3'-methly-4-dimethylaminoazobenzene으로 유도된 백서 간장암세포 원형질막 내 포도당 운반체의 특이성

Abstract

[한글]

포유 동물에서 포도당 운반에 관여하는 포도당 운반체는 세포에 따라 존재하는 종류가 다르고 이들의 물리화학적 성질과 표현 기전도 다르다고 알려져있다. 정립된 간장암 세포인 HepG2세포에서 적혈구형 포도당 운반체의 mRNA가 발견된 이래 여러 종류의 정립된 암

세포들(SK-Hep-1, Hep3B, Fao)에서도 적혈구형 포도당 운반체의 mRNA가 표현된다고 보고되어 있다. 따라서 화학 발암제에 의해 유도된 간장암 세포에서도 간장형 포도당 운반체보다는 적혈구형 포도당 운반체가 표현될 것이라는 가능성을 생각하게 되었다.

본 실험에서는 정상 백서 간장과 3'-methyl-4-dimethylaminoazobenzene (3'-Me DAB)으로 유도된 간장암 세포의 원형질막 및 low density microsomal fraction(LDM)에서 표현되

는 포도당 운반체들의 종류를 알아보기 위해 적혈구형과 간장형 포도당 운반체의 cDNA의 염기 배열 순서로부터 추론한 적혈구형 포도당 운반체의 아미노산 배열 순서 480에서 492번째까지의 13개 아미노산으로 구성된 peptide(pCT^^RBC; Glu-Glu-Leu-Phe-His-Pro-Leu-Gly-Ala-Asp-Ser-Gln-Val)와, 간장형 포도당 운반체의 513에서 522번째까지의 10개의 아미노산으로 구성된 peptide(pCT^^liver; Met-Glu-Phe-Leu-Gly-Ser-Ser-Glu-Thr-Val)를 solid phase peptide synthesis 법으로 합성하고 이에 대한 peptide 특이 항혈청을 New Zealand white 토끼에서 형성시켰으며 이 항혈청으로부터 affinity chromatography법으로 peptide 특이 항체를 정제 분리하였다.

이 두 항체를 이용하여 사람 적혈구막에서 준비한 protein depleted membrane(PDM)과 대조군 간장세포 원형질막을 전기 영동시킨후 western blot 법으로 분석해 본 결과 본 실험에서 준비한 peptide 특이 항체는 해당 포도당 운반체와는 특이하게 반응하나 다른 포

도당 운반체와는 교차 반응이 나타나지 않았다.

또한 이 두 항체를 이용하여 대조군 간장 및 간장암 세포의 원형질막과 LDM에 존재하는 포도당 운반체의 종류를 확인해 본 결과 적혈구형 포도당 운반체는 대조군 간장이나 간장암 세포에서 검출되지 않았다. 그러나 간장형 포도당 운반체는 대조군이나 간장암 세포

의 원형질막 및 LDM에서 다 같이 표현되나 양군간에 의의있는 양적변화는 없었다. 이러한 사실로 미루어 대조군 간장세포나 간장암세포의 원형질막 및 LDM 내에는 적혈구형 포도당 운반체가 존재하지 않거나 만일 존재한다 하더라도 양적으로 매우 적기 때문에 본 실험에서 제조한 항체로서는 검출할 수 없을 정도로 극소량이 존재할 것으로 사료된다. 또한 정상 간장 세포가 화학 발암제인 3'-Me DAB에 의해 간장암 세포로 변형되는 과정에서 포도당 운반체는 그 양이 증가하지도 않고 간장형으로부터 적혈구형으로 전환되지 않는

것으로 사료된다.

Identification of Glucose Transporters Expressed in the Control Rat Liver and

Hepatoma Induced by 3'-Methyl-4-dimethylaminoazobenzene

Soo Jeon Choi

Department of Medical Science, The Graduate School, Yonsei University

(Directed by Professor Yoon Soo Kim, M.D., Ph.D.)

The eukaryotic facilitated glucose transporters(GT) are different from type to

type with respect to the regulation of their expression, kinetic properties, and

physicochemical characteristics. Since the discovery of RBC type GT in HepG2 cells,

RBC type GT has been known to be expressed in several hepatoma cell lines including

SK-Hep-1, Hep3B, Fao. It has been suggested that the hepato-carcinogenesis is

closely related to the change in GT expression since the increased mRNA for RBC

type GT was observed in hepatoma cell lines.

In this study, two types of C-terminal peptides, one from 480 to 492 amino acid

sequence(Glu-Glu-Leu-Phe-His-Pro-Leu- Gly-Ala- Asp- Ser- Gln-Val) of RBC type GT

and the other from 513 to 522 amino acid

sequence(Met-Glu-Phe-Leu-Gly-Ser-Ser-Glu-Thr-Val) of liver type GT based on their

respective cDNA were synthesized and antisera against the peptides were prepared

through the New Zealand white rabbits to elucidate the differential expression of

the GT in control rat liver and in hepatoma induced by

3'-methyl-4-dimethylaminoazobenzene(3'-Me DAB). Antisera against the C-terminal

peptide of RBC type GT(pCT^^RBC) and liver type GT(pCT^^liver) reacted only with

corresponding types of GT in western blot analysis.

Peptide specific antibodies were purified from the polyclonal antisera by

affinity chromatography using each synthetic peptide, pCT^^RBC and pCT^^liver as

antigens. With these peptide specific antibodies, the expressions of RBC type GT

and liver type GT were analyzed in the plasma membrane(PM) and low density

microsomal fractions(LDM) of control rat liver and hepatoma. The expression of

liver type GT was detected only both in PM and in LDM of control rat liver and

hematoma, whereas the expressions of RBC type GT were not observed in the PM and

LDM of both groups. These results indicate that the RBC type GT may be absent or

too little to be detected by western blot in control rat liver and hepatoma, and

that the expression of liver type GT is not altered during hepatocarcinogenesis in

vivo.

[영문]

The eukaryotic facilitated glucose transporters(GT) are different from type to type with respect to the regulation of their expression, kinetic properties, and physicochemical characteristics. Since the discovery of RBC type GT in HepG2 cells, RBC type GT has been known to be expressed in several hepatoma cell lines including SK-Hep-1, Hep3B, Fao. It has been suggested that the hepato-carcinogenesis is closely related to the change in GT expression since the increased mRNA for RBC

type GT was observed in hepatoma cell lines.

In this study, two types of C-terminal peptides, one from 480 to 492 amino acid sequence(Glu-Glu-Leu-Phe-His-Pro-Leu- Gly-Ala- Asp- Ser- Gln-Val) of RBC type GT and the other from 513 to 522 amino acid sequence(Met-Glu-Phe-Leu-Gly-Ser-Ser-Glu-Thr-Val) of liver type GT based on their respective cDNA were synthesized and antisera against the peptides were prepared through the New Zealand white rabbits to elucidate the differential expression of

the GT in control rat liver and in hepatoma induced by 3'-methyl-4-dimethylaminoazobenzene(3'-Me DAB). Antisera against the C-terminal peptide of RBC type GT(pCT^^RBC) and liver type GT(pCT^^liver) reacted only with corresponding types of GT in western blot analysis.

Peptide specific antibodies were purified from the polyclonal antisera by affinity chromatography using each synthetic peptide, pCT^^RBC and pCT^^liver as antigens. With these peptide specific antibodies, the expressions of RBC type GT and liver type GT were analyzed in the plasma membrane(PM) and low density microsomal fractions(LDM) of control rat liver and hepatoma. The expression of liver type GT was detected only both in PM and in LDM of control rat liver and hematoma, whereas the expressions of RBC type GT were not observed in the PM and LDM of both groups. These results indicate that the RBC type GT may be absent or too little to be detected by western blot in control rat liver and hepatoma, and that the expression of liver type GT is not altered during hepatocarcinogenesis in vivo.