Synthesis and secretion of IgE by an established human myeloma cell line

The rate of IgE production in vitro and the cell population proliferation rate by the established human myeloma line 266 Bl have been studied quantitatively under various tissue culture conditions. The rate of extracellular IgE accumulation depended on the type of medium used, the cell density and the period of time elapsed after explantation. The maximum production rate of 8·1 × 10⁻¹² g IgE/cell/48 hr was noticed at cell densities <10⁶/30 ml and with the presence of feeder human skin fibroblasts or glia-like cells or with the use of conditioned media harvested from such cells. The rate of cell proliferation and secretion of IgE to the medium ran parallel suggesting that the IgE production is highest when cells are in the best physiological condition. During more than one year the rate of synthesis of IgE remained stable. This functionally stable human myeloma line is suitable for further studies on immunoglobulin biosynthesis at the cellular and subcellular level under the tissue culture conditions found optimal in this study.     

Functional human T cell-B cell hybridomas established from fusion of normal T cells and an EBV-transformed B cell line

Human T cell-B cell hybridomas containing 92 chromosomes were derived by fusing normal stimulated CD-depleted T cells with the EBV-transformed human B cell line 729-HF. These hybridomas coexpressed the T cell surface markers CD3, CD8, and CD2 and the B cell surface antigens CD19, CD20, CD23, DR, and DQ. They weakly and variably expressed surface IgM and TCR. Genomic analysis revealed the presence of rearranged Ig genes as well as beta-T cell receptor genes that could be ascribed to the B and T cell parent, respectively. Analysis of TCR rearrangement suggests that the T-B hybridomas are, in fact, subclones of a single dominant clone. Although the hybrids expressed CD8 and not CD4, following preincubation with PWM, some the of clones induced IgG synthesis from normal B cells while the parent B cell line failed to demonstrate this activity. Stimulation of the hybridomas with PMA down-regulated the T cell lineage-specific antigen display (CD3, CD8, and TCR) and increased IgM production from the hybrids without changing B cell surface antigen display. These hybridomas may be useful to dissect the steps involved in the ultimate commitment of a cell to the B or T cell lineages and will be made available to interested investigators.     

Karyotypic characterization of an established human hepatoma cell line HA22T/VGH

The karyotype of an established human hepatoma cell line HA22T/VGH was characterized by G-banding. A majority of the 200 cells counted had around 70 chromosomes at passage 24, and 60 at passage 338. Of the 50 cells karyotyped from each of passage 24 and passages 338-339, chromosomes #13 and #18 were absent. The presence of the Y chromosome was reduced dramatically from a mean value of 1.12/cell at passage 24 to 0.12/cell at passages 338-339. In general, most of the chromosomes--particularly chromosomes #5, #7, #9, #15, and #21--tended to be less represented in the course of propagation in vitro. The presence of multiple copies of a normal chromosome in a single cell was quite common for chromosomes #5 and #7 at both early and late passages. Numerous structural rearrangements of the chromosomes were observed.     

Characteristics of hematopoietic cell line established from human myelomonocytic leukemia

Summary The peripheral blood of an acute myelomonocytic leukemia patient has been cultured for 16 months. The culture is at present at the 140th population doupling level. The cultured cells have the characteristics of so-called lymphoblastoid cells and proliferate actively as individual cells in small clusters, or in large clumps consisting of large mononuclear cells. Some of these cells appeare to be lymphoid, but the majority are immature mononuclear cells with a tendency to lobulate. They gave a weakly positive peroxidase reaction at the beginning of cultivation, and have given a strongly positive esterase reaction persistently. The cytoplasm shows ciliary or tail-like projections as the cell matures. Complement (C₃) receptor and IgG receptors are found on the cell surface, and active phagocytosis is mannifest. Colloidal iron particles or viable red blood cells attached to the cell membrane suggesting possible differentiation to reticulum cells or macrophages. The cultured cells are mostly diploid but some cells show chromosome abnormality.Herpes type virus was found in the nucleus, cytoplasma and on the cell membrane. The transplantation of cultured cells to the cheek pouch of hamsters produced small tumors with histological findings resembling reticulum cell sarcoma.The authors wish to thank Miss Fujiko Yokoyama and Akiko Miyoshi for their technical assistance. This work was supported by a cancer research grant 49101 from Kawasaki Medical School.     

Secretion ofα 1-antitrypsin by an established human hepatoma cell line and by human/mouse hybrids

The human hepatoma cell line SK-HEP-1 has been shown by radioimmunoelectrophoresis to synthesize and secrete a protein which coprecipitates with human ₁-antitrypsin. This protein was indistinguishable from serum ₁-antitrypsin in terms of electrophoretic mobility, apparent subunit molecular weight (47,000), and binding to concanavalin-A. The protein identified as ₁-antitrypsin (₁AT) was secreted by seven clones derived from SK-HEP-1 and by twelve out of eighteen hybrid clones derived from the fusion of SK-HEP-1 with mouse RAG cells. There was no correlation between the expression of ₁AT and that of human enzymes assigned to sixteen different autosomes. There was an imperfect correlation between the expression of ₁AT and of the two chromosome 9 marker enzymes AK1 and AK3 (two discordant clones).     

A quantitative assay for measuring human foamy virus using an established indicator cell line

In order to improve the accuracy for detecting human foamy virus (HFV), an indicator cell line was established by co-transfecting baby hamster kidney-21 cells with two plasmids: one containing a G418 antibiotic resistance marker and the other including the luc gene which was placed downstream of the inducible HFV long terminal repeat promoter (from -533 to +20). Among 11 independent subclones, IdB14 was found to be stable with a low basal level of luciferase activity. Although the changes in luciferase activity in infected clones showed time-dependency and peaked at day 8, it is possible to differentiate infected and uninfected cells on day 2. The sensitivity of the foamy virus activated luciferase (FAL) assay was 400 times higher than the end-point syncytium formation by TCID(50). The HFV LTR promoter in the IdB14 cell line was specific for this virus. Moreover, a linear relationship was found between the MOI and the activated intensity of luciferase expression. These findings suggest that the FAL assay using the IdB14 indicator cell line is a simple and useful technique for rapid diagnosis and quantitation of active HFV infection.     

Replication of a cytoplasmic polyhedrosis virus in an established cell line of Trichoplusia in cells

The complete replication of a cytoplasmic polyhedrosis virus in an established cell line of the insect Trichoplusia ni was demonstrated. Virus produced in cultures was infectious to T. ni larvae.     

A human T cell line with an abnormal trisomy 2 karyotype established by coculture of peripheral lymphocytes with an HTLV-II-infected simian leukocyte cell line

A new human T cell in with a chromosomal abnormality (47, XY, +2), designated AS-IIA, was established by coculturing peripheral blood leukocytes of a healthy adult male with a lethally irradiated human T lymphotropic virus type II (HTLV-II)-infected simian leukocyted cell line (Si-IIA). A polymerase chain reaction method showed that this interleukin-2 (IL-2)-dependent cell line possessed the HTLV-II provirus genome; the cells also reacted with HTLV-II-positive human sera, anti-HTLV-I/II p24, and anti-HTLV-II gp46 antibodies. AS-IIA cells expressed the suppressor/cytotoxic T cell markers CD3+, CD4^-, CD25+, and HLA-DR+, with later conversion ot CD8^-. These cells showed better proliferation than other human HTLV-II-infected cell lines with normal karyotypes, but were not transplantable into severe combined immunodeficiency mice. Virus production from AS-IIA was confirmed not only by electron microscopic examination, which revealed mature and immature type C virus particels, but also by the capacity of the line to immortalize human T cells. These results suggest that HTLV-II shows broad tropism for T cells including CD4+ or CD8+, and that not only SI-IIA, but also AS-IIA, are goode sources of HTLV-II. The authors of the present study believe that AS-IIA may be a useful human T cell line for the investigation of HTLV-II in comarison with HTLV-I.     

Establishment and characterization of human metastatic hepatocellular carcinoma cell line

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with a poor prognosis. Recently, we established a HCC cell line from a metastatic HCC tumor. GTG banding analysis was performed and the karyotype showed that this metastatic HCC cell line is a hypertriploid (71-78 chromosomes) with a large marker chromosome containing a long homogeneously staining region (hsr). Comparative genomic hybridization was applied to characterize the chromosomal alterations in this metastatic HCC cell line. The results showed that the hsr was composed of amplified DNA sequences from 11q13. Further characterization of the hsr may lead to the isolation of the putative amplified oncogene at 11q13.     

Expression of macrophage inflammatory protein-3alpha in an endometrial epithelial cell line,HHUA, and cultured human endometrial stromal cells

It has been demonstrated that human endometrial epithelial cells (EEC) and stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. To evaluate the expression of macrophage inflammatory protein (MIP)-3alpha in endometrial cells, the production of MIP-3alpha by an EEC line, HHUA, and cultured ESC stimulated with various inflammatory mediators was evaluated by ELISA. Unstimulated HHUA and ESC constitutively secreted MIP-3alpha. Tumour necrosis factor-alpha and interleukin-1beta significantly stimulated the secretion of MIP-3alpha by HHUA and ESC. Lipopolysaccharide also stimulated the secretion of MIP-3alpha by ESC, but not by HHUA. These results show that the concentration of MIP-3alpha in the endometrium is modulated by these inflammatory mediators. MIP-3alpha may contribute to the normal and pathological processes of human reproduction by regulating the trafficking of immature dendritic cells and memory T lymphocytes into the endometrium.     

人肝癌细胞系中肿瘤相关基因MAGE的克隆

目的 克隆人肝癌细胞系HHCC中的肿瘤相关基因MAGE－1的全长cDNA。方法 根据GenBank中MAGE－1基因编码区，设计PCR引物，用RT-PCR手稿,人人肝癌细胞系HHCC中获得MAGE－1全长cDNA，双酶切后连接入质粒pUC19中，转化入大肠杆菌DH5α。挑选阳性克隆提取质粒，进行DNA序列分析，并将测得的核苷酸序列与GenBank中的DNA序列进行BLAST同源性分析。结果 获得MAGE－1全长cDNA,MAGE-6基因463bp片段，以及1个与MAGE－6基因编码区有93％同源性的片段，可能为MAGE家族中的新成员。结论 人肝癌细胞系HHCC可表达多种MAGE基因，可能有效的MAGE基因出现，为研究MAGE基因在HCC中的表达模式及其在HCC免疫治疗中的靶点提供了新的资料。     

Consistent chromosome 10 rearrangements in four newly established human hepatocellular carcinoma cell lines.

Cancer cell lines represent an invaluable resource for isolation of novel genes relevant to tumor pathogenesis and as in vitro models. In this study, we report on the successful establishment of four cell lines from hepatocellular carcinoma tissue. These cell lines, designated HKCI-1, HKCI-2, HKCI-3, and HKCI-4, have been growing continuously for more than 24 months and have been passaged more than 50 times. A comprehensive cytogenetic characterization on the primary tumors and the derived cell lines was achieved by the combined approach of spectral karyotyping and comparative genomic hybridization. Chromosomal imbalances from the primary tumors were also maintained in the cell lines and included gains of 1q, 6p, 7, 10p, 17q, and 20 and loss of 4q. Recurring translocations included t(X;11), t(1;10), t(4;16), i(5)(p10), t(7;21), t(8;17), t(9;22), i(10)(p10), t(14;20), t(16;22), and t(17;19). It was noteworthy that consistent chromosome 10 aberrations, in particular t(1;10)(q10;p10), were detected in all four cell lines. Furthermore, microsatellite analysis on primary tumor and derived cell lines indicated a common deleted region of 10q23-q26. The functional importance of chromosome 10 aberrations in relation to the pathogenesis of HCC is unknown; however, the high frequency with which such aberrations were maintained in the cell lines suggests proliferative advantages of 10q loss or 10p gain in the multistep development of hepatic neoplasia.     

Comparative immunohistochemical investigation of rat and human hepatocellular carcinomas

Abstract

Previously identified comparable morphological features of human and rat hepatoproliferative lesions were identified, including hepatocellular carcinomas (HCCs). In this study we identified similarities and differences in immunohistochemical (IHC) detection of some key proteins important in carcinogenesis that may link pathogenesis pathways of human and rat HCC. The comparative features of six IHC markers (Ki-67, beta-catenin, CD34, glutamine synthetase (GS), c-myc, and transforming growth factor alpha (TGF-alpha)), previously shown to be positive or altered in rodent and human HCC when compared to normal hepatocytes, were investigated. Glutamine synthetase (a hepatocellular enzyme) was strongly positive in 5/5 (100%) human and 4/5 (80%) rat HCCs examined. CD34 (an endothelial marker) and Ki-67 (a cell proliferation marker) were consistently positive in five human HCCs (5/5 for both markers) but weakly positive in only 2/5 and 3/5 rat HCCs, respectively. Beta-catenin, c-myc, and TGF-alpha were infrequently altered, with immunopositivity found in only one or two of either rat or human HCCs (a total of five each examined; beta-catenin: 1/5 rat samples, c-myc: 2/5 human samples, TGF-alpha: 1/5 rat samples positive). It was concluded that Ki-67, CD34, and GS, are most likely to be useful in studying the pathogenesis of HCC in rats and humans.     

人食管鳞癌细胞系EC9706的建立及其比较基因组杂交分析

目的 以中国男性食管鳞癌为来源，建立一个可良好传代的细胞系，从而提价七个有用的体外模式用于食管癌的研究。方法 采用组织块培养法，以新鲜的食管癌组织细胞系EC9706，做了初步的生物学特性观察，并采用比较基因组杂交的方法进行细胞遗传学的检测。结果 食管癌细胞系EC9706生长曲线显示其生长良好，易于培养。传代时间为26h，平皿集落形成率为91．9％，且能够在软琼脂中形成集落。经异种接种到裸鼠中均形成移植性肿瘤，肿瘤的病理诊断为中－低分化鳞状细胞癌。比较基因组杂交结果得出染色体1p1,q2-4,2p1,5p,7p14,7q21,11q1,11q2,20q扩增，其中5p表现出高水平的扩增。而染色体2p2,2q2,3p,4,9p,14,18,Xq缺失。结论 建立的细胞系EC9706可用于研究食管癌的癌变过程。     

Phenotypic and ultrastructural characterization of an epithelial cell line established from rat thymic cultures.

An epithelial cell line (TE-R 2.5) was established from a long-term culture of rat thymic epithelium. Its epithelial nature was confirmed using anti-cytokeratin (CK) monoclonal antibodies (mAb) and electron-microscopy. TE-R 2.5 cells were reactive with K 8.13, K 8.12, CK 8, R-MC 18, R-MC 19 and Mar 3 mAb and bind Ulex europaeus agglutinin I. Based on the results of this study it was concluded that they possess the phenotype of subcapsular/perivascular or medullary epithelium. This was in accordance with Western blot analysis of water-insoluble cell extracts showing the presence of 56,000, 52,000, 50,000 and 48,000 MW CK polypeptides. In addition, TE-R 2.5 cell line coexpressed CK and vimentin (a 57,000 MW polypeptide) which was demonstrated using dual immunohistochemistry and Western blot analysis. Electron microscopy demonstrated that TE-R 2.5 cells have all the characteristics of hypertrophic thymic epithelial cells (TEC) localized in situ exclusively in the medulla and thus further characterized this line as a type of medullary TEC. Finally, TE-4F10 mAb raised against an antigen of TE-R 2.5 cells selectively stained a subset of medullary TEC in situ including Hassall's corpuscles indicating again the medullary origin of this TEC line.     

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-Steiner's 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, alpha 1-acid glycoprotein, alpha 1-antitrypsin, ceruloplasmin, transferrin, complement C, fibrinogen, fibronectin, prothrombin, retinol-binding protein (serum type), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin and beta 2-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 14th 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.     

Replication and plaque formation of parainfluenza viruses in an established line of monkey kidney cells

All four types of parainfluenza virus produced distinct plaques in an established line of monkey kidney cells (LLCMK2) under agar overlay containing trypsin and DEAE dextran. Parallel titration of these viruses in LLCMK2 and primary cynomologous monkey kidney (MK) cells showed that LLCMK2 cells were about tenfold more sensitive than MK cells. When trypsin was added to the fluid medium, the virus yield in LLCMK2 cells was significantly higher than in MK cells.