|
ARTICLE |
CORRESPONDENCE Iannis Aifantis: iaifanti{at}medicine.bsd.uchicago.edu
![]() ![]() ![]() ![]() ![]() ![]() |
---|
M. Mandal and C. Borowski contributed equally to this work.
The interval between thymic entry of multipotent bone marrow lymphoid progenitors and thymic export of mature
In addition to its role in "selecting" for further development exclusively those progenitors containing a productive TCRß rearrangement, driving entry into the cell cycle, preventing further rearrangement of TCRß loci, and down-regulating surface expression of CD25, pre-TCR signaling is essential for survival of ß T lymphocyte progenitors (2). Analyses of mice unable to generate intact pre-TCR complexes (RAG/, CD3
/, SCID) revealed large increases in the proportion of DN thymocytes expressing an apoptotic surface phenotype, thereby linking the pre-TCR to thymocyte survival (3). Complementation with cytoplasmic tail mutant pT
molecules (4) or with a TCR
molecule (5) failed to rectify the survival defect in the DN compartment of pT
2/2 mice, implicating specifically the pT
component as crucial for pre-TCRinduced survival signals.
The prevalence of NF-B consensus sites among cis-elements controlling the expression of multiple antiapoptotic genes (for review see reference 6), in combination with the definitive placement of NF-
B as a distal effector of pre-TCR signaling (7, 8), establishes an indirect association between the pre-TCR and the survival of developing thymocytes. The search for distal mediators of pre-TCRinduced survival has met with little success. The ß selection process proceeds normally in progenitors lacking pro-survival BCL-2 family members BCL-2 or BCL-xL (9, 10), and neither BCL-2 nor BCL-xL transgenes restore
ß T lymphocyte development in pre-TCRdeficient mice (1113). T lymphocyte progenitors lacking the pro-survival Bcl-2 family member MCL-1 fail to progress through the DN2 to DN3 developmental transition, implicating MCL-1 as a crucial mediator of early cytokine-derived, rather than later pre-TCRderived, survival signals (14). Further refuting a critical role for BCL-2 is the observation that post-ß selection thymocytes contain lower levels of Bcl-2 protein than those that have not yet entered ß selection (15). However, a recent report presented conflicting evidence, suggesting that BCL-2 expression is induced by the pre-TCR in vitro (16).
Similarly, very little is known about the signaling pathways inducing death in pre-TCR thymocytes. The initial suggestion that the Fas-induced death pathway is essential for preT cell death has been challenged by recent studies of Newton et al. (17), in which the thymocyte profiles of RAG-1/lpr mice were found to be similar to those of their RAG-1/ littermates. Fas is a member of the TNF receptor family, whose members interact via their death domains with cytoplasmic adaptors including FADD and TRADD (18). The same investigators have shown that RAG-1/FADD/ preT cells differentiate and reach the double positive (DP) stage (17). These important observations suggested that FADD could be essential for the transduction of death signal within preT cells and fueled the search for an active pre-TCRdependent mechanism of cell death inhibition.
In this report, we direct our search toward antiapoptotic genes potentially targeted by pre-TCRinduced NF-B transcription factors. We identify the pro-survival A1 gene as a direct transcriptional target of both pre-TCR and NF-
B. Moreover, we connect transcriptional activation of A1 to PLC
, mobilization of Ca2+, and activation of protein kinase C (PKC) kinases. We also show that A1 expression inhibits preT cell death by suppressing the activation of the effector caspase-3. Finally, we show that A1 is up-regulated in both human and mouse preT cell leukemias, making it a possible target of the transformation event. Our observations suggest that through NF-
B activation and A1 expression, the pre-TCR ensures the promotion of physiological T cell development.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
BCL-2, MCL-1, and BCL-xL are not targets of the pre-TCR
Conflicting reports described the roles of BCL-2 and BCL-xL in preT cell death, and recent data indicated that yet another antiapoptotic BCL-2 family member, MCL-1, may regulate survival of thymocytes at an earlier, cytokine-mediated stage (14). To test whether pre-TCR signaling induces expression of these prosurvival factors, we performed real-time RT-PCR analysis using cDNA isolated from sorted DN3, DN4, and DP thymocytes. As evident from Fig. 2, none of these factors were induced by the pre-TCR. MCL-1 expression was high in DN3 cells. In agreement with previous reports, it decreased in DN4 and peaked again at the DP stage. BCL-xL expression was low in both DN3 and DN4 populations, peaking at the DP stage. BCL-2 expression seemed to be actively down-regulated upon pre-TCR expression, reaching a minimum at the DP stage (Fig. 2 A). Similar results showing no up-regulation of BCL-xL and MCL-1 and a down-regulation of BCL-2 were obtained using two distinct pre-TCR (SCIET27 and LR2) and pre-TCR+ (SCB29 and LR2wtpT) cell lines (Fig. 2 B and unpublished data; reference 19).
|
|
Activation of phospholipase C (PLC
), Ca2+, and NF-
B are critical for A1 induction
The above observations place A1 expression downstream of the pre-TCR, but the signaling components connecting pre-TCR signals with A1 induction remain unidentified. We initially addressed the role of NF-B in pre-TCRinduced A1 expression. As mentioned above, pre-TCR+ cell lines (SCB29 and LR2WTpT
), but not their pre-TCRdeficient counterparts (SCIET27 and LR2), contained abundant A1 transcripts. To inhibit NF-
B activation in SCB29 and LR2WTpT
cell lines, we generated a retroviral vector encoding a mutated nondegradable form of the inhibitor of NF-
B
(IKB
; reference 8). When expressed in pre-TCR+ SCB29 and LR2WTpT
cell lines, IKB
completely inhibited pre-TCRinduced A1 expression, suggesting an essential role of NF-kB in pre-TCRdependent up-regulation of A1 (Fig. 4 A). These observations are consistent with those of Voll et al. (8), who demonstrated that preT cellspecific expression of an IKB
transgene (driven by the p56lck promoter) slowed pre-TCRdriven
ß T lymphocyte development.
|
PKC activation is essential for A1 induction and survival
PLC activity generates both inositol triphosphate, which initiates Ca2+ mobilization, and diacylglycerol (DAG), which activates downstream kinases such as PKC. To address the potential role of PKC in pre-TCRinduced A1 expression, we expressed a constitutively active form of PKC (PKC
CAT) in pre-TCRdeficient (and thus A1) SCIET and LR2 cell lines. PKC activation strongly induced A1 expression in the SCIET (Fig. 5 A) and LR2 (not depicted) cell lines. To establish PKC specificity, we expressed an activated (myristoylated) form of PKB/Akt (Myr-Akt), another kinase involved in the transduction of pre-TCR signals (unpublished data). As shown in Fig. 5 A, Myr-Akt failed to up-regulate A1 expression, classifying pre-TCRinduced A1 expression as strictly dependent on PKC.
|
A1 as a regulator of preT cell survival
To study the function of A1 in the promotion of preT cell survival, we ectopically expressed A1d (the most commonly expressed A1 isoform) in SCIET preT cells (4). A1d and EGFP were expressed on a single transcript after infection with a bicistronic A1-IRES-EGFP retrovirus. This retrovirally encoded A1 protein was properly expressed and targeted to the mitochondria (not depicted). As preT cells sustain DNA breaks generated during ongoing recombination at their TCR loci, we investigated A1's ability to inhibit death induced by DNA-damaging ultraviolet irradiation. A1d expression efficiently suppressed irradiation-induced cell death in SCIET (Fig. 6 A) and LR2 (not depicted) preT cells. Mechanistically, the inhibition of cell death resulted from an A1-mediated reduction in caspase-3 activation (Fig. 6 B). Similar results were obtained with dexamethasone treatment as a death stimulus (not depicted).
|
To demonstrate that A1 is necessary for preT cell survival, we studied preT cell survival in the absence of A1 expression. The existence of three independent A1 isoforms precludes gene-targeting approaches. Multiple consecutive targeting steps would be necessary, as the genes encoding the three A1 isoforms are not all clustered together on chromosome 9. In addition, the significant conservation in both exon and intron sequences shared among the A1 isoforms would make design of gene-targeting vectors extremely difficult. Therefore, we used stable RNA interference to knock down A1 expression in a T lymphocytespecific manner. To eliminate concern about redundancy among A1 isoforms, we generated short interfering RNAs (siRNAs) capable of knocking down expression of all three A1 isoforms. These siRNAs were expressed using a self-inactivating retrovirus carrying a U6 Pol-II promoter able to drive expression of siRNAs. We tested three different siRNAs, and achieved optimal interference with siRNA-A (Fig. 7 A). Real-time RT-PCR analysis showed silencing efficiency at 7585% (Fig. 7 A). The effects of A1 deficiency on pre-TCR function were assessed in the pre-TCR+ N3-T cell line (Fig. 8 D). N3-T cells expressing each of the three different siRNAs (EGFP+) were mixed at a ratio of 50:50 with EGFPN3-T cells. As shown in Fig. 7 B, N3-T cells expressing A1-specific siRNAs exhibited a survival disadvantage, proportional in severity to the efficiency of A1 knockdown. This defect is due to loss of survival, as at any given time of the culture, the proportion of cells binding annexin V was two to four times higher in the population expressing A1-siRNA (siRNA-A) when compared with cells expressing a control EGFP-siRNA (not depicted). No significant differences in cell cycle progression were observed between cells expressing EGFP-siRNA and A1-siRNA constructs (not depicted). When infected with the same siRNAs, neither 3T3, 293T, nor LR2 cells (Fig. 7 C) showed survival defects, suggesting that the effect of A1 knockdown is specific to cells that specifically up-regulate A1 due to pre-TCR overexpression (Fig. 8). Moreover, BCL-xL and BCL-2 expression remained unaltered in N3-T cells that undergo apoptosis due to the A1 deficiency, suggesting that these two genes are not involved in the maintenance of N3-T cell survival (unpublished data).
|
|
A1 expression is a molecular target of preT cell transformation
Deregulated pre-TCR signaling triggers aggressive preT cell leukemia in mice (30, 31). Genes encoding molecules involved in pre-TCR signaling (p56Lck, ZAP-70, TCRß, CD3, and cyclin D3) are overexpressed in human T cell acute leukemias (T-ALLs) arising from early to late cortical thymocytes, which are characterized by the expression of the oncogenes HOX-11+ and TAL-1+. Thus, it is conceivable that the survival advantage conferred by pre-TCRinduced antiapoptotic genes like A1 may contribute to the transformation process (not as a bona fide oncogene but as a target of the transformation event). To connect pre-TCR and A1 expression to a precise window of thymocyte differentiation, we used the patient expression profile data presented by Ferrando et al. (32) to analyze the expression of pT
within the three different clusters of human T-ALL cases. As shown in Fig. 8 A, pT
expression is seen primarily within Hox-11+ early cortical thymocytes, rather than within LYL1+ CD34+ progenitors or TAL-1+ mature T cells. To link A1 expression with induction of T cell leukemia, we selected representative cases from each cluster of human T-ALL cases and performed quantitative real-time RT-PCR. As evident from Fig. 8 B, A1 expression correlated only with HOX-11 or TAL-1expressing samples. This is consistent with the notion that A1 is induced only after pre-TCR signaling and peaks at the DP stage of development. Interestingly, BCL-2 expression peaked early in development (in CD3 CD34+ samples) and declined after the appearance of the pre-TCR and A1 (Fig. 8 B). This observation is consistent with our previous expression analysis of A1 and BCL-2 during physiological T cell development (Figs. 2 and 3).
To complement these studies, we analyzed A1 expression in a mouse model of T cell leukemia. In this model, introduced by Bellavia et al. (33), T cellspecific expression of a constitutively active form of Notch-3 induces malignant transformation of thymocytes. We have chosen Notch-3 because "nearest neighbor" analysis of the T-ALL database used above had shown that pT and Notch-3 share a strikingly identical expression profile (unpublished data). The Notch transformation is strictly dependent on pre-TCR signaling (31). N3-T, an immature HSAhi CD25lo CD4 CD8 cell line derived from the thymus of a Notch-3 transgenic animal, most likely represents the pre-TCR+ DN3 to DN4 transitional stage of thymocyte development (Fig. 8 C; reference 33). Surface pre-TCR levels on N3-T cells are even higher than those on WT DN4 cells (Fig. 8 C). In line with our human T-ALL studies, N3-T cells expressed A1 at levels significantly higher that those in WT DN4 thymocytes or in DN cell lines (Fig. 8 D). As shown in Fig. 7 B, A1 is crucial for in vitro survival of N3-T cells, as A1 "knockdown" conferred a significant survival disadvantage.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
One can argue, however, that the data presented are equally consistent with the notion that A1 is a Notch target, as Notch can also activate the NF-kB pathway. This is an intriguing possibility that could provide insights in the mechanism of cooperation between Notch and the pre-TCR (37). However, multiple lines of evidence argue against this scenario and highlight the unique ability of the pre-TCR to regulate A1 expression. Initially, expression of activated forms of Notch-1 (N1-IC) in pre-TCR lines (SCIET27 and LR2) failed to up-regulate A1 expression (unpublished data). Moreover, specific mutation of the pre-TCR (cytoplasmic truncation) blocked any A1 expression without affecting Notch expression (Fig. 3). Finally, DN thymocytes from pT/ mice that overexpress active Notch-3 show activation neither of PKC nor of NF-
B, suggesting that the pre-TCR, rather than Notch, regulates the activity of this pathway in developing preT cells (unpublished data).
Why the pre-TCR uses A1 rather than the prototypical antiapoptotic molecules BCL-2 or BCL-xL is not yet understood. However, overexpression of BCL-2 is unable to restore normal developmental progression in pre-TCRdeficient pT2/2 (unpublished data) or RAG-1/ mice (11). The preferential use of A1 might be due to the antiproliferative properties ascribed to BCL-2. Introduction of a BCL-2 transgene into WT mice actually impaired the proliferation of TCRß-selected DN4 thymocytes (38). It was suggested that this antiproliferative effect depends on the ability of BCL-2 to bind to and sequester the calcium-dependent calcineurin phosphatase, thereby inhibiting NF-AT nuclear translocation and activation (39, 40). This interaction between BCL-2 and calcineurin depends on conserved regions in the BH4 domain of BCL-2, which are also present in BCL-xL and MCL-1. In contrast, all three A1 genes lack these conserved calcineurin interaction sites (unpublished data). Thus, BCL-2, BCL-xL, and MCL-1, but not A1, may prevent NF-AT activation, which is important for pre-TCR function (7). Consistent with this notion, Gonzalez et al. (28) recently have shown that overexpression of A1, but not BCL-2, allowed normal proliferation of mature activated T lymphocytes.
Our studies underline the role of PKC as an intermediate transducer of pre-TCR signals. PKC isoenzymes are grouped into two major subfamilies. The activation of conventional PKCs (cPKCs) as well as novel PKCs (nPKCs) typically involves recruitment to membranes and interaction with or allosteric activation by DAG. Ca2+ binding is an essential requirement for cPKCs, but not nPKCs (41). PKC, cPKC, PKC
, and nPKC, are the major PKCs expressed in thymocytes (41). In our experiments, PMA plus Iono treatment induced A1 expression in pre-TCR cells, whereas Iono alone did not. Also, PMA plus Iono in the presence of the ER Ca2+ chelator, TMB-8, induced neither A1 expression nor NF-
B activation. As induction of NF-
B activation and A1 expression required both PMA and Iono, it is tempting to suggest that pre-TCR signaling activates PLC
, which mobilizes Ca2+, which, along with DAG, activates only cPKC isoenzymes (i.e., PKC
). Consistent with this view is the lack of a pre-TCRdependent phenotype in mice lacking the PKC
isoenzyme (42). However, we cannot exclude that PKC
, although not absolutely essential, plays a secondary role amplifying PKC
-mediated signals to the nucleus.
A recent study illustrated PKC's importance in pre-TCRmediated allelic exclusion at the TCRß locus (43). Separate work demonstrated that although activation of PKC can partially restore DN to DP transition in RAG-1/ mice, it is not able to drive thymocyte expansion (a phenotype identical to those of mice reconstituted with A1+ RAG-1/ progenitors, presented here; reference 37). Perhaps the bifurcation in pre-TCR signaling that occurs downstream of the LAT/SLP-76 complex leads in one direction toward PKC-independent signals inducing thymocyte expansion (Raf/Ras, Akt) and in the other direction toward PKC-dependent signals establishing TCRß allelic exclusion and ensuring survival.
The critical role of A1 in transmitting pre-TCRinduced antiapoptotic signals and the previous correlation of deregulated pre-TCR signaling with T cell leukemia, prompted us to analyze the patterns of A1 expression in human T-ALL cases and in a Notch-induced model of mouse leukemia. We found a unique correlation between A1 expression and receipt of pre-TCR signals and have shown that A1 knockdown of A1 expression can affect survival of a preT cell leukemic line. We recently identified cyclin D3 as an essential mediator of pre-TCRinduced proliferative signals and suggested that cyclin D3 expression was essential for transformation of preT cells (44). A1 overexpression may cooperate with cyclin D3 to provide a survival signal rendering transformed preT cells resistant to apoptotic signals. These findings could have clinical importance because TAL1+ leukemias are resistant to chemotherapy potentially due to the overexpression of the antiapoptotic A1 (32).
The pre-TCR possesses the unique ability to override cell intrinsic (i.e., DNA damage-induced) and extrinsic (i.e., death receptorinduced) apoptotic stimuli. We describe a novel mechanism by which the pre-TCR meets this responsibility, namely transcriptional induction of A1. Our data do not exclude the possibility that the pre-TCR may use additional mechanisms of cell death inhibition or that non-pre-TCRinduced antiapoptotic factors (like BCL-xL or BCL-2) could assist A1 in the regulation of cell survival. Indeed, the pre-TCR could directly down-regulate the transcription of proapoptotic genes or it could regulate the stability and degradation of proapoptotic molecules at a posttranslational level, via phoshporylation and/or ubiquitination. These potential alternative regulation mechanisms as well as the extent of functional cooperation of A1 with other antiapoptotic genes during ß selection are currently under investigation.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patient material
Samples of cryopreserved lymphoblasts from children and young adults treated in Total Therapy studies XIXIII at St. Jude Children's Research Hospital were obtained with informed consent at the time of diagnosis before any chemotherapy was given. All protocols and consent forms were approved by the hospital's institutional review board, and informed consent was obtained from parents, guardians, or patients (as appropriate).
RT-PCR and quantitative RT-PCR
Total cellular RNA was isolated by using an RNeasy kit (QIAGEN). Each cDNA template was made from total RNA using the Superscript II-RT kit (Invitrogen). PCR reactions were performed with RedTaq (Sigma-Aldrich). Transcripts of all murine A1 isoforms were detected using two primer pairs: forward-1, 5'-cattaactggggaaggattgtgac-3', reverse-2 5'-gcagaaaagtcagccagccagatt-3', and forward-2, 5'-tcatgcatatccactccctggctgagc- 3', reverse-2, 5'-gtcctgtcatctgcagaaaagtcagcc-3'. Murine ß actin transcripts were detected using 5'-tggaatcctgtggcatccatgaaac-3' as the forward primer and 5'-taaaacgcagctcagtaacagtccg-3' as the reverse primer. For detection of antiapoptotic genes we used the following: BCL2F, 5'-atcttctccttccagcct-3'; BCL2R, 5'-tcattcaaccagacatgc-3'; MCL-1F, 5'-agagcgctggagaccctg-3'; MCL-1R, 5'-ctatcttattagatatgccagacc-3'; BCL-xL forward, 5'-tggagtcagtttagtgatgtc-3'; and BCL-xL reverse, 5'-gctcgattgttcccgtagag-3'.
Quantitative analysis of cDNA amplification was assessed by incorporation of SYBR Green into dsDNA. PCR reactions containing 1 ug cDNA template, 0.5 µM each of the primers, and SYBR Green PCR Master Mix (Applied Biosystems) were performed in a total volume of 25 µl. Gene expression was analyzed using the ABI PRISM 7700 Sequence Detector (Applied Biosystems) and ABI Prism Sequence Detection Software version 1.9.1 (Applied Biosystems). Normalization of samples was performed by dividing the value of the unknown gene by the value of the endogenous reference genes (ß actin). Quadruplicate reactions were performed using all cDNA samples. For the real-time PCR analysis using human leukemic samples, total RNA from cryopreserved lymphoblasts was extracted using the RNAqueous kit (Ambion).
The cDNA was analyzed by quantitative PCR using the SYBR Green RT-PCR Core Reagents (Applied Biosystems). Quantitative PCR analyses were performed with an ABI PRISM 7700 Sequence Detection System instrument (Applied Biosystems) in a total volume of 25 µl. Relative expression was calculated for each gene using the CT method. Expression levels were normalized according to expression of GAPDH housekeeping gene. We used the following primers: GAPDH forward, 5'-gaaggtgaaggtcggagt-3'; GAPDH reverse, 5'-gaagaggtgatgggatttct-3'; BCL2 forward, 5'-gattgtggccttctttgagttcg-3'; BCL2 reverse, 5'-cgtacagttccacaaaggcatcc-3'; BCL2-A1 forward, 5'-ccccggatgtggatacctataagg-3'; and BCL2-A1 reverse 5'-attttcccagcctccgttttgc-3'.
Microarray analysis.
Expression data from 43 T cell lymphoblastic leukemia samples reported by Yeoh et al. (46). Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling was also according to Yeoh et al. (46). Normalization of global fluorescence differences across arrays and calculation of gene expression values were preformed using dChip (http://www.dchip.org). Normalization of relative expression levels across all 43 samples and visualization of selected T cell differentiation marker genes (TCR-related genes) were performed using Gene Cluster (http://www.broad.mit.edu/cancer/software/genecluster2/gc2.html). Additional information concerning the samples and the microarray analysis can be found at: http://www.stjuderesearch. org/data/ALL1.
Flow cytometric analysis and cell sorting.
Anti-CD4 (L3T4), CD8 (53-6.7), CD25 (3C7), CD44 (IM7), B220 (RA3-6B2), CD11c (HL3), NK1.1 (PK136), TCRß (H57-597), TER-119 (TER-119), Mac-1 (M1/70), Gr-1 (RB6-8C5), and heat stable antigen (M1/69) mAbs were from BD Biosciences. These mAbs were directly coupled to FITC, PE, CYC, APC, or biotin. Surface marker expression on thymocytes and peripheral T cells was visualized using a FACSCalibur (Becton Dickinson) and analyzed with Flow-Jo and CELLQuest software. Cell sorting was performed using a Mo-Flo (DakoCytomation). DN thymocytes were isolated as described by Aifantis et al. (7).
Retroviral constructs and progenitor infections.
MigR1 (30) and a previously described moloney murine leukemia virusbased retroviral vector (4) were used for retroviral transduction experiments. PKCCAT (a constitutively active form of PKC) constructs were cloned upstream of the MigR1 IRES-GFP cassette (37). Mouse A1d cDNA was amplified from WT thymocyte cDNA using the following primers: A1XhoUp1, 5'-gcctcgagcaacagcctccagatatgattaggg-3' and A1BamLow1, 5'-cggatcctaaccattctcctgggagccaagg-3'. After amplification, the PCR product was sequenced and cloned in the XhoIBamHI sites of the moloney murine leukemia virusbased vector upstream of an IRES-EGFP cassette (4). Retroviral supernatants were generated as described previously (47).
Retrovirally expressed A1 siRNA.
We have used the Banshee-EGFP (48) retroviral vector (provided by J. Alberola-Ila, California Institute of Technology, Pasadena, CA) to express small interfering RNAs in preT cells. The cloning procedure has been described by Hernandez-Hoyos et al. (48). The following siRNA sequences were used: siA1-A, 5'-ggaaatgctctttctcctcaa-3'; siA1-C, 5'-gagcagattgccctggatgta-3'; and siA1-D, 5'-ggagaatggatacggcggaat-3'. After cloning, sequencing was used to ensure the insertion of the correct sequence. Concentrated retrovirus was generated as described above.
T cell differentiation in OP9-DL1 culture
2 x 103 sorted DN2 or DN3 thymocytes were placed in each well of a 24-well tissue culture plate containing a confluent monolayer of OP9-DL1 cells in DMEM (Life Technologies) supplemented with 15% FCS, penicillin/streptomycin, L-glutamine, and 5 ng/ml rIL-7 and rFlt3L (PeproTech) as described previously (27). The PKC inhibitor calphostin C (or DMSO) was added (100 nM) in wells. Differentiation was analyzed after 8 d, and viability was assessed by propidium iodide staining.
Active caspase-3 assay
SCIET27, SCIET27 transfected with PKCCAT, or SCIET27 transfected with A1 were irradiated (480 rads). At various time points after irradiation, 106 cells were harvested, washed, and fixed/ permeabilized. Active caspase-3 was detected using a PE-conjugated active caspase-3 mAb (BD Biosciences).
Western blot analysis
Rag-2/ sorted CD25+ or CD2544 (after anti-CD3 injections) cells were used. As controls for the caspase-3 activation, we have used embryonic fibroblasts from either WT or caspase-3/ mice. Total protein lysates were prepared as described by Aifantis et al. (7). The protein blots were probed with anti-cleaved caspase-3 (Cell Signaling) and anti-actin (Santa Cruz Biotechnology, Inc.) as an internal loading control.
Cytosolic Ca2+ measurements
For Ca2+ measurements, we have used 2 x 106 SCIET and SCB29 cells loaded with 1 µM Fura2-AM (Molecular Probes) dissolved in IMDM containing 1% FBS for 30 min at 37°C in the dark. The exact protocol followed is described elsewhere (7).
Nuclear extracts and electrophoretic mobility shift assay
510 x 106 SCIET cells were used for the preparation of nuclear protein extracts. The details of the exact protocol used can be found elsewhere (7).
![]() |
Acknowledgments |
---|
We would like to acknowledge the Saint Jude Cancer Center Support grant (CA-21765). This work was supported by the Leukemia Research Foundation, the Cancer Research Foundation, the Cancer Research Institute, and the V Foundation For Cancer Research (all to I. Aifantis). J.-C. Zuniga-Pflucker is supported by an Investigator award from the Canadian Institutes of Health Research (CIHR). M. Ciofani is supported by a Studentship award from the CIHR. I. Screpanti was supported by Associazione Italiana per la Ricerca sul Cancro. A. Ruiz-Vela is supported by a fellowship from the European Molecular Biology Organization. T. Palomero is a recipient of a fellowship from the Lady Tata Memorial Trust. C. Borowski was supported by a National Science Foundation Graduate Research Fellowship.
The authors have no conflicting financial interests.
Submitted: 16 September 2004
Accepted: 12 January 2005
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Related Article