Establishment and characterization of a human hepatocellular carcinoma cell line JHH-7 producing alpha -fetoprotein and carcinoembryonic antigen--changes in secretion of AFP and CEA from JHH-7 cells after heat treatment]

A human hepatocellular carcinoma cell line, JHH-7, was established from resected liver tumor of a 53 year old male with hepatitis B virus infection. JHH-7 was composed of polygonal epithelial cells and functionally synthesized and secreted human albumin, AFP, CEA and ferritin. No HBsAg was detected in the culture supernatant of JHH-7 cells. Changes of secretion of AFP and CEA from JHH-7 cells after heat treatment was studied using a temperature gradient incubator. Secretion of AFP decreased along with the inhibition of cell proliferation by heat treatment. Secretion of CEA, however, did not decrease even though the cells were damaged.     

Establishment and characterization of a human colonic cancer cell line expressing carbohydrate antigen 19-9 and carcinoembryonic antigen]

Colonic cancer cell strain KE43 was established from a human colonic cancer diagnosed histologically as a predominantly well differentiated adenocarcinoma with minute foci of poorly differentiated adenocarcinoma. The well differentiated adenocarcinoma cell line was identified as the major morphological picture in xenografts of KE43 and 58 in nude mice, but this changed to poorly differentiated adenocarcinoma in passage 105. Doubling time of this cancer cell line was 22.5 hours in passage 105. The modal numbers of chromosomes were 41 and 76. Cancer cells could be heterotransplanted in 100% of the nude mice. The tumor cells produced and secreted CA19-9, CEA and Laminin into the spent medium. This cell line appears to provide a useful system for studying colonic cancer in vivo and in vitro.     

Establishment and characterization of human pancreatic adenocarcinoma cell line SOJ producing carcinoembryonic antigen and carbohydrate antigen 19-9

A new tumor cell line derived from a human pancreatic exocrine adenocarcinoma was established in tissue culture and was transplantable in a nude mouse. In tissue culture, the neoplastic cells grew as epithelial-like, mucin-producing cells with a population doubling time of 50-70 hrs. Chromosomes ranged from 63 to 186 with a modal number of 77. Subcutaneous injection of 1 x 10(6) cultured neoplastic cells into nude mice resulted in tumor formation histologically closely resembling the original neoplasm. Ultrastructurally, the cell line showed characteristic ductal epithelium. Immunohistochemically, carcinoembryonic antigen (CEA). Carbohydrate Antigen 19-9 (CA19-9) and DU-PAN-2 antigen were demonstrated in the original tumor, the culture cells and the transplanted tumor. The cells secreted CEA (48.7 ng/1 x 10(5) cells/24 hrs) and CA19-9 (325 U/1 x 10(5) cells/24 hrs) in spent medium as well as sera of the nude mouse. This cell line has been passaged 30 times in vitro and maintained for more than one year. These characteristics will make the cell line SOJ a valuable tool in studying various aspects of biology of human pancreatic cancer.     

胃肝样腺癌的临床病理特征

目的探讨胃肝样腺癌（hepatoid adenocarcinoma of the stomach,HAS）的临床病理学特点、发生机制和免疫组化特征,以提高诊断水平。方法收集并分析5例胃肝样腺癌的临床特征和病理资料,采用HE染色、免疫组化方法,并复习文献。结果组织学胃肝样腺癌具有管状腺癌、粘液性癌和肝样分化腺癌的移行过渡,肿瘤细胞呈小梁状、巢团状,间质为丰富的血窦。免疫表型胃肝样腺癌AAT（5／5）、ACT（5／5）、CEA（5／5）、AFP（4／5）均为阳性,Heppar1灶性阳性（2／5）。结论胃肝样腺癌是一种少见的高度恶性、侵袭性强的特殊类型胃癌,组织学呈肝细胞样腺癌,免疫表型AFP、AAT和ACT阳性有助诊断。     

Control of apolipoprotein E secretion in the human hepatoma cell line KYN-2

Even though it is known that apolipoprotein E (apoE) is deeply involved in major age-related disorders such as atherosclerosis or Alzheimer's disease (AD), the control of cell-specific apoE expression is still poorly understood. We compared the apoE secretion as previously described in astrocytic cell17 to hepatic cell apoE secretion. We used the human hepatoma cell line KYN-2 to better delineate the characteristics of apoE secretion and to validate it with respect to the classical human hepatoma cell line HepG2. Interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) significantly inhibited, while IL-2, IL-6 and tumour necrosis factor-alpha (TNF-alpha) were inactive on apoE secretion by KYN-2 as well as HepG2 cells. Cholesterol and 25-OH cholesterol had no effect, while forskolin exerted a significant inhibitory effect, on apoE secretion in KYN-2 cells. Our results suggest that the KYN-2 cell line represents an appropriate cell model, and in any case an alternative model to the HepG2 cell line, to study the control of apoE secretion. The response of KYN-2 cells to both cytokines and cholesterol differs from that found in astrocytoma cells, suggesting that blood variations of apoE concentrations in AD may not reflect the dysregulations taking place in the brain.     

雌激素、孕激素对雌激素受体、孕激素受体阴性子宫内膜腺癌细胞系JEC裸鼠皮下移植瘤生长的影响

目的探讨一定浓度雌激素(E2)及孕激素(P)对雌激素受体(ER)和孕激素受体(PR)阴性的子宫内膜腺癌细胞系JEC裸鼠移植瘤的影响。方法选用人子宫内膜腺癌细胞系JEC为研究对象,Balb/c.nu裸鼠皮下注射8×105个/0.2mLJEC细胞,分别注射E2、P及NS连续一周,观察肿瘤的生长及病理学变化情况。结果各组肿瘤体积、瘤体重量及瘤细胞核的积分光密度值依次为E2组>NS组>P组,差异有统计学意义(P<0.01)。结论 ER和PR阴性的JEC细胞生长可受到雌激素和孕激素的调控,一定浓度的雌激素能促进瘤体的生长,而孕激素则对瘤体的生长有抑制效应。     

Characterization of a cell line (SW756) derived from a human squamous carcinoma of the uterine cervix

Summary An established cell line, SW756, derived from a primary squamous carcinoma of the uterine cervix is described by its morphology, ultrastructure, karyotype, genetic signature analysis, HLA typing, and tumorigenesis in the nude mouse. Cultured cells obtained from the SW756 derived nude mouse tumor also were studied for chromosome and isozyme markers. The original tumor was poorly differentiated carcinoma with minimal keratinization and is compared with that occurring in the nude mouse after the cultured cells were inoculated. The nude mouse tumor showed similar histological features, but better differentiation than the original tumor. Karyotype analysis of SW756 demonstrated a hyperdiploid stem line number and several marker chromosomes (MI-M6). No HeLa marker chromosomes were identified. The isozyme pattern for SW756 reported by others has been confirmed. The unique chromosome and isozyme features have been identified repeatedly in the cultured cells and, most importantly, in the post nude mouse culture. We recommend SW756 as a defined human tumorigenic cell line derived from a primary squamous carcinoma of the uterine cervix. This investigation was supported in part by Public Health Research Grant CA-06294 from the National Cancer Institute, Department of Health and Human Services.     

Immunohistochemical study of the appearance of some markers in liver adjoining hepatocellular carcinoma

The presence and distribution of AFP, AAT and HBsAg in peritumoral non-neoplastic hepatocytes (NNH) of 27 cases and, at the same time, in the neoplastic tissue of 37 liver cell carcinoma (HCC) were studied; AFP and HBsAg were more frequently found in NNH than in HCC cells; no differences were found for AAT. The presence of HBsAg also in normal liver without cirrhosis is probably best explained by its possible role in neoplastic transformation and by the inhibition of replication of the viruses AFP, considered to be expression of dedifferentiated cells, may possible be taken up by NNH for catabolic purposes.     

Establishment and characterization of a new human hepatocellular carcinoma cell line

Summary A human hepatocellular carcinoma cell line (FOCUS—Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular orgin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, α₁-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectble in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and it contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous infection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.     

Partial Biological Characterization of Cancer Stem-like Cell Line (WJ<Subscript>2</Subscript>) of Human Glioblastoma Multiforme

To provide suitable models for human GBM cancer stem cells in vitro and in vivo, and investigate their biological characteristics, a new human GBM cancer stem-like cell line, WJ2, was established in this experiment through serial passages from adherent monolayer culture to nonadherent tumor sphere culture in turns; Its partial biological characteristics were studied through cell proliferation and tumor sphere assay; cell cycle distribution, side population, and CD133 phenotype were analyzed with FCM. The expressions of CD133, Nestin, and GFAP of cancer stem-like cells and xenograft tumor cells were detected with RT-PCR and immunohistochemistry. Biological characterization, side population, CD133 phenotype and CD133 Nestin, BCRP-1, Wnt-1 gene expression revealed the stemness of this cancer stem-like cell line. Tumorigenicity heterotransplanted in nude mice; histopathological characteristics of xenograft tumor, and expressions of CD133, Nestin, and GFAP of xenograft tumor cells indicated that xenograft tumors recapitulated the phenotype and biological characterization of human primary GBM. All findings of this experimental study suggested that WJ₂ cancer stem-like cell line could accurately mimic human GBM cancer stem cell in vitro and in vivo; it would be useful in the cellular and molecular studies as well as in testing novel therapies of CSC-based anti-cancer therapies for human GBM.     

Establishment and characterization of a new human colon adenocarcinoma cell line: BCS-TC2

A new cell line designated as BCS-TC2 was established in culture from a primary human colon adenocarcinoma. This cell line has been in continuous culture over a 36-month period. The cells grow as a monolayer sheet, displaying areas with a multilayered pattern as well as single cells and free-floating aggregates. The morphological, immunological, and ultrastructural features of these cells are in agreement with their epithelial origin. The characterization of this cell line indicated a 38 hr doubling time, and a colony forming efficiency of 2% in semisolid media and 22% in liquid culture, at low cell densities. These cells produce low amounts of carcinoembryonic antigen in culture (0.1 ng of CEA/10⁶ cells). Sub-cutaneous injection into athymic mice shows that these cells have a non-tumorigenic capacity. Chromosomal analysis showed a karyotype 46 XX,-15, +der (15), inv (16) (p13::q13). BCS-TC2 cell line, which maintains in culture several characteristics of the original tumor, represents a useful model system for cell biology studies of primary and non-metastatic tumors.     

Development and characterization of a human cell line from an ovarian mixed mullerian tumor (carcinosarcoma)

Summary A cell line derived from a human ovarian carcinosarcoma was established in tissue culture and in nude mice. Two sublines, LDF and HDF, separated by discontinuous density centrifugation were also established from the parent line JoN. The cloning efficiency of the JoN line was 21%. Morphologic features of adenocarcinoma cells characteristic of the parent JoN cells were retained in the sublines and clones; all lines showed the same karyotype and DNA content (pseudodiploid and pseudotetraploid). Keratin, as demonstrated immunohistochemically, was strongly expressed in the parent line JoN and the xenograft tumor, but not at all in the LDF sublines and only moderately in the HDF sublines. Vimentin, however, was expressed in neither the parent line JoN nor the xenograft tumor, but was present in both sublines. Transglutaminase and plasminogen activator activity was high in the parent line JoN. Neither, sublines nor clones showed the same high enzyme activity as the parent line. It is concluded that this human tumor line JoN is comprised of epithelial cells, capable of multidirectional differentiation.     

Establishment and characterization of a human hepatocellular carcinoma cell line JHH-4

A human hepatocellular carcinoma cell line, JHH-4 was established from resected liver tumor. Morphological diagnosis of the original tumor was hepatocellular carcinoma, Edmondson type III. This cell line was composed of polygonal shaped cells. Subcellular organelle were observed in cytoplasm. Furthermore, bile canaliculi adhering junction was also remained at the cell surface. The growth rate of JHH-4 cell is slow, peaks of the chromosome number was 75 and 79, and plating efficiency was 3.0%. JHH-4 cell is transplantable to nude mouse. Furthermore, this cell line functionally synthesized and secreted human albumin, AFP and other proteins in vitro.     

Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium

Summary A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na₂SeO₃ (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linkedN-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium.α1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9±1.5 pmol · h⁻¹ · mg⁻¹ protein) than cultured with FBS-containing media (18.2±1.2 pmol · h⁻¹ · mg⁻¹ protein). These results have indicated that the selective increase ofα1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.     

Efficiency of composite laboratory tests in the diagnosis of liver malignancies

The utility of the markers CEA, beta-HCG, CA-50, alpha-fetoprotein (APF), ferritin, alkaline phosphatase (AP), its isoenzyme liver-1 (APL1), gamma-glutamyltransferase (gGT), its fast migrating isoenzyme (gGT1) and 5'nucleotidase (5'N) in differentiating liver malignancies and benign involvement was evaluated in the sera of 85 patients with hepatocellular carcinoma (HCC), 157 with chronic liver disease (CLD) and 91 with liver metastases (LM) derived from different tumors. The mean concentrations of all the parameters except CEA and GGT1 were significantly different in HCC and CLD, but a broad overlap existed in the two groups, so different cut-offs were considered to assess the positive and negative predictive values and test efficiency (Eff). The best results were observed considering AFP greater than 100 IU/m (Eff0.86), ferritin greater than 800 ng/ml (Eff0.69), CA-50 greater than 100 U/ml (Eff 0.63), beta-HCG greater than 10 mU/ml (Eff 0.61), AP greater than 300 IU/ml (Eff 0.66), the presence of APL1 (Eff 0.78), 5'N greater than 25 mU/ml (Eff 0.70), gGT greater than 100 mIU/ml (Eff 0.63). Among HCC patients 17% did not secrete AFP; in 26% the protein was less than 100 IU/ml and in 36% less than 400 IU/ml. Apart from AFP the most effective marker was APL1. At the above cut-offs more than three parameters were simultaneously positive in 71% of HCC and 9.9% of CLD. CEA, CA50, AFP were the only parameters that distinguished the HCC from the LM group; in the latter, APL1 was also a very sensitive marker (87%) for neoplastic involvement of the liver.     

Establishment and characterization of a human gall bladder carcinoma cell line NOZ

A human gall bladder carcinoma cell line was established from ascites of a patient of peritonitis carcinomatosa. The pathological diagnosis of this patient was adenocarcinoma tubular ++, moderately differentiated. This cell line was composed of polygonal, spindle and round shaped cells. Each cell types were cloned by single cell cloning technique and each cloned cell secreted CEA or Ferritin or none of them. The doubling time of cell number was 48 hours, and plating efficiency was 14-19%. NOZ cell was transplantable to nude mouse. The morphological feature of transplanted tumor was similar to the original one.     

Establishment and characterization of a human gestational choriocarcinoma cell line (HOCC)

Human gestational choriocarcinoma cell line (HOCC) was established from the mouse graft of choriocarcinoma. The HOCC cells were spindle or polygonal in shape and multi-nucleated giant cells, showing neoplastic and pleomorphic features. The cell proliferated stably, and the population doubling time was about 32 hours. The chromosome numbers showed a wide distribution of aneuploidy, the mode was in hypertriploid range and the marker chromosomes were recognized in the several generations. Heterotransplantation was easy, and subcutaneous transplantation of 1 X 10(7) cells in nude mouse formed a tumor composed of choriocarcinoma. It is most noteworthy characteristic of the cell line that it produced human chorionic gonadotropin (hCG) in an in vitro culture system and in vivo in nude mice.     

Unmasking of alpha-fetoprotein-specific CD4(+) T cell responses in hepatocellular carcinoma patients undergoing embolization

Necrosis of tumor cells can activate both innate and adaptive antitumor immunity. However, there is little information on the effects of necrosis-inducing cancer treatments on tumor-specific T cell immune responses in humans. We studied the effects of a necrosis-inducing treatment (embolization) on anti-alpha-fetoprotein (AFP)-specific CD4(+) T cell responses in hepatocellular carcinoma (HCC) patients and controls using an array of AFP-derived peptides. In this study, we show that AFP-specific CD4(+) T cell responses to three immunodominant epitopes in HCC patients were significantly expanded during (p < 0.0001) and after embolization (p < 0.002). The development of higher frequencies of AFP-specific CD4(+) T cells after treatment were significantly associated with the induction of >50% necrosis of tumor and an improved clinical outcome (p < 0.007). In addition, we identified two novel HLA-DR-restricted AFP-derived CD4(+) T cell epitopes (AFP(137-145) and AFP(249-258)) and showed that the CD4(+) T cells recognizing these epitopes produce Th1 (IFN-gamma and TNF-alpha) but not Th2 (IL-5)-type cytokines. AFP(137-145)-, AFP(249-258)-, and AFP(364-373)-specific CD4(+) T cells were detected in HCC patients but not in patients with chronic liver diseases or healthy donors. In conclusion; our study shows that induction of tumor necrosis by a conventional cancer treatment can unmask tumor rejection Ag cell-mediated immunity and provides a rationale for combining embolization with immunotherapy in HCC patients.     

Anticancer drugs are synergistic with freezing in induction of apoptosis in HCC cells

Cryotherapy has been shown to be an important therapeutic alternative to surgery in the treatment of hepatocellular carcinoma (HCC). Here, the influence of cryo-chemotherapy on HCC was examined in vitro using the human HCC cell line Bel-7402, a drug-resistant HCC cell line originating from Bel-7402 cells (Bel-7402/R), as well as two control cell lines, the HCC cell line SMMC-7721 and a colorectal tumor cell line HIC-251. Cells were treated with either exposure to different freezing temperatures (ranging from −15 to −80 °C for 20 min), exposure to sub-lethal concentrations of anticancer chemotherapy drugs or a combination of cryotherapy and chemotherapy. Cell viability and apoptosis under each condition were investigated. We found that the combined treatment resulted in increases in both cell death and apoptosis compared to either treatment alone. The increased level of apoptosis observed in Bel-7402 cells after cryo-chemotherapy was inhibited in the presence of caspase inhibitors. Furthermore, Bax expression was increased 2- to 3-fold in cells exposed to the combination treatment compared with cells treated by freezing or drugs alone. In contrast, Bcl-2 levels remained constant. Although Bel-7402/R cells originated from the Bel-7402 cell line, they were more sensitive to the freezing procedure than the parental cell line. The level of Bax expression in Bel-7402/R cells was also higher than that observed in the parental cell line. In addition, we found that Bel-7402/R cells had lower levels of survivin mRNA than the parental Bel-7402 cells, in both untreated and treated cells. In conclusion, our data show that in HCC cells, apoptosis induced by cryotherapy can be synergistically enhanced using anticancer drugs.     

Characterization of a human colonic adenocarcinoma cell line,LS123

Summary An epithelial cell line, LS123, was established in 1974 from the second in a series of three primary colonic tumors resected from a Caucasian female. The cell line is aneuploid, releases low concentrations of carcinoembryonic antigen (CEA), fails to grow progressively in nude mice, and forms colonies only in enriched semisolid medium developed for tumor stem cells. LS123 cells grow on confluent cell monolayers and in either low serum or serum-free medium. In the chick embryonic skin assay, LS123 cells grew as a well-differentiated abnormal colonic epithelium with little mitotic activity but with some indication of invasion. On floating collagen gels LS123 cells formed a one to three-cell-layer-thick undifferentiated epithelial sheet. The apparent low invasiveness of the cells of this line is supported by the patient's history of three primary colon tumors without systemic metastases during the past 30 yr. Therefore, although LS123 cells possess several properties associated with neoplasia, they have little invasive potential. Thus, LS123 cells may represent an important model for the study of human colon cancer. Presented in part at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, CA, June 6–10, 1982. The work has been partially supported by U.S. Public Health Service Grants CA23871 (L. P. R.), CA24024 and RCDAK04-CA00579 (B. H. T.), and CA27124 and CA22370 (B. D. K.); the latter was awarded through the National Large Bowel Cancer Project.