Abstract 902: XRCC1 induction after cisplatin treatment in head and neck squamous carcinoma cell lines: Evaluation using nanoimmunoassay
Authors: Schoeff Stephen S. , Barrett Dane M. , Teng James , Khalil Ashraf , Hubbard Matthew A. , Maxwell Anne K. , Allak Amir , Mendez Rolando E. , Axelrod Mark , Jameson Mark J. 
Published: May 2015

Abstract
The objective of this study was to use nanoimmunoassay (NIA) to quantify XRCC1 levels and correlate these with response to platinum-based chemotherapeutic agents in HNSCC cell lines. NIA is a developing technique that uses nanogram-scale samples to quantify specific proteins by immunoassay after separation by isoelectric focusing. Detection is more sensitive and quantification more accurate than with standard immunoblot techniques. XRCC1 is a base-excision repair protein that has been identified as a possible mediator of resistance to platinum-based chemotherapeutic agents, but its role and predictive utility have undergone limited investigation in head and neck squamous cell carcinoma (HNSCC). Detection of XRCC1 by NIA was first optimized. In lysates of HNSCC cell lines, XRCC1 was consistently identified at a pI of 5.45 ± 0.1. Peak intensity was improved by the addition of 7 M urea to the specimen prior to loading. In order to correct for sample and loading variability, and detection of a simultaneous internal loading control was also optimized. Under conditions that were optimal for detection of XRCC1, thioredoxin as consistently identified at a pI of 4.90 ± 0.1. Using 9 HNSCC cell lines, XRCC1 levels were evaluated using whole cell lysates in the untreated (basal) state as well as 24 h after treatment with cisplatin or vehicle. NIA was performed on 200 ng of whole cell lysate and XRCC1 and thioredoxin peak areas were quantified using the associated software. Proliferation assays were performed using alamarBlue to determine the IC50 for cisplatin in each cell line. Basal XRCC1 expression levels did not correlate with sensitivity to cisplatin. However, XRCC1 levels were noted to be significantly altered after treatment. When XRCC1 levels were normalized to the thioredoxin loading control, the ratio of treated to basal XRCC1 was correlated to the IC50 for cisplatin across all cell lines (R2=0.428). In cell lines identified as resistant to cisplatin, XRCC1 levels increased upon treatment, whereas in sensitive cell lines, levels remained unchanged. These NIA findings were confirmed using XRCC1 immunoblot with beta-actin as a loading control; XRCC1 levels obtained using this approach also correlated with the IC50 for cisplatin (R2=0.518). While timing and methodology need to be further elucidated, the change in XRCC1 protein level in response to a cisplatin challenge has the potential to be used as a biomolecular predictor of sensitivity that could direct treatment modality selection early in the course of therapy for HNSCC patients. The quantities of protein used allow for potential evaluation of fine-needle aspirates in patients undergoing treatment.
Citation Format: Stephen S. Schoeff, Dane M. Barrett, James Teng, Ashraf Khalil, Matthew A. Hubbard, Anne K. Maxwell, Amir Allak, Rolando E. Mendez, Mark Axelrod, Mark J. Jameson. XRCC1 induction after cisplatin treatment in head and neck squamous carcinoma cell lines: Evaluation using nanoimmunoassay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 902. doi:10.1158/1538-7445.AM2014-902