TOR1基因調控錯誤配對修復缺陷細胞於老化期間的基因穩定性

Abstract

老化會伴隨著細胞DNA受損或突變並提高罹癌的風險。先前研究已發現，將培養液中的葡萄糖濃度度由2%降低至0.5%的熱量限制培養方式，能減少錯誤配對修復系統缺陷細胞 (Mismatch repair, MMR) 的突變率，增加 HOM3基因穩定性，使細胞壽命得以延長。但熱量限制是否透過抑制TOR (Target of rapamycin) 和RAS的訊息傳遞路徑，而調控細胞內基因表現，提升細胞對抗活性氧屬 (Reactive oxygen species, ROS) 和環境壓力的能力，影響細胞對於DNA修復能力的影響仍需深入探討。首先，本研究發現，2%葡萄糖濃度 (正常) 培養下，老化期間MMR缺陷的msh2∆ 和msh3∆ 細胞剔除TOR1基因後，可達到與熱量限制降低突變率維持細胞存活數，延長細胞壽命的成效，因此，我們推論熱量限制可透過TOR路徑延長msh2∆和msh3∆的細胞壽命並增加基因穩定性，但tor1∆msh2∆msh3∆在熱量限制仍比正常培養條件下有明顯降低老化期間的細胞突變率的效果。其次，我們發現熱量限制培養條件可以顯著的降低細胞中的 .O2-含量，且熱量限制能使SOD1、CTT1和TSA1等，與抗氧化相關的基因表現量增加。更詳細探討發現，培養一日後，正常培養條件下僅有ras2Δ .O2- 含量顯著高於熱量限制培養條件，推論在第一日的熱量限制可能藉由抑制RAS途徑降低 .O2-的產生，而培養至第五日時，正常培養條件下無論是否剔除TOR1或是RAS2基因，細胞的，.O2- 明顯均高於熱量限制組，顯示熱量限制可透過TOR和RAS以外的途徑減少ROS，影響細胞壽命。最後，雖然已知抑制TOR和RAS可活化的下游RIM15，但從我們的結果看來，在正常或熱量限制培養條件下僅ras2∆的RIM15基因表現量並無差異，顯示熱量限制主要抑制RAS途徑調控RIM15基因表現。然而老化期間的ROS變化、是否主要由RAS途徑調控，以及msh2∆msh3∆細胞是否須同時透過抑制TOR和RAS的訊息傳遞路徑來維持老化期間基因的穩定性有待更進一步的驗證。

Mismatch repair (MMR) is a DNA repair system which is critical for the maintenance of genome stability. Defects in mismatch repair have been linked to colorectal and sporadic cancers. Calorie restriction (CR) has been shown to extend life span and increases stress resistance via TOR/SCH9 and RAS signaling pathways in various organisms. We have found that CR containing 0.5% glucose compared to normal treatment (2.0% glucose) can extend life span and promote HOM3 gene stability in MMR-defected cells during aging process in yeast previously. Here, we demonstrate tor1∆ can mimic CR condition and extend life span of msh2∆ and msh3∆ mutants with low mutation frequency. We also found the levels of reactive oxygen species (ROS) were significantly increased during aging and tor1∆ dramatically reduced the levels of ROS. However, tor1∆ could not completely reverse mutation rates in msh2∆msh3∆ mutants. According to these, we believe TOR1 gene can only partially regulates genome stability in MMR defected cells during aging. Whether RAS also affects the genome stability in MMR defected cells during aging needs to be further investigated.