KR100434080B1 - Human Sarcomatoid Cholangiocarcinoma and Clonorchiasis Sinenesis-associated Cholangiocarcinoma Cell Lines - Google Patents

Abstract

The present invention relates to a cell line of cholangiocarcinoma in the liver of the human body, and more particularly, to express malignant cholangiocarcinoma cell line (SCK) and epithelial cell antigen, which are dedifferentiated from the cholangiocarcinoma cells of the human liver and have sarcoma antigens and traits. It is related to liver dystomosis-related cholangiocarcinoma cell line (JCK) showing colony growth.

Cancer tissues were collected from four patients with hepatosarcoma cholangiocarcinoma, liver dystomosis-related cholangiocarcinoma and general cholangiocarcinoma, and placed in Opti-MEM (Gibco) medium and heated for 30 minutes at 2 mM glutamine and 0.5 mM sodium pyruvate at 56 ° C. 10% inactivated fetal calf serum is added, and penicillin, streptomycin, and metronidazole are incubated at 37 ° C. under carbonic acid gas of 5% -atmosphere 95% and 100% humidity. When the cancer cells are attached and cultured in a single layer at the bottom of the flask container and saturated, trypsin treatment removes the cancer cells and subcultures in another flask. The cells are then mixed with a freeze medium and frozen and stored in liquid nitrogen.

Human hepatosarcoma cholangiocarcinoma cell line (SCK) and hepatostoma related cholangiocarcinoma cell line (JCK) of the present invention may be usefully provided for the development of the cause and treatment of cholangiocarcinoma in the future.

Description

Human Sarcomatoid Cholangiocarcinoma and Clonorchiasis Sinenesis-associated Cholangiocarcinoma Cell Lines in Human Liver Sarcoma Cholangiocarcinoma Cell Line and Hepatostoma-associated Cholangiocarcinoma Cell Line

The present invention relates to a cell line of bile duct cell carcinoma in the human liver, and more particularly, to a malignant cholangiocarcinoma cell line (SCK) having antigens and traits that have been differentiated from the human bile duct carcinoma to cause sarcoma. The present invention relates to a liver dystomitis-related cholangiocarcinoma cell line expressing epithelial cell antigens and showing colony growth. It became.

In Korea, the incidence of cholangiocarcinoma is higher than in foreign countries due to liver dystomosis and oriental cholangitis, but it is difficult to detect early and clinically has a poor prognosis. However, more than 70% of surgical resections are impossible and resistant to conventional chemotherapy (Roberts SK et al., Gastroenterology, 1997; 112: 269-279). Therefore, it is necessary to identify the biological characteristics of cholangiocarcinoma in Korea for the early diagnosis of cholangiocarcinoma and the development of anti-cancer chemotherapeutic agents.

Although many hepatocellular carcinoma cell lines have been isolated and identified, only a few bile duct cell carcinoma cell lines have been reported (Homma S et al., Gastroenterology Japon, 1988, 114, 283-290). There are no bile duct cancer cell lines available for sale in the collection (ATCC). This may be due in part to the low incidence of bile ducts compared to hepatocellular carcinoma, and often containing dense fibrous tissue, making it difficult to separate sufficient live tumor cells from the mass, even if viable cells are isolated from the bile duct cancer and adhere to the culture flask. Even if contaminated fibroblasts fail to proliferate, they fail to passaging (Shimizu Y et al., Int J Cancer 1992; 52: 252-260). In addition, high-grade granulomatous cholangiocarcinoma with fast metastasis and poor prognosis has been reported rarely as about 5% of all bile duct cancers.

Therefore, the present invention not only isolates and identifies cell lines from patients with cholangiocarcinoma in Korea, but also secures a foundation for providing bile duct cancer cell lines by analyzing genes and biological characteristics thereof.

The present invention aims to provide isolation and identification and genetic and biological characteristics of de-differentiated cholangiocarcinoma cell line (SCK) and liver dystomoma-associated cholangiocarcinoma cell line (JCK) that caused sarcoma changes, thereby providing usefulness in the development of causes and treatments for cholangiocarcinoma. .

Figure 1 shows the findings showing a single-layered growth pattern in the flask on the inverted microscope during cell culture of the four cholangiocarcinoma cell lines isolated in Example 1 (1).

Figure 2 shows the histological staining and immunochemical staining performed on the tumor formed after inoculation to the shoulder skin of nude mice in order to see the tumorigenicity in the four types of cholangiocarcinoma cell line established in Example (1) of Example 1.

Figure 3 shows the analysis results of the p53 gene known as cancer suppression gene in the four types of cholangiocarcinoma established in (1) of Example 1.

Figure 4 shows the results of FHIT gene analysis known as a cancer suppressor gene in four types of cholangiocarcinoma cell line established in (1) of Example 1.

5 is a result of analyzing Fas receptor and Fas-L (pas ligand) expression in four types of cholangiocarcinoma cell lines established in Example (1).

6 shows the results of analyzing chromosomal karyotypes and mutations in four types of cholangiocarcinoma cell lines established in Example (1).

The present invention relates to a SCK cell line, an invasive and metastatic cholangiocarcinoma cell line expressing vimentin, a sarcoma antigen from bile duct cell carcinoma, and an epithelial cell antigen, a JCK cell line expressing epithelial cell antigen and showing colony growth. They were deposited with the Biotechnology Institute on January 2, 2001 with accession numbers (SCK: KCTC 0926BP, JCK: KCTC 0927BP).

The cholangiocarcinoma of the present invention is not contaminated with other cell lines and is a genetically unique cell line that can be used as a valuable material in future diagnostic treatment and prognosis of cholangiocarcinoma.

The present invention will be described in detail by the following examples. However, these examples do not limit the technical scope of the present invention.

<Example 1>:

[Isolation and Identification of Human Bile Duct Cancer Cell Line]

Cancer tissues from four patients with hepatic bile duct cancer were collected, cut, and put into Opti-MEM (Gibco) medium, and heated to 56 ° C for 30 minutes with 2 mM glutamine and 0.5 mM sodium pyruvate. 10% was added and incubated at 37 ° C. under carbonic acid 5% -95% and 100% humidity, containing penicillin (100 U / ml), streptomycin (100 μg / ml), metronidazole (0,008%). When the fibroblasts were observed during the culture, the fibroblasts were removed using a cell removal rod or, if necessary, using a G418 antibiotic (100 µg / ml).

The cancer cells were attached and incubated in a single layer at the bottom of the flask vessel, and when saturated, the cells were eliminated by trypsin treatment and the cancer cells were subcultured in two new flasks (1: 2 ratio) in the flask. At this time, the medium and culture conditions used for subculture were the same as the above medium and culture conditions. Some of the cells in each subculture step of subculture were mixed in a freeze medium and frozen in liquid nitrogen and stored.

As described above, the four types of intrahepatic bile duct cancer cell lines shown in FIG. 1 were purified and named SCK, JCK, Cho-CK, and Choi-CK, respectively.

In the following Figure 1, the four types of cholangiocarcinoma cell lines growing by attaching to the bottom of the flask as a single layer, A (upper left), are SCK fusiform and have low intercellular adhesion ability, and the double rate of division is about 12 hours, and B ( The upper right picture is JCK, with moderate intercellular adhesion and oval morphology. C (bottom left photo) and D (bottom right photo) are common cholangiocarcinoma cell lines Cho-CK and Choi-CK cell lines, respectively, with strong intercellular adhesions, growing in colonies, and doubling rate dividing by 36 hours.

Table 1 shows the characteristics of four types of cholangiocarcinoma-derived patients. In particular, patients with JCK cell lines are positive for liver dystomosis in feces, which is considered to be valuable as a study material for hepatobiliary-associated cholangiocarcinoma.

Table 1. Characteristics of patients derived from human cholangiocarcinoma cell line

Cell line Passage number Age / gender blood type Cancer Gandhistom cure Histopathology SCK 20 57 / M A IV Operation Differentiated Epithelial Cell Cancer JCK 20 59 / M O IV Stool Palliative care Differentiated Epithelial Cell Cancer Cho-CK 20 72 / M A IV Palliative surgery Differentiated Epithelial Cell Cancer Choi-CK 20 57 / M B IV Palliative surgery Low Differentiation Epithelial Cell Cancer

[Bile Duct Cancer Tumor Formation and Immunohistochemistry]

To measure the tumorigenic ability of bile duct cancer cell lines, the number of cancer cells was adjusted to 1 × 10 ⁶ cells / ml on the shoulders of 3-4 week-old nude mice (BALB / c, nu / nu, Kist) and inoculated with about 1 ml The breeding was observed aseptically. After about 4 weeks, the paraffin-embedded tissue block was excised when the diameter of the tumor was 1.5 cm or more, and then proceeded to hematoxylin-eosin (HE) staining to determine histologic features and to determine the specificity of the polyclonal antigen derived from bile duct cells. Immunohistochemical staining was performed. The results are summarized in Table 2. Tumors were formed in all cell lines, including SCK cell lines, and pathological examinations were performed on tumor masses. (b), Figure 2 (c), Figure 2 (d) is a photograph of the hematoxylin-eosin staining in each cell line ((a) is SCK, (b) is JCK, (d) is Cho-CK, (d) is Choi-CK, FIG. 2 (e), FIG. 2 (f), FIG. 2 (g), and FIG. 2 (h) are photographs subjected to special immunostaining ((e) are SCK-Vimentin, (f) is JCK-CK7, (g) is Cho-CK-CK19 and (h) is Choi-CK-CK7).

Tissue was chopped for subculture and cultured in the Opti-MEM medium described above. Epithelial cell antigen was negative for cytokeratin in SCK cell line, a sarcoma cholangiocarcinoma cell, and strongly positive for sarcoma cell antigen nonmentin.

Table 2. Pathological characteristics and immunochemical staining of liver cancer graft

Special Antibodies CK7 CK19 CK20 CEA Vimentin HE dyeing SCK - - - +/- ++ Sarcoma spindle cell carcinoma JCK ++ + - ++ - Highly differentiated epithelial cell carcinoma tissue Cho-CK + + - + (Local) - Differentiated epithelial cell carcinoma tissue Choi-CK +/- - - +/- - Low differentiated epithelial cell carcinoma tissue

Negative:-, positive: +, strongly positive: ++, weak positive or trace: +/-

[Variation of Cancer Suppressor Gene p53 in Mononuclear Cancer Cell Lines]

p53 gene mutations are the most common genetic mutations found in human cancers and found in about 60% of carcinomas (Hollistein M et al., Science 1991; 253: 49-53; Levin AJ et al., Nature 1991; 351: 453-456). Therefore, p53 mutations are predicted in bile duct cancer and are of great value in understanding the pathophysiology of cancer cells in the future. PCR single strand conformation polymorphysm (PCR SSCP) method (Wen WH et al., Int J Gynecol Pathol 1999; 1829-41) was used to detect p53 gene mutations. The p53 mutation site was determined by direct sequencing after PCR SSCP. PCR was performed using exon 3 to 10 sites as shown in Table 3 using the respective starter cells. PCR was performed at 0.12 μg DNA, 100 μM each dATP, dCTP, dGTP, and dTTP, 1.5 mM MgCl 2, 1.5 unit tack polymerase (Taq polymerase, Perkin-Elmer), 5 μCi [α-32p] dATP (NEN), 300 nM starter was used. DNA was denatured at 94 ° C. for 5 minutes and then subjected to PCR reaction cycle. In other words, 50 seconds denaturation at 94 ° C, contact at 55-62 ° C, expansion at 40 ° C for 72 seconds, circulating 30 times, and final expansion was performed for 5 minutes at 72 ° C. PCR products were developed by electrophoresis on 0.5% mutation detection promoting gel. To the X-ray film. The gel corresponding to the abnormal band was excised to elute DNA, re-amplified using a homologous starter using PCR, and the nucleotide sequence was determined using an ABI kit (Perkin Elmer).

Table 3. P53 Mutant Seeker Starter Sequences

Primer Sequence (5 '→ 3') Size of PCRproduct (bp) AnnealingTemperature (℃) Exon 3 3-F3-R TAG CAG AGA CCT GTG GGA AGCAGA GCA GTC AGA GGA CCA GGT 159 62 Exon 4 4A-F4A-R4B-F4B-R CGT TCT GGT AAG GAC AAG GGTCTG GGA AGG GAC AGA AGA TGAGTC GCC CCT GCA CCA GCA GCTAAG AAA TGC AGG GGG ATA CGG 304226 5862 Exon 5 5-F5-R CTG TTC ACT TGT GCC CTG ACAAC CAG CCC TGT CGT CTC TC 271 58 Exon 6 6-F6-R GCT GGA GAG ACG ACA GGG CTCAA CCA CCC TTA ACC CCT CC 228 62 Exon 7 7-F7-R CTT GCC ACA GGT CTC CCC AAAGG GGT CAG CGG CAA GCA GA 237 62 Exon 8 8-F8-R TTC CTT ACT GCC TCT TGC TTAGG CAT AAC TGC ACC CTT GG 231 55 Exon 9 9-F9-R AGC AAG CAG GAC AAG AAG CGGCA AAT GCC CCA ATT GCA GG 264 58 Exon 10 10-F10-R CGA TGT TGC TTT TGA TCC GTC AATC CTA TGG CTT TCC AAC CTA G 257 58

Mutations of p53 exon 3, 4, 5, 6, 7, 8, 9, and 10 in four cholangiocarcinoma cell lines showed that the codon 196 CGA (arginine) amino acid sequence in exon 6 was changed to the stop codon of TGA in JCK cell line. . Depletion of 292 AAA (lysine) amino acids was observed in exon 8 in the Cho-CK cell line, and a variation related to polymorphism in which the amino acid arginine (Arg) changed to proline (Pro) in exon 4 was found in the exon 4 cell line. No mutation was found in the SCK cell line. FIG. 3 is a PCR-SSCP gel X-ray photograph of JCK, Cho-CK and Choi-CK, showing a sequencing phenogram.

[Exploration of Cancer Suppressor Gene FHIT Mutation]

Fragile histidine triad (FHIT) gene has recently been known as a cancer suppressor gene in esophageal cancer, gastric cancer, lung cancer, head and neck cancer, and bowel cancer. -2115), but FHIT mutations in the cholangiocarcinoma cell line are not yet known. Total RNA was isolated from each cell line using a Tri reagent (Molecular Research Center), and a single-stranded cDNA was prepared using an oligo-d (T) 15 initiator. In order to detect FHIT gene mutations, 5U2 forward primers (5'-TCACTGGTTGAATACAGGA-3 ') and 3D2 reverse primers (5') were first identified as a single-initiator by nested PCR test. -TCACTGGTTGAATACAGGA-3 '), 100 mM dNTP, 1 X PCR polymerase mix, 1 unit Advantage Taq polymerase (Advantage Taq polymerase, Clontech) PCR reactor (Perkin-Elmer model 9700) ) PCR reaction was performed 25 times, and the PCR product was diluted to 20: 1, and 1 μl of the PCR product was again used as an inner primer (forward primer) 5U1 (5'TCCGTAGTGCTATCTACATC-3 '). PCR was performed using 3D1 reverse primer (5'CATGCTGATTCAGTTCCTCTTGG-3 ') and another pair of endogenous circulatory initiator MUR5 (5'-CTGTAAAGGTCCGTAGTGC-3') and retrograde initiator RP2 (( 5'ACAGGATGGTGAGATGAAGAAACTGC-3 ') (Thiagalingam S et al., Cancer Res 1996; 56: 2936-2939) The first starter pair should show the PCR product band corresponding to 707 bases, and the second starter pair should be the 747 base-sized band of the exon transcript of the normal gene. The first lane is the molecular weight size marker, the first and second lanes are normal standard cells, the fifth lane is the mutant cell line, the fourth sample is the SCK cell line, and the normal and mutant genes are homologous to the homologous chromosome. And Cho-CK were normal genes and Choi-CK showed mutated genes The results by the second initiator were consistent with each other as shown at the bottom of FIG.

[Fas and Fas-L expression]

Fas receptors expressed on cell membranes and Fas ligands (Fas-L) responding to these cells act to induce apoptosis in the immune surveillance system of cancer. The association of Fas receptors with anticancer drug-induced apoptosis has also been shown. Therefore, the review of Fas and Fas-L expression in bile duct cancer cell lines will be used as valuable data for future biological characteristics and chemotherapy strategies. As described above, cDNA was prepared by extracting total RNA from each cell line. -AACGTATCTGAGCTCTCTCTG-3) and 5-CAAGTCCAACTCAAGGTCCAT-3 retrograde initiators were used, and beta-actin was used as an internal control experiment. Beta-actin initiators used the circulating initiator CGTTCTGGCGGCACCACCAT-3 'and the retrograde initiator 5'-GCAACTAAGTCATAGTCCGC-3'. In each reaction cycle, PCR products were reacted 30 times by extending them for 1 minute at 57 ° C for 1 minute and at 72 ° C for 1 minute. The results are shown in FIG. 5 and the Fas receptor is expressed in all four cholangiocarcinoma cells, particularly in Cho-CK and Choi-CK. FasL expression was expressed to about the same level in three cell lines, but attenuated expression was observed in Cho-CK cell line.

Karyotype and Chromosome Analysis in Each Cell Line

Cells were grown 75-80% and harvested at a time when cell division was vigorous, and the cultures were exchanged the day before. Cells were separated from the flask by treatment with 0.1 μg / ml of colcemid and 2-3 minutes with 0.025% trypsin-EDTA 30 minutes-2 hours before harvest by the usual cytogenetic methods. The culture medium containing the cells was transferred to a test tube, centrifuged at 1,000 rpm for 5 minutes, the suspended solids were discarded, and the stock solution (0.075M KCl) was added and placed at 37 ° C. for 20 minutes. The slides were prepared after three exchanges with fixed solution (Methanol: acetic acid = 3: 1). Some of the slides were G-banded to analyze the chromosome, and slides for FISH were dehydrated with alcohol and stored at -20 ° C until use. The slides for G-banding were aged at 56 ° C for at least 12 hours, washed with PBS for 20-40 seconds with 0.05% trypsin, dyed for 10 minutes with 4% Giemsa, and then 1000. Observation was carried out under the magnification of the photographs and karyotype analysis. The results are summarized in Table 4 and the photographs are shown in FIG. 6 and in SCK cell lines there are 7, 2q31-q32, 5q11-q13, 8p, 9p, 12p and 13q11-q21 acquisitions, Y, 16, 18, 3pter There was a loss of -p14, 621-ter, 9p, 113q33-qter, and 18q21-qter chromosomes. In the JCK cell line, 9, 2p, 3q21-qter, 7q11-q22, 8q22-qter, 14q11-q22, 17q11-q21 and Yq chromosomal sites were obtained and 5,10, 15, 16, 18, 1p, 1q31-qter , 3p, 4q25-qter, 6p21-pter, 6p21-pter, 6q23-qter, 7p, 7q31-qer, 8p, 11q, 12p-q21, 14q23-qter, and 17p chromosome sites. Cho-CK cell lines have acquisitions of 4, 5, 9, 12, 16, 21, 1q, 7q11-q22, 8q, 12p, 14q11-q22, 15q21-qter, 17p11-qter, and 18p-q12 chromosomal sites and 22 , 6q23-qter, 7q31-qter, 8p, 14q23-qter, 15q10-q15, 17p12-pter and 18q21-qter chromosome sites were seen. In the Choi-CK cell line, there were acquisition of 7 and Xp chromosome sites and loss of 18, 3p13-p23, 5q11-q22 and 19p chromosome sites.

Table 4. Comparison of karyotypes in four types of cholangiocarcinoma cell lines

Cell line Combined G-banding and Rx-FISH karyotype SCK 52-60 <3n>, X, -X, -Y, dup (2) (q31q33) ins (2) (q21q31q33), del (3) (p14), del (5) (q13) x3, der (6 t (6; 13) (p21; q11) hsr (6) (p21) hsr (6) (q21), + 7, + 8, der (8; 14) (q10; q10) x3, i (12) (p10) x2, der (13) t (5; 13) (q11; q21), der (13) t (5; 13) (q13; q32) x2, -16, -18, der (18) t ( 12; 18) (q13; q21),-20, dup (20) (q11q13) t (20; 20) (q13; p11),-21 [cp44] / 98-110 <5n>, XXX, -Y, -Y, dup (2) (q31q33) ins (2) (q21q31q33), + i (2) (p10), del (3) (p10),-4, del (5) (q13) x6, + 6, der (6) t (6; 13) (p21; q11) hsr (6) (p21) hsr (6) (q21) x2, + 7, + 7, + 7, + 8, der (8; 14) ( q10; q10) x4, del (9) (p11), ins (9; 5) (q34; q13q31) dup (9) (q13q22) x2, i (9q),-10, -11, i (12) ( p10) x2, der (12; 14) (q10; q10),-13, der (13) t (5; 13) (q11; q21) x2, der (13) t (5; 13) (q13; q32 x2, ins (16; 15) (q22; q11q26),-18, der (18) t (12; 18) (q13; q21) x2, dup (20) (q11q13) t (20; 20) (q13 ; q11) x2 [cp20] JCK 54-60 <3n>, XXY, + Y, der (1; 14) (q10; q10) t (1; 14) (q25; q11) trp (14) (q11q21), der (2; 8) (p10 ; q10) dup (8) (q13q24), + ins (2) (q32; p11p15), + 3, der (3; 7) (q10; q10) del (3) (q12q21), der (3; 16) (q10; p10), inv (4) (p15q12) del (4) (q25) del (4) (q25),-5, der (6) t (6; 16) (p21; q12) hsr (6) (p21), del (6) (q23), der (7) t (7; 12) (q22; q11) trp (7) (q21q22), + der (9) t (9; Y) (p24; q12 ins (9; 7) (p22; q11),-10, -12, der (11; 12) (p10; p10), der (14) dup (14) (q13q22) ins (14; 7) (q22 ; q11q22) del (14) (q31), der (14) dup (14) (q13q22) ins (14; 7) (q22; q11q22) trp (14) (q11q21),-15, der (15) t ( 8; 15) (p11; q26),-16, der (17; 21) (q10; q10) x2, der (17) t (12; 17) (q22; q11), + dup (17) (q11q21) , -18, der (20) t (16; 20) (p13; p11), der (20) t (8; 20) (q22; p13) [cp51] Cho-CK 73-85 <3>, XXY, der (1; 17) (p10; q10) trp (7) (q11q22) t (q11q22) t (7; 15) (q22; q21) ins (7; 14) (q22 ; q11q22), + i (1) (q10), + 4, + 5, del (6) (q23), + der (7) trp (7) (q11q22) t (7; 15) (q22; q21) ins (7; 14) (q22; q11q22), + 8, der (8; 13) (q10; q10), i (8) (q10), + 9, + 12, + der (12; 18) (p10 p10) x2, der (13) t (13; 13) (p11; q22),-14, -15, + 16, + 17, del (17) (p11) x2, + del (18) (q12) x2,21,1-2dmin [cp49] Choi-CK 44-46, XY, del (3) (p13p21) x2, del (5) (q11q22), + 7, -18, der (19; 22) (q10; q10), der (20) t (X; 20 ) (p11; q13) [cp45] / 86-91, idemx2 [cp43]

The malignant cell lines SCK and liver dystomosis-associated cholangiocarcinoma cell line JCK, which are dedifferentiated from human cholangiocarcinoma of the present invention, may be advantageously used in the development of carcinogens and therapeutic agents in the future.

Claims (3)

Human cholangiocarcinoma cell line SCK characterized by dedifferentiation from human hepatic bile duct cancer and having sarcoma-changed antigens and traits. JCK (Global Engineering Research Institute Genetic Bank, Jan. 2, 2001, Accession No. KCTC 0927BP) delete