Different Types Of Cell Lines Research Articles

Modulation of function of three ABC drug transporters, P-glycoprotein (ABCB1), mitoxantrone resistance protein (ABCG2) and multidrug resistance protein 1 (ABCC1) by tetrahydrocurcumin, a major metabolite of curcumin

Many studies have been performed with the aim of developing effective resistance modulators to overcome the multidrug resistance (MDR) of human cancers. Potent MDR modulators are being investigated in clinical trials. Many current studies are focused on dietary herbs due to the fact that these have been used for centuries without producing any harmful side effects. In this study, the effect of tetrahydrocurcumin (THC) on three ABC drug transporter proteins, P-glycoprotein (P-gp or ABCB1), mitoxantrone resistance protein (MXR or ABCG2) and multidrug resistance protein 1 (MRP1 or ABCC1) was investigated, to assess whether an ultimate metabolite form of curcuminoids (THC) is able to modulate MDR in cancer cells. Two different types of cell lines were used for P-gp study, human cervical carcinoma KB-3-1 (wild type) and KB-V-1 and human breast cancer MCF-7 (wild type) and MCF-7 MDR, whereas, pcDNA3.1 and pcDNA3.1-MRP1 transfected HEK 293 and MXR overexpressing MCF7AdrVp3000 or MCF7FL1000 and its parental MCF-7 were used for MRP1 and MXR study, respectively. We report here for the first time that THC is able to inhibit the function of P-gp, MXR and MRP1. The results of flow cytometry assay indicated that THC is able to inhibit the function of P-gp and thereby significantly increase the accumulation of rhodamine and calcein AM in KB-V-1 cells. The result was confirmed by the effect of THC on [(3)H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [(3)H]-vinblastine in MCF-7 MDR in a concentration-dependent manner. This effect was not found in wild type MCF-7 cell line. The interaction of THC with the P-gp molecule was clearly indicated by ATPase assay and photoaffinity labeling of P-gp with transport substrate. THC stimulated P-gp ATPase activity and inhibited the incorporation of [(125)I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [(125)I]-IAAP to MXR was also inhibited by THC suggesting that THC interacted with drug binding site of the transporter. THC dose dependently inhibited the efflux of mitoxantrone and pheophorbide A from MXR expressing cells (MCF7AdrVp3000 and MCF7FL1000). Similarly with MRP1, the efflux of a fluorescent substrate calcein AM was inhibited effectively by THC thereby the accumulation of calcein was increased in MRP1-HEK 293 and not its parental pcDNA3.1-HEK 293 cells. The MDR reversing properties of THC on P-gp, MRP1, and MXR were determined by MTT assay. THC significantly increased the sensitivity of vinblastine, mitoxantrone and etoposide in drug resistance KB-V-1, MCF7AdrVp3000 and MRP1-HEK 293 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Taken together, this study clearly showed that THC inhibits the efflux function of P-gp, MXR and MRP1 and it is able to extend the MDR reversing activity of curcuminoids in vivo.

In vitro gene transfer to mammalian cells by the use of laser-induced stress waves: effects of stress wave parameters, ambient temperature, and cell type

Laser-mediated gene transfection has received much attention as a new method for targeted gene therapy because of the high spatial controllability of laser energy. We previously demonstrated both in vivo and in vitro that plasmid DNA can be transfected by applying nanosecond pulsed laser-induced stress waves (LISWs). In the present study, we investigated the dependence of transfection efficiency on the laser irradiation conditions and hence stress wave conditions in vitro. We measured characteristics of LISWs used for gene transfection. For NIH 3T3 cells, transfection efficiency was evaluated as functions of laser fluence and number of pulses. The effect of ambient temperature was also investigated, and it was found that change in ambient temperature in a specific range resulted in drastic change in transfection efficiency for NIH 3T3 cells. Gene transfection of different types of cell lines were also demonstrated, where cellular heating increased transfection efficiency for nonmalignant cells, while heating decreased transfection efficiency for malignant cells.

The influence of STAT5 antisense oligonucleotides on the proliferation and apoptosis of selected human leukaemic cell lines.

The signal transducers and activators of transcription--STAT5A and STAT5B--take part in the regulation of many essential physiopathological processes. They influence the cell cycle, apoptosis and the proliferation of different types of cell lines. The STAT5 proteins are induced in response to multiple haematopoietic cytokines. Because they are constitutively active in certain haemato-oncologic diseases, it is also suggested that they play an important role in leukaemogenesis. However, function of these proteins in haematopoietic cell transformation and proliferation is not clear. The aim of this study was to evaluate the impact of perturbation of STAT5 expression [using oligodeoxynucleotide (ODN) against STAT5 mRNA], on the clonogenicity and survival of selected human leukaemic cell lines, HEL, HL-60, K562, TF-1. We analysed the effect of ODN pre-treatment on the cell clonogenicity in methylcellulose cultures according to the time and the temperature of exposure. Moreover, we attempted to estimate apoptosis induced in examined cells, by flow cytometry using combined Annexin V-PI staining and the TUNEL method. We also applied the RT-PCR method to analyse Bax and Bcl-xL gene expression. We found that the perturbation of STAT5 expression with antisense oligonucleotides caused a decrease in the proliferative potential of human K562 and TF-1 cell lines. Also, we observed higher induction of apoptotic cell death in the K562 and TF-1 cells incubated with the antisense STAT5A ODNs. We did not notice any impact of ODNs on the HL-60 and HEL cells. Our studies using STAT5 antisense oligonucleotides showed that these proteins may be critical in the regulation of growth and apoptosis of some types of leukaemic blasts.

Dose-dependent modulation of apoptosis and cyclooxygenase-2 expression in human 1547 osteosarcoma cells by NS-398, a selective cyclooxygenase-2 inhibitor.

A selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, was shown to produce an anti-proliferative and pro-apoptotic effect on different types of cell lines. We describe the presence of COX-1 and COX-2 pathways in the human osteosarcoma 1547 cell line, as well as the conflicting effects of NS-398 (10, 50 and 100 microM) on programmed cell death, PGE2 release and COX-2 expression in 1547 cells cultured under apoptotic conditions. We demonstrate a link between the effects of 10 and 100 microM NS-398 on cell apoptosis, PGE2 release, and expression of COX-2 in 1547 cells undergoing apoptosis. At 10 microM, NS-398 acted as a selective COX-2 inhibitor moderately increasing apoptosis without any effect on COX-2 expression. In contrast, at 100 microM, NS-398 induced a cell cycle slowing or arrest, strongly enhanced COX-2 expression which was associated with a high PGE2 release and a marked decrease in apoptosis. This latter property of NS-398 at 100 microM in 1547 human osteosarcoma cells is novel compared to the described NS-398 pro-apoptotic effect on other cell lines.

The E-Box Motif, Recognized by Tissue-Specific Nuclear Factor(s), Is Important for BMP-4 Gene Expression in Osteogenic Cells

We analyzed the promoter activity of mouse BMP-4 gene in three different types of cell lines and demonstrated that position −786 to −691 of the 5′-flanking region of exon I plays a critical role for regulation of tissue specific expression of mouse BMP-4 gene in cells of an osteogenic lineage. By use of site-directed mutagenesis, we have established that the E-box, CATCTG, located within this 5′-flanking region, is essential for tissue-specific transcriptional activation of mouse BMP-4 gene and demonstrated that an osteogenic lineage-specific novel transcriptional factor(s) recognizes this E-box.

Widely Used Enhancer of Eukaryotic Expression Vectors Is Strongly and Differentially Regulated in Fibroblast, Myoblast, and Teratocarcinoma Cell Lines

The expression of transgenes in eukaryotic cells is a powerful approach in cell biology. In most cases, it is based on the activity of strong and constitutive viral cis-acting elements in eukaryotic expression vectors. Here we show that a widely used such element derived from an early gene of human cytomegalovirus is strongly and differentially regulated in mouse cell lines. We analyzed cytomegalovirus promoter-driven expression of stably transfected transgenes in growing, confluent, and differentiating mouse 3T3 fibroblasts, C2C12 myoblasts, and P19 teratocarcinoma cells. In the fibroblasts, transgene expression was strongly downregulated in confluent cultures and was upregulated in growing or confluent cultures by phorbol ester. In contrast, no downregulation by confluency, nor upregulation by phorbol ester, was detected in C2C12 cells. In addition, while marked upregulation was detected in differentiating myotubes, transgene expression was downregulated when differentiating teratocarcinoma cells assumed a neuronal phenotype. These results demonstrate the existence of drastic differences in the regulation of transgene expression in different types of cell lines, indicating that when studying transgene function in cells that are not growing exponentially, viral promoter-driven expression should not be taken for granted.

Chloroquine impairs the interferon-induced antiviral state without affecting the 2‘,5‘-oligoadenylate synthetase.

Chloroquine, a weak base which raises the pH in acidic cellular compartments such as lysosomes and endosomes, counteracts the induction by interferon of the antiviral state but not that of the 2',5'-oligoadenylate synthetase in three different types of cell lines (MDBK, WISH, and L929). Active interferon is recovered in crude extracts of cells which have been treated with interferon and chloroquine together, but not in extracts of cells treated with interferon alone, indicating that chloroquine has inhibited the intralysosomal proteolysis of interferon. A low pH-dependent event in the intracellular fate of interferon (perhaps its intralysosomal degradation) is, therefore, necessary for the establishment of the antiviral state but not for the induction of the 2',5'-oligoadenylate synthetase.

Studies on the mechanism of natural killer cell-mediated cytotoxicity. V. Lack of NK specificity at the level of induction of natural killer cytotoxic factors in cultures of human, murine, or rat effector cells stimulated with mycoplasma-free cell lines.

We have proposed that lysis of target cells by NK cells is mediated by NK cytotoxic factors (NKCF). According to our model, for a target cell to be NK-sensitive, it must be recognized by the NK cell, it must stimulate the release of NKCF, and it must be sensitive to lysis by these factors. This report examines whether the ability to stimulate release of NKCF is a characteristic restricted to NK-sensitive tumor cells or whether it is also a property of NK-resistant target cells. Many different types of cell lines were tested for their ability to stimulate release of NKCF in the human, rat, and murine systems. It was found that mycoplasma-free NK-sensitive cell lines, resistant cell lines, and Con A could stimulate the release of NKCF. Many different types of cell lines grown in suspension or in monolayers were found to be effective stimulators, including T or B lymphoid, myeloid, and those of histiocytic origin. Cells cultured in the absence of serum stimulated NKCF release, thus ruling out the possible involvement of serum components in stimulation. NKCF was also produced by xenogeneic combinations of effector and stimulator cells, demonstrating lack of species specificity in NKCF production. Factors stimulated by NK-resistant cell lines or by Con A exhibited the same NK target specificity as supernatants stimulated by NK-sensitive tumor cells. The finding that many different NK-resistant cell lines can stimulate the release of NKCF indicates that there is no apparent NK specificity at the level of induction of NKCF release from human, rat, or murine effector cells. Therefore, the NK specificity of a target cell is determined ultimately by its sensitivity to lysis by NKCF.

Expression of human hypoxanthine phosphoribosyl transferase in Chinese hamster cells treated with isolated human chromosomes

Chinese hamster cells deficient for the enzyme hypoxanthine phosphoribosyl transferase (HPRT) were incubated with isolated human metaphase chromosomes and 21 colonies were isolated in HAT medium. Three different types of cell lines were established from these clones. First, 4 cell lines had 10-30% of normal Chinese hamster HPRT activity with the same electrophoretic mobility as human HPRT. This HPRT activity remains detectable during at least 8 weeks of growth of the cells in nonselective medium. Second, 3 cell lines also had human-like HPRT with the same activity as the first type. This HPRT persists only if the cells are grown in HAT medium and disappears during 8 weeks of growth in nonselective medium. Third, other clones survived in HAT medium as well as in medium with 8-azaguanine. These cells had no detectable HPRT activity. Using differential chromosome staining techniques no recognizable human chromosome fragments were found in any of the cell lines.