Establishment and Characterization of a Woodchuck Hepatocellular Carcinoma Cell Line (WH44KA)

A continuous cell line was established from a hepatocellular carcinoma obtained from a woodchuck that was sero-positive for woodchuck hepatitis virus (WHV). The cell line, designated WH44KA, grows as an adhering monolayer with a doubling time of 36 hr in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells not only showed epithelial origin on light and electron microscopic examination but also possess biosynthetic markers of the latter, such as albumin and α-fetoprotein, which were demonstrated in cultured cells. When they were transplanted into athymic nude mice, tumors developed at the site of inoculation. These tumors were shown to he hepatocellular carcinoma, similar in morphology to the original tumor from which the WH44KA cells were derived. Chromosome analysis revealed a chromosome number ranging from 31 to 126, with a modal number of 35. Integration of WHV DNA was shown by Southern blot analysis. However, WHV surface antigen was not demonstrated in the cultured cells or supernatant medium. The WH44KA cell line appears to be a useful in vitro model for the study of virus-induced hepatocellular carcinoma.     

Establishment and characterization of a woodchuck hepatocellular carcinoma cell line (WH44KA)

A continuous cell line was established from a hepatocellular carcinoma obtained from a woodchuck that was sero-positive for woodchuck hepatitis virus (WHV). The cell line, designated WH44KA, grows as an adhering monolayer with a doubling time of 36 hr in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells not only showed epithelial origin on light and electron microscopic examination but also possess biosynthetic markers of the latter, such as albumin and alpha-fetoprotein, which were demonstrated in cultured cells. When they were transplanted into athymic nude mice, tumors developed at the site of inoculation. These tumors were shown to be hepatocellular carcinoma, similar in morphology to the original tumor from which the WH44KA cells were derived. Chromosome analysis revealed a chromosome number ranging from 31 to 126, with a modal number of 35. Integration of WHV DNA was shown by Southern blot analysis. However, WHV surface antigen was not demonstrated in the cultured cells or supernatant medium. The WH44KA cell line appears to be a useful in vitro model for the study of virus-induced hepatocellular carcinoma.     

Establishment and characterization of a chicken hepatocellular carcinoma cell line, LMH

A hepatocellular carcinoma cell line, LMH, has been established from a hepatocellular carcinoma induced in a male leghorn chicken by diethylnitrosamine. The cell line is characterized by well-differentiated morphological and biochemical features including the expression of glucose-6-phosphatase and canalicular ATPase activities and triploid karyotype with six marker chromosomes. The cells have been continuously propagated in culture for 5 yr and are now at about the 120th passage. Morphological change occurred in culture associated with gradual increase in growth rate at about the 40th passage. However, the biochemical and chromosomal features remained constant. This is the first established domestic fowl epithelial cell line and will allow comparative investigation of a number of parameters relevant to chicken hepatocarcinogenesis.     

Establishment of a cell line (NPC/HK1) from a differentiated squamous carcinoma of the nasopharynx

A long-term cell culture epithelioid cell line was established from a recurrent squamous carcinoma of the nasopharynx of a Chinese male 17 1/2 years after radiation therapy. The cell line, designated NPC/HK1, has been passed 72 times over a period 1 year. The cells have been shown by light and electron microscopies to be of the squamous epithelial type. When they were transplanted subcutaneously into the back of athymic nude BALB/c (nu/nu) mice, tumors developed at the sites of inoculation, which on histological examination were shown to be well-differentiated squamous carcinomas, similar in morphology to the recurrent human tumor from which they were derived. Karyotypic analysis of cells from the cell line demonstrates an aneuploid human type with a modal chromosome number of 74 with both numerical and structural aberrations. Viral particles or Epstein-Barr viral nuclear antigen (EBNA) has not been demonstrated in the cells from the primary culture or several of the subcultures tested. The presence of EBNA in touch smears prepared from the biopsy tissue was inconclusive. Infection of the subcultured cells with EBV from P3HR1 and B95-8 cells was unsuccessful.     

Experimental nude mouse model of human colorectal cancer liver metastases

A nude mouse model (BALB/c) was established for investigating the production of hepatic metastases by human colorectal carcinoma (HCC) cells. The malignant potentials of 3 different HCCs derived from a primary tumor (HCC-P4733), a lymph node metastasis (HCC-M14328), and a hepatic metastasis (HCC-M1410) were investigated following implantation into the spleens of athymic nude mice. Cells of the HCC-M1410 line produced extensive liver tumors in all mice by 30 days after injection. Cells of the HCC-M14328 and HCC-P4733 lines produced few liver tumors in the inoculated mice and then only after a 90-day period. Isozyme and karyotype analyses ascertained the human origin of all the tumors. Studies with [125I]IdUrd-labeled HT-29 carcinoma cells suggested that tumor cells reached the liver shortly after injection into the spleen. Thus the production of HCC tumors in livers of nude mice was determined by the ability of HCC cells to proliferate in the liver parenchyma rather than by the ability of the cells to reach the liver. The results suggest that the intrasplenic injection of HCC cells can provide a valuable model for the study of the biology and therapy of the liver metastases.     

Establishment and characterization of primary human pancreatic carcinoma in continuous cell culture and in nude mice.

Primary human panceratic exocrine adenocarcinoma has been established in tissue culture and as xenografts in immune-deficient nu/nu mice. The cell line has a doubling time of 36 h and grows as a confluent monolayer together with a constant population of free-floating cells. Evidence of tumourigenicity was provided by growth on an early diploid fibroblast monolayer and in soft agar, and as solid tumours in immune-deficient nu/mu mice. Chromosome analysis of the cultured cells confirmed their tumour origin. Xenografts established from the cell line or directly from primary tumour tissue have retained a similar histology to the original tumour on serial transplantation. An electrophoretic study of exportable pancreatic digestive enzymes and a number of intracellular enzymes has shown that the cell line and xenografts maintain a human intracellular enzyme profile, but do not produce pancreatic digestive enzymes.     

Detection of tumor antigens by syngeneic antiserum on in vitro-transformed tracheal epithelial cell line 2-10-1

Sera obtained from rats showing transplantation immunity to syngeneic, malignant epithelial cell line, 2-10-1, were used to identify antigens associated with transformation in vitro. The 2-10-1 cell line was derived from exposure of tracheal explants, in vitro, to the carcinogen MNNG. Tumorigenic passages of the cell line were shown to have common antigenic determinants, shared by independently transformed malignant tracheal epithelial cell lines, as well as antigenic determinants that appear to be limited to the immunizing cell line, 2-10-1. Care was taken to ensure that antibodies were not produced against viral antigens or non-specifically absorbed serum components. Early passages of the 2-10-1 cell line were not tumorigenic in athymic BALB/c (nu/nu) mice, but became malignant with serial passage in vitro. Antigenic specificities recognized by 2-10-1 immune sera were also present on early-passage 2-10-1 cells that had not as yet acquired the malignant phenotype. Such antigen expression must be an early event in neoplastic development.     

Development and characterization of a rat histiocyte-macrophage tumor line

A transplantable macrophage-like cell line has been obtained and established in W rats. This cell line is in its 85th passage and is perhaps the only established macrophage-like cell line that grows rapidly intraperitoneally in rats. The cells possess some of the typical characteristics of macrophages, such as adherence to glass, phagocytosis, presence of Fc receptors and C3d receptors, la determinants, leukocyte common antigen, lysozyme, non-specific esterase, and glycogen. The tumor also grows as solid when injected sc, intradermally, or intramuscularly. The cells have collagenase, tyrosine-specific protein kinase, and several other hydrolases. Histopathologic and ultrastructural observations suggest it to be a histiocytic tumor. The availability of a macrophage cell line of rat origin provides a useful experimental model to study different macrophage functions at the cellular and molecular level.     

Cytogenetic study on a human colorectal carcinoma cell line (HR-8348)

Cytogenetics of a human colorectal carcinoma cell line (HR-8348) established in China were described. The early (29th passage) and late passages (93th passage) of this cell line were used for chromosome analysis. HR-8348 was a cell line essentially with near triploid karyotype. The distribution of chromosome number was rather dispersed in the early passage, whereas it was concentrated to 65-70 range in the late passage. G-banding stain showed that the numerical distribution of different chromosomes was also dispersed in the early passage associated with abundant abnormal chromosomes. In the late passage, it turned to be more stable and the number of abnormal chromosomes reduced. In 60 metaphases analysed, 10 marker chromosomes were found. The frequency of M1, M3 and M2 was 100% in both early and late passages. The morphologic characteristics and their possible origin and role in the pathogenesis of colorectal carcinoma were discussed.     

Human pancreatic carcinoma (mia paca-2) in continuous culture: Sensitivity to asparaginase

An undifferentiated human pancreatic carcinoma has been established in continuous culture and is grown in Dulbecco's modified Eagle's medium fortified with 10% fetal calf serum and 2.5% horse serum. The established cell line (MIA PaCa-2) has a doulbing time of 40 h. The cells are large with abundant cytoplasm, exhibit a high degree of aneuploidy and have a tendency to grow on top of other cells. MIA PaCa-2 grows in soft agar with a colony-forming efficiency of 19%. Both MIA PaCa-2 cells and a cell line from another pancreatic carcinoma obtained from National Cancer Institute (NCI) are sensitive to asparaginase, a property not shared by several other human tumor cell lines tested.     

Characterization of cell lines derived from a multiply aneuploid human bladder transitional-cell carcinoma, UCRU-BL-13

A series of cultured cell lines (designated UCRU-BL-13) has been established from different serial passages of a multiply aneuploid human bladder transitional-cell carcinoma xenografted in nude mice. Serial passage of the xenografts in vivo and of the cell lines in vitro was accompanied by shifts in the tumor ploidy, with dominance of different major peaks. Despite this, the expression of tumor markers remained constant, and consistent chromosomal markers were observed both in the 8th xenograft passage and in a subline in tissue culture established over a year apart. Chromosomal numbers reflected the predominant aneuploid peaks observed; consistent numerical and structural changes included a marker derived from chromosome 1, 8p-, -10, 11q+, and 17q+. The cell line derived from the initial xenograft comprised a mixture of transitional, adenocarcinoma and squamous carcinoma cells in early passage, but adenocarcinoma cells were absent from later passages. The lines expressed the B-blood-group antigen, histocompatibility antigens, receptors for transferrin and EGF, and reacted with a series of monoclonal antibodies (MAbs) directed to malignant human epithelial cell lines. These lines provide a model for studying the evolution of tumor heterogeneity and drug resistance in bladder carcinoma exhibiting multiple aneuploidy.     

Establishment of a novel cell line derived from nasopharyngeal carcinoma

A novel epithelial cell line (designated as HNE-1), derived from nasopharyngeal carcinoma (NPC), was established and has passed more than 100 generations over one year. The NPC biopsy specimen was taken from a 27 year old man with poorly differentiated squamous cell carcinoma of the nasopharynx. The cultured cells showed polygonal shape and grew into multilayers under the inverted microscope. Electron microscopy showed that HNE-1 cells were characterized by bi-directional differentiation with some being poorly differentiated squamous carcinoma and the other poorly differentiated adenocarcinoma cells. A continuous positivity was showed by EBNA at subcultures 5-81. Tumorigenicity was demonstrated by heterotransplantation in BALB/c (nu/nu) mice, developing into well differentiated squamous carcinoma. Karyotype analysis showed aneuploidy with the modal chromosomal number 74-101 (5th-20th passages) and 15 marker chromosomes. The frequency of spontaneous sister chromatid exchange was very high in HNE-1 cells (87.6 +/- 0.4/cell).     

Establishment of an Epstein-Barr virus-negative B-cell lymphoma line from a Japanese Burkitt's lymphoma and its serial passage in hamsters.

An Epstein-Barr virus (EBV)-negative lymphoma line (JBL) was established in vitro from pleural effusion of an EBV-seropositive 29-year-old Japanese female with Burkitt's lymphoma. JBL cells as well as her original lymphoma cells bore monoclonal surface IgM with lambda light chains. The JBL line grew in single cell suspension with a doubling time of 30 hours. Attempts were made to serially transplant JBL cells in antilymphocyte serum-treated newborn hamsters; intraperitoneal implantation of 1-3 X 10(7) cells gave rise to invasive tumors in all recipients with death after 10 to 14 days. The hamster-passage line, now in the 9th passage, has been converted to an ascitic form with progression to leukemia in some animals. A "starry sky" pattern closely resembling the human tumor material was preserved in every tumor through serial animal passage.     

Visualization of oxidative processes at the cellular level during neutrophil-mediated cytotoxicity against a human hepatoma cell line, HCC-M.

Human neutrophil-mediated oxidative processes against a human hepatoma cell line, HCC-M, was visualized at the cellular level by using a silicon-intensified target camera and subsequently processing with a computer-assisted digital-imaging processor. Neutrophils were activated by a streptococcal preparation, OK-432. A hydroperoxide-sensitive tracer, dichlorofluorescein diacetate, was loaded in HCC-M and temporal and spatial changes of lipid peroxides in this cell after addition of stimulated neutrophils were analyzed. The luminol-dependent chemiluminescence activity of neutrophils was significantly enhanced and continued for at least 2 hr by stimulation with OK-432, and its activity was shown to be accumulated at the site where a neutrophil attached with HCC-M. The intensity of dichlorofluorescein fluorescence in HCC-M rapidly increased after adding stimulated neutrophils, and their reaction was significantly attenuated by superoxide dismutase. The number of non-viable cells was increased as the dichlorofluorescein fluorescence increase. It is suggested that oxidative stress may play an important role in neutrophil-mediated tumor-cell damage.     

Development of a 3T3-like line from an embryo culture of an inbred strain of Syrian golden hamster.

An embryo culture of an inbred strain of Syrian hamster developed into a permanent cell line under the "3T3" 3 X 10-5 cells/60-mm dish. The resulting cell line had properties very similar to those of the mouse 3T3 series and was named HAMS 3T3. The cell showed density-dependent inhibition of division with a saturation density of 1.0 to 1.2 x 10-6 cells/60-mm dish or 4.5 to 5.5 x 10-4 cells/sq cm. Addition of fresh medium containing 5 or 10% fetal calf serum to a confluent culture induced DNA synthesis in 18 hr with subsequent cell division. Cells were hyperdiploid with a mode of 45 chromosomes (80% of the cells). When cells at the 60th passage were injected into the skin or cheek pouch of an inbred hamster of the same strain as that from which they were derived, they produced a benign tumor that regressed after 3 weeks. Morphological transformation was obtained by infection with the Moloney strain of murine sarcoma virus.     

Enhancement of morphological and oncogenic transformation of mouse cells with 12-0-tetra-decanoyl phorbol-13-acetate following herpes sinplex virus type 2 infection

Sequential exposure of mouse embryo cells to HSV-2 and TPA gave rise to a synergistic enhancement of the transformation frequency. The transformants were selected for their ability to form dense foci of cells in medium containing 10% or 1% (low) fetal bovine serum. The average number of foci induced with HSV-2 followed by TPA was about 3 or 5 (in low serum) fold greater than that induced with HSV-2 alone. HSV-2 antigen could be detected in about 10% of transformed cells before 27th passage with immunofluorescence technique. Of two cell lines established from single focus, one designated BL which was preferable to form foci in subcultures was tumorigenic after 21th passage. All of the tumors were sarcomas with interlacing bundles of pleomorphic fibroblasts. The other, designated NP was nontumorigenic until 50th passage. The BL cell line was composed of two distict cell types, i. e., pigmented and unpigmented. No viral DNA sequences were detected in the cells of tumors derived from BL cell line.     

Changes of histological and biological features by serial passages in a human adenoid cystic carcinoma line transplantable in nude mice

The basic histologic patterns of adenoid cystic carcinoma (ACC) are classified into three types (tubular, cribriform and solid), but clinical significance of the histological type is unclear. We have successfully established a human tumor line derived from ACC that is serially transplantable in nude mice. This tumor showed an increased growth rate as the passage levels proceeded, and the histological type was changed from a cribriform pattern in the initial stage to a solid one. In this study, we investigated the relationship between histological type and biological characteristics by analyzing the serially transplantable ACC tumor model. As a result, the tumor growth rate at the 15th passage level was increased approximately 5-fold compared with that at the initial passage level. In the histological type, approximately 30% of the cribriform pattern in the initial level was changed to a solid one at the 15th passage level, and the PCNA labeling index was elevated 4-fold. Concomitant with this, expression of Ki-67, p53 and bcl-2 proteins was increased, and apoptotic cells were decreased as demonstrated by the TUNEL method. From these findings, it was suggested that cell proliferation and histological change of this ACC tumor are related to the inhibition of apoptosis. This tumor line would provide a useful model for investigating the biological behavior of ACC.     

ENHANCEMENT OF MORPHOLOGICAL AND ONCOGENIC TRANSFORMATION OF MOUSE CELLS WITH 12－O－TETRA－DECANOYL PHORBOL－13－ACETATE FOLLOWIN

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阿霉素诱导人肝癌细胞凋亡模型的建立

目的建立肝癌细胞凋亡模型,为进一步研究细胞凋亡分子机理打下基础。方法用抗癌药阿霉素诱导培养的人肝癌细胞株ＨＣＣ－９２０４肝癌细胞发生凋亡,用四唑盐比色试验（ＭＴＴ法）检测。结果研究发现,０．５μｇ／ｍｌ放线菌素Ｄ可明显抑制２０μｍｏｌ／Ｌ阿霉素对肝癌细胞凋亡的诱导作用。用２０μｍｏｌ／Ｌ阿霉素作用肝癌细胞３小时,换培养液继续培养１２小时后,肝癌细胞ＤＮＡ结构断裂,电泳呈梯状带型。肝癌细胞形态学改变出现膜小泡和凋亡小体形成等凋亡细胞特征。用流式细胞仪测定,出现典型的凋亡峰,凋亡细胞占７２．１％。用２０μｍｏｌ／Ｌ阿霉素诱导肝癌细胞凋亡后,细胞周期Ｇ１期、Ｓ期和Ｇ２期分别为７５．９％,２４．１％和０％,肝癌细胞活细胞率为３５．５％。结论用抗癌药阿霉素成功地诱导人肝癌细胞株ＨＣＣ－９２０４肝癌细胞发生凋亡,该细胞凋亡模型的建立,将有助于进一步探讨肝癌细胞凋亡的分子机制。     

ESTABLISHMENT OF A NOVEL CELL LINE (HNE_(1)) DERIVED FROM A NASOPHARYNGEAL CARCINOMA

A novel epithelial cell line, designated HNE1 was established from a biopsy specimen of a naso-pharyngeal carcinoma (NPC). Electron microscopic examination of the HNE1 cells demonstrated bi-directional differentiation, with some cells displaying features of poorly differentiated squamous cell carcinoma, while other cells appeared to have the morphology of poorly differentiated adenocarcinoma. The HNE1 cell line has been passaged more than 100 times over a period of one year. We recently reported that the Epstein-Barr virus (EBV) nuclear antigen (EBNA) was detected in a low pe rcentage of the HNE1 cells examined at subcultures 5-81; the cells were also shown to be EBV DNA positive. Tumorigenicity of the HNE1 cells was demonstrated by xentransplanta tion in athymic nude mice. The developed tumors were characterized as well-differentiated squamous cell carcinomas upon histological examination. Kar yotypic analysis of the HNE1 cells demonstrated an aneuploidy with a modal chromosomal number of 74 at passage