E and intracellular staining for common HNSCC biomarkers by fluorescen…

E and intracellular staining for common HNSCC biomarkers by Rosiglitazone fluorescently-labeled monoclonal antibodies and analysis by flow cytometry (Table 2). Compared to isotype controls, USC-HN1 displays significantly increased staining of FABP5, E-cadherin, and CD24. EGFr, c-Kit, and CD74 staining also showed an increased mean fluorescence intensity, whereas IL-13Ra, CD44v6 and CD133 did not show a significant increase.Cytogenetic analysisE-cadherin 0.Cytogenetic analysis of the USC-HN1 cell line was performed in collaboration with the City of Hope (Duarte, CA) (Figure 7). All mitotic cells analyzed from the USCHN1 cell line were clonally abnormal. The complex neartriploid clone was characterized by a modal number of chromosomes of 71 and by deletions involving chromosomes X, 3, 4, 6, and 7, additional material of unknown origin on chromosomes 8, 13, 17, and 19, a derivative chromosome 6 resulting from an unbalanced translocation with the long arm of chromosome 14, gains of chromosomes X, 7, 11, 15, and 19, losses of chromosomes 9, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/15501003 14, 18, 21, and 22, as well as gain of a large marker chromosome and a small suspected ring chromosome.*** > 1000 Control MFI ** 100-1000 Control MFI * < 100 Control MFI USC-HN1 cells in culture were stained for HNSCC-related cytokines and surface markers (left column) using monoclonal antibodies and analyzed by flow cytometry. Samples were run in duplicate. Percent of positive staining cells (middle columns) and mean fluorescence intensity (right columns) is shown for each antibody target and its isotype control.PCR Viral screen for HPV and EBVSince HPV and EBV are associated with malignancies of the head and neck, the USC-HN1 cell line was screened for these oncogenic viruses by PCR using consensus primers for HPV L1 and EBV EBNA2 genes as described previously [15,17]. The results showed that the USC-HN1 did not have specific amplifications for either the HPV L1 sequence or for the EBV sequence (Figure 8). HeLa cellsLiebertz et al. Head Neck Oncology 2010, 2:5 http://www.headandneckoncology.org/content/2/1/Page 9 ofFigure 7 Karyotype of USC-HN1. Karyotype of USC-HN1 cell line showing a near-triploid clone (modal 71) demonstrating features characteristic of head and neck cancer including multiple deletions (chromosomes X, 3, 4, 6, and 7), isochromosome formation, and many breakpoints around centromeres.(known HPV-positive cervical carcinoma) and SW579 (known HPV-negative thyroid carcinoma) were run in parallel as controls for both HPV consensus primer sets (MY09/MY11 and GP5+/GP6+). For the EBV screen, EBV + Raji and EBV - T-cell lymphoma TLBR-1 cell lines [17,20] were run in parallel as controls.Cytokine and oncogenes expressionThe cell line was also tested by qRT-PCR for its expression levels of pertinent oncogenes and cytokines. Run inparallel with RNA extracted from the human pharyngeal carcinoma FaDu cell line and compared to Universal Human Reference RNA, the overall expression levels of USC-HN1 closely paralleled that of FaDu, a documented HNSCC cell line (Table 3). Statistical analysis revealed significant differences between the expression levels of c-myc, VEGFa, TGF-b1, and IL-1b produced by USCHN1 and the FaDu cell line, and no significant differences between p53, Rb, c-Kit, VEGFc, COX2, TGF-b2, IL-4, IL-6, IL-8, and IL-10.Figure 8 PCR studies for the presence of oncogeneic viruses. (A) HPV detection using consensus primers GP5+/GP6+ (1a-4a) and MY09/ MY11 (1b-4b) demonstrating USC-HN1 (3a, 3b) is nega.