Establishment of a CSF-producing cell line from a human gastric carcinoma and characteristics of the CSF produced by that cell line

A human cell line producing colony-stimulating factor has been established in vitro from a human gastric carcinoma. The cell line was transplantable into nude mice which developed a marked neutrophilia. The cell line has been maintained for three years. The cells grew in a monolayered sheet and produced colony-stimulating factors that enhanced the formation of granulocyte and monocyte colonies in vitro with mouse bone marrow cells as the target and granulocyte colonies with human bone marrow cells as the target.     

Cellular retinaldehyde-binding protein-like (CRALBPL), a novel human Sec14p-like gene that is upregulated in human hepatocellular carcinomas, may be used as a marker for human hepatocellular carcinomas

Sec14p-like lipid-binding domain (SEC14 domain) is an evolutionarily conserved protein domain often found in secretory proteins, such as Saccharomyces cerevisiae phosphatidylinositol transfer protein Sec14p, and in lipid-regulated proteins, such as GTPase-activating proteins, guanine nucleotide exchange factors, and neurofibromin. We have cloned a novel human gene, cellular retinaldehyde-binding protein-like (CRALBPL), containing SEC14 domain from the cDNA library of human fetal brain. The RT-PCR expression pattern of 16 adult human tissues indicated that CRALBPL was only expressed in brain, while it was expressed in all of seven human carcinoma cell lines we used, especially in human gastric adenocarcinoma cell line, human rhabdomyoma cell line, human hepatocellular carcinoma (HCC) cell line, and human prostatic carcinoma cell line. Further, we found that CRALBPL has a remarkably more abundant RT-PCR expression pattern in human HCC cell lines than in normal human liver cell line, and the same result was gained when RT-PCR expression patterns between human HCC specimens and normal human liver specimens were compared. We also found that CRALBPL is located mainly in cytoplasm in human liver cell line L-02, which is consistent with the common function of Sec14p-like domain family. Our results show that CRALBPL may be used as a marker for human HCCs.     

Periodic fluctuations in proliferation of SV-40 transformed human skin fibroblast lines with prolonged lifespan

A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation. These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.Abbreviations I-SF immortalized human skin fibroblasts - T-SF transformed human skin fibroblasts - FBS fetal bovine serum     

Induction of the switch in the synthesis of immunoglobulin classes in the RPM 1-6410t lymphoblast cell line and its clones as affected by fetal calf serum factors

We have found that human lymphoblastoid cell line RPMI-6410t is a biochemical mutant for gene of thymidine kinase and has chromosome markers in the karyotype. Thus, this cell line can be used as a partner in somatic hybridization, in particular for producing hybridomas, synthesizing human monoclonal antibodies. We have discovered that line RPMI-6410t carries HLA-A2, -B7 and -B12 antigens of human histocompatibility complex on the cell surface. The cell membrane of this cell line contains immunoglobulins of M and D classes. RPMI-6410t cells secrete IgM molecules. It is demonstrated that induction of the switch of immunoglobulin heavy-chain classes by the factors of foetal calf serum takes place in the cells of RPMI-6410t line. Thus, the corresponding stage of B-lymphocytes differentiation in vivo is reproduced in 6410t line in vitro.     

Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line

In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.     

牛至油体外抗肿瘤活性研究

目的观察牛至油对肿瘤细胞株的生长抑制作用。方法采用MTT法检测不同浓度的牛至油对体外培养的多种肿瘤细胞株的生长抑制作用,计算半数抑制浓度(IC50)。结果不同浓度牛至油作用后,人肝癌细胞系HepG2、人子宫颈癌细胞系JTC-26和肺癌细胞系A549出现增殖阻滞。MTT法确定牛至油对肝癌HepG2的IC50为118μg/ml;人子宫颈癌JTC-26的IC50为118μg/ml;肺癌A549的IC50为59μg/ml。结论牛至油具有体外抗肿瘤活性。     

肉桂油成分分析及肉桂醛体外抗肿瘤活性研究

目的探讨肉桂醛对不同肿瘤细胞株的生长抑制作用。方法通过水蒸气蒸馏法提取肉桂油,用气相色谱—质谱联用仪进行肉桂油成分分析;采用噻唑蓝（MTT）比色法检测肉桂醛对体外培养的人宫颈癌细胞系HeLa细胞株、人肺癌细胞系A-549细胞株和人肝癌细胞系HepG2细胞株的生长抑制作用,计算半数抑制浓度（IC50）。结果肉桂油的收率为1.96%,分析了肉桂油中的10种成分,主要为肉桂醛,占总馏出峰面积的93.94%;肉桂醛能抑制人宫颈癌细胞系HeLa细胞、人肺癌细胞系A-549细胞和人肝癌细胞系HepG2细胞增殖,且呈剂量依赖性,IC50值分为0.20、0.36和0.73 mg/mL。结论肉桂醛具有体外抗肿瘤活性。     

A lymphoblastoid cell line with high phagocytic activity established from peripheral blood of a patient with chronic myelocytic leukemia in blast crisis

A human hematopoietic cell line (K-23-M) was established from a patient with chronic myelocytic leukemia in blast crisis. Morphologically, the cultured cells were lymphoblastoid cells that produced IgA and were Epstein-Barr viral nuclear antigen positive. But they showed high phagocytic activity to glutaraldehyde-treated sheep red cells and had properties of a monocyte or macrophage that included surface Fc receptors, alpha-naphthyl butyrate esterase positivity blocked by NaF, migration in soft agar and the ability to attach to a glass surface. Lysozyme secretion was absent, and chromosomes were diploid and Ph1 negative. This cell line is unique in that it has strong phagocytic activity. Its existence shows that lymphoblastoid cell line may be a more important cell line for the study of human hematopoietic cells than previously has been believed.     

Two distinct cytotoxic T lymphocyte subpopulations in patients with Vogt-Koyanagi-Harada disease that recognize human melanoma cells

The functional properties of cytotoxic lymphocytes from patients with Vogt-Koyanagi-Harada disease ( VKH ) specific for human melanoma cells (P-36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P-36 (SK-MEL-28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa-S3 cell line) or against a mouse melanoma cell line (B-16 cell line) originating from a C57BL/6 strain mouse or against the EL-4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P-36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti-human Leu-1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti-human Leu-2a or Leu-3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P-36 human melanoma cell line is, therefore, mediated by T cells bearing Leu-1+ Leu-2a+ or Leu-1+ Leu-3a+ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti-Leu-2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti-Leu-3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P-36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu-2a and Leu-3a.     

The symbiotically essential cbb(3)-type oxidase of Bradyrhizobium japonicum is a proton pump

The male germ line stem cell is the only cell type in the adult that can contribute genes to the next generation and is characterized by postnatal proliferation. It has not been determined whether this cell population can be used to deliberately introduce genetic modification into the germ line to generate transgenic animals or whether human somatic cell gene therapy has the potential to accidentally introduce permanent genetic changes into a patient's germ line. Here we report that several techniques can be used to achieve both in vitro and in vivo gene transfer into mouse male germ line stem cells using a retroviral vector. Expression of a retrovirally delivered reporter lacZ transgene in male germ line stem cells and differentiated germ cells persisted in the testis for more than 6 months. At least one in 300 stem cells could be infected. The experiments demonstrate a system to introduce genes directly into the male germ line and also provide a method to address the potential of human somatic cell gene therapy DNA constructs to enter a patient's germ line.     

Endocrine differentiation by a human rectal adenocarcinoma cell line (HRA-19)

Abstract. Colorectal epithelium is composed of a variety of cell types, including absorptive. mucous and endocrine cells. All of these cell types are thought to arise from stem cells located at the base of the crypt. However, the factors which control these differentiation pathways are poorly understood. In attempts to establish differentiated in vitro systems, one approach has been to grow primary human colorectal adenocarcinomas as cell lines. Some of these cell lines retain a sufficient number of the differentiated features of their tissue of origin to make them useful experimental systems for studying differentiation. This study describes the characterisation of such a cell line, the HRA-19 line. HRA-19 cells were derived from a primary human rectal adenocarcinoma. The cells grew as monolayers in vitro on tissue-culture plastic and remained pleomorphic even after 150 passages in vitro. Some colonies of cells expressed alkaline phosphatase activity, an enzyme normally expressed in vivo by absorptive cells of the upper crypt and surface epithelium. No evidence of differentiation into goblet or endocrine cells was obtained in monolayer cultures of HRA-19 cells. Xenografts of this cell line contained cells with the ultrastructural characteristics of absorptive and endocrine cells. These endocrine cells exhibited Grimelius silver staining, displayed formaldehyde-induced fluorescence and contained many basally located, electron-dense granules. When grown as monolayers, clones of this cell line retained the heterogeneity with respect to morphology and alkaline phosphatase expression of the parent cell line. It is proposed that this cell line is derived from malignant progenitor cells which retain the ability to differentiate. This cell line has properties not previously reported for a human colorectal adenocarcinoma cell line and will be useful for studying the control of differentiation in colorectal epithelial cells.     

Human tumor and rodent-human hybrid cells with an increased number of active human NORs.

A human fibrosarcoma line, HT1080-6TG, with a near diploid number of chromosomes, has an average of 7.3 chromosomes with an Ag-stained nucleolus organizer region (NOR). Cells of this line with an increased number of chromosomes have an increased number of Ag-stained NORs. This cell line has been used as the human parent in constructing mouse-human and rat-human hybrids that segregate rodent chromosomes. The hybrid ccell lines, which have 100 or more chromosomes per cell, show a proportionate increase in the number of Ag-stained NORs (means, 11.4--16.8). The frequency of association of acrocentric chromosomes increases in a similar fashion. There is no evidence of inactivation of human NORs in these cells.     

Genetics of the mammalian phenylalanine hydroxylase system. IV. Evidence of phenylalanine hydroxylase in a cultured human hepatoma cell line

We report here the identification of a cultured human hepatoma cell line which possesses an active phenylalanine hydroxylase system. Phenylalanine hydroxylation was established by growth of cells in a tyrosine-free medium and by the ability of a cell-free extract to convert [¹⁴C]phenylalanine to [¹⁴C]tyrosine in an enzyme assay system. This enzyme activity was abolished by the presence in the assay system of p-chlorophenylalanine but no significant effect on the activity was observed with 3-iodotyrosine and 6-fluorotryptophan. Use of antisera against pure monkey or human liver phenylalanine hydroxylase has detected a cross-reacting material in this cell line which is antigenically identical to the human liver enzyme. Phenylalanine hydroxylase purified from this cell line by affinity chromatography revealed a multimeric molecular weight (estimated 275,000) and subunit molecular weights (estimated 50,000 and 49,000) which are similar to those of phenylalanine hydroxylase purified from a normal human liver. This cell line should be a useful tool for the study of the human phenylalanine hydroxylase system.     

逆转录病毒载体介导入GM-CSF基因转染肿瘤细胞

本文应用逆转录病毒载体pZIP Neo SV(X)介导人GM-CSF基因转染肿瘤细胞获得表达。经Lipofectin将含有人GM-GSF基因的重组逆转录病毒载体pZIP-GM转染兼性病毒包装细胞系PA317,继之用病毒收获液感染人肝癌细胞SMMC7721和人胃癌细胞BGC-823,经GM-CSF依赖细胞株TF1测活和双抗夹心法ELISA测定表明:人GM-CSF基因在人肿瘤细胞中获得稳定高效表达。为进一步建立GM-CSF的转基因治疗模型提供了基础。     

Morphologic,immunologic, biochemical,and cytogenetic characteristics of the human glioblastoma-derived cell line,SNB-19

Summary Human glioma-derived cell cultures and lines have proven to be of significant value in the study of the basic properties that contribute to the highly malignant, invasive and angiogenic phenotype of glioblastoma multiforme tumors. It is frequently difficult to establish lines that retain glial tumor properties in long term culture. The SNB-19 cell line has maintained and exhibited properties of transformation, differentiation, autocrine growth response, and tumorigenesis while remaining in culture for over 13 yr and undergoing over 200 passages. This human line has been utilized in a wide range of studies related to the basic properties of human glioblastoma multiforme. In this report, we summarize the immunologic, biochemical, and cytogenetic properties of this versatile cell line and its utility for additional mechanistic investigation into the pathophysiology of the progression of human malignant gliomas.     

人表皮角质形成细胞自噬功能细胞模型的构建及鉴定

目的: 建立稳定表达GFP-LC3的人永生化角质形成细胞HaCaT细胞系。方法: 将构建的pcDNA3.1-GFP-LC3 真核表达载体转入HaCaT细胞,经G418筛选稳定表达的细胞系。HaCaT细胞中GFP-LC3的表达分别用荧光显微镜与Western blot方法检测,并利用该稳定表达的细胞系观察验证Rapamycin对细胞发生自噬透射电镜超微结构的变化。结果: 获得了3株转染并经G418反复筛选的HaCaT细胞系,在倒置荧光显微镜下观察可见绿色荧光细胞的表达率在95% 以上,Western blot结果证实了GFP-LC3融合蛋白的表达。Western blot和激光共聚焦显微镜均证明Rapamycin可以诱导自噬的发生。透射电镜细胞超微结构的观察表明Rapamycin可以有效地诱导HaCaT-LC3细胞自噬的发生。结论: 成功构建GFP-LC3稳定表达的HaCaT系,该细胞系可以作为研究人角质形成细胞自噬功能的一种细胞模型。     

Succinoyl trehalose lipid induced differentiation of human monocytoid leukemic cell line U937 into monocyte-macrophages

A novel type of succinoyl trehalose lipid (STL-1) prepared from n-hexadecane-culture ofRhodococcus erythropolis SD-74 markedly inhibited the growth of a human monocytoid leukemic cell line, U937, and induced its morphological alteration along a monocyte-macrophage lineage. STL-1 markedly increased differentiation-associated characteristics in macrophage, such as nitroblue tetrazolium reducing ability, appearance of Fc receptor, phagocytic activities in U937. Furthermore, U937 cells, which were activated with STL-1 exhibited cytotoxic activity against human lung carcinoma cell line A549. However, STL-1 did not affect growth of a normal human fetal lung cell line TIG-1. The individual components of STL-1, neither sugar moiety nor fatty acids in the free form, were effective at inducing the differentiation of U937 cell. From these results, we concluded that STL-1 has low cytotoxicity against normal human cells and the ester molecule itself is responsible for the activity of inducing differentiation of human monocytoid leukemic cell line U937 into monocyte-macrophage which results in the stimulation of the production of some cytotoxic substances.