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Cell lines which are infected with retrovirus are resistant to superinfection by a related retrovirus. Packaging of whole virions within synthetic lipid vesicles allows efficient infection of such resistant cell lines. This system is more efficient in introducing encapsulated virus into infected cells than into uninfected cells.
Cell surface expression of HLA class I (including non-classical HLA-G) in JEG3 (choriocarcinoma cell line) was blocked by stable transfection with the sequence encoding the Herpes simplex virus protein, infected cell peptide 47 (ICP47) inserted into a vector pCEP4. Intracellular expression of ICP47 protein in ICP47-transfected cells was demonstrated. The lack of HLA cell surface expression was likely to be due to blockage of peptide transport from the cytoplasm into the endoplasmic reticulum by ICP47. ICP47 is known to block the heterodimeric transporter associated with antigen processing (formed from TAP1 and TAP2). Western blotting with a polyclonal antibody to the C-terminus of TAP1 showed high expression of TAP1 in BeWo and JEG3, but not JAR cells, expression that was strongly upregulated by gamma-interferon. Gamma-interferon also upregulated the cell surface expression of HLA class I. TAP1 was strongly expressed in MC2 and MC3 extravillous cytotrophoblast cell lines immortalised with the SV40 large T antigen. The results suggest a role for non-classical HLA in the presentation of antigenic peptides to the immune system.
Cell lines derived from the major morphological types of human lung cancer were tested for their ability to utilize the SV40 enhancer/early promoter. These cell lines were transfected with a recombinant plasmid containing a reporter gene, coding for chloramphenicol acetyltransferase (CAT), under the control of the SV40 enhancer/early promoter. The transfected cells were then assayed for CAT activity. Non-small-cell carcinomas, especially squamous carcinomas, were found to be 2-3 orders of magnitude more efficient in utilizing the SV40 enhancer/early promoter than small-cell carcinomas. The presence of different SV40 enhancer/early promoter specific DNA-binding nuclear factors in squamous and small-cell carcinomas was demonstrated in gel mobility shift experiments. These observations seem to suggest that the set of transcriptional regulatory factors associated with squamous carcinomas may be distinct from that associated with small-cell carcinomas.
Cell lines derived from the rat suprachiasmatic nucleus (SCN) were screened for circadian clock properties distinctive of the SCN in situ. Immortalized SCN cells generated robust rhythms in uptake of the metabolic marker 2-deoxyglucose and in their content of neurotrophins. The phase relationship between these rhythms in vitro was identical to that exhibited by the SCN in vivo. Transplantation of SCN cell lines, but not mesencephalic or fibroblast lines, restored the circadian activity rhythm in arrhythmic, SCN-lesioned rats. Thus, distinctive oscillator, pacemaker, and clock properties of the SCN are not only retained but also maintained in an appropriate circadian phase relationship by immortalized SCN progenitors.
Cell contact with the extracellular matrix component, hyaluronan, plays a pivotal role in glioma cell invasion and proliferation. Although it is well established that glioma cells can bind hyaluronan to their surface via the expression of CD44, the cellular responses following ligand-receptor interaction remain poorly understood. Given that a large proportion of human high grade gliomas over express the epidermal growth factor receptor (EGFR) and ErbB2, this study aimed to investigate whether an interaction exists between CD44 and these receptor tyrosine kinases. Here we present evidence that CD44 co-immunoprecipitates with EGFR and ErbB2 in the glioma cell lines U87MG and SMA560. Hyaluronan treatment mediated the rapid and transient phosphorylation of extracellular signal regulated kinases 1 and 2 (ERK1 and ERK2) in glioma cell lines. This response to hyaluronan was augmented by the co-expression of EGFR. EGFR also differentially modified the hyaluronan induced expression of a number of genes associated with cellular invasion and proliferation. Northern blot analysis demonstrated that genes encoding urokinase type plasminogen activator (uPA), urokinase type plasminogen activator receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinases (TIMP-1) and c- myc were up-regulated in response to hyaluronan. Furthermore, zymographic analysis revealed increased levels of uPA in the conditioned medium of hyaluronan stimulated cells. These results indicate a novel functional relationship between CD44 and EGFR in glioma cell lines. The capacity of CD44 to form stable complexes with receptor tyrosine kinases may provide a versatile system for the regulation of cellular invasion and proliferation that allows hyaluronan to activate signal transduction pathways and modulate gene expression via an EGFR-dependent manner. These findings provide new insights into the mode by which hyaluronan regulates the malignant phenotype and also suggest a role for EGFR-CD44 interactions in glial tumorigenesis.
Cells of the weakly CD14 positive human B cell line RPMI 8226, clone 1, will mobilize NF-kappaB (p50/p65 and p50/p50) proteins and produce TNF mRNA when stimulated with lipopolysaccharide (LPS). When such cells are precultured with a low amount of LPS (50-250 ng/ml) for 3 - 4 days followed by a secondary stimulation with a high dose of LPS (1 microg/ml) then the cytokine expression is strongly reduced, i. e. the cells have become tolerant. Western blot analysis of proteins of the NF-kappaB/rel family demonstrates cytoplasmic p50 and p65 for naive B cells plus a low level of p52. While with tolerance induction the pattern of p50 and p65 proteins remains essentially unchanged, the LPS tolerant 8226 cells show a dramatic increase of both p52 protein and its p100 precursor in the cytosol. This p52 is found strongly upregulated in Western blots of extracts from purified nuclei of tolerant cells. Also, gelshift analysis with the -605 kappaB motif of the human TNF 5'-region shows an additional high mobility complex in LPS tolerant cells -a complex that is supershifted with an anti-p52 antibody. Functional analysis with the -1064 TNF 5'-region in front of the luciferase reporter gene demonstrates that transactivation of the TNF promoter is strongly reduced in tolerant cells. Also, overexpression of p52 will suppress activity of TNF promoter reporter gene constructs. Taken together these data show that tolerance to LPS in the human RPMI 8226 B cell line involves upregulation of the p52 (NF-kappaB2) gene, which appears to be instrumental in the blockade of TNF gene expression.
This study was aimed to investigate the effects and the mechanism of mangiferin on chronic myeloid leukemia cell lines K562 cells in vitro. The antiproliferation effects of mangiferin on K562 leukemia cells were tested by tetrazolium salt (MTT) method; the apoptosis induced by mangiferin on K562 cell line was explored by means of cell morphology, DNA gel electrophoresis and flow cytometry. The changes in bcr/abl gene expression was detected by using reverse transcriptase (RT)-PCR. The results showed that five different concentrations of mangiferin (25 - 200 micromol/L) dose-dependently and time-dependently inhibited the proliferation of K562 cells, and induced apoptosis in K562 cell line. RT-PCR revealed that bcr/abl gene expression was down-regulated when K562 cells had been treated with different concentrations of mangiferin. In conclusion, mangiferin remarkably inhibits the proliferation of K562 leukemia cells in vitro, and induces apoptosis in K562 cell line probably through down-regulation of bcr/abl gene expression.
The gene encoding for p16ink4a, a negative regulator of transition between G1 and S phase, is homozygously deleted in a large proportion of acute lymphoblastic leukaemias (ALL). Transfer of p16ink4a gene in several solid tumour cell lines with functional pRb and lacking both p16ink4a alleles has resulted in a dramatic reduction of cell proliferation, and the aim of this work was to confirm this effect in leukaemic (especially ALL) cell lines. We tested the proliferation in liquid medium and in soft agar after transfer of p16ink4a gene by a retroviral vector in leukaemic cell lines with homozygous p16ink4a gene deletion (K562, CEM, Jurkat cell lines) or with p16ink4a gene hemizygous deletion and a point mutation inactivating the remaining allele (HL60 cell line). The viral titre obtained after transfection of PA317 amphotropic packaging cell line, which has a p16ink4a gene homozygous deletion, was low, suggesting that p16ink4a gene expression could impair viral production of retroviral packaging cell lines derived from the NIH3T3 cell line. After retroviral transfer of p16ink4a in cell lines and G418 selection in liquid medium, a strong cell proliferation inhibition was observed for K562, CEM and Jurkat, but no inhibition was seen for HL60. A strong growth reduction in soft agar was also observed with p16ink4a-transduced CEM, Jurkat and K562 cells, with a moderate growth reduction in the HL60 cell line. The growth inhibition in liquid culture, of K562 and Jurkat cell lines, was confirmed by electroporation transfer of the p16ink4a gene. Our findings show that p16ink4a gene transfer has a growth-inhibitory effect in leukaemic cell lines with p16ink4a gene homozygous deletion. These data suggest that p16 could be a suitable gene for gene therapy in ALL.
Cell plating density influences the growth pattern of human squamous cell carcinoma (SCC) cell lines in culture. SCC lines were used to study factors responsible for these effects. Experiments in which the medium was changed daily or in which conditioned medium was used showed that the growth-supporting factors were not found in the media. However, when cells were plated in dishes in which the same cell line had been grown to confluence and removed by scraping, logarithmic growth began immediately. The effect was not seen on areas of the plate that had been covered with coverslips or in dishes where fibroblasts had been cultured. Antibodies to fibronectin, laminin, and a variety of epidermal cell antigens were used to determine the nature of the growth-stimulating substances remaining on the dish following cell removal. Results indicate that an extracellular matrix with similarities to basement membranes was present on the conditioned dishes.
OBJECTIVE: Inhibition of somatostatin on cell proliferation via somatostatin receptor subtype 2 (SS-R2), suggested this receptor subtype may be involved in tumor growth. Present study is to demonstrate whether SS-R2 was expressed in tumor cell lines extensively as well as in human gastric epithelia immortalized cell line. Meanwhile, the effect of p53 mutation on SS-R2 expression in gastric cancer cell line was also elucidated. METHODS: Full-length human somatostatin receptor subtype 2 cDNA was cloned by RT-PCR. Using cDNA as the probe and RT-PCR, expression of mRNA for SS-R2 in 9 cancer cell lines and human gastric immortalized cell line was detected. RESULTS: Data revealed that most cancer cell lines but a pancreatic cancer cell line expressed SS-R2 mRNA. CONCLUSION: Regardless whether p53 mutation or not in human gastric cancer cell lines, SS-R2 mRNA was transcribed.
N-myc gene amplification of the gynecological malignant tumor cell lines and a neuroblastoma cell line was studied by the Southern hybridization method along with the production of neuron-specific enolase (NSE) by these cell lines. N-myc amplification and NSE production were observed side by side in three cell lines: neuroblastoma cell line HSNB, endodermal sinus tumor cell line HAEST, and malignant teratoma cell line HUOT. However, N-myc amplification and NSE production disappeared gradually following successive passages of the HAEST and HUOT lines. With respect to the HUOT line, these parameters disappeared along with the cells of nervous origin. N-myc amplification and NSE production were not observed in nine other cell lines.
Cells from the Y79 human retinoblastoma cell line were examined by immunofluorescence immunocytochemistry using an antiserum against insulin. All the cells showed intense staining, indicating the presence of insulin-like immunoreactivity in these cells. Our observations suggest that insulin may play an important role in the metabolism of retinoblastoma cells and that it may be possible to use this cell line as an in vitro model for studies on the action of insulin in the metabolism of human retinal cells.
Cell survival, induction of apoptosis, and micronucleus formation have been examined in non-transformed human amnion fluid fibroblast-like (AFFL) cells and in a human squameous cell carcinoma (SCL-II) cell line after exposure to the Auger electron emitter (65)Zn and after external low-LET radiation. Cellular uptake and subcellular distribution of (65)Zn(2+) were studied in vitro and the absorbed radiation dose was calculated applying analytical dosimetry models. Auger electrons generated during decay of (65)Zn induced a prominent decrease in cell survival and increased the levels of apoptotic as well as micronucleated cells when compared to external low-LET irradiation. Relative biological effectiveness has been determined for cell survival (RBE approximately 4), micronucleus formation (RBE approximately 2) and apoptosis induction (RBE approximately 5-8) in SCL-II cells and for micronucleus formation (RBE approximately 4-5) and apoptosis induction (RBE approximately 6-10) in AFFL cells, respectively. This demonstrates a general enhanced biological effectiveness of (65)Zn in both investigated cell lines when compared to external low-LET radiation. The distribution pattern of intracellular Zn(2+) was found to be non-uniform, showing enhanced amounts of Zn(2+) in the perinuclear region and low amounts inside the cell nucleus, suggesting a major energy deposition close to the nuclear envelope.
Cells produce a heterogeneous population of extracellular vesicles and particles (EVPs). Small extracellular vesicles (sEVs or exosomes) are lipid membrane-enclosed vesicles with sizes ranging from 30 to 150 nm. Here, we present a protocol to isolate and characterize EVPs from the conditioned medium of cell lines. We describe steps for cell culture, conditioned media collection, differential ultracentrifugation, and EVP characterization. We then detail procedures for proteomic and biodistribution analyses. For complete details on the use and execution of this protocol, please refer to Yeung et al.1.
Cell motility is an important factor in the metastatic process that can be affected by environmental conditions. A quantitative study was made of the relationship between cell motility and the colonization potential of a mouse colon adenocarcinoma cell line (MCA-38). MCA-38 cells grown in culture did not produce hepatic or pulmonary colonies following ileocolic or tail vein injection, respectively. In contrast, MCA-38 cells adapted to grow in the mouse produced colonies in both organs. The motility of the MCA-38 cells that did not produce colonies, as determined by the depth of penetration into cellulose nitrate filters (8 micron pore size), was significantly less than that of MCA-38 cells with colony-forming potential. Return to in vitro growth resulted in both a loss of colonization potential and a reduction in motility. In this system, secondary organ colonization and in vitro cell motility are positively correlated, suggesting an association between cell motility and metastatic potential.
Cell lines 4A4 and 2C5 are the respective metastatic and non-metastatic variants of the human mammary carcinoma cell line MDA-MB-435 in the nude mouse system. We compared the transcriptional profile of approximately 5000 full-length genies using the Affymetrix HuGene FL Array technology. We have shown that the metastatic phenotype is mediated by different functional categories of genes, e.g. genes involved in immune response, genes responsible for tumor antigens, genes involved in migration and invasion, genes involved in mediating signal transduction, genes responsible for transcription factors, genes involved in phospholipid signaling, genes involved in modulation of extracellular matrix and cytoskeleton, genes with a cell-type specific mode of expression and genes which do not fit into the subclasses as defined above. Our results suggest an important role of Autocrine Motility Factor (AMF) as a mediator of metastasis in this system.
Cell lines were established from three hamster melanomas. One was a spontaneous melanotic tumour which lost its ability to produce pigment. Two were induced with DMBA (9.10 dimethyl 1,2-benz/a/anthracene). One of these was pigmented. The two amelanotic lines (CHT-1 and 2) produced highly malignant amelanotic tumours after reimplantation of tissue culture cells. Electron microscopy showed that melanin forming organelles were absent. Tyrosinase activity was also absent. The line established from the pigmented tumour (CHT-8) retained its pigment production for the first seven transfers. Cells from these cultures produced slow growing pigmented tumours. Cells from the 7th to the 35th transfer, a period of 28 weeks, failed to produce tumours but cells from the 36th and subsequent transfers produced slow growing amelanotic tumours. The change in tumorigenicity was not related to changes in the growth rate of the cells in vitro: this remained constant after the 11th transfer generation. Tyrosinase activity and a whole range of melanin forming organelles were present in cells of transfers 1 to 7 but absent from subsequent transfers. Type A and H virus particles were present in the two amelanotic cell lines, CHT-1 and 2. Although the two amelanotic lines produced highly malignant tumours the loss of a differentiated character--melanin production--was not invariably associated with increased malignancy. Three cell lines should provide a good system for studying the relationship between tumour differentiation and growth.
Cell viability assays are important tools in oncological research and clinical practice to assess the tumor cell sensitivity of individual patients. The purpose of this study was to demonstrate the comparability of 3 widely used assays (MTT, ATP, calcein assays) by principal component analysis. The study included 4 different cytostatics (cisplatin, docetaxel, doxorubicin, vinblastine) and 3 different human cancer cell lines (MCF-7, A2780, doxorubicin resistant A2780adr). Ninety-three percent of the total variance of all variables included in the principal component analysis (resulting from 3 cell lines and 3 assays) could be explained by 1 principal component. Factor loadings were > 0.937 except for the variable MTT-A2780adr, which was 0.872. These results indicate the similarity of the 3 assays. A 2nd principal component analysis included literature data and showed accordance of data from this study and the literature. The MTT assay was further improved as a high-throughput screening-capable assay. The ATP assay is able to detect effects of cytostatics already after 1 h incubation. The determination of resistance factors allowed to differentiate cytostatics into P-gp or non-P-gp substrates. In conclusion, this study provides improved microplate reader-based cell viability assays and sets a statistically solid basis for a future comparison of data obtained in different laboratories by any of the 3 assays.