Establishment and characterization of a cell line derived from a spontaneous murinelung carcinoma (M109)

Summary The Madison lung (M109) tumor cell line, initiated from a “spontaneous”, anaplastic murine lung carcinoma, has been propagated continuously in vitro for more than 300 cell generations. Cytogenetic analysis revealed a mouse karyotype with a mode of 78 chromosomes (2n=40). Three distinct marker chromosomes were identified by trypsin-giemsa banding. The cells piled up in culture and had a short generation time and high plating efficiency. Electron microscopy revealed highly undifferentiated cells with little rough endoplasmic reticulum, an abundance of free polysomes, the presence of few and often oddshaped mitochondria, lipid bodies and phagocytic vacuoles. Virus particles of the C-type were found frequently. The subcutaneous transplantation of M109 cultured cells at a relatively low cell inoculum produced highly metastatic tumors in syngeneic BALB/c mice.     

高转移潜力食管癌细胞株的建立及其生物学特征

目的建立具有高转移潜力食管癌细胞株并研究其生物学特征。方法将食管癌细胞系EC109细胞悬液异位移植到SCID小鼠胃壁,约3个月后或动物濒临死亡时处死,行病理学解剖,将肉眼可见的纵隔淋巴结转移瘤块接种于SCID鼠皮下扩增,然后取小鼠皮下瘤组织块进行细胞培养,得到性状稳定的细胞株NMC109后,用MTT法分析细胞生长曲线,Western bloting法检测与细胞分裂增殖能力密切相关的TopoⅡα表达,酶谱法检测MMP-2和MMP-9的活性,划痕实验和Transwell体外移动实验检测细胞的移动能力。结果与母本细胞EC109相比,所获得的细胞株NMC109其增殖能力和TopoⅡα表达明显增强,MMP-9的活性明显升高,移动能力明显增强。结论获得了具有高转移潜力的食管癌细胞株。     

Cadmium transport through type II alveolar cell monolayers: contribution of transcellular and paracellular pathways in the rat ATII and the human A549 cells

Cadmium (CD) transport in alveolar type II (ATII) cells has been studied using two in vitro models widely used to investigate lung function: primary cultures of rat ATII cells and the human cell line A549. Nonlinear regression analyses of the uptake time-course of (109)Cd revealed: a zero-time accumulation, a fast process of accumulation which proceeds within minutes, and a much slower process which takes hours. This three-step mechanism was characterized by different parameter values under dishes-or filter-growth conditions. A higher initial uptake rate (v(i)) and equilibrium accumulation (A(max)) of (109)Cd were found in the rat ATII cells; these differences were not related to a higher level of adsorption onto the external surface of the cell membrane. Specific transport systems of similar capacity but different affinity (threefold higher in rat cells) were characterized. A significant transepithelial transport of (109)Cd, with similar P(coeff) in both cell models, could not be exclusively related to cellular metal release. Results on 3H-mannitol permeability together with (109)Cd efflux data strongly suggest a greater contribution of the paracellular pathways in Cd transport through A549 cell monolayers. These differences in transport properties between the two lung cell models may modify the dose-response curve for Cd toxicity.     

Isolation and Characterization of Subpopulations of Rat Ascites Hepatoma Cell Line of AH109A with Different Metastatic Potentials

Rat ascites hepatoma cell line of AH109A proved to be divided into two subpopulations with different invasive and metastatic potentials, when cultured in the medium containing allogeneic rat sera. One population adheres to the culture dish, actively extending pseudopodia, and the other remains in a floating state. Utilizing this character, we have separated these two populations. After three successive separation steps, adhesive AH109A cells and floating AH109A cells were obtained. Adhesive AH109A cells proliferated more rapidly and invaded more actively than did floating AH109A cells. Adhesive AH109A cells metastasized mainly to lung, while floating AH109A cells to mesentery, when intravenously injected into tail veins. Histological studies revealed that adhesive AH109A cells showed lymphatic metastases to lung. These results suggest that the two populations separated from parental AH109A cells provide good models for the study of tumor invasion and tissue-specific metastasis and that adhesive AH109A cells can be used for the creation of lymphatic metastasis model of rats.     

Establishment and characterization of two fetal fibroblast cell lines from the yak

The objective of this work was to not only establish two fetal fibroblast cell lines from yak lung and ear tissue using a primary explant technique and cell cryogenic preservation technology but also check for their quality and biological characteristics. The cells showed typical morphologic characteristics of fibrous and long spindle appearance. Outgrowth of fibroblast-like cells from the lung and ear explants was around 2 and 3 d, and reaching 90% confluence level was in the ninth day and the thirteenth day, respectively. Biological analysis showed that the average viability of the lung fibroblast cells (ear fibroblast cells) was 97.5% (95.0%) before freezing and 91.0% (89.5%) after thawing. Analysis of the growth of the fifth passage culture revealed an ??S??-shaped growth curve with the population doubling times of 30 h for lung fibroblast cell line and 35 h for ear fibroblast cell line. Karyotyping indicated the chromosome number of yak was 2n?=?60, comprising 29 pairs of autosomes and one pair of sex chromosomes (XY). All somatic chromosomes were telocentric autosomes except that the two sex chromosomes were submetacentric. Assays for bacteria, fungi, and mycoplasmas were negative. Immunocytochemical staining showed that the cells were positive for the expression of vimentin and negative for the expression of cytokeratin. In conclusion, two yak fetal fibroblast cell lines (YFLF and YFEF) from lung and ear explants are successfully established in culture. It will not only preserve the genetic resources of yaks at the cellular level but also provide valuable materials for somatic cell cloning and transgenic research.     

The influence of a substrate including extracellular matrix proteins on karyotypic variability in two cell lines of Indian muntjac skin fibroblasts

The influence of cell-culture conditions on numerical and structural karyotypic variability has been investigated in two Indian muntjac skin fibroblast “markerless” cell lines, M and MT. The cells were cultivated on a substrate consisting of extracellular matrix proteins (ECMs) synthesized by human mesenchymal stem cells (SC5-MSC). The character of cell distribution for the chromosome number of the cell line M changed after cultivation for 1 and 4 days as compared to control cells, which were cultured on a hydrophilic surface without ECM coating. These changes involve a significant decrease in frequency of cells with the modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Many new types of additional structural variants of the karyotype (SVK) appear. The MT cell line, differing from M line in the number of homologous chromosomes, exhibited a character of cell distribution similar to that of the M line for the chromosome number for only 1 day after cultivation on the ECM substrate, but not after 4 days under the same culture conditions, when no difference from the control cells was observed. Further cultivation of MT cells for 8 days did not change the character of cell distribution for the chromosome number relative to the control variant. The observed alterations seem to be due to disturbances in the correct chromosome-segregation process, which were caused by an abrupt shift in the cell-culture conditions. Analysis of the structural karyotypic variability revealed a significant increase in frequency of chromosomal aberrations in the M cell line for 1 and 4 days in culture on the ECM substrate as compared to the control cells. The frequency of dicentric chromosomes (telomeric associations) was increased and constituted more than 50% of all chromosome aberrations. No increase in frequency of chromosome aberrations was observed for MT cells cultured under the same conditions. It can be suggested that the differing by the karyotypic structure, but the genetically identical cell lines have different response to the substrate. In contrast to the M line, in the MT line, a fast normalization of numerical karyotypic characteristics and no enhancement of structural karyotypic variability takes place. This provides a possibility to cultivate an MT cell on the given protein substrate while maintaining a balanced karyotypic structure characteristic of MT cell line.     

Adenovirus vector-mediated FAM176A overexpression induces cell death in human H1299 non-small cell lung cancer cells

FAM176A (family with sequence similarity 176 member A) is a novel molecule related to programmed cell death. A decreased expression of FAM176A has been found in several types of human tumors in including lung cancers. In the present study, we investigated the biological activities of FAM176A on the human non–small cell lung cancer cell line H1299 cells. We constructed a recombinant adenovirus 5-FAM176A vector (Ad5-FAM176A) and evaluated the expression and anti-tumor activities in vitro. Cell viability analysis revealed that the adenovirus-mediated increase of FAM176A inhibited the growth of the tumor cells in a dose- and time-dependent manner. This inhibitory effect was mediated by both autophagy and apoptosis that involved caspase activation. In addition, cell cycle analysis suggested that Ad5-FAM176A could induce cell cycle arrest at the G2/M phase, all of which suggested that adenovirus-mediated FAM176A gene transfer might present a new therapeutic approach for lung cancer treatment. [BMB Reports 2014; 47(2): 104-109]     

Induction of G2/M cell cycle arrest and apoptosis by a benz[f]indole-4,9-dione analog in cultured human lung (A549) cancer cells

A synthetic benz[f]indole-4,9-dione analog, 2-amino-3-ethoxycarbonyl-N-methylbenz[f]indole-4,9-dione (SME-6), showed a potent growth inhibition of a panel of human cancer cell lines. The mechanism of action study revealed that the growth inhibitory effect of SME-6 was highly related to the induction of G2/M cell cycle arrest and apoptosis in human lung cancer cells (A549). These data may provide new information for understanding the mechanisms by benz[f]indole-4,9-diones-mediated antitumor activity.     

Establishment and characterization of a lung cancer cell line,SMC-L001, from a lung adenocarcinoma

Lung cancer cell lines are a valuable tool for elucidating lung tumorigenesis and developing novel therapies. However, the majority of cell lines currently available were established from tumors in patients of Caucasian origin, limiting our ability to investigate how cancers in patients of different ethnicities differ from one another in terms of tumor biology and drug responses. In this study, we established a human non-small cell lung carcinoma cell line, SMC-L001, and characterized its genome and tumorigenic potential. SMC-L001 cells were isolated from a Korean lung adenocarcinoma patient (male, pStage IIb) and were propagated in culture. SMC-L001 cells were adherent. DNA fingerprinting analysis indicated that the SMC-L001 cell line originated from parental tumor tissue. Comparison of the genomic profile of the SMC-L001 cell line and the original tumor revealed an identical profile with 739 mutations in 46 cancer-related genes, including mutations in TP53 and KRAS. Furthermore, SMC-L001 cells were highly tumorigenic, as evidenced by the induction of solid tumors in immunodeficient mice. In summary, we established a new lung cancer cell line with point mutations in TP53 and KRAS from a Korean lung adenocarcinoma patient that will be useful for investigating ethnic differences in lung cancer biology and drug response.     

Cadmium transport through type II alveolar cell monolayers: contribution of transcellular and paracellular pathways in the rat ATII and the human A549 cells

Cadmium (Cd) transport in alveolar type II (ATII) cells has been studied using two in vitro models widely used to investigate lung function: primary cultures of rat ATII cells and the human cell line A549. Nonlinear regression analyses of the uptake time-course of ¹⁰⁹Cd revealed: a zero-time accumulation, a fast process of accumulation which proceeds within minutes, and a much slower process which takes hours. This three-step mechanism was characterized by different parameter values under dishes-or filter-growth conditions. A higher initial uptake rate (v_i) and equilibrium accumulation (A_max) of ¹⁰⁹Cd were found in the rat ATII cells; these differences were not related to a higher level of adsorption onto the external surface of the cell membrane. Specific transport systems of similar capacity but different affinity (threefold higher in rat cells) were characterized. A significant transepithelial transport of ¹⁰⁹Cd, with similar P_coeff in both cell models, could not be exclusively related to cellular metal release. Results on ³H-mannitol permeability together with ¹⁰⁹Cd efflux data strongly suggest a greater contribution of the paracellular pathways in Cd transport through A549 cell monolayers. These differences in transport properties between the two lung cell models may modify the dose-response curve for Cd toxicity.     

Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells

In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.     

Increase in a 55-kDa keratin-like protein in the nuclear matrix of rat liver cells during proliferative activation

We have identified a protein (p55) with a molecular weight of 55 kDa and a pI of 6.2, which was strongly increased in the nuclear matrix of rat liver cells during proliferative activation. This protein is highly insoluble since it could not be solubilized either by detergents or by alkaline extraction. We have obtained three partial amino acid sequences which revealed that p55 has a high homology with cytokeratins. Polyclonal antibodies raised against p55 were used to carry out Western blot and immunocytochemical studies which indicated that p55 was localized only in the nuclei, specifically in the nuclear matrix. Autoradiographic experiments revealed that not all the cells presenting an increase in p55 incorporated [3H]thymidine, indicating that this protein is not related to DNA replication. Immunocytochemical studies also revealed that during mitosis p55 is localized surrounding the chromosomes and associated with the mitotic apparatus, suggesting that p55 is involved in the separation of chromosomes during cell division.     

Evidence for a relatively random array of human chromosomes on the mitotic ring

We used fluorescence in situ hybridization (FISH) to study the positions of human chromosomes on the mitotic rings of cultured human lymphocytes, MRC-5 fibroblasts, and CCD-34Lu fibroblasts. The homologous chromosomes of all three cell types had relatively random positions with respect to each other on the mitotic rings of prometaphase rosettes and anaphase cells. Also, the positions of the X and Y chromosomes, colocalized with the somatic homologues in male cells, were highly variable from one mitotic ring to another. Although random chromosomal positions were found in different pairs of CCD-34Lu and MRC-5 late-anaphases, the separations between the same homologous chromosomes in paired late-anaphase and telophase chromosomal masses were highly correlated. Thus, although some loose spatial associations of chromosomes secondary to interphase positioning may exist on the mitotic rings of some cells, a fixed order of human chromosomes and/or a rigorous separation of homologous chromosomes on the mitotic ring are not necessary for normal mitosis. Furthermore, the relative chromosomal positions on each individual metaphase plate are most likely carried through anaphase into telophase.     

Requirement of human chromosomes 19, 6 and possibly 3 for infection of hamster x human hybrid cells with baboon M7 type C virus.

The replication pattern of the endogenous baboon type C virus M7 was studied in 29 primary Chinese hamster × human hybrid clones generated with leukemic cells from two different patients with acute lymphoblastic or myeloblastic leukemia. There was no evidence of viral particulate RDDP or M7 antigen before viral infection. M7 virus replicated in human and some hybrid cells but not in Chinese hamster cells, indicating that M7 requires dominantly expressed human gene(s) for replication. Enzyme and cytogenetic analyses show that a gene(s) coded for by human chromosome 19 is necessary for M7 infection of these hybrids. Detailed cytogenetic correlations revealed, however, that the chromosome 19+/M7 + hybrid clones with intact chromosomes also had copies of chromosomes 3 and 6. Previously, Bevi, the putative integration site for M7 virus, has been assigned to human chromosome 6. Many clones with various combinations of chromosomes 3 and 6 lacked chromosome 19, however, and failed to replicate exogenously applied M7 virus, while tests of 27 secondary clones showed that M7 markers co-segregated with chromosome 19 markers. These findings all confirm the need for a chromosome 19-coded function in Chinese hamster × human hybrids. In addition, the yield of viral particulate RDDP produced into the culture fluid was 50–100 fold less per viral antigen-positive cell in the hybrids compared with human cells. Thus some form of regulation of viral components exists in the hybrid cells. When the virus replicating in hybrid cells was transferred back to human cells, this regulation was relaxed and the yield of RDDP per FA(+) cell greatly increased. We conclude that human chromosomes 6 and 19 code for functions involved in M7 virus metabolism, and we cannot exclude a function coded for by chromosome 3.     

Cell cycle related behavior of a chromosomal scaffold protein in MDCK epithelial cells

Because the mechanisms that govern mitosis are a key to the understanding of cell growth, the proteins associated with chromosomes specifically during this phase have received thorough attention. In the present work we report an Mr 58000 protein in MDCK epithelial cells, recognized by a monoclonal antibody (LFM-1) that decorates chromosomes during M-phase. Cell fractionation methods followed by immunoblotting and immunofluorescence showed that this protein is associated with the nuclear fraction. Biochemical extraction procedures on isolated metaphase chromosomes from nocodazole-synchronized cells indicated that the Mr 58000 protein behaves as a chromosomal scaffold protein, that is, it remains in the pellets after high salt (2 M NaCl) or 3–5 diiodosalicylic acid treatments, even in DNAse pre-digested samples. In addition, confocal microscopy of those chromosomes revealed the LFM-1 epitopes distributed on the external surface and the axis of chromatids. Parallel analysis of interphase nuclei revealed LFM-1 epitopes inside G1-, but excluded from G2-phase nuclei. These results were independently confirmed on nuclei sorted by flow cytometry and in cell populations synchronized by release of G1-/S-phase hydroxyurea arrest. The Mr 58000 and a minor Mr 38000 protein (which was enriched only in mitotic chromosomes of synchronized cells) were analyzed by Edman degradation. They shared the sequence at the amino-terminal end but failed to show total homology with known proteins. These results suggest that LFM-1 antigens fit some of the predictions of the licensing factor model, and may have a role in cell cycle dependent events.     

Lentiviral delivery of a shRNA sequence analogous to miR-4319/miR-125-5p induces apoptosis in NSCLC cells by arresting G2/M phase

In this study, we explored the therapeutic potential of microRNA (miR) analogs against non–small-cell lung cancer (NSCLC) using lentiviral delivery of short hairpin RNA (shRNA). By using A549 as a model cell line, we used analogs and mimics of miR-4319/miR-125-5p to target the tumorigenic RAF1 gene. Lentiviral vectors carrying shRNA of a highly efficient miRNA analog of miR-4319/miR-125-5p, Analog2, were constructed to infect A549 cells. Our results showed that, compared with the noncancerous bronchial epithelial cell line 16HBE, lentivirus delivering Analog2 shRNA induced significant G2/M arrest and subsequent apoptosis in A549 cells, but not in 16HBE cells. Western blot analysis revealed that key factors regulating cell cycle were downregulated following RAF1 inhibition. In vivo xenograft experiments showed that lentivirus carrying Analog2 shRNA markedly decreased tumor size. Therefore, lentiviral delivery of Analog2 shRNA is a valid RNA interference-based treatment against NSCLC with high potency and specificity.     

沉默单核细胞趋化蛋白-1基因降低人食管癌EC109细胞的迁移及侵袭能力

单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)是白色脂肪细胞分泌的炎症趋化刺激因子,属于趋化因子CC亚族,可促进肿瘤血管形成和细胞外基质降解,从而促进肿瘤细胞的浸润与转移。沉默MCP-1基因可显著抑制恶性肿瘤生长及转移,但其作用的分子机制尚不完全清楚。本研究应用小干扰RNA技术沉默人食管癌EC109细胞中MCP-1表达。细胞划痕试验显示,与对照组相比,沉默MCP-1基因可明显抑制食管癌EC109细胞迁移能力。Transwell 侵袭实验显示,沉默MCP-1基因后,EC109细胞侵袭能力降低。Western 印迹试验和RT-PCR试验揭示,沉默MCP-1基因后,细胞中MMP-7、MMP-9、TGF-β1及VEGF表达水平显著下降。研究结果提示,沉默MCP-1基因可通过抑制MMP-7、MMP-9、TGF-β1及VEGF表达,降低癌细胞迁移及侵袭能力。