인체 간암세포(PLC/PRF/5)에서 N-ras 암유전자의 발현과 염기서열 결정

Abstract

[한글]

간암은 우리 나라에서는 가장 흔한 악성 종양의 하나로서 최근에 여러가지 새로운 치료법이 개발되어 임상에서 시도되고 있으나 조기절제술 이외에는 적절한 치료 방법이 아직 없다. 간암 발생의 원인은 확실치 않으나 역학적 및 실험적 보고에 따르면 원발성 간암이

만성 간질환, 특히 간경화증과 관련이 많으며 그 원인인자로 B형 간염 바이러스, C형 간염 바이러스, 마이코톡신(aflatoxin B)과 성홀몬 등이 거론되고 있다. 세포암유전자중 N-γas 유전자의 활성화는 혈액종양 이외에 폐암 및 간암 세포에서도 일어나고 있음이 보고되어 있으나 이 유전자의 활성화 양상은 조직과 세포의 종류에 따라 다르며 이 유전자의 활성화가 간암 발생에 어떻게 관여하는지에 대한 구체적인 지식은 미흡한 실정이다.

본 연구에서는 B형 간염 바이러스 표면 항원을 생성하는 인체 간암 세포주인 PLC/PRF/5(Alexander cell)와 원발성 간암 조직 및 정상 간조직에서 발현되는 N-γas mRNA를 slot blot hyridization과 Northern blot hybridization을 시행하여 간암세포에서의 N-γas 유

전자 발헌 정도를 평가한 결과 간암세포(PLC/PRF/5), 원발성 간암 조직 및 정상 간조직에서 발현되는 N-γas mRNA의 현저한 양적 차이는 나타나지 않았다.

간암세포(PLC/PRF/5)에서 N-γas 유전자의 구조적 변이 여부를 알아보기 위하여 다른 암세포에서 주로 변이가 관찰되고 있는 codon 12번과 13번, 그리고 61번의 염기 서열을 확인하고자 하였다. Codon 12번파 13번을 포함한 exon 1과 codon 61번을 포함한 exon 2를

중합 효소연쇄 반응으로 증폭시키고 이를 Ml3(mp19) vector에 subcloning하여 염기 서열을 결정한 결과 간암세포(PLC/PRF/5)의 N-γas exon 1에는 변이가 관찰되지 않았고, exon 2에 위치한codon 61번의 두 번째 염기인 A가 G로 변이되어 있었다. 즉 61번 codon의 정

상적 염기 서열인 CAA가 CGA로 바뀌어서 N-γas에서 발현되는 단백질(p21)의 61번째 아미노산인 gluta-mine이 arginine으로 치환되었음을 알 수 있었다.

이상과 같은 실험 결과는 인체 간암세포(PLC/PRF/5)의 N-γas는 codon 61번에 변이를 일으켜 활성화되어 있음을 알 수 있으며 N-γas의 활성화는 이 유전자에서 발현되는 단백질(p21)의 양적 증가에 기인하는 것이 아니고 이 유전자에서 발현되는 N-γas 단백질(p21

)의 61번째 아미노산인 glutamine이 arginine으로 치환되어 나타나는 구조적 변형에 의한 생물학적 변화에 기인하는 것으로 사료된다.

Expression and sequence determination of N-γas oncogene in human hepatocellular

carcinoma cell(PLC/PRF/5)

Choo-Yon Cho

Department of Medical Science The Graduate School, Yonsei University

(Directed by Professor Yoon Soo Kim)

Hepatocellular carcinoma is one of the maior malignant diseases, in Africa and

Asia including Korea. Although there are some epidemiological and experimental

evidences of correlations between the incidence of hepatocellular carcinoma and

chronic hepatitis, especially liver cirrhosis, the real mechanism of

hepatocar-cinogenesis has not been unveiled. The causative factors associated with

hepatocellular carcinoma are thought to be related to hepatitis B virus(HBV),

hepatitis C virus(HCV), mycotoxin(aflatoxin B) and sex hormones.

It has been reported that N-γas is activated in hepatocellular carcinoma and

lung carcinoma cells as well as hematopoietic cells, and that the pattern of the

activation varied with the type of tissues and cells. However, the detailed

knowledge of the involvement of the activated N-γas oncogene in the hepatocellular

carcinogenesis is still obscure.

In the present study, N-γas mRNA expressions in human hepatocellular carcinoma

cell PLC/PRF/5(Alexander cell), human primary hepatocarcinoma and normal liver

tissues were measured and compared. There were no remarkable differences in the

N-γas mRNA contents between PLC/PRF/5, human primary hepato carcinorna tissue and

normal liver tissue.

In order to determine the base sequences of codon 12, 13 and 61 of N-γas in

PLC/PRF/5, in which point rnutations were frequently observed in various tumors,

axon 1 and exon 2 of N-reswere amplified by polymerase chain reaction, subcloned

into Ml3(mp19) and sequenced.

No mutations were observed in exon 1 in PLC/PRF/5 cells as well as in normal and

primary hepatocarcinoma cells. However, A to G point mutation at second base of

codon 61 of N-γas in PLC/PRF/5 cell was observed. The change of CAA of normal

sequence of codon 61 to CGA indicates the substitution of glutamine at 61st amino

acid of N-γas protein(p21) to arginine.

In conclusion, that N-γas is activated by a point mutaiton at codon 61 and the

activation of thegene is not a consequence of a quantitative increase of N-γas

Protein(p21) but a consequence of aqualitative ohange due to the amino acid

substitution of glutamine to arginine at 61st position of N-γas protein.

[영문]

Hepatocellular carcinoma is one of the maior malignant diseases, in Africa and Asia including Korea. Although there are some epidemiological and experimental evidences of correlations between the incidence of hepatocellular carcinoma and

chronic hepatitis, especially liver cirrhosis, the real mechanism of hepatocar-cinogenesis has not been unveiled. The causative factors associated with hepatocellular carcinoma are thought to be related to hepatitis B virus(HBV), hepatitis C virus(HCV), mycotoxin(aflatoxin B) and sex hormones.

It has been reported that N-γas is activated in hepatocellular carcinoma and lung carcinoma cells as well as hematopoietic cells, and that the pattern of the activation varied with the type of tissues and cells. However, the detailed knowledge of the involvement of the activated N-γas oncogene in the hepatocellular carcinogenesis is still obscure.

In the present study, N-γas mRNA expressions in human hepatocellular carcinoma cell PLC/PRF/5(Alexander cell), human primary hepatocarcinoma and normal liver tissues were measured and compared. There were no remarkable differences in the

N-γas mRNA contents between PLC/PRF/5, human primary hepato carcinorna tissue and normal liver tissue.

In order to determine the base sequences of codon 12, 13 and 61 of N-γas in PLC/PRF/5, in which point rnutations were frequently observed in various tumors, axon 1 and exon 2 of N-reswere amplified by polymerase chain reaction, subcloned into Ml3(mp19) and sequenced.

No mutations were observed in exon 1 in PLC/PRF/5 cells as well as in normal and primary hepatocarcinoma cells. However, A to G point mutation at second base of codon 61 of N-γas in PLC/PRF/5 cell was observed. The change of CAA of normal sequence of codon 61 to CGA indicates the substitution of glutamine at 61st amino

acid of N-γas protein(p21) to arginine.

In conclusion, that N-γas is activated by a point mutaiton at codon 61 and the activation of thegene is not a consequence of a quantitative increase of N-γas Protein(p21) but a consequence of aqualitative ohange due to the amino acid substitution of glutamine to arginine at 61st position of N-γas protein.