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P21 tumor protein의 특성 연구

P21 tumor protein의 특성 연구
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대학원 약학과
이화여자대학교 대학원
P21 which is also called as P21 in human and TCTP (Translationally controlled tumor protein) is a growth-related tumor protein. P21 is a cytosolic acidic and calcium binding protein and known to be regulated by mitogens, at the level of transcription as well as translation. In this study we showed that the P21 mRNA existed in all of human 16 organs with pancreas expressing the highest level. We found that P21 mRNA expression was variable among in the six cervical cancer cell lines (HeLa, Me180, HT3, C33A, Caski, SiHa). The P21 mRNA increased 2.16 times in Caski and 2.98 times in Me180 than SiHa. We also tested, if breast cancer tissues from three patients and cervical cancer tissues from two patients express more P21 mRNA than normal tissues. We found that the P21 mRNA increased 1.4 times in one breast cancer patients but not in other patients, and 2.4 times in one cervical cancer patient but not in the other patient. This suggests that the P21 may be involved in the tumorigenesis of breast and cervix in some cases but not always. We tested if the level of P21 mRNA is regulated by EFG using Me180 and Caski. We found that the P21 mRNA level was not changed by EGF in Me180 and Caski cell lines. We also tested if P21 mRNA from HeLa cell line is regulated by insulin. The P21 mRNA level of HeLa cells was decreased by 50% after 10 min but recorved to that of control after 24 hrs. We investigated whether the P21 mRNA is regulated during cell cycle. The result was that the P21 mRNA from Yeast (Saccharomyces Cerevisiae) was not changed during cell cycle, G1, S, G2 and cytokinesis. We tested if (Na,K)ATPase binds with P21. We found that P21 interacted with (Na,K)ATPase, KD was 8.5×10^-7 M^-1 and interacted with itself, KD was 1.49×10^-7 M^-1. P21 has potential phosphorylation site at the residue of SIK (98-100AA). Therefore, we looked for protein kinases that might phosphorylate the P21 by affinity chromatography. We found that the PKC βI and βII but not α, γ, δ, ε, ζ,λ were bound to the P21 by Western blotting, suggesting that they most likely phosphorylate the potential residue SIK of the P21. ; Growth related tumor protein인 P21은 P23, TCTP (translational controlled tumor protein)이라고도 불리며 transcription과 translation level에서 모두 조절된다. P21 mRNA는 사람의 16개 조직 (심장, 뇌, 태반, 허파, 간, 근육, 신장, 이자, 비장, 흉선, 전립선, 고환, 자궁, 소장, 대장, 백혈구) 모두에서 존재하며 비장에서 다른 조직보다 3-4배 높게 나타났다. 자궁 경부암 세포 6종류 (C33A, Caski, HeLa, HT3, Me180, SiHa) 중에서 EGFR 발현이 다른 세포보다 높은 Me180, Caski에서 P21 mRNA의 양이 많았다. In vivo에서, 즉 3명의 유방암 환자와, 2명의 자궁 경부암 환자의 암 조직과 정상 조직 간의 P21 mRNA 양을 비교해 본 결과 1명의 유방암 환자와 1명의 자궁 경부암 환자에서 각각 1.4배, 2.4배 높은 P21 mRNA가 나타났다. 이로써 모든 경우는 아니지만 암조직과 P21 mRNA 양이 연관되었다고 예상 할 수 있다. EGF를 Me180, Caski에 처리시 P21 mRNA 양의 변화가 나타나지 않았지만 HeLa 세포에 insulin 처리 결과 10분 후 P21 mRNA가 50% 감소하였다가 24시간 후 거의 정상 수치로 되돌아 왔다. 세포주기 G1, S, G2, cytokinesis 중 P21 mRNA 양은 변하지 않았으므로 P21 mRNA의 발현 조절이 transcription level에는 일어나지 않거나 cell cycle과는 무관하게 transcription된다고 예상된다. 세포질에 위치하는 P21, cofilin은 세포막에 위치하는 (Na,K)ATPase와 상호작용하는 데, P21과 (Na,K)ATPase는 8.5×10^-7 M^-1 KD이었고 cofilin과 (Na,K)ATPase의 9.01×10^-9 M^-1 KD이었다. 또한 P21과 P21 상호간에는 1.49×10^-7 KD이었다. P21의 98-100번째 amino acid에는 SIK라는 Ser/Thr kinase인 PKC의 인산화 잔기가 있는 데 affinity chromatography 실험 결과 12개의 PKC isoform중에서 PKC βI, βII가 P21에 결합하였다. 그러므로 PKC βI, βII가 P21을 인산화시킬 가능성을 제시하고 있다.
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