HPV Microarrays
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Supplementary Methods Tissue sample process. Each tissue sample was cryosectioned, and selected sections stained with hematoxylin/eosin, and reviewed to determine tumor content, pathological status, and freedom from necrosis and freezing artifacts. Epithelial cells from all normal samples and tumor cells from HNC or CC samples with less than 80% tumor were laser capture microdissected from adjacent sections using a PixCell II LCM system (Arcturus, Mountain View, CA) (Supplementary Table 2). For guidance, an adjacent section was briefly stained with hematoxylin to visualize tissue structure. RNA amplification and labeling. Total RNA was extracted from sectioned and/or microdissected samples as follows: 1 ml of TRIzol (Invitrogen, Carlsbad, CA) was added to each tissue sample, homogenized by passing through a 20 gauge needle, and added to 0.2 ml chloroform. After centrifugation at 20,000 xg for 20 min at 4oC, RNA in the aqueous phase was precipitated with an equal volume of isopropanol for 30 min at 4oC, pelleted, and washed twice with cold 70% ethanol. Double strand (ds) cDNA was synthesized from this RNA using a SuperScript ds cDNA synthesis kit (Invitrogen) and T7 promoter-linked oligo (dT)24. Complementary RNA (cRNA) was synthesized from T7 promoter-linked ds cDNA using a MEGAscript high transcription kit (Ambion, Austin, TX). To obtain a sufficient cRNA for ≥2 microarray hybridizations, this amplification process was repeated. Second round cRNA was biotin labeled using a BioArray High Yield RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, NY) and stored at -80oC until hybridized. cRNA quality and quantity was determined by gel electrophoresis and UV spectrophotometry. HPV microarrays. HPV arrays included probes representing all 37 sequenced mucosotropic HPV genotypes (6, 11, 12, 13, 16, 17, 18, 22, 30, 31, 32, 33, 34, 35, 38, 39, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 66, 67, 68, 69, 70, 72, 83), human β-actin and GAPDH as 1 positive controls, cell differentiation markers (PCNA, K8, K1, K10, Filaggrin, Suprabasin, Loricrin, Sp4), known cervical cancer markers (p16, p53, RB1, hTERT, Cyclin D1, IFITM1, ISGF3, MCM4, MCM5, MCM6, MCM7, USP3), and selected rice genome sequences as a negative controls. NCBI BLAST Searches were used to remove probes with predicted cross- reactivity to other genes. Microarray hybridization. Biotin-labeled cRNA was dissolved in fragmentation buffer (40mM Tris-Acetate, pH 8.1, 100mM potassium acetate, 30mM magnesium acetate) and incubated at 95oC for 25 min. 10 µg of cRNA was used for Affymetrix microarray hybridization and scanning at the University of Wisconsin Biotechnology Gene Expression Center. HPV arrays were washed and blocked following the epoxy slide manufacturer’s washing and blocking protocol (Genomic Solutions). 10 µg cRNA was mixed with 200 µl of array hybridization buffer (Genomic Solutions) and added to the HPV array covered with a hybridization chamber (Schleicher & Schuell, Keene, NH). After hybridization at 50oC for 24 h, the array was washed sequentially with 0.1% SDS plus 2X SCC, 1X SCC, and 0.2X SCC at 50oC, then hybridized to streptavidin-Cy3 at room temperature for 15 min, followed by washing with 2X SCC, 1X SCC, and 0.5X SCC at room temperature. The dry array was scanned using an Agilent DNA microarray scanner (Agilent, Palo Alto, CA) and data were analyzed using Axon GenePix Pro 5.1 microarray image analysis software (Molecular Devices, Sunnyvale, CA). Quantitative reverse transcriptase-PCR. First strand DNA was synthesized from 15 µg of total RNA from tissues or cultured cells using oligo (dT) 15 and SuperScript reverse transcriptase. Quantitative PCR (qPCR) was performed with 0.5 µg of cDNA using a QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA) and an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA). Oligonucleotide primers (Supplementary Table 1) were designed using the Primer3 primer design program (1), 2 synthesized by Integrated DNA Technologies (Coralville, IA) and used at 0.5 µM for PCR amplification for 40 cycles of 15 s denaturation at 95°C, 30 s annealing at 55°C, and 30 s polymerization at 72°C, following 10 min pre-incubation at 95°C. Plasmid standards containing each gene used in qPCR were produced by TA cloning PCR amplified fragments into pGEM-T Easy vector (Promega). Immunohistochemistry and western blotting. All steps were at room temperature unless otherwise specified. For immunohistochemistry, cryosectioned tissue was incubated successively in 4% paraformaldehyde for 15 min, 3% H2O2/methanol for 10 min, 10 mM citrate buffer (pH6.0) at 95oC for 20 min, and 2M HCl for 20 min. Following blocking with 2.5% horse serum, tissue sections were incubated successively with mouse anti-PCNA antibody (PC10, Chemicon International, Temecula, CA) or mouse anti-Cdc7 (DCS-341, Sigma, St. Louis, MO) for 2 hr, biotin-conjugated anti-mouse IgG (Vector Laboratories, Burlingame, CA) for 30 min, and ABC reagent (Vector Laboratories) for 30 min. Tissue sections were developed with DAB reagent (Vector Laboratories) for 2 min and counter- stained with hematoxylin. IHC images were analyzed and quantified as described previously (Zhang et al.). Briefly, the developed brown color of the PCNA signal was digitally extracted from other spectral components of the image, converted to a gray scale image, and the gray scale densities of five random fields within the tumor tissue of each sample were measured using Image J and averaged. Fig. 4 shows representative images and a plot of the numerical values for all tested samples, which included 11 HPV+ cancers (6 HPV+HNCs and 5 - HPV+CCs) and 10 HPV HNCs. Approximately one million cultured cells were used for western blotting. Cell pellets were lysed with lysis buffer (10 mM Tris, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA) plus complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Proteins fractionated by SDS polyacrylamide gel electrophoresis transferred to polyvinylidene difluoride paper (Amersham). SYCP2, HPV E7, and actin protein bands were visualized by chemiluminescence using goat anti-human SYCP2 (T-16, Santa Cruz 3 Biotechnology, Santa Cruz, CA), mouse anti-HPV16 E7 ( (ED17, Santa Cruz and 8C9, Zymed, South San Francisco, CA), and rabbit anti-actin (I-19, Santa Cruz) antibodies, respectively, and secondary antibodies (donkey anti-goat IgG (Santa Cruz), donkey anti- mouse IgG (Jackson ImmunoResearch), or goat anti-rabbit IgG (Jackson ImmunoResearch) conjugated with horseradish peroxidase. Tissue culture. Normal human immortalized keratinocytes (NIKSTM, obtained from Lynn Allen Hoffman) that differentiate faithfully, NIKS16, 18, 31 derivatives stably transfected with the indicated HPV DNA genomes, and HaCaT cells were maintained as described (2-4). Primary cultures of human cervical epithelial cells were established from fresh human cervical tissue obtained after hysterectomy due to fibroids or endometriosis (5). Cells were maintained in serum free MCDB153-LB medium (K-SFM, Invitrogen, Carlsbad, CA). Secondary cultures were infected (6) for 3 h with retroviruses expressing the neomycin resistance gene alone (pLXSN) or with HPV16 E6, E7, or E6/E7 genes. Cells were selected in G418 (200 µg/ml for 2 days) and subcultured once prior to RNA extraction. For RNA extraction, subconfluent monolayers were washed with prewarmed PBS, lysed with Trizol (Invitrogen), and extracted with chloroform to remove protein and DNA (6). Primary cervical keratinocytes transfected with HPV16 were a kind gift from Dr. Aloysius Klingelhutz (7). - HPV array hybridization was carefully optimized using RNA from known HPV+ and HPV keratinocyte cell lines such as NIKS (previously named BC1-Ep/SL cells) (17), NIKS-16 (HPV16) (3), NIKS-18 (HPV18), NIKS-31 (HPV31), W12E clone 20850 (HPV16) (4), CaSki (HPV16) (8), and HaCaT cells (9) References 1. Rozen, S., and Skaletsky, H.J. (2000). 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Characterization of Normal Human Exocervical Epithelial Cells Immortalized in Vitro by Papillomavirus Types 16 and 18 DNA. Cancer Res, 48: 4620- 4628, 1988. 6. Nees, M., Geoghegan, J. M., Hyman, T., Frank, S., Miller, L., and Woodworth, C. D. Papillomavirus Type 16 Oncogenes Downregulate Expression of Interferon-Responsive Genes and Upregulate Proliferation-Associated and NF-{kappa}B-Responsive Genes in Cervical Keratinocytes. J. Virol., 75: 4283-4296, 2001. 7. Sprague, D. L., Phillips, S. L., Mitchell, C. J., Berger, K. L., Lace, M., Turek, L. P., and Klingelhutz, A. J. Telomerase activation in cervical keratinocytes containing stably replicating human papillomavirus type 16 episomes. Virology, 301: 247-254., 2002. 8. Baker CC, Phelps WC, Lindgren V, Braun MJ, Gonda MA, Howley PM. Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J Virol 1987;61:962-71. 9. Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 1988;106:761-71. 5 Supplementary Figure Legends Supplementary Figure S1. LCM decision with hematoxylin and eosin staining.