營養缺乏對神經瘤母細胞轉殖入PPP2R2B的影響

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2011

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蛋白磷酸酶2A (PP2A) 是絲胺酸/酥胺酸去磷酸酶,由催化次單元、骨架次單元與調控次單元所組成的三體蛋白。其中PPP2R2B (Bβ)是特別作用在腦部的調控單元,會經由選擇性剪接後,生成兩種蛋白異構物:Bβ1與Bβ2。前者表現於細胞質中,後者表現於粒線體。過去研究顯示,異常表現PP2A的細胞與神經退化疾病的發生有關。過去本實驗室以SK-N-SH轉殖入Bβ1基因與Bβ2基因建立穩定細胞株。實驗顯示Bβ1與Bβ2是藉由細胞自噬(autophagy)方式維持細胞生存。過去的研究中發現,這類轉殖的穩定細胞會受外來氧化壓力的影響,並在細胞自噬相關基因失活時提高其細胞存活。 過去的研究中顯示,細胞株在飢餓壓力下,會以細胞凋亡維持細胞存活。本計畫以HBSS (Hank’s Balanced Salts Solution) 作為細胞飢餓壓力的來源。研究結果顯示轉殖Bβ1的SK-N-SH細胞在HBSS環境會促使細胞自噬。並試以細胞自噬抑制劑20μM 3-MA處理,發現3-MA可抑制因飢餓壓力所造成的細胞自噬,進而減少後續的細胞凋亡,而使細胞的存活提高,但是抑制lysosome及autophagosome的抑制劑Bafilomycin A1,則沒有減少後續細胞凋亡的效果。此外,添加胺基酸glutamine可以減少的細胞自噬,進而降低細胞凋亡,但非必需胺基酸(NEAA)則無此效果。 此項研究可對於研究神經細胞異常表現PPP2R2B後,在不同環境壓力下,提供更好的研究模式及瞭解細胞自噬生成機制。
Protein phosphatase 2A (PP2A) is a heterotrimeric Ser/Thr phosphatase that consists of a catalytic C, scaffolding A and a variable regulatory subunit. The brain-specific regulatory subunit gene, Bβ (PPP2R2B), encodes two splicing regulatory variants that target the enzyme isoforms to mitochondria (Bβ2) and cytoplasm (Bβ1), respectively. Previously, we have demonstrated stable cell lines by transferring Bβ1 or Bβ2 into human SK-N-SH cell respectively can be accomplished and maintained. Autophagy promotes cell survival in all the established clones. To test how Bβ clones respond to starvation, the cells were exposed to Hank’s Buffered Salt Solution (HBSS). Cell death was induced in Bβ1 clones, but not in Bβ2 clones, following six hr starvation. Suppression of autophagy using pharmacological inhibitor, 3-methyladenine (3-MA), can rescue autophagic cell death caused by starvation and reduce autophagy-related apoptosis. On the other hand, Bafilomycin A1 did not rescue autophagy- related apoptosis. Further, amino acid supplement glutamine partialy reduced autophagic cell death by HBSS. Therefore, we showed that cells with ectopically expressed regulatory subunit PPP2R2B of the holoenzyme PP2A were predisposed to autophagy. Starvation induced autophagic cell death in Bβ1 clones with cytoplasmic PPP2R2B, but not in Bβ2 clones that is related to apoptosis. The results promised a model for studying the mechanism and function of aberrant PPP2R2B expression in neuronal cells under starvation.

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蛋白磷酸酶2A, 細胞自噬, 細胞凋亡, 飢餓壓力, 營養缺乏, PP2A, PPP2R2B, Autophagy, Apoptosis, Nutrient Deprivation, starvation

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