Tumor suppression by chromosome 11 is not due to cellular senescence.

Previous hybrid studies involving fusion of normal with immortal human cells indicated that the phenotype of cellular senescence is dominant and that immortality results from recessive changes in normal growth regulatory genes. We have further assigned 28 different immortal human cell lines to at least four complementation groups for indefinite division. In order to identify the chromosomes involved in regulating cell proliferation, we have introduced single human chromosomes by microcell fusion into immortal human cells representative of the different complementation groups. Our results demonstrate that the introduction of chromosome 11, implicated in tumor suppression, does not cause cellular senescence in three different immortal human cell lines tested.     

Relationship between mitogen-regulated protein (MRP) and proliferin (PLF), a member of the prolactin/growth hormone family

Mitogen-regulated protein (MRP) is a glycoprotein secreted by Swiss murine 3T3 cells whose levels are increased 63-fold or more over the controls by growth factors. The sequence of a 226-bp MRP cDNA clone showed that a region close to the C terminus of MRP is identical to a sequence found in the cDNA-encoding proliferin (PLF). PLF, cloned from Balb/c 3T3 cells, is a member of the prolactin/growth-hormone family. Here we show that MRP and PLF are also antigenically identical. Antiserum raised against purified MRP specifically immunoprecipitated PLF secreted by CV-1 cells that had been transfected with PLF cDNA in an SV40 vector. Also, fibroblast growth factor (FGF) specifically increased the amount of PLF poly(A)+ RNA in Swiss 3T3 cells. We have previously shown that FGF increases the amount of MRP and MRP mRNA synthesized by the same cells. The anti-MRP antiserum recognized both unglycosylated and glycosylated forms of MRP and PLF. The unglycosylated and glycosylated forms of PLF had the same Mr values as those of the unglycosylated (21,500) and glycosylated (34,000) forms of MRP. However, the anti-MRP antiserum did not recognize mouse prolactin and anti-mouse prolactin antibody did not recognize MRP. Evidently, MRP/PLF is an immunologically distinct member of the prolactin/growth-hormone family of secreted, intercellular regulators.     

Establishment and characterization of three immortal bovine muscular epithelial cell lines

We have established three immortal bovine muscular epithelial (BME) cell lines, one spontaneously immortalized (BMES), the second SV40LT-mediated (BMEV) and the third hTERT-mediated (BMET). The morphology of the three immortal cell lines was similar to that of early passage primary BME cells. Each of the immortal cell lines made cytokeratin, a typical epithelial marker. BMET grew faster than the other immortal lines and the BME cells, in 10% FBS-DMEM medium, whereas neither the primary cells nor the three immortal cell lines grew in 0.5% FBS-DMEM. The primary BME cells and the immortal cell lines, with the exception of BMES, made increasing amounts of p53 protein when treated with doxorubicin, a DNA damaging agent. On the other hand, almost half of the cells in populations of the three immortal cell lines may lack p16(INK4a) regulatory function, compared to primary BME cells that were growth arrested by enforced expression of p16(INK4a). In soft-agar assays, the primary cells and immortal cell lines proved to be less transformed in phenotype than HeLa cells. The three immortal epithelial-type cell lines reported here are the first cell lines established from muscle tissue of bovine or other species.     

Correlation between complementation group for immortality and DNA synthesis inhibitors

Previous studies had demonstrated that a DNA synthesis inhibitor(s) was produced by senescent but not young human diploid fibroblasts (HDF). Analysis of immortal human cell lines led to the finding that SUSM-1, carcinogen-treated immortal human liver fibroblast cells, expressed a potent inhibitor of DNA synthesis that was active in proliferation-competent young HDF but did not affect the SUSM-1 cell line itself. To determine whether one mechanism of escape from senescence to the immortal phenotype involved the loss of response to such DNA synthesis inhibitors, we initiated the present study analyzing a larger number of immortal human cell lines representative of the four complementation groups for indefinite division identified to date. We have found a correlation between the assignment of a cell line to Complementation Group D and the production of DNA synthesis inhibitors coupled with inability to respond to the inhibitory factors. We have also observed a correlation between the ability of immortal cell lines to respond to such DNA synthesis inhibitory factors and assignment to Complementation Group B. These data suggest DNA synthesis inhibitors are involved in the limited lifespan of normal cells and that the immortalization process may involve alterations in the activity of or response to such inhibitors.     

Regulation of MARCKS and MARCKS-related protein expression in BV-2 microglial cells in response to lipopolysaccharide

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) have been implicated in membrane-cytoskeletal events underlying cell adhesion, migration, secretion, and phagocytosis. In BV-2 microglial cells, lipopolysaccharide (LPS) elicited a dose-dependent increase in mRNA of both MRP (sixfold) and MARCKS (threefold) with corresponding increases in [3H]myristoylated and immunoreactive protein levels. LPS also produced significant increases in protein kinase C (PKC)-beta twofold and PKC-epsilon (1.5-fold). Pro-inflammatory cytokines produced by activated microglia (IL-1beta, IL-6, TNF-alpha) did not mimic LPS effects on MARCKS or MRP expression when added individually or in combination. LPS and IFN-gamma produced a synergistic induction of iNOS but not MARCKS or MRP. Induction of MARCKS and MRP by LPS was completely blocked by inhibitors of NF-kappaB (PDTC) and protein tyrosine kinases (herbimycin A), partially blocked by the p38 kinase inhibitor SB203580, and unaffected by the MEK inhibitor PD98059. LPS induction of iNOS was considerably more sensitive to all these inhibitors. The Src kinase inhibitor PP2 had no effect, while the closely related inhibitor PP1 actually increased LPS induction of MARCKS and MRP. Our results suggest that MARCKS and MRP may play an important role in LPS-activated microglia, but are not part of the neuroinflammatory response produced by cytokines.     

Opposite and selective effects of epidermal growth factor and human platelet transforming growth factor-beta on the production of secreted proteins by murine 3T3 cells and human fibroblasts

Growth regulators such as epidermal growth factor (EGF) and type beta transforming growth factor (TGF-beta) regulate the synthesis and secretion of certain proteins by cells in culture. The secretion pattern of each cell line and the effect of growth regulators on the secretion pattern are unique. EGF increased the secreted and intracellular levels of mitogen-regulated protein (MRP) and major excreted protein (MEP) by Swiss 3T3 cells. MRP is related by sequence to prolactin. MEP is a thiol protease located intracellularly in the lysosomes. EGF also selectively induced a 52,000-dalton mitogen-induced protein (MIP 52) secreted by human fibroblasts. Two types of TGF-betas were tested for their effects on the expression of secreted proteins in mouse and human fibroblasts: TGF-beta from human platelets and a growth inhibitor (GI/TGF-beta) secreted by BSC-1 cells. Each selectively decreased the levels of the two secreted proteins induced by growth factors in mouse embryo 3T3 cells and one secreted protein induced by growth factors in human fibroblasts. Platelet TGF-beta and GI/TGF-beta also induced one 48,000-dalton protein secreted by human fibroblasts. Synthesis of DNA and the incorporation of [35S]methionine into total protein in Swiss 3T3 cells were not affected by platelet TGF-beta or GI/TGF-beta. Thus, the inhibitory effect of platelet TGF-beta on the synthesis and secretion of these three proteins is due to a specific effect of platelet TGF-beta on the regulation of MRP and MEP that does not interfere with the ability of EGF to stimulate DNA or protein synthesis.     

miR-126 enhances the sensitivity of non-small cell lung cancer cells to anticancer agents by targeting vascular endothelial growth factor A

Increasing evidence suggests that hsa-miR-126 (miR-126) is down-regulated in non-small cell lung cancer (NSCLC) cell lines and the restoration of miR-126 impairs tumor cell proliferation, migration, invasion, and survival by targeting specific molecules. Here, we reported for the first time that miR-126 was involved in regulating the response of NSCLC cells to cancer chemotherapy. After transfected A549 cells with miR-126 mimic or inhibitor, we found that an elevated level of miR-126 was significantly associated with a decreased half maximal inhibitory concentration of adriamycin (ADM) and vincristine, an increased accumulation of ADM, down-regulation of vascular endothelial growth factor A (VEGFA) and multidrug resistance-associated protein 1 (MRP1), and inactivation of the Akt signaling pathway. Furthermore, enhanced expression of miR-126 suppressed the growth of A549 xenograft and inhibited the expression of VEGFA and MRP1. miR-126 could efficiently down-regulate VEGFA expression through the interaction with the VEGFA 3'-untranslated region, whereas restoration of VEGFA could partially attenuate the suppression of MRP1 by miR-126. However, LY294002, an inhibitor of the PI3K/Akt signaling pathway, diminished this effect, suggesting that enhanced expression of miR-126 increased the sensitivity of NSCLC cells to anticancer agents through negative regulation of a VEGF/PI3K/Akt/MRP1 signaling pathway.     

Stimulation of the release of two glycoproteins from mouse 3T3 cells by growth factors and by agents that increase intralysosomal pH

Peptide growth factors selectively increase the amount of mitogen-regulated protein (MRP) and major excreted protein (MEP) released by mouse 3T3 cells. $\text{Balb}\text{c}$ 3T3 cells release mainly MEP and Swiss 3T3 cells release mainly MRP. Fibroblast growth factor, epidermal growth factor, nerve growth factor, serum, and concanavalin A increase the extracellular appearance of both MEP and MRP, but to different extents. Several agents that have been shown to, or would be expected to increase, intralysosomal pH also selectively increase the release of MEP and MRP from both $\text{Balb}\text{c}$ and Swiss 3T3 cells. The effective agents are monensin, nigericin, ammonium chloride, methylamine, chloroquine, and high extracellular pH.     

Three steps to the immortality of cancer cells: senescence,polyploidy and self-renewal

Background

Hepatocellular carcinoma (HCC) exhibits strong intrinsic and acquired drug resistance which is the main obstacle to chemotherapy. Overexpression of ATP binding cassette (ABC) proteins correlates with activation of mitogen activated protein kinase (MAPK) pathway in HCC. Here, we systematically investigated the inhibition of MAPK pathway and its role in regulating HCC cell growth as well as ABC proteins MRP1 and MRP3 expression.

Methods

The Raf1 kinase inhibitor (GW5074) and different MEK inhibitors (U0126 and AZD6244) were used to treat HCC cells to identify their effects on HCC cell growth and ABC proteins expression in vitro. Cell viability tests were performed after the treatment of MAPK pathway inhibitors and in combination with gemcitabine or doxorubicin. Western blot was applied to assess the changes of MAPK pathway and protein expression of MRP1 and MRP3. Flow cytometry was used to measure intracellular doxorubicin accumulation after the treatment of MEK inhibitors.

Results

Both Raf1 inhibitor (GW5074) and MEK inhibitors (U0126 and AZD6244) suppressed HCC cell growth in a dose dependent manner. Pre-treatment of MEK inhibitor U0126 or AZD6244 sensitized HCC cells to gemcitabine or doxorubicin based chemotherapy. Raf1 inhibitor GW5074 had no effect on MRP1 and MRP3 protein expression. Treatment of gemcitabine or doxorubicin activated phosphorylated ERK and induced the upregulation of MRP1 and MRP3. MEK inhibitors U0126 and AZD6244 deactivated phosphorylated ERK, decreased endogenous MRP1 expression, reversed gemcitabine or doxorubicin induced MRP1 and MRP3 upregulation, and increased the intracellular doxorubicin accumulation.

Conclusion

This study provides evidence that MEK inhibitors sensitize HCC cells to chemotherapy by increasing intracellular chemodrug accumulation. MEK inhibirors U0126 and AZD6244 reduced MRP1 as well as MRP3 expression, and may contribute partially to the sensitization. The combination of MEK inhibitor and conventional chemotherapy may offer new therapeutic option for the treatment of resistant HCC.     

Selective stimulation by mitogens of incorporation of 35S-methionine into a family of proteins released into the medium by 3T3 cells

Serum and three mitogens for mouse embryo 3T3 cells—fibroblast growth factor from brain, fibroblast growth factor from pituitary, and epidermal growth factor—specifically stimulate the synthesis and release into the medium by these cells of a group of proteins that travel together on SDS gel electrophoresis and that are detected by ³⁵S-methionine labeling. These proteins, designated mitogen-releasable proteins (MRPs), have a median, monomer molecular weight on SDS polyacrylamide gel electrophoresis of 34,000 daltons (30,000–38,000 daltons). Our evidence indicates that these proteins comprise a family of glycoproteins, probably with a common polypeptide backbone. The observations supporting this conclusion are that MRPs give a diffuse pattern of bands upon SDS gel electrophoresis; travel as a single, diffuse band when resolved by electrophoresis in the absence of SDS; adsorb to a pea-lectin-sepharose column and can be eluted with α-methyl mannose; and can be labeled metabolically with ³H-mannose. In addition, in the presence of tunicamycin, MRPs are not made—instead, a smaller molecular weight (22,000 dalton), and apparently homogeneous, protein appears. We believe this 22,000 dalton protein to be the unglycosylated form of MRP. Further support for this idea comes from our observation that treatment of MRPs with endoglycosidase H produces a protein with a molecular weight slightly greater than 22,000 daltons. The effect of mitogens on DNA synthesis and MRP release are correlated in the following ways. First, serum factors are required for both responses. Second, in 3T3 cells transformed by SV40, Moloney and Kirsten viruses that do not synthesize DNA in response to FGF, MRPs are not released in response to FGF. Third, in untransformed 3T3 cells, the dose-response curves for fibroblast growth factor on MRP release and thymidine incorporation are closely correlated. Fourth, insulin, a poor mitogen for 3T3 cells, does not enhance MRP release. Fifth, stimulation of MRP release by epidermal growth factor or fibroblast growth factor is inhibited by hydroxyurea and butyrate, both inhibitors of DNA synthesis in these cells. Sixth, if the mitogen is removed at any time during the 20 hr preincubation period, the effect on MRP release observed between 20 and 24 hr is severely diminished.     

Simian virus 40 small tumor antigen activates AKT and telomerase and induces anchorage-independent growth of human epithelial cells

Human keratinocytes immortalized by full-length or early-region simian virus 40 (SV40) DNA grow in agarose and form tumors in nude mice, in contrast to keratinocytes immortalized by the E6/E7 genes of human papillomaviruses. To determine the molecular basis for this biological difference in growth, we have used the individual SV40 oncogenes (large T antigen [LT] and small t antigen [st]) and human papillomavirus oncogenes (E6/E7) to study the progression of human epithelial cells from the nonimmortal to the immortal state as well as from the immortal to the anchorage-independent state. Transfection of primary human foreskin keratinocytes with LT did not immortalize cells but did extend the in vitro life span and produced cells that were resistant to calcium- and serum-induced terminal differentiation. Cells transfected with st alone did not passage beyond vector-transfected keratinocytes. The simultaneous expression of LT- and st-immortalized keratinocytes occurred without evidence of crisis and, as anticipated, these immortal cells were anchorage- independent for growth. Interestingly, we found that keratinocytes expressing both LT and st, but not keratinocytes with LT alone, exhibited increased phosphorylation of the protein kinase AKT. In addition, AKT activation was paralleled by an increase in telomerase activity. Addition of st to anchorage-dependent keratinocytes, expressing either LT (nonimmortal) or E6/E7 (immortal), converted the cells to anchorage independence, with similar accompanying increases in AKT phosphorylation and telomerase activity. However, it was not possible to induce keratinocyte growth in agarose with activated AKT and/or overexpressed hTERT, indicating that these newly defined st-induced activities are not sufficient for progression to the anchorage-independent state.     

FUT family mediates the multidrug resistance of human hepatocellular carcinoma via the PI3K/Akt signaling pathway

The fucosyltransferase (FUT) family is the key enzymes in cell-surface antigen synthesis during various biological processes such as tumor multidrug resistance (MDR). The aim of this work was to analyze the alteration of FUTs involved in MDR in human hepatocellular carcinoma (HCC) cell lines. Using mass spectrometry (MS) analysis, the composition profiling of fucosylated N-glycans differed between drug-resistant BEL7402/5-FU (BEL/FU) cells and the sensitive line BEL7402. Further analysis of the expressional profiles of the FUT family in three pairs of parental and chemoresistant human HCC cell lines showed that FUT4, FUT6 and FUT8 were predominant expressed in MDR cell lines. The altered levels of FUT4, FUT6 and FUT8 were responsible for changed drug-resistant phenotypes of BEL7402 and BEL/FU cells both in vitro and in vivo. In addition, regulating FUT4, FUT6 or FUT8 expression markedly modulated the activity of the phosphoinositide 3 kinase (PI3K)/Akt signaling pathway and MDR-related protein 1 (MRP1) expression. Inhibition of the PI3K/Akt pathway by its specific inhibitor wortmannin, or by Akt small interfering RNA (siRNA), resulted in decreased MDR of BEL/FU cells, partly through the downregulation of MRP1. Taken together, our results suggest that FUT4-, FUT6- or FUT8-mediated MDR in human HCC is associated with the activation of the PI3K/Akt pathway and the expression of MRP1, but not of P-gp, indicating a possible novel mechanism by which the FUT family regulates MDR in human HCC.     

Loss of p53 protein during radiation transformation of primary human mammary epithelial cells.

The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated gamma-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletion within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G1 arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells.     

Relationship of LRP-human major vault protein toin vitro and clinical resistance to anticancer drugs

Multidrug resistance (MDR) has been related to two members of the ABC-superfamily of transporters, P-glycoprotein (Pgp) and Multidrug Resistance-associated Protein (MRP). We have described a 110 kD protein termed the Lung Resistance-related Protein (LRP) that is overexpressed in several non-Pgp MDR cell lines of different histogenetic origin. Reversal of MDR parallels a decrease in LRP expression. In a panel of 61 cancer cell lines which have not been subjected to laboratory drug selection, LRP was a superior predictor forin vitro resistance to MDR-related drugs when compared to Pgp and MRP, and LRP's predictive value extended to MDR unrelated drugs, such as platinum compounds. LRP is widely distributed in clinical cancer specimens, but the frequency of LRP expression inversely correlates with the known chemosensitivity of different tumour types. Furthermore, LRP expression at diagnosis has been shown to be a strong and independent prognostic factor for response to chemotherapy and outcome in acute myeloid leukemia and ovarian carcinoma (platinum-based treatment) patients. Recently, LRP has been identified as the human major protein. Vaults are novel cellular organelles broadly distributed and highly conserved among diverse eukaryotic cells, suggesting that they play a role in fundamental cell processes. Vaults localise to nuclear pore complexes and may be the central plug of the nuclear pore complexes. Vaults structure and localisation support a transport function for this particle which could involve a variety of substrates. Vaults may therefore play a role in drug resistance by regulating the nucleocytoplasmic transport of drugs.Abbreviations LRP Lung Resistance-related Protein - MVP Major Vault Protein - MDR Multidrug resistance - MRP Multidrug resistance-associated Protein - NPC Nuclear Pore Complex - Pgp P-glycoprotein     

Modification of expression of stem cell factor by various cytokines.

The local production of stem cell factor (SCF) may be an important mechanism for regulating proliferation, differentiation, and migration of various cells bearing c-kit receptors, and might be susceptible to the cytokines that serve in inflammation and tissue repair. We have demonstrated that in three murine cell lines, Balb/3T3A31, MC3T3-E1, and C3H-2K, which constitutively produced SCF with different quantity, the SCF mRNA expression was greatly enhanced in response to basic fibroblast growth factor (bFGF) or transforming growth factor beta1 (TGF-beta1). The study was carried out by in situ hybridization utilizing nonradioactive oligonucleotide probes and quantitative image analysis. Leukemia inhibitory factor (LIF) or interleukin-4 (IL-4) moderately increased SCF mRNA in all cell lines, but IL-3 did not. The dot-blot enzyme-linked immunosorbent assay (ELISA) further confirmed that SCF protein production in these cell lines and bone marrow stromal cells was markedly enhanced by TGF-beta1, although TGF-beta1 suppressed the proliferation of all these cells. bFGF also enhanced the SCF production in these cell lines, but did not in bone marrow stromal cells, suggesting a difference in their susceptibility to the cytokine. Our results suggest that TGF-beta1 and bFGF potentially modulate the biological function of cells bearing c-kit receptors through the modulation of SCF production in fibroblasts.     

Applications of immortalized cells in basic and clinical neurology

Immortalized cell lines can serve as model systems for studies of neuronal development and restoration of function in models of neurological disease. Cell lines which result from spontaneous or experimentally-induced tumors have been used for these purposes. More recently, the techniques of genetic engineering have resulted in the production of cell lines with specific desired characteristics. This has been accomplished by insertion of a desired gene into a pre-existing immortal cell or by immortalizing primary cells. The production of immortal cell lines using temperature-sensitive immortalizing genes offers an additional method of controlling gene expression, and thereby controlling cell proliferation and differentiation. In the nervous system, these techniques have produced immortal cell lines with neuronal and glial properties.     

Shared and specific functions of Arfs 1–5 at the Golgi revealed by systematic knockouts

ADP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arf1/3) and class II (Arf4/5) Arfs, either individually or in combination. Most knockout cell lines were viable with slight growth defects only when lacking Arf1 or Arf4. However, Arf1+4 and Arf4+5 could not be deleted simultaneously. Class I Arfs are nonessential, and Arf4 alone is sufficient for viability. Upon Arf1 deletion, the Golgi was enlarged, and recruitment of vesicle coats decreased, confirming a major role of Arf1 in vesicle formation at the Golgi. Knockout of Arf4 caused secretion of ER-resident proteins, indicating specific defects in coatomer-dependent ER protein retrieval by KDEL receptors. The knockout cell lines will be useful tools to study other Arf-dependent processes.     

Relation between the regulation of DNA synthesis and the production of two secreted glycoproteins by 12-O-tetradecanoylphorbol-13-acetate in 3T3 cells and in phorbol ester nonresponsive 3T3 variants

12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, acts similarly to growth factors by selectively increasing the rate of production of the secreted proteins, mitogen regulated protein (MRP) and major excreted protein (MEP) by murine 3T3 cells. MRP, a 34 kilodalton (kDa) glycoprotein, is a member of the prolactin-growth hormone family of proteins. MEP, a 39 kDa glycoprotein, is a lysosomal thiol protease that is also secreted. The aim of our investigation was to determine the relation between increases in MRP and MEP production and the initiation of DNA synthesis in response to mitogens. The TNR-9 cell line is a variant of 3T3 cells in which growth factors, but not TPA and teleocidin, stimulate DNA synthesis and cell division. Using [35S]methionine to metabolically label proteins and SDS polyacrylamide gel electrophoresis to resolve the proteins, we found that growing cultures of 3T3 and TNR-9 cells responded equally well to TPA and teleocidin with increased rates of production of MRP and MEP. By contrast, the responses of quiescent TNR-9 cells to these tumor promoters in the increased production of MRP and MEP was greatly diminished compared with quiescent 3T3 cells. The changes in production of MRP in response to tumor promoters in quiescent and growing cells paralleled similar changes in the level of MRP mRNA. In summary, the ability to TPA and teleocidin to increase the rate of production of MRP and MEP correlated with the ability of these tumor promoters to stimulate DNA synthesis in quiescent 3T3 and TNR-9 cells. Evidently the biochemical condition that distinguishes TNR-9 from 3T3 cells and that limits the ability of tumor promoters to stimulate the production of MEP and MRP, and perhaps also DNA synthesis in TNR-9 cells occurs only when the cells are quiescent.     

Gangliosides do not affect ABC transporter function in human neuroblastoma cells

Previous studies have indicated a role for glucosylceramide synthase (GCS) in multidrug resistance (MDR), either related to turnover of ceramide (Cer) or generation of gangliosides, which modulate apoptosis and/or the activity of ABC transporters. This study challenges the hypothesis that gangliosides modulate the activity of ABC transporters and was performed in two human neuroblastoma cell lines, expressing either functional P-glycoprotein (Pgp) or multidrug resistance-related protein 1 (MRP1). Two inhibitors of GCS, D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (t-PPPP) and N-butyldeoxynojirimycin (NB-dNJ), very efficiently depleted ganglioside content in two human neuroblastoma cell lines. This was established by three different assays: equilibrium radiolabeling, cholera toxin binding, and mass analysis. Fluorescence-activated cell sorting (FACS) analysis showed that ganglioside depletion only slightly and in the opposite direction affected Pgp- and MRP1-mediated efflux activity. Moreover, both effects were marginal compared with those of well-established inhibitors of either MRP1 (i.e., MK571) or Pgp (i.e., GF120918). t-PPPP slightly enhanced cellular sensitivity to vincristine, as determined by 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide analysis, in both neuroblastoma cell lines, whereas NB-dNJ was without effect. MRP1 expression and its localization in detergent-resistant membranes were not affected by ganglioside depletion. Together, these results show that gangliosides are not relevant to ABC transporter-mediated MDR in neuroblastoma cells.