Publication Date

4-25-2021

Document Type

Other

First Advisor

Bode, Barrie P.

Degree Name

B.S. (Bachelor of Science)

Legacy Department

Department of Biological Sciences

Abstract

Our laboratory has used the CRISPR-Cas9 gene editing system to eliminate the expression of the SLC1A5 gene, which encodes the ASCT2 amino acid transporter, which is implicated in driving the glutamine-dependent growth of several cancers. At last year’s URAD event, the isolation of clones confirmed for knockout (KO) of ASCT2 in two human hepatocellular carcinoma (HCC) cell lines – Huh7 and SK-Hep1 - was described. As these ASCT2 knockout (ASCT2KO) cell lines are currently being deployed in studies assessing the role of this transporter in tumorigenesis, we asked two fundamental questions: 1) Does the removal of antibiotic selective pressure – under which ASCT2KO lines are selected – result in the re-appearance of ASCT2 in populations or selected clones of Huh7 and SK-Hep1? 2) Does the continuous exposure of populations or isolated clones of Huh7 or SK-Hep1 to lethal antibiotics used in selection of ASCT2KO lines lead to increased expression of the Multi-Drug Resistance Transporter MDR1? These two questions are fundamental to the interpretation of tumorigenesis results. ASCT2 and MDR1 expression was assessed by western blot analysis, and growth of the lines were measured by the colorimetric MTT assay. The results indicate that the SK-Hep1 cell lines targeted for ASCT2KO exhibited no transporter expression in the absence or presence of antibiotic selective pressure. Conversely, Huh7 populations targeted for ASCT2KO unexpectedly exhibited enhanced ASCT2 expression under antibiotic selection. Huh7 ASCT2KO clones in contrast showed no re-emergence of ASCT2 expression, suggesting that each was successfully “knocked out” for this transporter. Likewise, SK-Hep1 exhibited no MDR1 expression, but Huh7 significantly expressed this drug resistance transporter; its abundance was increased by Puromycin, the antibiotic used for selection of CRISPR-Cas9-mediated ASCT2KO lines. This drug-induced expression of MDR1 was blunted in Huh7 ASCT2KO lines, suggesting that ASCT2 might play a role in drug resistance in cancerous cells. Finally, studies were done to test the growth rate of Huh7 clonal cells lines compared to the population, as well as study the growth of the Huh7 and Sk-Hep ASCT2KO populations when grown in the presence of two drug therapies-Metformin and Sorafenib. It was found that the clonal cell population initially showed a slower growth rate, however the rate increased after 72 hours to become consistent with the population and control. It was also observed that Metformin and Sorafenib slightly slowed the growth of Huh7 cells, but neither drug had an effect on Sk-Hep cells overall. These results suggest that while ASCT2 elimination does not affect growth, it may render targeted cancer cells more vulnerable to certain chemotherapies. Further studies can be designed to test this hypothesis.

Extent

21 pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

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