678.