담췌암 환자의 Lewis 항원과 CA19-9 상승에 관여하는 인자 규명

Abstract

[한글]

담도 및 췌장암 환자에 있어서 Cancer Antigen (CA) 19-9는 가장 흔하게 이용되는 종양 표지자로 sialyl Lewis a (sLea)의 탄수화물구조를 가지고 있어 Lewis system과 연관성을 갖고 있다. CA19-9의 생합성 과정에 있어서 Lewis 항원이 없는 경우에는 CA19-9이 형성되지 않고 전구 물질인 sialyl-Lec (DUPAN-2) 만을 생성하게 되며 β3Gal-T가 없는 경우에는 type I chain이 전혀 형성되지 않아 CA19-9 및 DUPAN-2 둘 다 형성 할 수 없게 된다. 그러나 생합성 과정에 관여하는 효소의 유전자 변이 유무, Lewis 항원의 유전자형과 표현형간의 차이에 따라 CA19-9치가 달라질 수 있어 이에 대한 보정이 필요하다. 그러므로 Lewis 항원과 관련하여 CA19-9치 상승에 관여하는 인자를 규명하고자 8개의 사람 췌장암 세포주를 대상하여 β3Gal-T 존재 유무를 확인, Lewis 항원의 유전자형과 표현형 및 sLea 의 표현형의 결정, 및 CA19-9과 DUPAN-2치를 측정하여 다음과 같은 결과를 얻었다. 모든 세포주에서 β3Gal-T가 존재하고 Lewis 유전자형 le/le인 세포주는 없어 모든 세포주에서 CA 19-9의 분비가 가능하나 표현형 검사상 Le(a+b+)인 Capan-2, CFPAC-1와 표현형 검사상 Le(a-b-)이지만 sialyl Lea의 존재를 확인된 BxPC-3에서 CA19-9의 증가를 보였다. 그렇지만 표현형 검사상 Le(a-b-)이고 sialyl Lea 의 존재가 확인되지 않은 세포주에서 CA19-9은 검출되지 않았으나 DUPAN-2의 치는 일정하지 않았다. 이상의 실험 결과를 종합하여 볼 때 CA 19-9치의 상승은 단순히 유전자의 존재보다는 유전자 발현의 증가가 CA19-9 수치의 증가에 상관관계가 있는 것으로 판단되고 조직 내에서 유전형보다는 표현형에 의해 좌우되는 것으로 보아 방해 항체(blocking antibody)의 존재의 가능성을 시사하며 표현형상 Lewis 항원이 검출되지 않는 경우 DUPAN-2치가 일정치 않아 또 다른 효소인 ST3Gal에 대한 연구가 필요할 것으로 사료된다.

[영문]

CA19-9 in serum is a well-known tumor marker, which is frequently used for the clinical diagnosis of pancreatic cancer. The oligosaccharide on the CA19-9 epitope is a sialylated Lewis A blood group antigen. At least three glycosyltransferase

are required for the synthesis of the sLea epitope. First,

N-acetylglucosamine-β1,3-galactosyltransferase (β3Gal-T) transfers a galactose (Gal) to acetylglucosamine(GlcNAc) with a β 1,3 -linkage, resulting in the synthesis of a type 1 chain, Galβ1,3GlcNAc. Second, galactose-α2,3-sialyltransferase (ST3Gal) transfers a sialic acid(SA) to the Gal residue of the type 1 chain with an α2,3-linkage, resulting in sialyl-type 1

(sialyl Lewis c; sLec). Third, α1, 3/4 -fucosyltransferase (Fuc-III, FUT3, Lewis enzyme) transfers a fucose (Fuc) to the GlcNAc residue of the sialyl-type a 1 chain with an α1,4-linkage to complete the synthesis of α2,3Galβ1,3 (Fucα1,4) GlcN. Without Lewis antigen, not CA19-9 but Sialyl-Lec (DUPAN-2) is produced in the pathway on the synthesis of CA19-9. If there is no β3Gal-T which makes type 1 chain, both CA19-9 and DUPAN-2 can not be made. Depending on the variations of glycosyltransferase gene and the differences between the phenotypes and genotypes of Lewis antigen, the values of CA19-9 could be different. However, the exact mechanism on this remains unknown. The 8 pancreatic cancer cell lines (BxPC-3, Capan-2, CFPAC-1, HPAC, Capan-1, AsPC-1, MIA PaCa-2, PANC-1) were investigated to identify the factors which would increase CA19-9 related to Lewis antigen. The β3Gal-T was detected by reverse transcriptase polymerase chain reaction (RT-PCR).

The phenotypes and genotypes of Lewis antigen were determined by flow cytometry analysis and restriction fragment length polymorphism (RFLP), respectively. The phenotypes of sLea were assessed by flow cytometry analysis and the sLea on supernatants was detected by sodium dodecyl sulfate - polyacrylamide gel

electrophoresis (SDS-PAGE). CA19-9 and DUPAN-2 on supernatants were measured by enzyme immunoassay. /From all cell lines, CA19-9 productions were possible since all of them had β3Gal-T and were no genotypical Lewis negative (le/le). The elevation of CA19-9 was noted on Capan-2 and CFPAC-1 which were phenotypically

Lewis positive (Le(a+b+)) as expected. Interestingly, it was also elevated in the BxPC-3 even though the line was known to be phenotypically Lewis negative (Le(a-b-)). Sialyl Lea appeared to play an important role in this phenomenon. Although CA 19-9 was not detected in the phenotypically negative pancreatic cell

line without sialyl Lea, the levels of DUPAN-2 were variable, not increased in these cell lines different from what we expected. It was revealed that an elevated CA19-9 was related with increased expression of Lewis gene, not merely the existence of the gene. It was conceivable that there might be a blocking antibody interfering the expression of Lewis gene. Further investigations on the role of ST3Gal are warranted to explain the mechanisms of the variable levels of DUPAN-2 in phenotypically Lewis negative cell lines.