JAK/STAT-deficient cell lines

Mutant cell lines B3 and B10, which are unresponsive to both interferon (IFN)-alpha and IFN-gamma, and line B9, which does not respond to IFN-gamma stimulation, are described. The mutants were submitted to fluorescence-activated cell sorting (FACS) from a cellular pool, which was obtained from the parental cell line 2C4 after several rounds of mutagenesis. The unresponsiveness to IFN stimulation was observed both in terms of expression of cell surface markers (CD2, class I and II HLAs) and mRNA expression of IFN-stimulated genes (2'-5' oligoadenylate synthetase (OAS), 9-27, and guanylate binding protein (GBP)). Genetic crossing of B3, B9 and B10 with U3 (STAT1-), gamma2a (JAK2-) and U4 (JAK1-) mutants, respectively, did not restore IFN responsiveness to the hybrid cell lines. However, when these cell lines were crossed with the same mutants, but using the pairwise crosses B3 x U4, B9 x U3 and B10 x U3, the cell hybrids recovered full IFN responsiveness. The present genetic experiments permitted us to assign the mutant cell lines B3, B9 and B10 to the U3, gamma2 and U4 complementation groups, respectively. These conclusions were supported by the analysis of IFN-stimulated genes in the mutants.

Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

Intercellular contact-dependent survival of human A549, NCI-H596 and NCI-H520 non-small cell lung carcinoma cell lines

In the present study, we examined the relationship between cell phenotype and cell survival of three human non-small cell lung carcinoma cell lines (A549, NCI-H596 and NCI-H520). Cells in exponential growth at various densities were incubated for 24 h at 37ºC in a 5% CO2 humidified atmosphere and then exposed to UV radiation for 1 min (256 nm, 40 W, source-to-target distance 100 cm). After two days the surviving cells were quantified by sulforhodamine ß staining and DNA fragmentation assay. The differences in UV sensitivity at 60 x 10³ cells/cm² among the cell lines were not related to the proliferative state of the cells but to the extent of intercellular contact. In contrast to A549 and NCI-H596, irradiated NCI-H520 cells presented lower DNA fragmentation and an aggregated cell culture phenotype even prior to confluence, suggesting that a contact-effect mechanism provides further protection against UV radiation.

Fingerprinting of cell lines by directed amplification of minisatellite-region DNA (DAMD)

The development of in vitro propagation of cells has been an extraordinary technical advance for several biological studies. The correct identification of the cell line used, however, is crucial, as a mistaken identity or the presence of another contaminating cell may lead to invalid and/or erroneous conclusions. We report here the application of a DNA fingerprinting procedure (directed amplification of minisatellite-region DNA), developed by Heath et al. [Nucleic Acids Research (1993) 21: 5782-5785], to the characterization of cell lines. Genomic DNA of cells in culture was extracted and amplified by PCR in the presence of VNTR core sequences, and the amplicons were separated by agarose gel electrophoresis. After image capture with a digital camera, the banding profiles obtained were analyzed using a software (AnaGel) specially developed for the storage and analysis of electrophoretic fingerprints. The fingerprints are useful for construction of a data base for identification of cell lines by comparison to reference profiles as well as comparison of similar lines from different sources and periodic follow-up of cells in culture.

TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines

Measurement of telomerase activity in clinically obtained tumor samples may provide important information for use as both a diagnostic marker and a prognostic indicator for patient outcome. In order to evaluate telomerase activity in tumor tissue without radiolabeling the product, we developed a simple telomeric repeat amplification protocol-silver-staining assay that is less time-consuming, is safe and requires minimal equipment. In addition, we determined the sensitivity of the silver-staining method by using extracts of telomerase-positive thyroid carcinoma cell lines which were serially diluted from 5,000 to 10 cells. Telomerase activity was also assayed in 19 thyroid tumors, 2 normal controls and 27 bone marrow aspirates. The results indicate that the technique permits the detection of telomerase activity from 5000 to as few as 10 cells. We propose that it could be immediately applicable in many laboratories due to the minimal amount of equipment required.

Modulation of rhodopsin gene expression and signaling mechanisms evoked by endothelins in goldfish and murine pigment cell lines

Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of vasoconstrictor peptides, which regulate pigment migration and/or production in vertebrate pigment cells. The teleost Carassius auratus erythrophoroma cell line, GEM-81, and Mus musculus B16 melanocytes express rhodopsin, as well as the ET receptors, ETB and ETA, respectively. Both cell lines are photoresponsive, and respond to light with a decreased proliferation rate. For B16, the doubling time of cells kept in 14-h light (14L):10-h darkness (10D) was higher compared to 10L:14D, or to DD. The doubling time of cells kept in 10L:14D was also higher compared to DD. Using real-time PCR, we demonstrated that SRTX S6c (12-h treatment, 100 pM and 1 nM; 24-h treatment, 1 nM) and ET-1 (12-h treatment, 10 and 100 pM; 24- and 48-h treatments, 100 pM) increased rhodopsin mRNA levels in GEM-81 and B16 cells, respectively. This modulation involves protein kinase C (PKC) and the mitogen-activated protein kinase cascade in GEM-81 cells, and phospholipase C, Ca2+, calmodulin, a Ca2+/calmodulin-dependent kinase, and PKC in B16 cells. Cells were kept under constant darkness throughout the gene expression experiments. These results show that rhodopsin mRNA levels can be modulated by SRTXs/ETs in vertebrate pigment cells. It is possible that SRTX S6c binding to the ETB receptors in GEM-81 cells, and ET-1 binding to ETA receptors in B16 melanocytes, although activating diverse intracellular signaling mechanisms, mobilize transcription factors such as c-Fos, c-Jun, c-Myc, and neural retina leucine zipper protein. These activated transcription factors may be involved in the positive regulation of rhodopsin mRNA levels in these cell lines.

Tetramethylpyrazine potentiates arsenic trioxide activity against HL-60 cell lines

The objective of this study was to evaluate the effects of tetramethylpyrazine (TMP) in combination with arsenic trioxide (As2O3) on the proliferation and differentiation of HL-60 cells. The HL-60 cells were treated with 300 µg/mL TMP, 0.5 µM As2O3, and 300 µg/mL TMP combined with 0.5 µM As2O3, respectively. The proliferative inhibition rates were determined with MTT. Differentiation was detected by the nitroblue tetrazolium (NBT) reduction test, Wright’s staining and the distribution of CD11b and CD14. Flow cytometry was used to analyze cell cycle distribution. RT-PCR and Western blot assays were employed to detect the expressions of c-myc, p27, CDK2, and cyclin E1. Combination treatment had synergistic effects on the proliferative inhibition rates. The rates were increased gradually after the combination treatment, much higher than those treated with the corresponding concentration of As2O3 alone. The cells exhibited characteristics of mature granulocytes and a higher NBT-reducing ability, being a 2.6-fold increase in the rate of NBT-positive ratio of HL-60 cells within the As2O3 treatment versus almost a 13-fold increase in the TMP + As2O3 group. Cells treated with both TMP and As2O3 expressed far more CD11b antigens, almost 2-fold compared with the control group. Small doses of TMP potentiate As2O3-induced differentiation of HL-60 cells, possibly by regulating the expression and activity of G0/G1 phase-arresting molecules. Combination treatment of TMP with As2O3 has significant synergistic effects on the proliferative inhibition of HL-60 cells.

Berberine induces apoptosis via ROS generation in PANC-1 and MIA-PaCa2 pancreatic cell lines

Pancreatic cancer is the fourth leading cause of cancer death. Gemcitabine is widely used as a chemotherapeutic agent for the treatment of pancreatic cancer, but the prognosis is still poor. Berberine, an isoquinoline alkaloid extracted from a variety of natural herbs, possesses a variety of pharmacological properties including anticancer effects. In this study, we investigated the anticancer effects of berberine and compared its use with that of gemcitabine in the pancreatic cancer cell lines PANC-1 and MIA-PaCa2. Berberine inhibited cell growth in a dose-dependent manner by inducing cell cycle arrest and apoptosis. After berberine treatment, the G1 phase of PANC-1 cells increased by 10% compared to control cells, and the G1 phase of MIA-PaCa2 cells was increased by 2%. Whereas gemcitabine exerts antiproliferation effects through S-phase arrest, our results showed that berberine inhibited proliferation by inducing G1-phase arrest. Berberine-induced apoptosis of PANC-1 and MIA-PaCa2 cells increased by 7 and 2% compared to control cells, respectively. Notably, berberine had a greater apoptotic effect in PANC-1 cells than gemcitabine. Upon treatment of PANC-1 and MIA-PaCa2 with berberine at a half-maximal inhibitory concentration (IC50), apoptosis was induced by a mechanism that involved the production of reactive oxygen species (ROS) rather than caspase 3/7 activation. Our findings showed that berberine had anti-cancer effects and may be an effective drug for pancreatic cancer chemotherapy.

Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

The establishment of two novel bovine cell lines by transfection with the putative transforming region of bovine adenovirus type 3

Human adenoviruses (Ads), members of the family adenoviridae, are medium-sized DNA viruses which have been used as valuable research tools for the study of RNA processing, oncogenic transformation, and for the development of viral vectors for use in gene delivery and immunization technology. The left 12% of the linear Ad genollle codes for products which are necessary for the efficient replication of the virus, as well as being responsible for the forlllation of tumors in animallllodels. The establishlllent of the 293 cell line, by immortalization of human embryonic kidney cells with th~ E1 region of Ad type S (AdS), has facilitated extensive manipulation of the Ads and the development of recombinant Ad vectors. The study of bovine adenoviruses (BAVs), which cause mild respiratory and gastrointestinal infections in cattle has, on the other hand, been limited primarily to that of infectivity, immunology and clinicallllanifestations. As a result, any potential as gene delivery vehicles has not yet been realized. Continued research into the molecular biolo~gy of BAVs and the development of recolllbinant vectors would benefit from the development of a cell line analogous to that of the 293 cells. In an attelllpt to establish such a cell line, the recombinant plaslllid pKC-neo was constructed, containing the left 0-19.7% of the BAV type 3 (BAV3) genome, and the selectable marker for resistance to the aminoglycoside G418, a neomycin derivative. The plasmid construct was then used to transfect both the Madin-Darby bovine kidney (MDBK) -iicell line and primary bovine lung cells, after which G418-resistant foci were selected for analysis. Two cell lines, E61 (MDBK) and E24 (primary lung), were subsequently selected and analysed for DNA content, revealing the presence of the pKC-neo sequences in their respective genomes. In addition, BAV3 RNA transcripts were detected in the E61 cells. Although the presence of E1 products has yet to be confirmed in both cell lines, the E24 cells exhibit a phenotype characteristic of partial transformation by E1. The apparent immortalization of the primary lung cells will permit exploitation of their ability to take up exogenous DNA at high efficiency.

A Survey of hMLH1 mRNA Splicing Profiles in Human Cell Lines: Comparing Primary Cultured Fibroblasts Before and After Oxidative Stress and Transiently versus Stably Demethylated Colon Cancer Derived Cell Lines

Most human genes undergo alternative splicing and loss of splicing fidelity is associated with disease. Epigenetic silencing of hMLH 1 via promoter cytosine methylation is causally linked to a subset of sporadic non-polyposis colon cancer and is reversible by 5-aza-2' -deoxycytidine treatment. Here I investigated changes in hMLHI mRNA splicing profiles in normal fibroblasts and colon cancer-derived human cell lines. I established the types and frequencies of hMLHI mRNA transcripts generated under baseline conditions, after hydrogen peroxide induced oxidative stress, and in acutely 5-aza-2' -deoxycytidine-treated and stably derepressed cancer cell lines. I found that hMLHI is extensively spliced under all conditions including baseline (50% splice variants), the splice variant distribution changes in response to oxidative stress, and certain splice variants are sensitive to 5- aza-2' -deoxycytidine treatment: Splice variant diversity and frequency of exon 17 skipping correlates with the level of hMLHI promoter methylation suggesting a link between promoter methylation and mRNA splicing.

Escherichia coli STb toxin induces apoptosis in intestinal epithelial cell lines

La toxine stable à la chaleur de type b (STb) est une des toxines produites par les souches Enterotoxigenic Escherichia coli (ETEC) impliquée dans le développement de la diarrhée. Une étude antérieure par Goncalves et al. (2009) a démontré que les cellules ayant internalisé la toxine STb démontraient une morphologie qui rappelle l’apoptose. Le changement du potentiel membranaire observé par Goncalves et al. (2009) nous a incité à vérifier la capacité de la toxine STb à induire l’apoptose des cellules HRT-18 et IEC-18 par la voie intrinsèque. Les cellules HRT-18 et IEC-18 ont été traitées avec de la toxine purifiée pour une durée de 24 heures puis ells ont été récoltées et examinées pour des caratéristiques de l’apoptose. L’activation des caspases-9 et -3, mais pas de la caspase-8, a été observée dans les deux lignées cellulaires à l’aide des substrats fluorescents spécifiques pour chaque caspase. L’ADN extrait des cellules HRT-18 et IEC-18 a révélé une fragmentation lorsque migré sur gel d’agarose. La condensation et la fragmentation des noyaux ont été observées en microscopie à fluorescence suite à une coloration de l’ADN au Hoechst 33342. Les indices apoptotiques des cellules HRT-18 et IEC-18 traitées avec des quantités croissantes de STb montrent une dose-réponse pour les deux lignées. L’activation de la caspase-9 est une indication que la voie intrinsèque de l’apoptose est activée dans les cellules HRT-18 et IEC-18. L’absence de l’activation de la caspase-8 démontre que la voie extrinsèque n’est pas impliquée dans la mort cellulaire médiée par STb.

Establishment of Shrimp Cell Lines: Perception and Orientation

Development of continuous shrimp cell lines for effective investigation on shrimp viruses remains elusive with an arduous history of over 25 years. Despite presenting challenges to researchers in developing a cell line, the billion dollar aquaculture industry is under viral threat. Advances in molecular biology and various gene transfer technologies for immortalization of cells have resulted in the development of hundreds of cell lines from insects and mammals, but yet not a single cell line has been developed from shrimp and other marine invertebrates. Though improved growth and longevity of shrimp cells in vitro could be achieved by using modified growth media this did not make any leap to spontaneous transformation; probably due to the fact that shrimp cells inhibited neoplastic transformations. Oncogenic induction and immortalization are considered as the possible ways, and an exclusive medium for shrimp cell culture and an appropriate mode of transformation are crucial. In this review status of shrimp cell line development and its future orientation are discussed

Effects of 17β-estradiol and tamoxifen on the induction of chromosomal abnormalities and on the expression of her2 gene in breast cancer cell lines

This study covers an area of great importance in the research of breast cancer, related to the study of the effects of both estrogens (E2) and anti-estrogens (Tamoxifen) on chromosomes and of modulation of gene expression. Considering that breast cancer is a very heterogeneous disease and that patients respond differently to treatment, the identification of chromosomal abnormalities as well as genes responsive to 17β-estradiol (E2) and Tamoxifen (TAM) could provide the necessary framework to understand the complex effects of this hormone in target cells and could explain, at least in part, the development of cellular resistance to TAM treatment and the subsequent best therapeutic option. In this order of ideas, we determined the effects of E2 and TAM on the chromosomes and on the modulation of gene expression in four breast cancer cell lines, which represent three of the five subtypes of breast cancer known at present. The results are presented in six chapters - each one has a group of the results achieved around the cytogenetic characteristics and gene expression profiles of four cell lines and the effects of E2 and TAM incubation on those. The first chapter describes the main features of breast cancer, furthering the use and effects of E2 and TAM treatment.

A human pancreatic ribonuclease variant kills cancer cells by apoptosis and reduces the expression of P-glycoprotein in MDR cell lines

En aquesta tesi s'han estudiat les propietats antitumorals d'una variant de la ribonucleasa pancreàtica humana anomenada PE5 que incorpora un senyal de localització nuclear. Aquest estudi mostra que PE5 indueix l'apoptosi de les cèl·lules tractades i que aquesta mort és independent de l'activitat de p53. A més, l'efecte citotòxic no es veu afectat per un fenotip de resistència a múltiples drogues. Les dades també mostren que l'activitat citotòxica de PE5 és selectiva per a cèl·lules tumorals in vitro i que la capacitat citotòxica de les dues ribonucleases és semblant. S'ha estudiat l'efecte d'aquestes dues ribonucleases sobre el cicle cel·lular, l'activació de diferents caspases i l'expressió de proteïnes relacionades amb l'apoptosi i el cicle cel·lular. Els resultats indiquen que PE5 i l'onconasa maten les cèl·lules a través de mecanismes diferents. A més, PE5 però no l'onconasa, redueix l'acumulació de glicoproteïna-P en dues línies cel·lulars resistents a múltiples drogues.