NUT Monoclonal Antibody Production
A GST fusion protein containing amino acids 450–700 of human NUT was used to immunize New Zealand rabbits (Cell Signaling Technology, Inc. (CST), Danvers, MA). Positive immuno-reactive rabbits were identified by Western blotting and IHC and chosen for rabbit monoclonal development. Three lead monoclonal antibodies were chosen for further clinical validation. The NUT antibody is being prepared for commercial release and will be available from CST.
The BRD4-NUT-expressing cell line, TC797, has been described previously(9
). All other lines were obtained from the American Type Culture Collection (Manassas, Va.). TC797 and 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA.) supplemented with a solution containing 10% bovine growth serum (Hyclone, Logan, Utah), 2 mM L-glutamine (Gibco), 100 U of penicillin G/ml, and 100 mg of streptomycin/ml. The A549, A673 and MCF7 cell lines were acquired through ATCC and grown as recommended by the supplier.
Expression plasmids, siRNA, and transient transfection
A cDNA encoding FLAG-BRD4-NUT was assembled in the plasmid pcDNA3 (Invitrogen, Carlsbad, CA) as described(5
). A small interfering RNA (siRNA) duplex designed against human NUT
, (5'- AAACTCAGAACTTTATCCTTACCTGTCTC –3’), and scrambled siRNA (Silencer
® Negative Control #1) were purchased from Applied Biosystems/Ambion (Austin, Tx). Transfection of pcDNA3-FLAG-BRD4-NUT into 293T cells was performed using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Transfection of siRNA into TC797 cells was performed by electroporation as described(5
). Briefly, siRNA (680nM in 100ul solution R) was transfected into 3×106
cells using the Nucleofector II instrument and program T-27 (Amaxa Inc., Gaithersburg, MD).
Fluorescence in situ Hybridization
Dual-color FISH assays were performed on formalin-fixed paraffin-embedded 4µm tissue sections as described(3
). Probes used for the 15q14 NUT
breakpoint, flanking a 181kb region containing NUT
, included the 3’ telomeric BAC clones 1H8 and 64o3, and the 5’ centromeric clones 412e10 and 3d4. Probes used for the 19p13.1 BRD4
breakpoint were the 5’ centromeric BAC clone 187l3 and the 3’ telomeric BAC clone 87m17. The probe spanning NUT, BAC clone 122p18, was used to detect the cryptic NUT breakpoint in a bring-together assay with 5’ centromeric BAC clone 187l3. Sections in which >80% of cells contained hybridization signals in four areas (200 cells/area) were considered adequate for interpretation.
FISH for NUT rearrangement was evaluable in 481 cases. This included one author’s (CAF) collection of cases (N = 141, Group 1, below), a head and neck tumor microarray (N = 327, from Group 2 below), and selected testicular and ovarian germ cell tumors (N = 13, from Group 2 and 3 below).
IHC was performed on 5 µm sections prepared from formalin-fixed, paraffin-embedded primary tumors. To stain for NUT, following deparaffinization and rehydration, sections were subjected to antigen retrieval in Dako pH 9.0 solution (Dako USA, Capinteria, CA) in a steam pressure cooker (BioCare Medical, Walnut Creek, CA). Other antigen retrieval buffers that were tested and determined to be less effective in producing optimal signal/noise on control tissues included citrate buffer, pH6, and EDTA buffer, pH8 (both from Zymed-Invitrogen). After washing in distilled water and treatment with Peroxidase Block (Dako) for 5 min to quench endogenous peroxidase activity, sections were incubated with primary rabbit monoclonal anti-NUT (9.2ug/ml) in antibody diluent (Dako) for 1 hr, washed in 50 mM Tris-HCl (pH 7.4), and incubated with horseradish peroxidase–conjugated secondary antibodies (Envision detection kit, DAKO USA). Staining was developed through incubation with diaminobenzidine (DAB), and sections were counterstained with hematoxylin.
The results of IHC staining were interpreted independently by two pathologists (CAF and JCA), who were both blind to the FISH results. Cases were scored based on the extent of nuclear immunoreactivity in the tumor cells. Cases with unequivocal nuclear staining in the majority of the tumor cells were considered positive. Consensus was reached in all discrepant cases through dual review and discussion.
Reverse Transcriptase Polymerase Chain Reaction
RNA was extracted from fresh human peripheral lymphocytes, TC797 cells, frozen human testis, and dysgerminoma (Fig.3a) using Trizol according to the manufacturer’s instructions (Invitrogen). cDNA was synthesized using ArrayScript reverse trancriptase and random decamers (Ambion/ Applied Biosystems, Inc.) according to the manufacturer’s instructions. PCR was performed using primer sets A (NUT 750fwd - 5’-GCTGAAGCCCACTATGACCCTGGAG-3’, NUT 994rev - 5’-TGGAGGCTGCCTTCTTCGGAATGTA-3’) and B (NUT 750fwd, NUT 1289rev - 5’-TCTGCCAGAAATTGAGGGTGAATGA-3’), which cross intron 3–4 and 3–5 of NUT, respectively.
To make cell-blocks, cultured 797 cells transfected with either siRNA specific for NUT or control siRNA were collected by trypsinization (Invitrogen), washed with PBS, and fixed for 20 min in 10% buffered formalin. After repeated washing with PBS, fixed cell pellets were suspended in warmed Histogel according to the manufacturer’s instructions (Invitrogen). After gelation was complete, the pellets were paraffin-embedded and used as controls during IHC test development.
Archival tumor sections (4µm thick, formalin-fixed, paraffin-embedded) were collected from four different sources. Group 1 (N = 141) consisted of one author’s (CAF) collection of cases, all of which have been evaluated for NUT rearrangement by FISH, and includes 28 known NMCs. Group 2 consisted of several non-commercial tumor microarrays (TMA, N = 674 cases): a head and neck squamous cell carcinoma TMA (provided by EBS, N = 442 cases, 2 cores/ neoplasm), that included 327 predominantly smoking-related carcinomas; a miscellaneous carcinoma TMA (provided by EBS, N = 165 cases, 4 cores/ neoplasm); and two male germ cell tumor TMAs (provided by MAR, N = 67 cases, 2 cores/ neoplasm). Group 3 was a collection of ovarian germ cell tumors (provided by MCC, N = 34). Group 4 was a commercial TMA of common carcinomas (US Biomax MC2081 TMA, N = 208 tissues, 1 core/ neoplasm (photos of cores available at http://www.biomax.us/tmaimage.php?catalognum=MC2081
); US Biomax, Rockville, MD). Studies were performed in accordance with IRB protocol 2000-P-001990/6; BWH.