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Browsing by Author "Halme, Milla Marjaana"

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  • Halme, Milla Marjaana (2022)
    Colorectal cancer is one of the most common cancers in the world, and in 2020 it was the cause of nearly 1 million deaths. A major reason for the high death rate is treatment resistance; eventually, almost all colorectal cancer patients with metastatic disease stop responding to chemotherapy. The problem of treatment resistance is not specific to this type of cancer, but it is a widespread issue for all cancer treatments. Chemotherapy resistance is the sum of several cellular and non-cellular factors that together enable sustained cell growth despite the treatment. The non-cellular factors are related to the tumor microenvironment, whereas the cellular factors are related to changes in gene expression, which facilitate e.g. the repair of drug-induced damage or lead to changes in drug metabolism. Lately, researchers have turned their interest to translational control in chemotherapy resistance. This is because translational control plays a major role in stress adaptation. During cellular stress, global translation rates are reduced and those messenger RNAs that are most important for cell survival are translated efficiently. Moreover, translation is fine-tuned by transfer RNA (tRNA) modifications. These modifications are chemical groups that are added to the ribose and the nucleobase of the tRNA molecule, and they affect all aspects of tRNA function, ranging from the structure and stability of the molecule to reading frame maintenance and rate of translation. tRNA modifications are dynamic and change in response to the cellular state, thus facilitating adaptation by translational control. Given the major role of translational control and tRNA modifications in cellular stress responses, their role in the chemotherapy response and adaptation should be thoroughly investigated. The aim of this thesis was to study how chemotherapy affects translation and tRNA modifications in a colon adenocarcinoma model. The cell lines SW480 (from a primary colorectal tumor) and SW620 (from a metastasis) were treated with 5-fluorouracil, oxaliplatin, and leucovorin (FOLFOX), a common combination of chemotherapeutics used in colorectal cancer treatment. The cells were subjected to long-term cyclic treatment as well as 24 h pulse treatment. Chemotherapy resistant cell lines were established by increasing the concentration of FOLFOX for each round of treatment. The effect on translation was studied by polysome profiling, which revealed that FOLFOX treatment causes immediate translational stress, as evidenced by the “shoulders” in the polysomal fractions in the profiles of the pulse treated cells. We hypothesized that these shoulders represent halfmers, polyribosomes without the large subunit. No difference was observed between the long-term treated cells and controls, possibly indicating that the cells had adapted to FOLFOX. The resistant cells exhibited slightly reduced translational activity, which might be due to altered function of ribosomes following the exposure to 5-fluorouracil. Changes in tRNA modification levels were quantified by liquid chromatography mass spectrometry. Several anticodon loop modifications exhibited altered levels after the pulse treatment. In addition, 5-FUrd, a metabolite of 5-fluorouracil, was incorporated into the tRNA. The long-term treated or resistant cells exhibited no differences in the modification levels. In conclusion, this study provided insights on the immediate effects of FOLFOX treatment on translation. This constitutes the first step towards understanding how RNA-based regulatory mechanisms may contribute to the effect and possible resistance to chemotherapy.