BIRC7- and RTEL1-knockdown in the human colorectal cancer cell line HT29 with chromosomal 20q amplification : effects on cell proliferation and cell death
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Colorectal cancer (CRC) is one of the most frequently occurring cancer types in the world. The most common chemotherapeutic drug used for treatment has been 5-fluorouracil (5-FU), but development of patient drug resistance is a major obstacle to successful treatment. The 20q13 amplicon, which is frequently detected in sporadic CRC, has been suggested to be the home of one or more oncogenes important for tumor progression and drug response. We investigated two genes, BIRC7 and RTEL1, in this amplicon. Elevated expression of the apoptotic suppressor BIRC7 has been shown to be associated with aggressive tumors, poor response to chemotherapeutic treatment, and shorter survival time. Loss of, or inactivation, of the tumor suppressor RTEL1 may be a driving force for genomic instability, as it helps to maintain genomic stability. We performed FISH analysis by hybridizing the human CRC cell lines HCT116 and HT29 with a centromere 20 probe to determine whether chromosome 20 was amplified in these cell lines. SiRNA-mediated knockdown of BIRC7 and RTEL1 was performed to investigate changes in specific cellular phenotypes, with and without 5-FU-treatment. Assessments of cellular phenotypes were accomplished using Western blotting and various proliferative and apoptotic markers. Impact of knockdown and drug treatment on cell cycle progression was assessed by flow cytometry. We found that chromosome 20 was amplified in HT29, BIRC7 was overexpressed, and HT29 expressed both isoforms of BIRC7 (α and β). The HCT116 cell line had no chromosome 20 amplification, no BIRC7 overexpression, and only isoform β was expressed. Knockdown of BIRC7 in HT29 seemed to be time-dependent and isoform-specific, and we confirmed that the anti-apoptotic isoform α was dominated by the pro-apoptotic β (Tβ) when expressed together, as in HT29. In HCT116 we did not detect knockdown within the experimental time window studied. No RTEL1 expression was detected under any experimental conditions due to antibody problems. Knockdown effects were thus studied by assessing specific phenotypes. 5-FU induced apoptosis was observed in HCT116 for both genes, while HT29 seemed less affected by 5-FU-treatment. Knockdown of BIRC7 or RTEL1 did not sensitize cells towards 5-FU-treatment, but rather increased cell viability in both cell lines. In HT29-cultures the overexpression of BIRC7 seemed rather to inhibit 5-FU-induced apoptosis. Drug response was more likely affected by the cell lines’ TP53-genotype and mismatch repair status, among others.
Master i biomedisin
UtgiverHøgskolen i Oslo og Akershus