Trafficking and localization of platinum complexes in cisplatin-resistant cell lines monitored by fluorescence-labeled platinum

Cisplatin is a chemotherapeutic agent commonly used in the treatment of a wide variety of malignant tumors. Resistance to cisplatin represents a major obstacle to effective cancer therapy because clinically significant levels of resistance quickly emerge after treatment. Based on previous studies indicating abnormal plasma membrane protein trafficking in cisplatin-resistant (CP-r) cells, Fluorescence (Alexa Fluor)-labeled cisplatin was used to determine whether this defect altered the trafficking and localization of cisplatin by comparing drug sensitive KB-3-1 and KB-CP-r cells. Alexa Fluor-cisplatin was readily internalized and localized throughout the KB-3-1 cells, but overall fluorescence decreased in KB-CP-r cells, as detected by flow cytometry (FACS) and confocal microscopy. Only punctate cytoplasmic staining was observed in KB-CP-r cells with less fluorescence observed in the nucleus. Colocalization experiments with a Golgi-selective stain indicate the involvement of Golgi-like vesicles in initial intracellular processing of Alexa Fluor conjugated cisplatin complexes. As detected using an antibody to Alexa Fluor-cisplatin, cisplatin complex-binding proteins (CCBPs) were reduced in membrane fractions of single-step cisplatin-resistant KB-CP.5 cells, and increased in the cytoplasm of KB-CP.5 cells compared to KB-3-1 cells. CCBPs localized to lower density fractions in KB-CP.5 cells than in KB-3-1 cells as determined by iodixanol gradient centrifugation. In summary, inappropriate trafficking of CCBPs might explain resistance to cisplatin in cultured cancer cells, presumably because membrane binding proteins for cisplatin are not properly located on the cell surface in these cells, but are instead trapped in low density vesicles within the cytoplasm.     

Codominance of cisplatin resistance in somatic cell hybrids

Intrinsic or acquired resistance to cisplatin in cancer cells remains a major obstacle to successful chemotherapy. The clinically relevant genetic and molecular mechanisms of resistance have not yet been identified. Cisplatin-resistant (CP-r) human KB epidermoid carcinoma cell lines (HeLa) resistant to varying levels of cisplatin after single and multiple selection steps are cross-resistant to other platinum compounds and to methotrexate. Intraspecies hybrids of the sensitive and KB CP-r cells were fused with HeLa D98(OR) CP-s, hypoxanthine-aminopterin-thymidine (HAT) sensitive, ouabain resistant, to determine whether cisplatin resistance is dominant or recessive. Cell-cell hybridization between the sensitive cells and single-step or two-step KB CP-r cells both indicated codominance of cisplatin resistance compared to hybrids between sensitive cell lines (D98(OR)xKB). The hybrids between sensitive cell lines (D98xKB) and a single-step CP-r KB cell line (D98xKB-CP.5) also were cross-resistant to carboplatin and methotrexate. In addition, the relatively slower growth rate of CP-r cells appears to be dominant. In the two-step CP-r KB cell line, KB-CP1, resistance is no more dominant than in the single-step CP-r KB cell line, KB-CP.5, suggesting that one of the two steps of resistance in KB-CP1 may not be dominant. These dominance data suggest that it might be possible to identify one or more genes responsible for cisplatin resistance by gene transfer from a resistant cell line to a sensitive cell line.     

A toxin-resistant mouse L-cell mutant defective in protein transport along the secretory pathway.

Using methods designed for isolation of mutants defective in receptor-mediated endocytosis, a novel L-cell mutant was obtained that exhibits resistance to three different protein toxins as well as alterations in secretion. This mutant, LEFIC, is resistant to modeccin, Pseudomonas exotoxin, and ricin. These toxins, which enter the cytoplasm via receptor-mediated endocytosis, are thought to penetrate into cells at the level of late endosomes or the trans Golgi network. Early endosomal acidification appears to be normal in the mutant based on its accumulation of iron from transferrin and its sensitivity to diphtheria toxin A chain-transferrin conjugate. Within the secretory pathway two delays in transport of vesicular stomatitis virus (VSV) G protein were observed in LEFIC: a 20-30 min delay in acquisition of Endo H resistance and a 1-2 hr delay in appearance of newly synthesized G protein on the cell surface. Movement of endogenous proteins along the secretory pathway was also affected in LEFIC. Fibronectin secretion was delayed by 15 min, and membrane proteins were delayed in arrival at the cell surface. The phenotype of LEFIC is consistent with a defect in a component or compartment shared by both the late endocytic and constitutive secretory pathways.     

Glycosylphosphatidylinositol-anchored proteins are not required for crosslinking-mediated endocytosis or transfection of avidin bioconjugates into biotinylated cells

Even though glycosylphosphatidylinositol (GPI)-anchored proteins lack direct structural contact with the intracellular space, these ubiquitously expressed surface receptors activate signaling cascades and endocytosis when crosslinked by extracellular ligands. Such properties may be due to their association with membrane microdomains composed of glycosphingolipids, cholesterol and some signaling proteins. In this study, we hypothesize that GPI proteins may be required for crosslinking-mediated endocytosis of extracellular bioconjugates. To test this hypothesis, we first biotinylated the surface membranes of native K562 erythroleukemia cells versus K562 cells incapable of surface GPI protein expression. We then compared the entry of fluorescently labeled avidin or DNA condensed on polyethylenimine-avidin bioconjugates into the two biotinylated cell populations. Using fluorescence microscopy, nearly 100% efficiency of fluorescent avidin endocytosis was demonstrated in both cell types over a 24 h period. Surprisingly, plasmid DNA transfer was slightly more efficient among the biotinylated GPI-negative cells as measured by the expression of green fluorescence protein. Our findings that GPI proteins are not required for the endocytosis of avidin bioconjugates into biotinylated cells suggest that endocytosis associated with general membrane crosslinking may be due to overall reorganization of the membrane domains rather than GPI protein-specific interactions.     

Sorting nexin 27 interacts with multidrug resistance-associated protein 4 (MRP4) and mediates internalization of MRP4

Multidrug resistance-associated protein 4 (MRP4/ABCC4) makes a vital contribution to the bodily distribution of drugs and endogenous compounds because of its cellular efflux abilities. However, little is known about the mechanism regulating its cell surface expression. MRP4 has a PDZ-binding motif, which is a potential sequence that modulates the membrane expression of MRP4 via interaction with PDZ adaptor proteins. To investigate this possible relationship, we performed GST pull-down assays and subsequent analysis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This method identified sorting nexin 27 (SNX27) as the interacting PDZ adaptor protein with a PDZ-binding motif of MRP4. Its interaction was confirmed by a coimmunoprecipitation study using HEK293 cells. Knockdown of SNX27 by siRNA in HEK293 cells raised MRP4 expression on the plasma membrane, increased the extrusion of 6-[(14)C]mercaptopurine, an MRP4 substrate, and conferred resistance against 6-[(14)C]mercaptopurine. Cell surface biotinylation studies indicated that the inhibition of MRP4 internalization was responsible for these results. Immunocytochemistry and cell surface biotinylation studies using COS-1 cells showed that SNX27 localized to both the early endosome and the plasma membrane. These data suggest that SNX27 interacts with MRP4 near the plasma membrane and promotes endocytosis of MRP4 and thereby negatively regulates its cell surface expression and transport function.     

Mass spectrometry analysis of the native protein complex containing actinin-4 in prostate cancer cells

Actinin-4 was originally identified as an actin-binding protein associated with cell motility and cancer invasion and metastasis. However, actinin-4 forms complexes with a large number of different partner proteins and is speculated to have several distinct functions depending on its partner. The level of actinin-4 expression was found to be significantly lower in prostate cancer cells than in non-cancerous basal cells, and restoration of actinin-4 expression inhibited cell proliferation by prostate cancer cell line 22RV1. Immunoprecipitation and mass spectrometry analysis revealed that actinin-4 forms native complexes with several partner proteins in 22RV1 cells, including with beta/gamma-actin, calmodulin, the clathrin heavy chain, non-muscular myosin heavy chain, heterogeneous nuclear ribonucleoprotein A1, and Ras-GTPase-activating protein SH3 domain-binding protein. Clathrin is a coat protein that covers the internalized membrane pit that forms during early endocytosis. We found that other clathrin-related and unrelated cargo proteins, including dynamin, adaptin-delta, beta subunit of neuronal adaptin-like protein, and p47A, also interact with actinin-4. Immunofluorescence microscopy revealed that dynamin and clathrin co-localized with actinin-4 at the sites of membrane ruffling, and transfection of actinin-4 cDNA facilitated the transport of transferrin into perinuclear endosomes. Endocytosis terminates signaling evoked by cell surface receptors and regulates the recycling of receptors and ligands. We identified a panel of proteins whose expression and/or subcellular localization was regulated by actinin-4 by performing organelle fractionation and ICAT-LC-MS/MS. The decreased expression of actinin-4 protein in prostate cancer cells may cause aberrations in the intracellular trafficking of various cell surface molecules and contribute to carcinogenesis.     

Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins

We have established four cell lines derived from the human KB carcinoma cell line which express high-level multiple drug resistance. One of these lines was selected for resistance to colchicine, one was selected for resistance to colchicine in the presence of the tumor promoter, mezerein, one for resistance to vinblastine, and one for resistance to adriamycin. All of these cell lines are cross-resistant to the other selective agents. The development of multidrug resistance in these cultured human carcinoma cells is associated with a limited number of specific protein alterations revealed by high resolution two-dimensional gel electrophoresis and Western blot analysis. These protein alterations in multidrug-resistant lines include the decreased prevalence of members of a family of proteins of molecular mass 70,000 to 80,000 daltons, pI 4.8-5.0, the increased synthesis of a protein of molecular mass 21,000 daltons, pI 5.0, in the colchicine-resistant cell lines only, and the increased expression of a 170,000-dalton protein in membrane preparations from all of the resistant cells. The loss of the 70,000- to 80,000-dalton proteins in the multidrug-resistant lines, which can also be demonstrated by immunoprecipitation of these proteins with specific antisera, is associated with a loss of translatable mRNA for these proteins. These studies suggest that only a limited number of protein changes occur in multidrug-resistant cell lines.     

Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis

Cellular activity depends to a large extent on membrane bilayer dynamics. Many processes, such as organelle biogenesis and vesicular transport, rely on alterations in membrane structure and shape. It is now widely accepted that intracellular membrane curvature generation and remodelling is mediated and regulated by protein action, and the mechanisms behind the processes are currently being revealed. Here, we will briefly discuss the key principles of membrane deformation and focus on different endocytic events that use various kinds of proteins to shape the plasma membrane into transport carriers. The entry routes are adopted to make sure that a vast variety of molecules on the cell surface can be regulated by endocytosis. The principles for membrane sculpting of endocytic carriers can be viewed either from a perspective of rigid coat budding or of flexible opportunistic budding. We will discuss these principles and their implications, focusing on clathrin-dependent and -independent carrier formation and the proteins involved in the respective pathways.     

Membrane protein changes in an L1210 leukemia cell line with a translocation defect in the methotrexate-tetrahydrofolate cofactor transport carrier

We report on membrane protein changes in an L1210 leukemia cell line with a highly specific defect in the function of the methotrexate (MTX)-tetrahydrofolate cofactor transport carrier. This clonal line, MTXrA, made 100-fold resistant to MTX, was derived in a single step and exhibited stable resistance over 120 generations in the absence of drug. The transport defect was associated with a 10-fold decrease in influx Vmax without a change in influx Km. There was no difference between the MTXrA and parent lines in the levels or affinities of specific cell surface binders for MTX nor in the labeling of the 44-kDa membrane protein upon treatment with the specific affinity label, N-hydroxysuccinimide ester of tritiated MTX. Consistent with impaired carrier function was the observation that trans-stimulation of MTX influx by intracellular 5-formyltetrahydrofolate observed in the parent line was not demonstrated in the MTXrA line. The transport defect was highly specific for the MTX-tetrahydrofolate cofactor transport carrier. Initial uptake rates for 5-fluoro-2'-deoxyuridine and 2-deoxyglucose were unchanged and influx and net transport of alpha-aminoisobutyric acid were, in fact, increased. There was no cross-resistance of this line to phenylalanine mustard or cytosine arabinoside, agents that utilize specific amino acid and nucleoside transport carriers, respectively. SDS-polyacrylamide gel electrophoresis of purified plasma membrane preparations stained with Coomassie Blue revealed several protein differences between the parental and MTXrA lines. Most prominent is a band at approximately 190 kDa which ran with slightly greater mobility than a lesser staining band in the parent line. [3H]Borohydride labeling of cells also identified a distinct protein peak in the MTXrA line at approximately 190 kDa eliminated by prior treatment of cells with neuraminidase. Absence of expression of protein or mRNA related to the multidrug resistance gene as well as lack of cross-resistance to daunorubicin or trimetrexate indicate that this mechanism of resistance to MTX is completely unrelated to the multidrug resistance phenomenon observed with high molecular weight heterocyclic compounds. These data represent the first demonstration of membrane protein differences in a highly resistant L1210 murine leukemia cell line with a marked unique defect in MTX transport which appears to be related to impaired mobility of the tetrahydrofolate-cofactor carrier. Further studies are now required to elucidate the possible role of one or more of these proteins in the transport defect.     

Cisplatin stabilizes a multimeric complex of the human Ctr1 copper transporter: requirement for the extracellular methionine-rich clusters

Cisplatin is a highly effective cancer chemotherapy agent. However, acquired resistance currently limits the clinical utility of this drug. The human high affinity copper importer, hCtr1, and its yeast and murine orthologues have been shown to mediate the uptake of cisplatin. This transporter is located at the plasma membrane under low copper conditions, and excess copper concentrations stimulate its endocytosis and degradation. In this study we further examined the link between cisplatin and hCtr1 by examining whether cisplatin can also stimulate the endocytosis and degradation of hCtr1. The steady-state location of hCtr1 and its endocytosis from the plasma membrane were not altered by cisplatin treatment. Unexpectedly, cisplatin treatment of a cell line expressing hCtr1 revealed the time- and concentration-dependent appearance of a stable hCtr1 multimeric complex, consistent with a homotrimer, which was not observed following copper treatment of these same cells. Mutagenesis studies identified two methionine-rich clusters in the extracellular amino-terminal region of hCtr1 that were required for stabilization of the hCtr1 multimer by cisplatin, suggesting that these sequences bind cisplatin and form crosslinks between hCtr1 polypeptides. Treatment with the metal chelator dimethyldithiocarbamate disassembled the hCtr1 multimer following cisplatin exposure, suggesting that platinum was an integral component of this complex. These studies provide the first evidence for a direct interaction between cisplatin and the hCtr1 protein and establish that cisplatin and copper have distinct biochemical consequences on this transporter.     

Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal

After synaptic vesicles fuse with the plasma membrane and release their contents, vesicle membrane proteins recycle by endocytosis and are targeted to newly formed synaptic vesicles. The membrane traffic of an epitope-tagged form of VAMP-2 (VAMP-TAg) was observed in transfected cells to identify sequence requirements for recycling of a synaptic vesicle membrane protein. In the neuroendocrine PC12 cell line VAMP-TAg is found not only in synaptic vesicles, but also in endosomes and on the plasma membrane. Endocytosis of VAMP-TAg is a rapid and saturable process. At high expression levels VAMP-TAg accumulates at the cell surface. Rapid endocytosis of VAMP-TAg also occurs in transfected CHO cells and is therefore independent of other synaptic proteins. The majority of the measured endocytosis is not directly into synaptic vesicles since mutations in VAMP-TAg that enhance synaptic vesicle targeting did not affect endocytosis. Nonetheless, mutations that inhibited synaptic vesicle targeting, in particular replacement of methionine-46 by alanine, inhibited endocytosis by 85% in PC12 cells and by 35% in CHO cells. These results demonstrate that the synaptic vesicle targeting signal is also used for endocytosis and can be recognized in cells lacking synaptic vesicles.     

Involvement of MKP-1 and Bcl-2 in acquired cisplatin resistance in ovarian cancer cells

Although cisplatin is a very effective anticancer agent against several types of cancer including ovarian cancer, the mechanisms of acquired resistance are not fully understood. By chronically exposing cisplatin to ovarian cancer cell lines, we established two cisplatin-resistant cell lines OV433 and TOV112D. Our results indicate that the mechanisms underlying their cisplatin resistance are distinct. In OV433 cells, cisplatin resistance is associated with increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). By knocking down MKP-1 expression by siRNA or inhibiting MKP-1 expression by its pharmacological inhibitor triptolide, cisplatin-resistant OV433 cells became cisplatin-sensitive and subsequently increased cisplatin-induced apoptosis. In TOV112D cells, on the other hand, acquired cisplatin resistance is associated with increased levels of Bcl-2 protein. By inhibiting the activity of Bcl-2 protein with its pharmacological inhibitor gossypol or knocking down Bcl-2 expression by siRNA, cisplatin-resistant TOV112D cells became cisplatin-sensitive and subsequently increased cisplatin-induced apoptosis. Therefore, our data suggest that the mechanisms of acquired cisplatin resistance vary among ovarian cancer cells, which involve up-regulation of molecules associated with the cell survival pathways.     

Proteomics profile changes in cisplatin-treated human ovarian cancer cell strain

Ovarian cancer is an important gynecological tumor, with the third highest morbidity rate and highest mor-tality rate in gynecological cancers, of which the 5-year survival rate remains at only 19%―38.7%[1]. Tumors occurring in ovary can hardly be diagnosed in the early stages for the dormant start, and approxi-mately 70% patients cannot get the final diagnosis until the advanced stages. Surgical operation and post-operational chemotherapy based on cisplatin have become the primary treatment …     

Proteomics profile changes in cisplatin-treated human ovarian cancer cell strain

To compare the alterations in proteomes between cisplatin-treated and -untreated human ovarian cancer SKOV3 cells, and to explore the feasibility of proteomics in research about antitumor mechanisms of agents, SKOV3 cells were exposed to cisplatin (6 ώg/mL) for 6 h. Then, the cells were collected and solubilized and global proteins were extracted by lysis buffer; two-dimensional electrophoresis was conducted with the IPG readystrips as carriers; the gels were stained with Coomassie blue and alterations between gels were compared by PDQuest. Eventually, 11 spots with significant differences were selected and excised and the proteins were identified by PMF and MS/MS analysis. The results revealed that exposure to cisplatin could notably increase expressions of some proteins, such as tropomyosin family, actin family, triosephosphate isomerase family, and HSP60, etc.; while expressions of some other proteins decreased, such as enolase family, etc. Those proteins were involved in cellular energy metabolism, transformation, apoptosis and morphologic maintenance, which suggested that alterations of those physiological processes might be involved in anti-tumor mechanism of cisplatin.     

Resistance to paclitaxel in a cisplatin-resistant ovarian cancer cell line is mediated by P-glycoprotein

The IGROVCDDP cisplatin-resistant ovarian cancer cell line is also resistant to paclitaxel and models the resistance phenotype of relapsed ovarian cancer patients after first-line platinum/taxane chemotherapy. A TaqMan low-density array (TLDA) was used to characterise the expression of 380 genes associated with chemotherapy resistance in IGROVCDDP cells. Paclitaxel resistance in IGROVCDDP is mediated by gene and protein overexpression of P-glycoprotein and the protein is functionally active. Cisplatin resistance was not reversed by elacridar, confirming that cisplatin is not a P-glycoprotein substrate. Cisplatin resistance in IGROVCDDP is multifactorial and is mediated in part by the glutathione pathway and decreased accumulation of drug. Total cellular glutathione was not increased. However, the enzyme activity of GSR and GGT1 were up-regulated. The cellular localisation of copper transporter CTR1 changed from membrane associated in IGROV-1 to cytoplasmic in IGROVCDDP. This may mediate the previously reported accumulation defect. There was decreased expression of the sodium potassium pump (ATP1A), MRP1 and FBP which all have been previously associated with platinum accumulation defects in platinum-resistant cell lines. Cellular localisation of MRP1 was also altered in IGROVCDDP shifting basolaterally, compared to IGROV-1. BRCA1 was also up-regulated at the gene and protein level. The overexpression of P-glycoprotein in a resistant model developed with cisplatin is unusual. This demonstrates that P-glycoprotein can be up-regulated as a generalised stress response rather than as a specific response to a substrate. Mechanisms characterised in IGROVCDDP cells may be applicable to relapsed ovarian cancer patients treated with frontline platinum/taxane chemotherapy.     

Fractionated irradiation can induce functionally relevant multidrug resistance gene and protein expression in human tumor cell lines

The molecular basis of radiotherapy-related multidrug resistance (MDR) is still unclear. Here we report on a study investigating the effect of fractionated irradiation on expression of the MDR-associated proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and lung resistance-related protein (LRP), the respective mRNAs, and the functional consequences. Cells of six colon and five breast cancer cell lines were irradiated with a total dose of 27 Gy, five fractions of 1.8 Gy per week. The mRNA expression was measured by quantitative RT-PCR, protein levels and drug sensitivity to cisplatin, doxorubicin and bendamustine were assessed by flow cytometry. Breast cancer cell lines showed enhancement of the mRNAs encoding for P-gp, MRP1 and LRP in comparison to nonirradiated cells. No up-regulation of the three mRNA species was observed in the colon cancer cell lines. After irradiation, three breast cancer cell lines showed an up-regulation of LRP, one line an up-regulation of MRP1, and four lines a small up-regulation of P-gp. In the colon cancer cell lines, radiation induced significant enhancement of all three proteins. In comparison to controls, the irradiated cells lines showed a significant resistance to cisplatin, doxorubicin and bendamustine. This study confirms the prior reports of enhancement of P-gp and MRP1 after irradiation, which is accompanied by a multidrug resistance phenomenon, but in addition proposes a novel mechanism in the appearance of MDR after radiation-induced enhancement of LRP.     

Transforming growth factor-alpha and beta-amyloid precursor protein share a secretory mechanism

Cleavage and release of membrane protein ectodomains, a regulated process that affects many cell surface proteins, remains largely uncharacterized. To investigate whether cell surface proteins are cleaved through a shared mechanism or through multiple independent mechanisms, we mutagenized Chinese hamster ovary (CHO) cells and selected clones that were unable to cleave membrane-anchored transforming growth factor alpha (TGF-alpha). The defect in TGF-alpha cleavage in these clones is most apparent upon cell treatment with the protein kinase C (PKC) activator PMA, which stimulates TGF-alpha cleavage in wild-type cells. The mutant clones do not have defects in TFG-alpha expression, transport to the cell surface or turnover. Concomitant with the loss of TGF-alpha cleavage, these clones have lost the ability to cleave many structurally unrelated membrane proteins in response to PMA. These proteins include beta-amyloid precursor protein (beta-APP), whose cleavage into a secreted form avoids conversion into the amyloidogenic peptide A beta, and a group of cell surface proteins whose release into the medium is stimulated by PMA in wild type CHO cells but not in mutants. The mutations prevent cleavage by PKC- dependent as well as PKC-independent mechanisms, and thus affect an essential component that functions downstream of these various signaling mechanisms. We propose that regulated cleavage and secretion of membrane protein ectodomains is mediated by a common system whose components respond to multiple activators and act on susceptible proteins of diverse structure and function.