Nucleocytoplasmic traffic disorder induced by cardioviruses

Some picornaviruses, for example, poliovirus, increase bidirectional permeability of the nuclear envelope and suppress active nucleocytoplasmic transport. These activities require the viral protease 2A(pro). Here, we studied nucleocytoplasmic traffic in cells infected with encephalomyocarditis virus (EMCV; a cardiovirus), which lacks the poliovirus 2A(pro)-related protein. EMCV similarly enhanced bidirectional nucleocytoplasmic traffic. By using the fluorescent "Timer" protein, which contains a nuclear localization signal, we showed that the cytoplasmic accumulation of nuclear proteins in infected cells was largely due to the nuclear efflux of "old" proteins rather than impaired active nuclear import of newly synthesized molecules. The nuclear envelope of digitonin-treated EMCV-infected cells permitted rapid efflux of a nuclear marker protein. Inhibitors of poliovirus 2A(pro) did not prevent the EMCV-induced efflux. Extracts from EMCV-infected cells and products of in vitro translation of viral RNAs contained an activity increasing permeability of the nuclear envelope of uninfected cells. This activity depended on the expression of the viral leader protein. Mutations disrupting the zinc finger motif of this protein abolished its efflux-inducing ability. Inactivation of the L protein phosphorylation site (Thr47-->Ala) resulted in a delayed efflux, while a phosphorylation-mimicking (Thr47-->Asp) replacement did not significantly impair the efflux-inducing ability. Such activity of extracts from EMCV-infected cells was suppressed by the protein kinase inhibitor staurosporine. As evidenced by electron microscopy, cardiovirus infection resulted in alteration of the nuclear pores, but it did not trigger degradation of the nucleoporins known to be degraded in the poliovirus-infected cells. Thus, two groups of picornaviruses, enteroviruses and cardioviruses, similarly alter the nucleocytoplasmic traffic but achieve this by strikingly different mechanisms.     

Intracellular Redistribution of Truncated La Protein Produced by Poliovirus 3Cpro-Mediated Cleavage

The La autoantigen (also known as SS-B), a cellular RNA binding protein, may shuttle between the nucleus and cytoplasm, but it is mainly located in the nucleus. La protein is redistributed to the cytoplasm after poliovirus infection. An in vitro translation study demonstrated that La protein stimulated the internal initiation of poliovirus translation. In the present study, a part of the La protein was shown to be cleaved in poliovirus-infected HeLa cells, and this cleavage appeared to be mediated by poliovirus-specific protease 3C (3C^pro). Truncated La protein (dl-La) was produced in vitro from recombinant La protein by cleavage with purified 3C^pro at only one Gln₃₅₈-Gly₃₅₉ peptide bond in the 408-amino-acid (aa) sequence of La protein. The dl-La expressed in L cells was detected in the cytoplasm. However, green fluorescence protein linked to the C-terminal 50-aa sequence of La protein was localized in the nucleus, suggesting that this C-terminal region contributes to the steady-state nuclear localization of the intact La protein in uninfected cells. The dl-La retained the enhancing activity of translation initiation driven by poliovirus RNA in rabbit reticulocyte lysates. These results suggest that La protein is cleaved by 3C^pro in the course of poliovirus infection and that the dl-La is redistributed to the cytoplasm. dl-La, as well as La protein, may play a role in stimulating the internal initiation of poliovirus translation in the cytoplasm.     

Facilitated nuclear transport of histone H1 and other small nucleophilic proteins.

Upon microinjection into the cytoplasm, three small nonnuclear (extracellular or mitochondrial) proteins diffused into nuclei of chilled or energy-depleted cells. In contrast, the facilitated transport of two large nuclear localization signal (NLS)-containing proteins was reversibly arrested by chilling or energy depletion. Surprisingly, the transport of two small nucleophilic proteins, histone H1 and P(Lys)-cytochrome c (cytochrome c cross-linked with synthetic peptide NLSs), was also arrested by either chilling or energy depletion. In situ titration studies indicate that the transport arrest of H1 in chilled cells is mediated by a cytoplasmic receptor. Therefore, even though they are potentially able to diffuse into nuclei, histones and other small NLS-containing proteins are localized by a receptor-mediated process that precludes their diffusion through the nuclear pores.     

Entry of poliovirus into cells is blocked by valinomycin and concanamycin A

Poliovirus contains a virus particle devoid of a lipid envelope that does not require an intact pH to enter into susceptible cells. Thus, the blockade of pH gradient generated in endosomes is not sufficient to impede the translocation of poliovirus particles to the cytoplasm, suggesting that translocation takes place at the plasma membrane. Measuring both viral protein synthesis and eIF4G-1 cleavage mediated by poliovirus protease 2A has been used to monitor productive entry of poliovirus into cells. Translation of the input poliovirus RNA produces enough 2A(pro) to cleave eIF4G-1, providing a sensitive assay to estimate poliovirus RNA delivery to the cytoplasm followed by its translation. Combination of concanamycin A, a vacuolar proton-ATPase inhibitor, and valinomycin, an ionophore that promotes K(+) efflux from cells, powerfully prevented poliovirus infection. Moreover, modifying the ionic conditions of the culture medium (increasing the concentration of K(+) and decreasing the concentration of Na(+)), together with concanamycin A, also significantly interfered with poliovirus entry. These findings suggest that poliovirus RNA requires an intact concentration of K(+) ions inside the cells to be uncoated and to gain access to the cytoplasm. In addition, the actual contribution of concanamycin A (as well as other inhibitors of endocytosis) to the total inhibition of productive poliovirus entry points to the idea that at least some percentage of polioviral subparticles translocates from the endosomes.     

A switching system regulating subcellular localization of nuclear proteins using a viral protease

We explored a novel approach to the functional regulation of nuclear proteins; altering their subcellular localization. To anchor a nuclear protein, beta-galactosidase with the nuclear localization signal of SV40 (nbeta-gal), within the cytoplasm, nbeta-gal was fused to the transmembrane domain of granulocyte colony-stimulating factor receptor (G-CSFR), a membrane protein. To liberate the nbeta-gal portion from the fusion protein, we used a protease derived from a plant virus, whose recognition sequence was inserted between the G-CSFR and nbeta-gal. Western analysis showed that the chimeric protein was cleaved in the presence of the protease in 293 cells and that the fusion protein without the recognition sequence remained intact. This chimeric protein was localized exclusively in the cytoplasm as visualized by X-gal staining and immunofluorescence microscopy. In contrast, when expressed together with the protease, beta-gal was predominantly detected in the nuclei. Moreover, we isolated 293-cell clones constitutively expressing the protease, indicating that this protease is not cytotoxic. These results suggest that the viral protease-mediated alteration of subcellular localization can potentially regulate the function of nuclear proteins.     

Poliovirus 2A(pro) induces the nucleic translocation of poliovirus 3CD and 3C' proteins

Poliovirus genomic RNA replication, protein translation, and virion assembly are performed in the cytoplasm of host cells. However, this does not mean that there is no relationship between poliovirus infection and the cellular nucleus. In this study, recombinant fluorescence-tagged poliovirus 3CD and 3C' proteins were shown to be expressed mainly in the cytoplasm of Vero cells in the absence of other viral proteins. However, upon poliovirus infection, many of these proteins redistributed to the nucleus, as well as to the cytoplasm. A series of transfection experiments revealed that the poliovirus 2A(pro) was responsible for the same redistribution of 3CD and 3C' proteins to the nucleus. Furthermore, a mutant 2A(pro) protein lacking protease activity abrogated this effect. The poliovirus 2A(pro) protein was also found to co-localize with the Nup153 protein, a component of the nuclear pore complexes on the nuclear envelope. These data provide further evidence that there are intrinsic interactions between poliovirus proteins and the cell nucleus, despite that many processes in the poliovirus replication cycle occur in the cytoplasm.     

Autographa californica multiple nucleopolyhedrovirus nucleocapsid protein BV/ODV-C42 mediates the nuclear entry of P78/83

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-c42 (orf101; c42), which encodes a 41.5-kDa viral nucleocapsid protein with a putative nuclear localization signal (NLS) motif at the C terminus, is a highly conserved gene among members of the Baculoviridae family. C42 is demonstrated to be essential for AcMNPV propagation and can bind to nucleocapsid protein P78/83, a viral activator for the actin-related protein 2/3 (ARP2/3) complex to initiate nuclear actin polymerization, which is essential for viral nucleocapsid morphogenesis during AcMNPV infection. Here, we report the identification of a novel pathway through which c42 functions in nucleocapsid assembly. Cotransfection of Sf9 cells with c42 and p78/83 plasmids demonstrated that C42 was capable of recruiting P78/83 to the nuclei of uninfected cells and that the NLS motif of C42 was essential for this process. To validate this nuclear relocation mode in bacmid-transfected cells, a c42-disrupted bacmid (vAc(c42ko-gfp)) and rescued bacmids with wild-type c42 (vAc(c42res-gfp)) or with NLS coding sequence-mutated c42 (vAc(c42nls-gfp)) were prepared. By immuno-staining, P78/83 was found to be localized in the cytoplasm of either vAc(c42ko-gfp)- or vAc(c42nls-gfp)-transfected cells, whereas P78/83 was relocated to the nuclei of vAc(c42res-gfp)-transfected cells. Furthermore, F-actin-specific staining confirmed that there was no actin polymerization activity in the nuclei of either vAc(c42ko-gfp)- or vAc(c42nls-gfp)-transfected cells, which might be attributed to the absence of nuclear P78/83, an activator of the ARP2/3 complex to initiate nuclear actin polymerization. We therefore hypothesize a mode of action where C42 binds to P78/83 in the cytoplasm to form a protein complex and cotransports to the nucleus under the direction of the NLS motif in C42 during AcMNPV infection.     

Inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus

Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.     

Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1.

Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymphocyte granule proteinase granzyme B (graB) and is thought to protect cytotoxic lymphocytes and bystander cells from graB-mediated apoptosis. Following uptake into cells, graB promotes DNA degradation, rapidly translocating to the nucleus, where it binds a nuclear component. PI-9 should therefore be found in cytotoxic lymphocyte and bystander cell nuclei to ensure complete protection against graB. Here we demonstrate by microscopy and subcellular fractionation experiments that PI-9 is present in the nuclei of human cytotoxic cells, endothelial cells, and epithelial cells. We also show that the related serpins, PI-6, monocyte neutrophil elastase inhibitor (MNEI), PI-8, plasminogen activator inhibitor 2 (PAI-2), and the viral serpin CrmA exhibit similar nucleocytoplasmic distributions. Because these serpins lack classical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively. Large (approximately 70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleocytoplasmic distributions to the parent proteins, indicating that nuclear import is active. By contrast, CrmA-GFP is excluded from nuclei, indicating that CrmA is not actively imported. In vitro nuclear transport assays show that PI-9 accumulates at a rate above that of passive diffusion, that it requires cytosolic factors but not ATP, and that it does not bind an intranuclear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We conclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.     

The Mex67p-mediated nuclear mRNA export pathway is conserved from yeast to human

Human TAP is an orthologue of the yeast mRNA export factor Mex67p. In mammalian cells, TAP has a preferential intranuclear localization, but can also be detected at the nuclear pores and shuttles between the nucleus and the cytoplasm. TAP directly associates with mRNA in vivo, as it can be UV-crosslinked to poly(A)+ RNA in HeLa cells. Both the FG-repeat domain of nucleoporin CAN/Nup214 and a novel human 15 kDa protein (p15) with homology to NTF2 (a nuclear transport factor which associates with RanGDP), directly bind to TAP. When green fluorescent protein (GFP)-tagged TAP and p15 are expressed in yeast, they localize to the nuclear pores. Strikingly, co-expression of human TAP and p15 restores growth of the otherwise lethal mex67::HIS3/mtr2::HIS3 double knockout strain. Thus, the human TAP-p15 complex can functionally replace the Mex67p-Mtr2p complex in yeast and thus performs a conserved role in nuclear mRNA export.     

MPF localization is controlled by nuclear export.

In eukaryotes, mitosis is initiated by M phase promoting factor (MPF), composed of B-type cyclins and their partner protein kinase, CDK1. In animal cells, MPF is cytoplasmic in interphase and is translocated into the nucleus after mitosis has begun, after which it associates with the mitotic apparatus until the cyclins are degraded in anaphase. We have used a fusion protein between human cyclin B1 and green fluorescent protein (GFP) to study this dynamic behaviour in real time, in living cells. We found that when we injected cyclin B1-GFP, or cyclin B1-GFP bound to CDK1 (i.e. MPF), into interphase nuclei it is rapidly exported into the cytoplasm. Cyclin B1 nuclear export is blocked by leptomycin B, an inhibitor of the recently identified export factor, exportin 1 (CRM1). The nuclear export of MPF is mediated by a nuclear export sequence in cyclin B1, and an export-defective cyclin B1 accumulates in interphase nuclei. Therefore, during interphase MPF constantly shuttles between the nucleus and the cytoplasm, but the bulk of MPF is retained in the cytoplasm by rapid nuclear export. We found that a cyclin mutant with a defective nuclear export signal does not enhance the premature mitosis caused by interfering with the regulatory phosphorylation of CDK1, but is more sensitive to inhibition by the Wee1 kinase.     

Differential targeting of nuclear pore complex proteins in poliovirus-infected cells

Poliovirus disrupts nucleocytoplasmic trafficking and results in the cleavage of two nuclear pore complex (NPC) proteins, Nup153 and Nup62. The NPC is a 125-MDa complex composed of multiple copies of 30 different proteins. Here we have extended the analysis of the NPC in infected cells by examining the status of Nup98, an interferon-induced NPC protein with a major role in mRNA export. Our results indicate that Nup98 is targeted for cleavage after infection but that this occurs much more rapidly than it does for Nup153 and Nup62. In addition, we find that cleavage of these NPC proteins displays differential sensitivity to the viral RNA synthesis inhibitor guanidine hydrochloride. Inhibition of nuclear import and relocalization of host nuclear proteins to the cytoplasm were only apparent at later times after infection when all three nucleoporins (Nups) were cleaved. Surprisingly, analysis of the distribution of mRNA in infected cells revealed that proteolysis of Nup98 did not result in an inhibition of mRNA export. Cleavage of Nup98 could be reconstituted by the addition of purified rhinovirus type 2 2A^pro to whole-cell lysates prepared from uninfected cells, suggesting that the 2A protease has a role in this process in vivo. These results indicate that poliovirus differentially targets subsets of NPC proteins at early and late times postinfection. In addition, targeting of interferon-inducible NPC proteins, such as Nup98, may be an additional weapon in the arsenal of poliovirus and perhaps other picornaviruses to overcome host defense mechanisms.     

Increased nuclear traffic chaos in hyphae of Aspergillus nidulans: molecular characterization of apsB and in vivo observation of nuclear behaviour

Filamentous fungi are model microorganisms for studying nuclear migration in eukaryotic cells. Two genes, apsA and apsB (=anucleate primary sterigmata), were identified in Aspergillus nidulans that affect nuclear distribution in hyphae and specifically block conidiophore development at the metula stage when mutant. Here we describe the cloning, sequencing and molecular analysis of apsB. The gene encodes a 121 kDa coiled-coil, hydrophilic protein that was localized in the cytoplasm. No protein-protein interaction was detected between ApsB and ApsA, a membrane-associated, previously identified protein. An apsB null mutant was characterized by video epifluorescence microscopy using strains that express green fluorescent protein (GFP) in nuclei. With this novel approach, we have discovered a new mutant phenotype and have found that nuclei display an increased chaotic movement in older hyphal compartments that results in clustering and an uneven distribution of these organelles. These results suggest a regulatory role of ApsB in nuclear migration.     

The adaptor protein TRADD activates distinct mechanisms of apoptosis from the nucleus and the cytoplasm

TNFR1 associated death domain protein (TRADD) contains an N-terminal TRAF binding domain and a C-terminal death domain along with nuclear import and export sequences that cause shuttling between the cytoplasm and nucleus. The death domain of TRADD contains the nuclear import sequence and expression of the core death domain (nuclear TRADD) results in exclusive nuclear localization and activation of a distinct apoptotic pathway. Cytoplasmic TRADD activates apoptosis through Fas-associated death domain protein (FADD) and caspase-8 activation that was blocked by caspase inhibitors or dominant-negative FADD. These inhibitors did not inhibit death induced by nuclear TRADD, which could only be inhibited by combining caspase inhibitors and a serine protease inhibitor. The pathway activated by nuclear TRADD requires caspase-9 catalytic activity. However, apoptosis activating factor deficiency confers only partial protection from death. This pathway represents an alternate means by which TRADD can regulate cell death independently of FADD and caspase-8 that occurs from the nucleus rather than the cytoplasm.     

Perforin-dependent nuclear entry of granzyme B precedes apoptosis, and is not a consequence of nuclear membrane dysfunction

Killer lymphocytes utilize the synergy of a membranolytic protein, perforin, and the serine protease granzyme B (grB) to induce target cell apoptosis, however the mechanism of this synergy remains incompletely defined. We have previously shown that perforin specifically induces the redistribution of cytoplasmic grB into the nucleus of dying cells, however a causal role for nuclear targeting of grB in cell death has not been demonstrated. In the present study, we used confocal laser scanning microscopy (CLSM) to determine whether the nuclear accumulation of fluoresceinated (FITC-) grB precedes or is a consequence of apoptosis. Two distinct and mutually exclusive cellular responses were observed in FDC-P1 cells: (i) up to 50% of the cells rapidly accumulated FITC-grB in the nucleus (maximal at 7 min; t1/2 of 2 min) and underwent apoptosis; (ii) the remaining cells took up FITC-grB only into the cytoplasm, and escaped apoptosis. Under these conditions, DNA fragmentation was not observed for at least 13 min, indicating nuclear accumulation of grB preceded the execution phase of apoptosis. Furthermore, nuclear import of grB proceeded through an intact nuclear membrane, as the nuclei of cells whose cytoplasm was pre-loaded with 70 kDa FITC-dextran excluded dextran for up to 90 min while still undergoing apoptosis in response to perforin and grB. These findings indicated that perforin-induced nuclear accumulation of grB precedes apoptosis, and is not a by-product of caspase-induced nuclear membrane degradation. The cell membrane lesions formed by perforin in these experiments were not large enough to permit a 13 kDa protein (yeast cdk p13suc) access into the cytoplasm, but an 8 kDa protein (bacterial azurin) was able to equilibrate between the cytosol and the exterior. Therefore, transmembrane pores large enough to allow passive diffusion of grB (32 kDa) into the cell are not necessary for apoptosis. Rather, a perforin-dependent signal results in a redistribution of grB from the cytoplasm to the nucleus, where it may contribute to the nuclear changes associated with apoptosis.     

A lily pollen ASR protein localizes to both cytoplasm and nuclei requiring a nuclear localization signal

LLA23, an abscisic acid-, stress- and ripening-induced (ASR) protein, was isolated previously from lily ( Lilium longiflorum ) pollen. Close examination of the C-terminus of this ASR protein revealed the presence of basic regions reminiscent of a nuclear localization signal (NLS). Fluorescence microscopy studies using green fluorescent protein (GFP) fusion proteins indicated that the bipartite NLS in LLA23 exhibited nuclear localization properties. Accordingly, mutations in the NLS motifs of LLA23 defined two regions, either of which was necessary for partial nuclear targeting and both of which were required for complete nuclear localization. In addition, oligonucleotide-directed mutagenesis identified lysine residues within the NLS necessary for nuclear localization. Immunogold localization confirmed that the protein was located to both the cytoplasm and nucleus of generative and vegetative cells of pollen grains; the generative nuclei showed the highest number of LLA23 labelling. The possible function of ASR proteins in both the cytoplasm and nuclei of pollen grains is discussed.     

The KASH domain protein MSP-300 plays an essential role in nuclear anchoring during Drosophila oogenesis

During late stages of Drosophila oogenesis, the cytoplasm of nurse cells in the egg chamber is rapidly transferred ("dumped") to oocytes, while the nurse cell nuclei are anchored by a mechanism that involves the actin cytoskeleton. The factors that mediate this interaction between nuclei and actin cytoskeleton are unknown. MSP-300 is the likely Drosophila ortholog of the mammalian Syne-1 and -2 and C. elegans ANC-1 proteins, contained both actin-binding and nuclear envelope localization domains. By using an antibody against C-terminus of MSP-300, we find that MSP-300 is distributed throughout the cytoplasm and accumulates at the nuclear envelope of nurse cells and the oocyte. A GFP fusion protein containing the C-terminal region of MSP-300 is also sufficient to localize protein on the nuclear envelope in oocytes. To eliminate the maternal gene activity during oogenesis, we generated homozygous germ-line clones of a loss-of-function mutation in msp-300 in otherwise heterozygous mothers. In the mutant egg chambers that develop from such clones, cytoplasmic dumping of nurse cells is severely disturbed. The nuclei of nurse cells and the oocyte are mislocalized and the usually well-organized actin structures are severely disrupted. These results indicate that maternal MSP-300 plays an important role in actin-dependent nuclear anchorage during cytoplasmic transport.     

The major apoptotic pathway activated and suppressed by poliovirus

Cells respond to poliovirus infection by switching on the apoptotic program, implementation of which is usually suppressed by viral antiapoptotic functions. We show here that poliovirus infection of HeLa cells or derivatives of MCF-7 cells was accompanied by the efflux of cytochrome c from mitochondria. This efflux occurred during both abortive infection (e.g., interrupted by guanidine-HCl and ending with apoptosis) and productive infection (leading to cytopathic effect). The former type of infection, but not the latter, was accompanied by truncation of the proapoptotic protein Bid. The virus-triggered cytochrome c efflux was suppressed by overexpression of Bcl-2. Both abortive and productive infections also resulted in a decreased level of procaspase-9, as revealed by Western blotting. In the former case, this decrease was accompanied by the accumulation of a protein with the electrophoretic mobility of active caspase-9. In contrast, in the productively infected cells, the latter protein was absent but caspase-9-related polypeptides with altered mobility could be detected. Both caspase-9 and caspase-3 were shown to be essential for the development of such hallmarks of virus-induced apoptosis as chromatin condensation, DNA degradation, and nuclear fragmentation. These and some other results suggest the following scenario. Poliovirus infection activates the apoptotic pathway, involving mitochondrial damage, cytochrome c efflux, and consecutive activation of caspase-9 and caspase-3. The apoptotic signal appears to be amplified by a loop which includes secondary processing of Bid. The implementation of the apoptotic program in productively infected cells may be suppressed, however, by the viral antiapoptotic functions, which act at a step(s) downstream of the cytochrome c efflux. The suppression appears to be caused, at least in part, by aberrant processing and degradation of procaspase-9.     

Detection of P-glycoprotein in the nuclear envelope of multidrug resistant cells

P-glycoprotein is a plasma membrane efflux pump which is responsible for multidrug resistance of many cancer cell lines. A number of studies have demonstrated the presence of P-glycoprotein molecules, besides on the plasma membrane, also in intracellular sites, such as the Golgi apparatus and the nucleus. In this study, the presence and function of P-glycoprotein in the nuclear membranes of human breast cancer cells (MCF-7 WT) and their multidrug resistant variants (MCF-7 DX) were investigated. Electron and confocal microscopy immunolabelling experiments demonstrated the presence of P-glycoprotein molecules in the nuclear membranes of MCF-7 DX cells. Moreover, the labelling pattern was strongly dependent on pH values of the incubation buffer. At physiological pH (7.2), a strong labelling was detected in the cytoplasm and the nuclear matrix in both sensitive and resistant MCF-7 cells. By raising the pH to 8.0, the P-glycoprotein molecules were easily detected in the cytoplasm (transport vesicles and Golgi apparatus), plasma and nuclear membranes exclusively in MCF-7 DX cells. Furthermore, drug uptake and efflux studies, performed by flow cytometry on isolated nuclei in the presence of the P-glycoprotein inhibitor cyclosporin A, suggested the presence of a functional P-glycoprotein in the nuclear membrane, but not in the nuclear matrix, of drug resistant cells. Therefore, P-glycoprotein in the nuclear envelope seems to represent a further defense mechanism developed by resistant cells against antineoplastic agents.