Abstract
Normal human colonic epithelial cells (HCECs) are not immortalized by telomerase alone but also require CDK4. Some human cell types growth-arrest due to stress- or aberrant signaling-induced senescence (stasis). Stasis represents the consequences of growth conditions culture that are inadequate to maintain long-term proliferation. Overexpressed CDK4 titers out p16 and allows cells to ignore the growth arrest signals produced by stasis. To identify factors contributing to the inadequate culture environment, we used a 62,000-member shRNA library to knock down factors cooperating with human telomerase reverse transcriptase (hTERT) in the immortalization of HCECs. Knockdown of Klotho gamma (KLG; also known as KLPH and LCTL) allowed hTERT to immortalize HCECs. KLG is one isoform of the Klotho family of factors that coordinate interaction between different FGF ligands and the FGF receptor. We also found that knockdown of KLG induced another member of the Klotho family, Klotho beta (KLB). Induction of KLB was maintained and could activate ERK1/2 in immortalized cells. Supplementation of the culture medium with the KLB ligand FGF19 had a similar effect on hTERT-expressing HCECs as knockdown of KLG regarding both immortalization and down-regulation of the tumor suppressor Klotho alpha. Together, these data suggest that KLB is an important regulator in the immortalization of HCECs by facilitating FGF19 growth factor signaling.
Highlights
Human colonic epithelial cells (HCECs) show premature growth arrest due to p16 proteins under normal culture conditions [8]
To identify factors that might be contributing to stasis, we infected HCEC2-human telomerase reverse transcriptase (hTERT) cells with a 62,000-member shRNA library
We have demonstrated that knockdown of Klotho gamma (KLG) overcame stasis and allowed hTERT to immortalize two different colonic epithelial cell strains, HCEC1-hTERT and HCEC2hTERT
Summary
Cell Culture and RNA Preparation—HCEC strains 1 and 2 (HCEC1 and HCEC2) were derived and maintained as. Total RNA was isolated from 1 ϫ 106 cells using an RNeasy mini kit (Qiagen) according to the manufacturer’s procedures. Production of Virus-packaged shRNA Library—The Open Biosystems pGIPZ viral shRNA library was produced as described following the manufacturer’s protocol with the following modifications. We divided the 62,000-member library into pools of 20,000 shRNAs and infected three sets of duplicate plates of HCEC2hTERT cells (seven to eight population doublings (PDs)) prior to when they would enter stasis. After day 5, these dishes were plated at low density (10,000 cells/15-cm plate) and cultured until control HCEC2-hTERT cells reached stasis. Detection of mRNA Levels—First-strand cDNA of HCEC mRNA was synthesized using 1 g of total RNA in a 20-l reverse transcriptase reaction mixture as recommended by the manufacture (Roche Applied Science).
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