Abstract 2905: Induction of hTERT and increased proliferative potential in conditionally reprogrammed normal bronchial epithelial cells
Authors: Craig Daniel J. , Zolondek Rose T. , Zhang Xiaolu , Yeo Jiyoun , Crawford Erin L. , Willey James C. 
Published: July 2016

Background. Based on increasing evidence from this laboratory and genome wide association studies (GWAS), single nucleotide polymorphisms (SNPs) responsible for inter-individual variation in normal bronchial epithelial cell (NBEC) cis-regulation of antioxidant, DNA repair, and cell cycle control genes are key determinants of lung cancer risk. Thus, there is a need for NBEC culture methods that enable extended population doublings without genetic alteration to enable experimental investigation of putative cis-regulatory SNPs in NBEC. Toward this goal, we assessed the effect of previously reported conditional reprogrammed culture (CRC) conditions on regulation of human telomerase reverse transcriptase (hTERT) transcript abundance and proliferative potential in NBEC. Methods. NBEC were obtained by bronchoscopic brush from eight individuals after obtaining informed consent to an IRB-approved protocol. NBEC were incubated in three different culture conditions: bronchial epithelial cell growth media (BEGM) only, co-cultured with irradiated mouse embryonic fibroblasts (IRR-MEF) + Rho kinase inhibitor (ROCKi) in BEGM, and conditioned BEGM + ROCKi. Media were changed every three days and cells were passaged and sub-cultured after ten days. Human telomerase reverse transcriptase (hTERT) was measured in three individuals after each passage in triplicate via qPCR. The proliferative capacity of all eight individuals was assessed using cell count and morphology at passage &gt;3. Results. Co-culturing NBEC with IRR-MEF in BEGM supplemented with ROCKi produced a highly proliferative cell population while maintaining lineage commitment evident after removal of CRC conditions. Transcript abundance of hTERT was elevated 6.5-fold in NBEC in co-cultured conditions and 4.3-fold in NBEC in conditioned media compared to BEGM alone. Cell count in CRC conditions were up to 22-fold higher compared to BEGM alone. Cells were passaged and sub-cultured up to passage 4, followed by being frozen down in cell culture freezing media for further assessment. Conclusion. NBEC hTERT transcript abundance was up-regulated and cell population proliferative potential was extended in CRC conditions. It is likely that hTERT functions to protect the ends of linear chromosomes in dividing cells, enabling increased cell divisions while maintaining normal genome. These cell populations will be used in future studies to assess the effect of putative cis-regulatory single nucleotide polymorphisms (SNPs) on gene expression in NBEC.

Citation Format: Daniel J. Craig, Rose T. Zolondek, Xiaolu Zhang, Jiyoun Yeo, Erin L. Crawford, James C. Willey. Induction of hTERT and increased proliferative potential in conditionally reprogrammed normal bronchial epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2905.