Masafumi Maruiwa

A NEW HUMAN PLEOMORPHIC HEPATOCELLULAR CARCINOMA CELL LINE, KYN‐2

A new human hepatocellular carcinoma (HCC) cell line, KYN‐2, has been established from a surgical specimen obtained from a 52‐year‐old Japanese male HCC patient. The originally resected HCC was classified as pleomorphic HCC corresponding to Edmondson‐Steiners grade III with a thick trabecular to solid arrangement. The cell line has been maintained for 17 months through 35 passages. Morphologically, the KYN‐2 cells have retained the characteristics of the original HCC, being pleomorphic and composed of various types such as cells with relatively small, polygonal, eosinophilic cytoplasm and oval‐shaped nuclei with a marked tendency to pile up, flat cells with abundant clear cytoplasm and oval‐shaped nuclei, and many multinucleated giant cells, proliferating in a pavement‐like cell arrangement. Some junctional complexes and a number of microvilli are evident between the cells by electron microscopy. Functionally, these cells were found to secrete albumin, α,‐acid glycoprotein, α1‐antitrypsin, ceruloplasmin, transferrin, complement C, fibrinogen, fibronectin, prothrombin, retinol‐binding protein (serum type), α‐fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin and β2‐microglobulin in chemically defined medium (CDM). The secretion of AFP and CEA is apparently dependent upon culture medium and passage. The doubling time of cells growing in serum‐containing medium at the 14 th passage was 84 h, and those of cells in serum‐containing medium, HB101 (serum‐free medium) and CDM at late passage were 28,68, and 42 h, respectively. Chromosome analysis revealed that the chromosome number ranged from 56 to 69 without a mode, and the presence of marker chromosomes. HB virus DNA sequence was not detected by hybridization analysis. The tumorigenicity of KYN‐2 cells was identified by development of tumors in nude mice after subcutaneous injection of the cells; the tumors showed an appearance basically similar to that of the original HCC. Thus, these findings suggest that the KYN‐2 cell line is available as a new human HCC cell line and should be useful for various studies on HCC. ACTA PATHOL JPN 38: 953‐966, 1988.

Establishment and characterization of a new human extrahepatic bile duct carcinoma cell line (KMBC)

A new human extrahepatic bile duct carcinoma cell line (KMBC) was established from a serially transplanted tumor in nude mice that originated from a surgically resected tumor from a 73‐year‐old Japanese man; the cell line has been maintained for 5 five years. KMBC cells proliferate in a monolayered sheet with a population doubling time of 30 hours. Chromosome number was distributed in a range from 37 to 44, with modal numbers of 40 and 41. KMBC cells and the reconstituted tumor in a nude mouse showed moderately to poorly differentiated adenocarcinoma and possessed various functional characteristics of extrahepatic bile duct carcinoma. KMBC cells secreted carbohydrate antigen 19‐9, tissue polypeptide antigen, carcinoembryonic antigen, ferritin, β2‐microglobulin, fibronectin, and α2‐macroglobulin and produced glutamic oxaloacetic transaminase and alkaline phosphatase. KMBC is the second established cell line that originated from a human extrahepatic bile duct carcinoma in the world literature, and it will be applicable to various experiments.

A new human cholangiocellular carcinoma cell line (KMC-1)

We have recently established a cholangiocellular carcinoma (CCC) cell line, designated KMC-1, from a nude mouse subcutaneous tumor which developed after inoculation of a surgically resected peripheral type CCC from a 62-year-old Japanese male patient. KMC-1 cells grew over a 26-month period and passaged 57 times. These cells retained the morphologic characteristics of both the original tumor and the subcutaneous tumor in the nude mouse, which mainly consisted of irregular tubules and invaded surrounding interstitial tissue in part with an indurate pattern. KMC-1 cells grew in a monolayer pavement-like cell arrangement with tubular formation in part. Some cells and/or glands had a mucin-like substance inside. The doubling time of KMC-1 cells growing in serum-containing medium was 54 h at passage 31. Cell growth in serum-free medium was slow but steady. The number of chromosomes was distributed in range from 73 to 83 with modes of 76 and 78. KMC-1 cells secreted some tumor markers such as DUPAN-2, CA125, TPA, hCG, CA19-9 and ferritin, however, the secretion of DUPAN-2, and CA19-9 and ferritin were only detectable in serum-containing and serum-free medium, respectively. These findings suggest that KMC-1 cells will provide a variety of experimental models for research on CCC and the mechanisms of tumor marker secretion.

Establishment and characterization of a new human renal cell carcinoma cell line (KRC/Y)

SummaryA new renal cell carcinoma (RCC) cell line (KRC/Y) has been established from a surgical specimen of a 41-yr-old Japanese female patient with RCC composed of both clear cells and granular cells. This cell line has been maintained for more than 15 mo. through 45 passages with a stable growth, KRC/Y cells have clear or eosinophilic polygonal cytoplasm and round to oval nuclei with one or two nucleoli, and proliferate in a pavementlike cell arrangement with a lack of contanct inhibition. By electron microscopy, these cells contain abundant fat droplets and glycogen granules or well-developed organells or both, which were also observed in the original tumor. The doubling time of these cells at the 15th passage was 73 h. The chromosome number was from 37 to 45 with a hypodiploid modal number of 42. Tumorigenicity was identified by tumor formation after subcutaneous injections of KRC/Y cells in nude mice, which showed close resemblance to the original tumor by light and electron microscope observations.

HETEROTRANSPLANTATION OF AN ALPHA‐FETOPROTEIN‐PRODUCING HUMAN GALLBLADDER CARCINOMA INTO NUDE MICE

An alpha‐fetoprotein (AFP)‐producing human gallbladder carcinoma showing direct invasion into the liver was transplanted into BALB/c‐nu/nu nude mice. Although patient serum levels of AFP and carcinoembryonic antigen (CEA) were within normal limits, they were elevated to 1,040 ng/ml and 22.1 ng/ml, respectively, after cholecystectomy. Prominent liver metastasis was demonstrated by diagnostic imaging techniques shortly after the operation. Pathologically, the resected tumor consisted of papillotubular adenocarcinoma and the part which had Invaded the liver showed a solid growth pattern with no papillo‐tubular structure. The transplanted tumor showed both papillo‐tubular and solid growth patterns, in which positive reactions for AFP, CEA, ferritin (FER), carbohydrate antigen 19‐9 (CA 19‐9), albumin (ALB) and fibrinogen (FIB) were confirmed by the avidin‐biotin‐peroxidase complex method. Serum levels of AFP, CEA, CA 19‐9, β2‐microglobulin (BMG) and FER were elevated in the nude mice bearing tumor transplants. Twenty‐five percent of the serum AFP from nude mice with tumor transplants bound with concanavalin A (Con A), suggesting that the tumor was of gastrointestinal rather than hepatic origin.

Expression of CD44 Molecule in Gastric Cancer

The expression of the CD44 molecule was investigated in 111 cases of a resected gastric cancer, and also in 5 gastric cancer cell lines. An immunohistochemical study using frozen sections revealed that the mucosal lymphocytes and the pyloric glands deep in the normal mucosa reacted to the anti-CD44 monoclonal antibody. 54 cases (48.6%) were stained on the cell surface involving the intercellular aspect. Well-differentiated cancer showed the highest frequency of CD44 expression, among the various histological types. Moreover, cases with extensive vascular invasion frequently expressed CD44 (75%) and CD44-positive cases had a significantly higher rate of metastasis to the liver than did negative cases. Flowcytometric analysis revealed expression of CD44 on the cell surface in one of 5 examined cell lines.