Um scc 1

This study was approved by the Institutional Review Board at the University of Louisville. Formalin-fixed paraffin-embedded (FFPE) specimens of hgOED (N = 38) were obtained from the University of Louisville Oral Pathology Laboratory (Louisville, KY, USA) from April 2003 to February 2015 as previously described [3 (link)]. Fresh, frozen or FFPE tissues from cases of HNSCC (N = 50) were collected from 2006 to 2015 from the Cancer Database and Specimen Repository at the James Graham Brown Cancer Center (Louisville, KY, USA). University of Michigan Squamous Cell Carcinoma (UMSCC) oral cavity cancer cell lines (HPV-negative [UMSCC-1]; HPV-positive [UMSCC-47 and UMSCC-104]) [20 (link), 50 (link), 60 (link)] were purchased from EMD Millipore Corporation (Temecula, CA, USA) and cultured by standard protocols. Cervical cancer cell lines (CaSki and SiHa) were cultured as suggested by the American Type Culture Collection (Manassas, VA, USA).

Two head and neck cancer cell lines were use in this study. Both were purchased through Merck Millipore (Darmstadt Germany).
A. UM-SCC-1 was isolated from a recurrent squamous cell carcinoma in the mouth floor of a 73-year-old male. The cell line originated from the laboratory of Dr. Thomas Carey at the University of Michigan and is negative for the Human Papilloma Virus (HPV).
B. UM-SCC-47 is also a squamous cell carcinoma but derived from a primary tumour of the lateral tongue. This cell line also originated from the laboratory of Dr. Thomas Carey at the University of Michigan and is HPV positive for HPV type 16. Oncogenicity is conferred through the expression of viral oncoproteins E6 and E7 [35 (link)].
Cell lines were cultured in T75 flasks (Sigma-Aldrich^® Darmstadt DE) as a monolayer using RPMI 1640 medium (Sigma-Aldrich^® Darmstadt DE) supplemented with 10% foetal calf serum (FCS), 10 mM HEPES, 12.5 μg/ml penicillin and 16 μg/ml gentamycin. Cell flasks were incubated in a humidified atmosphere at 37°C containing 5% CO₂ and passaged after reaching exponential growth prior to confluency. Both cell lines were tested for the presence of mycoplasma (biotool.com B3903, Madrid ES) and found to be negative.

Three cell lines representing each HPV status were selected. UM-SCC-47, UPCI-SCC-154, and UPCI-SCC-090 are HPV positive. UM-SCC-17a, UM-SCC-22a and UM-SCC-1 are HPV negative (Table 1). UM-SCC-22a, UM-SCC-47, UM-SCC-17a and UM-SCC-1 were sourced through Merck Millipore (Darmstadt, Germany). UPCI-SCC-154 (ATCC^® CRL-3241TM) and UPCI-SCC-090 (ATCC^® CRL-3239TM) were sourced from ATCC (Manassas, VA, USA). To reflect characteristic mutational status of the HPV groups, the chosen HPV negative cell lines have TP53 mutations and HPV positive retain wild type expression. Cell lines were grown in Dulbecco’s Modified Eagle’s Medium with 4500 mg/L glucose (Sigma-Aldrich^® Darmstadt, Germany) 10% fetal bovine serum (FBS), 10 mM HEPES, 100 U/mL penicillin and 0.1 mg/mL streptomycin (Sigma-Aldrich^® Darmstadt, Germany). Cell cultures were grown in T25 flasks (Greiner Bio-One, Frickenhausen, Germany) and incubated at 37 °C in humidified atmosphere with 5% CO₂.