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• W2025708651 abstract "The molecular mechanisms by which the anti-HER2 antibodies trastuzumab and its murine equivalent 4D5 inhibit tumor growth and potentiate chemotherapy are not fully understood. Inhibition of signaling through the phosphatidylinositol 3-kinase (PI3K)-AKT pathway may be particularly important. Treatment of breast cancer cells that overexpress HER2 with trastuzumab inhibited HER2-HER3 association, decreased PDK1 activity, reduced Thr-308 and Ser-473 phosphorylation of AKT, and reduced AKT enzymatic activity. To place the role of PI3K-AKT in perspective, gene expression was studied by using Affymetrix microarrays and real time reverse transcription-PCR. Sixteen genes were consistently down-regulated 2.0–4.9-fold in two antibody-treated breast cancer cell lines. Fourteen of the 16 genes were involved in three major functional areas as follows: 7 in cell cycle regulation, particularly of the G2-M; 5 in DNA repair/replication; and 2 in modifying chromatin structure. Of the 16 antibody-regulated genes, 64% had roles in cell growth/maintenance and 52% contributed to the cell cycle. Direct inhibition of PI3K with an inhibitor markedly reduced expression of 14 genes that were also affected by the antibody. Constitutive activation of AKT1 blocked the effect of the anti-HER2 antibody on cell cycle arrest and on eight differentially expressed genes. The antibody enhanced docetaxel-induced growth inhibition but did not increase the fraction of apoptotic cells induced with docetaxel alone. In contrast, the antibody plus docetaxel markedly down-regulated two genes, HEC and DEEPEST, required for passage through G2-M. Thus, anti-HER2 antibody preferentially affects genes contributing to cell cycle progression and cell growth/maintenance, in part through the PI3K-AKT signaling. Transcriptional regulation by anti-HER2 antibody through PI3K-AKT pathway may potentiate the growth inhibitory activity of docetaxel by affecting cell cycle progression. The molecular mechanisms by which the anti-HER2 antibodies trastuzumab and its murine equivalent 4D5 inhibit tumor growth and potentiate chemotherapy are not fully understood. Inhibition of signaling through the phosphatidylinositol 3-kinase (PI3K)-AKT pathway may be particularly important. Treatment of breast cancer cells that overexpress HER2 with trastuzumab inhibited HER2-HER3 association, decreased PDK1 activity, reduced Thr-308 and Ser-473 phosphorylation of AKT, and reduced AKT enzymatic activity. To place the role of PI3K-AKT in perspective, gene expression was studied by using Affymetrix microarrays and real time reverse transcription-PCR. Sixteen genes were consistently down-regulated 2.0–4.9-fold in two antibody-treated breast cancer cell lines. Fourteen of the 16 genes were involved in three major functional areas as follows: 7 in cell cycle regulation, particularly of the G2-M; 5 in DNA repair/replication; and 2 in modifying chromatin structure. Of the 16 antibody-regulated genes, 64% had roles in cell growth/maintenance and 52% contributed to the cell cycle. Direct inhibition of PI3K with an inhibitor markedly reduced expression of 14 genes that were also affected by the antibody. Constitutive activation of AKT1 blocked the effect of the anti-HER2 antibody on cell cycle arrest and on eight differentially expressed genes. The antibody enhanced docetaxel-induced growth inhibition but did not increase the fraction of apoptotic cells induced with docetaxel alone. In contrast, the antibody plus docetaxel markedly down-regulated two genes, HEC and DEEPEST, required for passage through G2-M. Thus, anti-HER2 antibody preferentially affects genes contributing to cell cycle progression and cell growth/maintenance, in part through the PI3K-AKT signaling. Transcriptional regulation by anti-HER2 antibody through PI3K-AKT pathway may potentiate the growth inhibitory activity of docetaxel by affecting cell cycle progression. The human epidermal growth factor receptor 2 (HER2, 1The abbreviations used are: HER2, human epidermal growth factor receptor 2; PI3K, phosphatidylinositol 3-kinase; RT, reverse transcription; PCNA, proliferating cell nuclear antigen; FC, fold change; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; hIgG, human IgG; m, mammalian. 1The abbreviations used are: HER2, human epidermal growth factor receptor 2; PI3K, phosphatidylinositol 3-kinase; RT, reverse transcription; PCNA, proliferating cell nuclear antigen; FC, fold change; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; hIgG, human IgG; m, mammalian. also known as c-Neu or ErbB-2) encodes a 185-kDa transmembrane tyrosine kinase growth factor receptor. The ligand that binds to the homodimers of HER2 has not yet been identified. Rather, HER2 functions as a preferred co-receptor to form heterodimers with HER1 (epidermal growth factor receptor), HER3, or HER4. Of these heterodimers, HER2-HER3 is particularly important for intracellular signaling (1Citri A. Skaria K.B. Yarden Y. Exp. Cell Res. 2003; 284: 54-65Crossref PubMed Scopus (475) Google Scholar). HER2 signaling has been linked to a variety of cellular responses to growth factors under both normal and pathophysiological conditions. HER2 signaling is required not only during normal development of the mammary gland but also during development of the glia, neurons, and heart (1Citri A. Skaria K.B. Yarden Y. Exp. Cell Res. 2003; 284: 54-65Crossref PubMed Scopus (475) Google Scholar, 2Stern D.F. Exp. Cell Res. 2003; 284: 89-98Crossref PubMed Scopus (107) Google Scholar). Amplification of the HER2 gene and overexpression of HER2 protein have been documented in ∼30% of breast and 15% of ovarian cancers (3Pegram M.D. Konecny G. Slamon D.J. Cancer Treat. Res. 2000; 103: 57-75Crossref PubMed Scopus (174) Google Scholar). In many (but not all) reports, HER2 overexpression has been associated with a more aggressive course of disease. Although the underlying mechanisms for this association are still not well characterized, HER2 overexpression has been linked to increased proliferation and invasiveness (4Holbro T. Civenni G. Hynes N.E. Exp. Cell Res. 2003; 284: 99-110Crossref PubMed Scopus (519) Google Scholar). HER2 is currently one of the best defined targets for specific therapy. The substantially greater expression of HER2 on cancer cells than on normal epithelial tissues permits selective targeting of malignant cells. HER2 is expressed on the cell surface where it can interact with ligands and antibodies (1Citri A. Skaria K.B. Yarden Y. Exp. Cell Res. 2003; 284: 54-65Crossref PubMed Scopus (475) Google Scholar, 2Stern D.F. Exp. Cell Res. 2003; 284: 89-98Crossref PubMed Scopus (107) Google Scholar, 3Pegram M.D. Konecny G. Slamon D.J. Cancer Treat. Res. 2000; 103: 57-75Crossref PubMed Scopus (174) Google Scholar, 4Holbro T. Civenni G. Hynes N.E. Exp. Cell Res. 2003; 284: 99-110Crossref PubMed Scopus (519) Google Scholar). Trastuzumab, a monoclonal antibody directed against the extracellular domain of HER2, is therapeutically active in HER2-positive breast carcinomas (3Pegram M.D. Konecny G. Slamon D.J. Cancer Treat. Res. 2000; 103: 57-75Crossref PubMed Scopus (174) Google Scholar). Clinical trials in HER2-positive patients with breast cancer have demonstrated that targeted therapy with trastuzumab in conjunction with cytotoxic chemotherapy (such as platinum compounds, taxanes, and anthracyclines) improves time to disease progression and overall survival (3Pegram M.D. Konecny G. Slamon D.J. Cancer Treat. Res. 2000; 103: 57-75Crossref PubMed Scopus (174) Google Scholar, 5Arteaga C.L. Breast Cancer Res. 2003; 5: 96-100Crossref PubMed Scopus (54) Google Scholar). Clinical trials have also documented an increased risk for cardiotoxicity when trastuzumab is combined with anthracyclines, suggesting that HER2 signaling may contribute to normal heart function. The mechanisms by which trastuzumab affects growth of HER2-positive cancer cells and enhances sensitivity to chemotherapy are not fully understood. Anti-HER2 antibody can down-regulate the HER2 receptor and prevent cleavage of the extracellular domain of the receptor (13Baselga J. Albanell J. Molina M.A. Arribas J. Semin. Oncol. 2001; 28: 4-11Crossref PubMed Google Scholar). Receptors are, however, generally re-expressed within a matter of hours, and binding of anti-HER2 antibody can also alter intracellular signaling by enhancing kinase activity and preventing heterodimer formation. Our group and others have demonstrated that anti-HER2 monoclonal antibodies exert inhibitory effects on HER2-overexpressing breast cancer cells through induction of G1 cell cycle arrest associated with induction of p27Kip1 and reduction of CDK2 (6Le X.-F. McWatters A. Wiener J. Mills G.B. Bast Jr., R.C. Clin. Cancer Res. 2000; 6: 260-270PubMed Google Scholar, 7Neve R.M. Sutterluty H. Pullen N. Lane H.A. Daly J.M. Krek W. Hynes N.E. Oncogene. 2000; 19: 1647-5166Crossref PubMed Scopus (133) Google Scholar, 8Pietras R.J. Poen J.C. Gallardo D. Wongvipat P.N. Lee H.J. Slamon D.J. Cancer Res. 1999; 59: 1347-1355PubMed Google Scholar, 9Sliwkowski M.X. Lofgren J.A. Lewis G.D. Hotaling T.E. Fendly B.M. Fox J.A. Semin. Oncol. 1999; 26: 60-70PubMed Google Scholar, 10Lane H.A. Beuvink I. Motoyama A.B. Daly J.M. Neve R.M. Hynes N.E. Mol. Cell. Biol. 2000; 20: 3210-3223Crossref PubMed Scopus (270) Google Scholar, 11Yakes F.M. Chinratanalab W. Ritter C.A. King W. Seelig S. Arteaga C.L. Cancer Res. 2002; 62: 4132-4141PubMed Google Scholar). We have further shown that post-translational regulation of p27Kip1 plays a critical role in the anti-HER2 antibody-mediated G1 cell cycle arrest and tumor growth inhibition (12Le X.-F. Claret F.X. Lammayot A. Tian L. Deshpande D. LaPushin R. Tari A.M. Bast Jr., R.C. J. Biol. Chem. 2003; 278: 3441-3450Google Scholar). Of the post-translational mechanisms, we have shown that modulation of the phosphorylation of p27Kip1 protein is the mechanism by which anti-HER2 antibody up-regulates the protein (12Le X.-F. Claret F.X. Lammayot A. Tian L. Deshpande D. LaPushin R. Tari A.M. Bast Jr., R.C. J. Biol. Chem. 2003; 278: 3441-3450Google Scholar). Anti-HER2 antibodies that inhibit tumor growth also prevent HER2-HER3 interaction and inhibit the PI3K-AKT signaling pathway (6Le X.-F. McWatters A. Wiener J. Mills G.B. Bast Jr., R.C. Clin. Cancer Res. 2000; 6: 260-270PubMed Google Scholar, 11Yakes F.M. Chinratanalab W. Ritter C.A. King W. Seelig S. Arteaga C.L. Cancer Res. 2002; 62: 4132-4141PubMed Google Scholar, 16Le X.-F. Vadlamudi R. McWatters A. Bae D.S. Mills G.B. Kumar R. Bast Jr., R.C. Cancer Res. 2000; 60: 3522-3531PubMed Google Scholar, 17Hermanto U. Zong C.S. Wang L.H. Oncogene. 2001; 20: 7551-7562Crossref PubMed Scopus (57) Google Scholar). As the PI3K-AKT pathway is critically important to cell survival signaling, inhibition of the PI3K-AKT pathway may explain, in part, the ability of trastuzumab to enhance paclitaxel-induced apoptosis (20Lee S. Yang W. Lan K.H. Sellappan S. Klos K. Hortobagyi G. Hung M.C. Yu D. Cancer Res. 2002; 62: 5703-5710PubMed Google Scholar). Trastuzumab also suppresses DNA repair capacity (18Pietras R.J. Pegram M.D. Finn R.S. Maneval D.A. Slamon D.J. Oncogene. 1998; 17: 2235-2249Crossref PubMed Scopus (354) Google Scholar) through as yet unknown pathways, contributing to the ability of the antibody to enhance the anti-tumor effect of DNA-damaging agents such as cisplatin (18Pietras R.J. Pegram M.D. Finn R.S. Maneval D.A. Slamon D.J. Oncogene. 1998; 17: 2235-2249Crossref PubMed Scopus (354) Google Scholar) and radiotherapy (19Pfaffl M.W. Nucleic Acids Res. 2001; 29: 2002-2007Crossref Scopus (24586) Google Scholar). In vivo, trastuzumab inhibits angiogenesis and induces antibody-dependent cellular cytotoxicity (13Baselga J. Albanell J. Molina M.A. Arribas J. Semin. Oncol. 2001; 28: 4-11Crossref PubMed Google Scholar, 14Kono K. Takahashi A. Ichihara F. Sugai H. Fujii H. Matsumoto Y. Cancer Res. 2002; 62: 5813-5817PubMed Google Scholar), potentially contributing to its activity. Loss or blockade of the FcγRIII receptor on leukocytes has been shown to severely impair the anti-tumor effect of trastuzumab in vivo (15Clynes R.A. Towers T.L. Presta L.G. Ravetch J.V. Nat. Med. 2000; 6: 443-446Crossref PubMed Scopus (2264) Google Scholar), indicating involvement of Fc-receptor-dependent mechanisms. Of the several mechanisms proposed for the action of anti-HER2 antibodies, interruption of the PI3K-AKT pathway may be critical for enhancing sensitivity to docetaxel and other cytotoxic drugs. Our current study explored the mechanisms of action of the anti-HER2 antibody and the impact of the antibody on activation of AKT as well as on sensitivity to docetaxel. Anti-HER2 antibody alone inhibited HER2-HER3 association, decreased PDK1 activity, reduced Thr-308 phosphorylation of AKT, and reduced AKT enzymatic activity. We have used a pharmacogenomic approach to compare the global changes that occur after treatment with anti-HER2 antibody and after treatment with the chemical inhibitor of PI3K. Treatment with anti-HER2 antibody decreased the expression of 16 genes. Fourteen of these 16 genes contribute to the following three different areas of cell function: cell cycle regulation, DNA repair/replication, and modification of chromatin structure. Direct inhibition of PI3K markedly decreased the expression of 14 genes regulated by the anti-HER2 antibody. Conversely, dominant active AKT prevented cell cycle G1 arrest and down-regulation of cell cycle genes induced by anti-HER2 antibody. A combination of anti-HER2 antibody and docetaxel exerted additive growth inhibition against breast cancer cell lines that overexpressed HER2. The combination did not increase the fraction of apoptotic cells induced with docetaxel alone but markedly down-regulated two genes that participate in cell cycle regulation, HEC and DEEPEST, required for passage through G2-M. Cell Culture—The human breast cancer cell lines, SKBr3 and BT474, were obtained from the American Type Culture Collection (ATCC, Manassas, VA). SKBr3 cells were grown in complete medium that contained RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (Sigma), 2 mm l-glutamine, 100 units/ml of penicillin, and 100 μg/ml streptomycin in humidified air with 5% CO2 at 37 °C. BT474 cells were grown in complete medium containing Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 1 mm sodium pyruvate (Sigma), 100 units/ml penicillin, and 100 μg/ml streptomycin. For all experiments, cells were detached with 0.25% trypsin, 0.02% EDTA. For cell culture, 2–6 × 105 exponentially growing cells were plated into 100-mm tissue culture dishes or 3 × 103 into 96-well plates in complete medium. After culture overnight in complete medium, cells were treated with anti-HER2 antibody 4D5 at 5–10 μg/ml (for SKBr3) or trastuzumab at 10 μg/ml (for BT474) in complete medium at 37 °C for 24 (for SKBr3) or 48 h (for BT474). Monoclonal antibody MOPC21 served as control antibody for 4D5 and was used at 5–10 μg/ml in SKBr3 cells. Human IgG served as control antibody for trastuzumab and was used at 10 μg/ml in BT474 cells. Reagents—Anti-HER2 murine monoclonal antibody 4D5 and humanized monoclonal antibody trastuzumab (Herceptin®) were kindly provided by Genentech (South San Francisco, CA). MOPC21 murine myeloma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). MOPC21 cells were grown in the peritoneal cavities of BALB/c mice to produce ascites fluid, and the immunoglobulin was purified as reported previously (25Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1381) Google Scholar). A control IgG1 was purchased from Calbiochem and further dialyzed against sterile cold PBS to eliminate sodium azide. Antibodies reactive with phospho-Ser-473 AKT, phospho-Thr-308 AKT, and total AKT as well as an AKT kinase assay kit were purchased from Cell Signaling Technology, Inc. (Beverly, MA). A monoclonal antibody to PCNA was purchased from BioGenex (San Ramon, CA). An antibody to AKT1 and a PDK1 kinase assay kit were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). An antibody reactive with HER2 (for Western blotting) was purchased from Oncogene Research Products (Cambridge, MA). An antibody to HER3 (for Western blotting and immunoprecipitation) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A monoclonal antibody to β-actin was purchased from Sigma. Recombinant human heregulin β1 (hereafter named heregulin) was obtained from NeoMarkers, Inc. (Fremont, CA). Affymetrix Human Genome U95Av2 Gene Chips that permitted measurement of the expression of 12,000 human genes were purchased from Affymetrix (Santa Clara, CA). Preparation of Total RNA—SKBr3 cells were treated with 4D5 (10 μg/ml) or MOPC21 (10 μg/ml) for 24 h. BT474 cells were treated with trastuzumab (10 μg/ml) or hIgG (10 μg/ml) for 48 h. Total RNA was then extracted from the treated SKBr3 or BT474 cells using the TRIzol reagent (Invitrogen). Procedures were performed according to the manufacturers' recommendation. The purity of RNA was assessed by absorption at 260 and 280 nm (values of the ratio of A260/A280 of 1.9–2.1 were considered acceptable) and by ethidium bromide staining of 18 S and 28 S RNA on gel electrophoresis. RNA concentrations were determined from the A260. Only samples of intact RNA were used for subsequent Affymetrix and RT-PCR analysis. Preparation of Fragmented cRNA for Affymetrix Analysis—A total of 15 μg of total RNA were used in the first-strand cDNA synthesis with T7-(dT)24 primer (GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24) (Proligo, Boulder, CO) by Superscript II (Invitrogen). The second-strand cDNA synthesis was carried out at 16 °C by adding Escherichia coli DNA ligase, E. coli DNA polymerase I, and RNase H into the reaction. This was followed by the addition of T4 DNA polymerase to blunt the ends of newly synthesized cDNA. Double-stranded cDNA was then purified by phase lock gel (Eppendorf, Westbury, NY) with phenol/chloroform extraction. The purified cDNA was then used as templates in an in vitro transcription to produce cRNA labeled with biotin using the BioArray High Yield RNA transcript labeling kit from Enzo Diagnostics, Inc. (Farmingdale, NY). The procedure was carried out according to the manufacturer's recommendation, and cRNA was further purified with a Qiagen RNeasy mini kit (Valencia, CA). Approximately 15 μg of cRNA was fragmented by incubating in a buffer containing 200 mm Tris acetate (pH 8.1), 500 mm potassium acetate, and 150 mm magnesium acetate at 95 °C for 30 min. Agarose gel electrophoresis was performed before the synthesis of cRNA and after the fragmentation of cRNA to ensure the quality of the samples. Only intact, high quality cRNA samples were used for subsequent array hybridization. Affymetrix Oligonucleotide Array Hybridization and Data Acquisition—cRNA hybridization to the human U95A arrays was performed at the M. D. Anderson Cancer Center Gene Microarray Facility by using an Affymetrix GeneChip System (Affymetrix, Santa Clara, CA). The fragmented cRNA was hybridized with pre-equilibrated Affymetrix chips at 45 °C for 16 h. Hybridized chips were then washed in a fluidic station with nonstringent buffer (6× SSPE (1× SSPE is 0.18 mol/liter NaCl, plus 0.015 mol of sodium citrate), 0.01% Tween 20, and 0.005% antifoam) for 10 cycles and with stringent buffer (100 mmol/liter MES, 0.1 mol/liter NaCl, and 0.01% Tween 20) for 4 cycles, and stained with streptavidin-phycoerythrin. This was followed by incubation with biotinylated mouse anti-avidin antibody. The chips were scanned in an Agilent ChipScanner to detect hybridization signals. Average target intensity was set at 500 arbitrary units. Each array was assessed for quality and stability by examining replicate copies of the same gene at different locations on the array. To ensure the quality of the cRNA samples and of the Affymetrix GeneChips, quality control experiments were performed using test chips, and the same cRNA sample before the test samples were processed. Further details are available from the CCSG shared resources web site (www.mdanderson.org/departments/dnamicroarray). The raw data (hybridization data) generated by MAS 5.0 were imported into Microsoft Excel and transferred to our Department of Biostatistics for analysis. Statistical Analysis of Affymetrix Array Data—Before analyzing the Affymetrix array data, every Affymetrix Hu95Av2 Gene Chip from our experiments underwent the following quality control checks: 1) scanner alignment and the proper dicing of images into correct cells; 2) overall chip brightness; and 3) spatial variation. Scanner alignment was checked by using the alternating pattern of positive and negative control cells on the border of each GeneChip (Affymetrix, Santa Clara, CA). The intensities of positive and negative controls were plotted as a function of border position to obtain visual confirmation that each image had been correctly aligned. Brightness was examined by looking at the histograms of detection p values (provided by Affymetrix MAS 5.0) for each array. Detection p values measure how likely a transcript was expressed at a level to be called present on the array. As a general rule, chips are flagged if less than 10% of probe sets are detected at the p = 0.01 level. Spatial variation is not easily detected one chip at a time, so we compared the log transformed median corrected ratio (Z) for each cell between each combination of chips A and B (Z = log2(A/B) – median (log2(A/B)). The range in Z was additionally constrained to enhance visual artifacts on the slide. All checks were passed for each chip. Two array methods were used to analyze differential gene expression. A standard Affymetrix MAS 5.0 statistical analysis tool package was used for each probe set to measure fold change (FC) on the log2 scale, a 95% confidence interval for FC on the log2 scale, a p value for detection used to make the presence or absence calls for each gene, and a p value for detecting change or differential expression between samples. The default standard Affymetrix change calls were not used. Here a probe set was considered differentially expressed if the change p value was very small, the detection p value in at least one sample being compared was small, and the absolute lower bound of log2(FC), the log ratio, exceeded 0.8, corresponding to at least a 1.75-fold change. A second method designated the position-dependent nearest neighbor model was developed by Dr. Li Zhang at the Department of Biostatistics, M.D. Anderson Cancer Center (17Hermanto U. Zong C.S. Wang L.H. Oncogene. 2001; 20: 7551-7562Crossref PubMed Scopus (57) Google Scholar). The position-dependent nearest neighbor model (available at the following web site: odin.mdacc.tmc.edu/~zhangli/PerfectMatch/) relies on a relationship between DNA base pair stacking energies and probe binding efficiencies. Only a perfect match is used to estimate the intensity of probe set. Cross-hybridization is accounted for in model estimation. Remarkable reproducibility between replicate samples has been attained at our Gene Microarray Core Facility (21Zhang L. Miles M.F. Aldape K.D. Nat. Biotechnol. 2003; 21: 818-821Crossref PubMed Scopus (266) Google Scholar). Zhang et al. (21Zhang L. Miles M.F. Aldape K.D. Nat. Biotechnol. 2003; 21: 818-821Crossref PubMed Scopus (266) Google Scholar) recommend using as criteria for differential expression between two chips a |log2 ratio| >0.8 and mean log2 signal >7.9. The contrasts were limited to comparison between treated versus untreated samples in each cell line. Subsequently, a comparison of gene expression was made between methods and cell lines. The results of contrasts with Affymetrix software include the following: 1) detection p values; 2) log ratios with 95% confidence intervals; and 3) change p values (one-sided). The p values are from one-sided tests of up-regulation in expression. The results of contrasts with Zhang's Perfect Match software include the following: 1) mean Log2 signal and 2) log2 ratio. The results for the four contrasts described above were exported to Excel spreadsheets. Although log ratio >0.8 produced some consistency, we have used log ratio >1.0, corresponding to a 2-fold change, as the final selection criteria for the differentially expressed genes in this study. Reverse Transcription-PCR Analysis—To verify the analysis results (Table I) from Affymetrix chip hybridization, total RNA was reversetranscribed with a random hexamer or T7-(dT)24 primer (Invitrogen). An aliquot (50 ng of total RNA) of the first strand cDNA was used as a template for PCR. Two genes (DEEPEST and H4FG listed in Table I) that lacked the validated primer sets and probes for real time PCR were examined in this study with regular RT-PCR. Oligonucleotide sequences of the primer sets used in this study are as follows: mitotic spindle coiled-coil protein (DEEPEST), sense (S)-AGCTGGAACAGGACCTAGCA and antisense (AS)-TCTGGGTAAGCTGGCAGAGT; H4 histone family, member G (H4FG), S-TAAGGTGCTCCGGGATAACA and AS-CCCTGACGTTTTAGGGCATA; and glyceraldehyde phosphate dehydrogenase (GAPDH), S-GAGTCAACGGATTTGGTCGT and ASTTGATTTTGGAGGGATCTCG. To monitor better the amplification efficiency and to control experimental errors, a duplex PCR that simultaneously amplified two genes, one internal control GAPDH and one gene of interest (DEEPEST or H4FG) in the same tube, was adopted. Duplex PCR was carried out in 50 μl containing 50 ng of cDNA, 50 pmol of each primer (25 pmol for H4FG primer set), 20 mm (NH4)2SO4, 75 mm Tris-HCl (pH 8.8), 1.5 mm MgCl2, 0.01% (v/v) Tween 20, and 0.2 mm each of dATP, dCTP, dGTP, and dTTP, using 1.25 units of Taq polymerase (Invitrogen). The following conditions were used: 95 °C, 3 min followed by 25 (H4FG) or 35 (DEEPEST) cycles of denaturation (95 °C, 30 s), annealing (59 °C (DEEPEST) or 62 °C (H4FG), 30 s), and extension (72 °C (DEEPEST) or 68 °C (H4FG), 45 s). The reaction was incubated at 72 °C for 10 min at the conclusion of the PCR cycle. The resulting PCR product was analyzed by ethidium bromide-agarose gel electrophoresis. Bands were subjected to densitometric analysis and were normalized to expression of the internal control GAPDH. All validation experiments using duplex PCR were performed by two independent technicians and confirmed in both SKBr3 and BT474 cell lines. Two end cycle numbers were used for DEEPEST (35 (better) and 40) and H4FG (25 (better) and 30). To exclude any possible contamination or errors, a positive control and a negative control were included in each experiment.Table IDifferential expression of genes that are induced by anti-HER2 antibodyGene nameGene symbolFold changeaFold change represents expression ratio of anti-HER2 antibody-treated sample over control antibody-treated sample in average sample.FunctionSKBr3BT474Pleckstrin homology-like domain, family APHLDA2-2.42-2.44Fas expressionKIAA0186 gene productKIAA186-2.93-2.67UnknownCDC28 protein kinase 2CKS2-2.01-2.72Cell cycleHigh mobility group protein 2HMG2-2.46-2.73ChromatinMitotic spindle coiled-coil related proteinDEEPEST-2.21-3.00Cell cycle, spindleTopoisomerase (DNA) II αTOP2A-2.01-3.01DNA replicationUbiquitin-conjugating enzyme E2CUBE2C-2.05-3.36Cell cycle, mitosisFlap structure-specific endonuclease 1FEN1-2.18-3.36DNA repair and synthesisProliferating cell nuclear antigenPCNA-2.02-3.41DNA replication and repairReplication factor C (activator 1) 4RFC4-2.01-3.46DNA replication and repairMitotic checkpoint kinase Mad3LMAD3L-2.24-3.57Cell cycle, mitosisThymidylate synthetaseTYMS-2.21-3.77DNA replicationZW10 interactantZWINT-2.32-3.92Cell cycle, spindleSerine/threonine kinase 15STK15-2.42-4.28Cell cycle, spindleHighly expressed in cancerHEC-2.56-4.72Cell cycle, spindleH4 histone family, member GH4FG-2.60-4.88Chromatina Fold change represents expression ratio of anti-HER2 antibody-treated sample over control antibody-treated sample in average sample. Open table in a new tab Quantitative Real Time RT-PCR Analysis—To validate gene expression changes, quantitative real time RT-PCR analysis was performed with an Applied Biosystems Prism 7900HT Sequence Detection System using TaqMan® universal PCR master mix according to the manufacturer's specifications (Applied Biosystems Inc., Foster City, CA) for the 14 genes listed in Table I for which validated TaqMan Gene Expression Assays are available. The TaqMan probes and primers for CKS2 (assay identification number Hs00829071_s1), HEC (assay identification number Hs00196101_m1), MAD3L (assay identification number Hs00176169_m1), STK15 (assay identification number Hs00269212_m1), UBE2C (assay identification number Hs00853610_g1), ZWINT (assay identification number Hs00199952_m1), FEN1 (assay identification number Hs00748727_s1), PCNA (assay identification number Hs00427214_g1), RFC4 (assay identification number Hs00427469_m1), TOP2A (assay identification number Hs00172214_m1), TYMS (assay identification number Hs00426591_m1), HMG2 (assay identification number Hs00357789_g1), KIAA186 (assay identification number Hs00221421_m1), and PHLDA2 (assay identification number Hs00169368_m1) were assay-on-demand gene expression products (Applied Biosystems). Human GAPDH gene was used as endogenous control (Applied Biosystems, catalog number 4326317E). The gene-specific probes were labeled by using reporter dye FAM, and the GAPDH internal control probe was labeled with a different reporter dye VIC at the 5′ end. A nonfluorescent quencher and the minor groove binder were linked at the 3′ end of probe as quenchers. The thermal cycler conditions were as follows: hold for 10 min at 95 °C, followed by two-ste" @default.
• W2025708651 created "2016-06-24" @default.
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• W2025708651 date "2005-01-01" @default.
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• W2025708651 title "Genes Affecting the Cell Cycle, Growth, Maintenance, and Drug Sensitivity Are Preferentially Regulated by Anti-HER2 Antibody through Phosphatidylinositol 3-Kinase-AKT Signaling" @default.
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