Patient Samples, Cytogenetics, and Cell Lines
Patients were enrolled in protocols approved by the Baylor College of Medicine and University of Texas MD Anderson Cancer Center Institutional Review Boards that include clinical sample collection, linkage to clinical data, and banking of samples for research purposes. DNA was prepared using the QIAamp DNA blood mini kit (Qiagen, Valencia, CA, 51106), Gentra PUREGENE Cell and Tissue Kit (Gentra, Valencia, CA, 158788), or DNA STAT60 (IsoTex Diagnostics, Friendswood, TX, TL-4200) and RNA was prepared using the RNeasy mini kit (Qiagen, 74104) or RNA STAT60 (IsoTex Diagnostics, CS-110) according to the manufacturer’s instructions. MH500 [sample from the t(17;19) subject] RNA was processed using Ultraspec RNA (Biotecx, Houston, TX, BL-10−100
). Protein for reverse phase protein array was prepared as previously described (Kornblau et al., 2009
Cell pellets from subject MH500 were prepared and karyotype and fluorescence in situ hybridization (FISH) analyses were performed as previously published (Poland et al., 2007
KG-1 cells were cultured in Iscove’s modified Dulbecco’s medium (Gibco Life Technologies, Inc., Carlsbad, CA, 12200036) with 20% bovine growth serum (BGS; Hyclone, Logan, UT, SH30541.03) and U937 cells were cultured in RPMI 1640 (Gibco Life Technologies, Inc., 31800-022) with 10% BGS.
The t(17;19) translocation breakpoint regions were initially narrowed with directly labeled FISH probes as in Poland et al., 2007
: LSI dual-color PML/RARA probe (Vysis, Abbott Park, IL, 32-191009) for 17q and the LSI dual-color to 19q13/19p13 probe (Vysis, 32-231004) for 19q. Indirectly labeled BAC FISH was performed as in Poland et al., 2007
with the following modifications: BACs RP11-380H7 and CTD-3149D2 were labeled with biotin and used to mark the p-arm of chromosomes 17 and 19, respectively. The following probes, listed in order from centromere to telomere, were labeled with digoxigenin and used to map the translocation breakpoints; chromosome 17: RP11-506H21, RP11-110F1, RP11-113K1, RP11-112H10, RP11-481M4, RP11-118K23, RP11-561K8, RP11-721P9, CTB-61P23, RP11-489G5, RP11-52B5, RP11-90L11 and chromosome 19: RP11-84C16, RP11-568L16, RP11-43N16.
The sequences of the primers used in the following experiments can be found in .
Experimental Primer Sequences
One microgram of genomic DNA from MH500 or U937 was digested with 20 units of BclI for 2 hr at 50°C. The Universal Vectorette System (Sigma-Genosys, St. Louis, MO, UVS1) was used to perform vectorette PCR. The vectorette library was created per the manufacturer’s recommendation and half was diluted with 100 μl water. PCR was then performed using 2 μl of diluted library, a primer specific for exon 2 of MPO (MPO ex2), and the vectorette primer. The PCR product was diluted 1:100 with sterile water and 1 μl was used as the template for nested PCR with the nested exon 2 MPO primer (MPO n-ex2) and the nested vectorette primer. Bands unique to MH500 were gel purified using the QIAquick gel extraction kit (Qiagen, 28706), TOPO cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA, K4550-01SC) and transformed into TOP10F’ competent cells as recommended by the manufacturers. DNA from positive clones was prepared using the QIAprep spin miniprep kit (Qiagen, 27106) and sequenced.
PCR was performed using genomic DNA from MH500 or the wild-type lymphoblastoid cell line (LCL) 4G3 and the following primer pairs surrounding the translocation breakpoints: MPO F + ZNF342 R and ZNF342 F + MPO R. The reaction contained 1× AccuTaq buffer, 2 mM dNTP, 200 ng template DNA, 2.5 μM each primer, and 2.5 U/μl AccuTaq polymerase (Sigma, St. Louis, MO, D8045). The PCR program was as follows: one cycle of 96°C for 2 min; 30 cycles of 94°C for 10 sec, 68°C for 1.5 min; one cycle of 68°C for 2 min. The PCR products were gel purified using QIAquick PCR purification kit (Qiagen, 28106) as recommended by the manufacturer and sequenced.
ZNF342 RNA Expression Analysis
SYBR green quantitative reverse transcriptase PCR (qRT-PCR) was performed for the following genes: GEMIN7, CLPTM1, LOC284352, SFRS16, ZNF342, RELB, MPO, TRAPPC6A, and NKPD1. Levels of expression were compared between MH500, three additional AML samples, one normal bone marrow, and KG-1 and U937 cell lines. The QuantiTect SYBR Green RT-PCR kit (Qiagen, 204143) was used and the 25 μl reaction contained 1× QuantiTect SYBR Green RT-PCR master mix, 1.6 μM each primer, 0.25 μl QuantiTect RT Mix, and 100 ng template RNA. The PCR program was as follows: one cycle of 50°C for 30 min; one cycle of 95°C for 15 min; 45 cycles of 95°C for 15 sec, 55°C for 30 sec, 72°C 30 sec. Each reaction was performed in triplicate for the gene of interest and ACTB for each patient sample.
To perform TaqMan qRT-PCR, cDNA was generated from 1 μg patient RNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, 4374966) with RNase inhibitor according to the manufacturer’s recommendation. PCR (Applied Biosystems, 4304437) was performed in duplicate with 1 μl of cDNA and either primers for ACTB or ZNF342 in a 25 μl reaction containing 1× TaqMan Universal PCR Master Mix, 500 nM F primer, 500 nM R primer, and 250 nM TaqMan probe. The PCR program was as follows: one cycle of 50°C for 2 min; one cycle of 95°C for 10 min; 45 cycles of 95°C for 15 sec, 50°C for 1 min. The primers used are as follows: ACTB F, ACTB R, ACTB TaqMan, ZNF342 F, ZNF342 R, and ZNF342 TaqMan.
Quantitation of Murine Gene Expression
levels were based on published data from Chambers et al. (2007)
quantitated in murine hematopoietic populations by RNA hybridization to Affymetrix (Santa Clara, CA) MOE430 2.0 microarrays as previously published (Chambers et al., 2007
To sequence the three exons of the ZNF342 gene we used four sets of primers, each set included M13F and M13R sequence: ex1F + ex1R, ex2F + ex2R, ex3 − 1F + ex3 − 1R, ex3 − 2F + ex3 − 2R. The reaction contained 1× GoTaq Flexi buffer, 2.5 mM MgCl2, 0.2 mM dNTP, 0.5 mM F + R primer mix, 1.25U GoTaq polymerase (Promega, Madison, WI, M8291), 100 ng genomic DNA template, and 1 M betaine (Sigma, B0300). The PCR program was as follows: one cycle of 96°C for 2 min; 5 cycles of 95°C for 30 sec, 60°C for 30 sec, 72°C for 45 sec; 35 cycles of 95°C for 30 sec, 55°C for 30 sec, 72°C for 45 sec; one cycle of 72°C for 5 min. Reactions were sequenced using M13F and M13R primers.
Reverse Phase Protein Array Assembly and Printing Method
For quantification purposes, five serial dilutions (1:2 dilution steps) of each protein lysate were arrayed in 384 well plates (Genetix, Boston, Massachusetts). Samples were printed onto nitrocellulose coated glass slides (FAST Slides, Schleicher & Schuell BioScience, Inc. USA, Keene, NH) using an Aushon Biosytems 2470 Arrayer (Aushon BioSystems Inc., Burlington, MA) with 175 micron pins and a single touch. The samples were printed in replicate, printed side by side. Based on the sample concentration of 1 × 104 cells per microliter and a printing volume of 2 nanoliter/touch, we estimate that the spots ranged from 85 cell equivalents of protein in undiluted, with ~5 cell protein equivalents in the most diluted (1:16) spot. To permit topographical normalization, a sample of protein prepared from 11 pooled cell lines, printed in five serial dilutions or a negative control sample, was printed at the end of each row of patient sample, creating a grid across the whole slide. Each slide contained 6,912 dots.
Antibody Detection and Array Staining
A detailed description of the array methodology including antibody staining and detection has been published (Tibes et al., 2006
) and the experiment was performed as published. A rabbit polyclonal antibody (AB) against ZNF342 (Abcam, Cambridge, MA, ab51265) at a concentration of 1:1,000 was added for 1–2 hr with frequent rotation. A biotinylated secondary antibody (anti-rabbit), diluted 1:15,000 and used for signal amplification, was added for 1 hr. Subsequently, the array slides were incubated using the DAKO (Copenhagen, Denmark) signal amplification system using catalyzed reporter deposition of substrate to amplify the signal detected by the primary antibody (Hunyady et al., 1996
). Slides were incubated in streptavidine-biotin-peroxidase and biotinyl-tyramide/hydrogen peroxide reagents. Finally, 3,3′-diaminobenzidine tetrachloride (DAB) was cleaved by tyramide-bound horseradish peroxidase, giving a stable brown precipitate with excellent signal-to-noise ratio. This technique is sensitive and reproducible to the femtomolar range as reported (Charboneau et al., 2002
; Tibes et al., 2006
Survival analysis for RPPA data were performed using Statistica, version 6 (Statsoft, Tulsa, OK).