Establishment and characterization of human malignant lymphoma cell line derived from uterine cervix

Human uterine cervical malignant lymphoma (B-cell type) was cultured and the cell line (HIUML) was newly established. The HIUML cells were round in shape and had a tendency to make floating clusters. The cells had a smooth surface or protrusion on the margin of the cytoplasm, and proliferate in floatation. The population doubling time was about 32 hours and 42 or more passages were successfully observed in two years. The HIUML cells were not transplantable into nude mice but were successfully done in the cheek pouch of hamster with formation of malignant lymphoma. Epstein-Barr virus was detected in the HIUML cells.     

Establishment and characterization of a brain‐cell line from kelp grouper Epinephelus moara

A new brain‐cell line, EMB, was developed from kelp grouper Epinephelus moara, a cultured marine fish. The EMB cells were subcultured for more than 60 passages. The cells were cultured in Leibovitz's L‐15 medium (L15) supplemented with antibiotics, foetal bovine serum (FBS), 2‐mercaptoethanol (2‐ME) and basic fibroblast growth factor (bFGF). The cells could grow at 18–30° C, with the maximum growth between 24 and 30° C. The optimum FBS concentration for the cells growth ranged between 15 and 20%. Chromosome analysis indicated that the modal chromosome number was 48 in the cells at passage 45. After being transfected with pEGFP‐N3 plasmid, the cells could successfully express green fluorescence protein (GFP), implying that this cell line can be used for transgenic studies. A significant cytopathic effect (CPE) was observed in the cells after infection with Singapore grouper iridovirus (SGIV) or red spotted grouper nervous necrosis virus (RGNNV) and the viral replication was confirmed by quantitative real‐time PCR (qrt‐PCR) assay, which suggested EMB's application potential for studies of SGIV and RGNNV.     

Establishment and characterization of a fish‐cell line from the brain of Japanese flounder Paralichthys olivaceus

A new brain‐cell line derived from Japanese flounder Paralichthys olivaceus (POBC) was established. POBC was subcultured for 67 passages over the course of 420 days. The cultured cells were primarily epithelioid‐like. Chromosome analysis revealed the cell line to possess the normal P. olivaceus diploid karyotype of 2n = 48t (telocentric chromosomes). The cells exhibited the astrocyte marker glial fibrillary acidic protein by immunocytochemistry, and significant fluorescent signals were observed when the cells were transfected with green fluorescent protein reporter plasmid. The established POBC would be ideal material for the study of function of fish ependyma, the central neuroendocrine system and endocrine disruptors in the marine environment.     

Inhibition of PI3-kinase-Akt pathway enhances dexamethasone-induced apoptosis in a human follicular lymphoma cell line

Glucocorticoids are commonly used in the treatment of various lymphoid malignancies. In the present study, we show that dexamethasone (Dex) induced depolarization of mitochondrial membrane, release of cytochrome c and DNA fragmentation in a human follicular lymphoma cell line, HF28RA. New protein synthesis was required before Dex-induced mitochondrial changes, and the kinetics of the apoptotic events correlated with the upregulation of the Bim protein. Furthermore, we studied whether specific inhibitors of known survival pathways would potentiate Dex-induced apoptosis. Our results show that inhibition of PKC and ERK pathways had no effect on apoptosis. In contrast, inhibition of PI3-kinase or Akt markedly enhanced Dex-induced apoptosis. The enhancement was seen at the mitochondrial level, and the kinetics of apoptosis was notably accelerated. In addition, inhibition of PI3-kinase did not alter levels of Bax, Bcl-2, Bcl-X(L) or Bim proteins in mitochondria but caused translocation of the pro-apoptotic protein Bad to mitochondria. However, inhibition of PI3-kinase-Akt pathway and subsequent translocation of Bad to mitochondria did not induce apoptosis itself. Based on these results and our current understanding of Bim and Bad action, it seems that both proteins play a synergistic role in this process. Thus, these results indicate that inhibitors of PI3-kinase-Akt pathway might be combined in future with glucocorticoids to improve the treatment of lymphoid malignancies.     

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.     

Gonadotropes in a novel rat pituitary tumor cell line,RC-4B/C. Establishment and partial characterization of the cell line

Summary An epithelial cell line (RC-4B/C) was established from a pituitary adenoma obtained from a 3-yr-old (ACI/fMai × F344/fMai)F1 male rat. Before Year 5 in vitro, RC-4B/C cells could not be viably recovered from cryogenic storage. Recovery of viable cells from cryogenic storage in Year 5 was associated with a more transformed phenotype, including the appearance of endogenous C-type rat retroviral particles. The ultrastructural appearance of the cells was similar to that of differentiated anterior pituitary cell; the cultured cells contained numerous, electron dense, secretory granules, Golgi complexes, and extended arrays of rough endoplasmic reticulum. Immunocytochemical study showed that all cell types present in the rat anterior pituitary gland were present in the cell line. The percentage of luteinizing hormone beta (LHβ) cells in the cell line was higher (19.9%) and that of growth hormone cells was lower (12.2%) than in normal male rat pituitary, whereas the cell line contained a comparable percentage of follicle stimulating hormone beta (FSHβ), prolactin (PRL), ACTH, and thyrotropin beta cells. Radioimmunoassay data demonstrated the PRL content of the cells was comparable to that of normal male rat pituitary gland, whereas the content of LH and FSH was 70- and 800-fold lower, respectively. Assay of specific receptor sites for gonadotropin releasing hormone (GnRH) using Scatchard plots of the data established the RC-4B/C cells contained GnRH receptor sites of the same affinity as in the pituitary gland, but of twofold lower capacity. These data suggest the RC-4B/C cell line warrants further study as a model for the induction and maintenance of the gonadotropic function of the pituitary gland. An abstract of portions of these results was presented at the 8th International Congress of Endocrinology, Kyoto, Japan, 1988. This work was supported in part by grants DK-17631 (E.H.L.), CA-24145 (W.G.B.), CA-31102 (H.G.B.), AG-01753 (D.E.H.) and HD-1778 (M.T.D.) from the National Institutes of Health, Bethesda, MD, and by a grant from the Association pour la Recherche sur le Cancer, France (M.J.). The NIH is not responsible for the contents of this publication nor do the contents necessarily represent the official views of that agency. Jolanta Polkowska was a recipient of a Foundation Simone et Cino del Duca grant.     

A novel LED‐based 2D‐fluorescence spectroscopy system for in‐line monitoring of Chinese hamster ovary cell cultivations – Part I

A new two‐dimensional fluorescence sensor system was developed for in‐line monitoring of mammalian cell cultures. Fluorescence spectroscopy allows for the detection and quantification of naturally occurring intra‐ and extracellular fluorophores in the cell broth. The fluorescence signals correlate to the cells’ current redox state and other relevant process parameters. Cell culture pretests with twelve different excitation wavelengths showed that only three wavelengths account for a vast majority of spectral variation. Accordingly, the newly developed device utilizes three high‐power LEDs as excitation sources in combination with a back‐thinned CCD‐spectrometer for fluorescence detection. This setup was first tested in a lab design of experiments study with process relevant fluorophores proving its suitability for cell culture monitoring with LOD in the μg/L range. The sensor was then integrated into a CHO‐K1 cell culture process. The acquired fluorescence spectra of several batches were evaluated using multivariate methods. The resulting batch evolution models were challenged in deviating and “golden batch” validation runs. These first tests showed that the new sensor can trace the cells’ metabolic state in a fast and reliable manner. Cellular distress is quickly detected as a deviation from the “golden batch”.     

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.     

Establishment and characterization of a novel in vitro angiogenesis model using a microvascular endothelial cell line, F-2C, cultured in chemically defined medium

Summary The behavior of vascular endothelial cells (EC) is an important factor in the processes involved in angiogenesis, but the regulatory mechanisms of angiogenesis, especially underlying the tubulogenesis by EC are not yet clear. Although a number of in vitro experimental models of tubulogenesis have been developed by use of cultured EC, most of those models are too complex to be easily handled and further, the culture media are usually supplemented with serum, creating problems in interpretation of experimental results. To generate a simple in vitro angiogenesis study model under serum-free culture conditions, we adapted a murine microvascular endothelial cell line, F-2, to a chemically defined medium, Cos Medium 001, and successfully established a subline of F-2, designated F-2C, which revealed a unique growth pattern. In Cos Medium 001, F-2C proliferates in a cobblestone pattern at an early growth stage, but, at a late growth stage, spontaneously differentiates to form three-dimensional honeycomblike tubular structures without the supplementation of any specific factors. The cell aggregation activity of F-2C in the presence of Ca²⁺ was much greater than that of F-2. The amount of subendothelial matrix deposited by F-2C was significantly higher than that by F-2, and increased prominently after the F-2C cells reached the differentiating stage of tubulogenesis. These findings indicate that F-2C is a new EC line in which tubulogenesis is spontaneously induced by the marked deposition of basement membrane analog to the subendothelial matrix and by the enhancement of presumable cadherin activity. We suggest that this cell line, F-2C, represents a simple and useful in vitro angiogenesis model.     

Chromosomal and proteome analysis of a new T24‐based cell line model for aggressive bladder cancer

Cell line models aid in understanding cancer aggressiveness. The aim of this study was the establishment of a metastatic variant (T24M) of the T24 bladder cancer cell line and its initial characterization at chromosomal and proteomic levels. T24M were spontaneously developed in mice from T24 cells, following cycles of subcutaneous injections and culture in vitro. Transwell migration assays and injections in mice revealed increased migration and tumorigenic properties of T24M compared to the T24 cells. Cytogenetic analysis demonstrated that T24M retained several karyotypic characteristics of the parental cells and also acquired novel chromosomal aberrations related to aggressive bladder cancer. Proteomic analysis of the T24 and T24M cells by 2‐DE and MS led to the generation of their 2‐DE proteomic map and revealed differences in multiple proteins. These include proteases of the lysosomal and proteasome degradation pathways, mitochondrial and cytoskeletal proteins. The 2‐DE findings were confirmed by immunoblotting of cell lysates and immunohistochemistry of bladder cancer tissue sections for cathepsin D and activity assays for proteasome. Collectively, our results suggest that the T24M cells reflect many known chromosomal and proteomic aberrations encountered in aggressive bladder cancers but also provide access to novel findings with potentially clinical applications.     

Establishment and characterization of a cell line (SS78) from a human renal cell carcinoma

Summary A new cell line, SS78, was established from a primary renal cell carcinoma of a Caucasian male. The tissue was dispersed with collagenase, and viable cells were separated by flotation on a Ficoll-Hypaque gradient. In culture, the SS78 cells retained a distinct epithelial morphology, and no fibroblastlike cells were seen. The cultured cells were aneuploid with a modal chromosome number of 80 and had several marker chromosomes. Inoculation of the cultured cells into athymic nude mice caused tumors at the sites of inoculation. This research was supported in part by Grants CA 15972 and CA 14930 from the National Cancer Institute through the National Bladder Cancer Project and by the Medical Research Service of the Veterans Administration.     

Establishment and characterization of human glioblastoma cell line (HUBT-n)

We succeeded in primary culture of 3 in 4 cases of glioblastomas. The long-term passage cultures were not done from the primary cultures of original tumor, but glioblastoma cell line (HUBT-n) was established from a xenograft of nude mouse. This line grew well without interruption for 4 years and was subcultivated over 120 times. The cells were spindle like or round in shape and neoplastic and pleomorphic features contained glial fibrillar acid protein (GFAP) and S-100 protein and grew multilayering without contact inhibition. A bough-shaped long projection was noted from a small cell. One of the characteristics of the HUBT-n cells was existence of well developed intermediate filaments in their cytoplasm. The cells proliferated rapidly, and the population doubling time was about 32 hours. The chromosome number showed a narrow distribution of diploid range. Abnormal constitution was observed in all cells by G-band karyotyping. The culture cells were easily transplanted into the subcutis of nude mouse and produced the tumor resembling the original tumor.     

一株新的人肾癌原代细胞系的建立及其生物学特性鉴定

目的:取肾癌病人标本建立一株新的人肾癌细胞系,初步对该细胞系进行鉴定,为进一步的肾癌基础研究提供实验模型.方法:2008-2009年间共采集20例肾癌新鲜手术标本,于手术后1 h内将每例标本切取四块0.5cm× 0.5 cm× 0.5 cm组织块,分别包埋于2只裸鼠的右后肢皮下及背部皮下,连续传代3次,取移植瘤体外培养,记录细胞株的生长曲线,测定细胞集落形成率,对细胞进行DNA含量测定,进行染色体分析及病理学检查.结果:其中1只裸鼠皮下移植瘤生长,继续传代,肿瘤生长速度明显加快.取移植瘤标本体外培养得到肾癌细胞系XJG-9201.形态结构,分化程度与原发瘤一致,染色体众数为65.细胞群体倍增时间为38.2h,细胞周期分析G1期62.7%,G2期11.2%,S1期25.3%,集落形成率为70%.结论:肾癌细胞系XJG-9201与原发癌保持相同的生物学特性,体外连续传代1年以上传115代.细胞形态不变,生长周期恒定,已成为一个稳定的细胞系.     

Establishment and characterization of the RMG-V cell line from human ovarian clear cell adenocarcinoma

A cell line, designated as RMG-V, was established from a patient with clear cell adenocarcinoma of the ovary. The cell line has grown without interruption and has been propagated continuously by serial passaging (more than 36 times) over 5 years. The cells are spindle-shaped, display neoplastic and pleomorphic features, and grow in a jigsaw puzzle-like arrangement while forming monolayers without contact inhibition. These cells proliferate rapidly, and the population doubling time is about 15.5 hours. The number of chromosomes ranges between 77 and 85, with a modal number of 83.     

Establishment and characterization of a cell line from embryos of the Indianmeal moth,Plodia interpunctella

A cell line, UMN-PIE-1181, initiated in November, 1981, from embryos of a malathion-resistant strain of Indianmeal moth, Plodia interpunctella, was in the 83rd passage on January 28, 1985. The line consists of single, small, fibroblastlike cells that are polyploid with chromosome numbers ranging from 56 to 180. Growth rate is dependent on seeding density, there being no growth at or below seeding densities of $\text{2 ×}\text{10}{}^5\text{5}\text{,}\text{ml}$; optimum growth requires a fetal bovine serum concentration of at least 5%. Twenty-nine isozymes were examined. Five enzymes from the cell lines resolved well and subsequently were compared to enzymes extracted from 4-day-old embryos and other life stages of the insects. Phosphomannose isomerase, malic enzyme, malate dehydrogenase, phosphoglucose isomerase, and glucose-6-phosphate dehydrogenase in extracts from the cultured cells and from the insects had identical patterns. Two bands for glutamate-oxalacetate transaminase, present in the cell line, were not observed in the tissue extracts. Furthermore, lactate dehydrogenase from the cultured cells appeared as four bands but was not detectable in any of the samples run from the various life stages of the insects.     

Establishment and characterization of a noradrenergic adrenal chromaffin cell line, tsAM5NE, immortalized with the temperature-sensitive SV40 T-antigen

We established a clonal adrenal medullary cell line, named tsAM5NE, from transgenic mice harbouring the temperature-sensitive Simian virus 40 large T-antigen gene, under the control of the tyrosine hydroxylase promoter. tsAM5NE cells conditionally grew at a permissive temperature of 33°C and exhibited the noradrenergic chromaffin cell phenotype. To understand the characteristics of tsAM5NE cells, we first examined the responsiveness of the cells to ligands of the GDNF (glial cell line-derived neurotrophic factor) family. tsAM5NE cells proliferated at the permissive temperature of 33°C in response to either GDNF or neurturin, but not artemin or persephin. At the non-permissive temperature of 39°C, GDNF or neurturin caused tsAM5NE cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, LIF (leukaemia inhibitory factor) did not affect the GDNF-mediated cell proliferation at 33°C, but promoted the survival and differentiation of GDNF-treated cells at 39°C. In the presence of GDNF plus LIF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal markers, indicating that the cells had indeed undergone neuronal differentiation. Thus, we demonstrated that tsAM5NE cells had the capacity to terminally differentiate into neuron-like cells in response to GDNF plus LIF when the oncogene was inactivated by the temperature shift. Thus, this cell line provides a useful model system for studying the mechanisms regulating neuronal differentiation.