Nucleocytoplasmic sorting of macromolecules following mitosis: fate of nuclear constituents after inhibition of pore complex function

PtK2 cells in which pore complex-mediated transport is blocked by microinjection early in mitosis of a monoclonal antibody (specific for an Mr 68,000 pore complex glycoprotein) or of wheat germ agglutinin (WGA) complete cytokinesis. However, their nuclei remain stably arrested in a telophase-like organization characterized by highly condensed chromatin and the absence of nucleoli, indicating a requirement for pore-mediated transport for the reassembly of interphase nuclei. We have now examined this requirement more closely by monitoring the behavior of individual nuclear macromolecules in microinjected cells using immunofluorescence microscopy and have investigated the effect of microinjecting the antibody or WGA on cellular ultrastructure. The absence of nuclear transport did not affect the sequestration into daughter nuclei of components such as DNA, DNA topoisomerase I and the nucleolar protein fibrillarin that are carried through mitosis on chromosomes. On the other hand, lamins, snRNAs and the p68 pore complex glycoprotein, all cytoplasmic during mitosis, remained largely cytoplasmic in the telophase-arrested cells. Electron microscopy showed the nuclei to be surrounded by a double-layered membrane with some inserted pore complexes. In addition, however, a variety of membranous structures with associated pore complexes was regularly noted in the cytoplasm, suggesting that chromatin may not be essential for the postmitotic formation of pore complexes. We propose that cellular compartmentalization at telophase is a two-step process. First, a nuclear envelope tightly encloses the condensed chromosomes, excluding non-selectively all macromolecules not associated with the chromosomes. Interphase nuclear organization is then progressively restored by selective pore complex-mediated uptake of nuclear proteins from the cytoplasm.     

Nucleus-specific and temporally restricted localization of proteins in Tetrahymena macronuclei and micronuclei

Labeled nuclear proteins were microinjected into the cytoplasm of Tetrahymena thermophila. Macronuclear H1, calf thymus H1, and the SV40 large T antigen nuclear localization signal linked to BSA accumulated specifically in macronuclei, even if cells were in micronuclear S phase or were nonreplicating. The way in which histone H4 localized to either the macronucleus or the micronucleus suggested that it accumulates in whichever nucleus is replicating. The inability of the micronucleus to accumulate Tetrahymena H1 or heterologous nuclear proteins, even at a period in the cell cycle when it is accumulating H4, suggests that it has a specialized transport system. These studies demonstrate that although the mechanism for localizing proteins to nuclei is highly conserved among eukaryotes, it can differ between two porecontaining nuclei lying in the same cytoplasm.     

核定位信号及其分析策略

真核细胞核膜上的核孔复合体 (nuclear pore complex, NPC) 是细胞核内外进行物质交换的主要通道, 分子量较小的化合物可自由通过NPC或采取被动扩散的方式进入细胞核, 而分子量为50 kD以上的蛋白质则只能通过主动转运进入细胞核. 以这种方式进入细胞核的 蛋白质必须在其氨基酸序列上拥有特殊的核定位信号(nuclear localization signal, NLS)以被相应的核转运蛋白(karyopherins) 识别. 核定位信号具有多样性, 包括经典核定位信号(classical NLS,cNLS), 内输蛋白β2识别的核定位信号（又称PY模体-NLS）和其它类型的NLS. 每一类NLS具有相似的特征, 但并不具有完全保守的氨基酸组成. 不同的NLS, 往往对应着各不相同的核输入机制. 而对同一蛋白质来说, 也可能同时拥有几个功能性的NLS. 研究核定位信号一方面可以帮助揭示新的大分子物质核转运机制, 另一方面也有助于发现一些蛋白质的新功能. 本文对常见NLS的分类进行了总结, 并介绍了两种常用的NLS预测软件及鉴定NLS的一般策略.     

Nuclear transport of H1 histones meets the criteria of a nuclear localization signal—mediated process

We have studied the nuclear transport of H1 histones using the digitonin permeabilization assay system in order to establish the transport requirements for H1 translocation to the nucleus. Using HeLa cells and fluorescence-labeled calf thymus H1, we show that the H1 nuclear transport in permeabilized cells requires the addition of cytoplasmic extract. Furthermore, it can be blocked by energy depletion and by chilling or by addition of wheat germ agglutinin or by nonhydrolyzable GTP analogs. Thus, the import of H1 histones follows the criteria established for nuclear import mediated by nuclear localization signals (NLS). The distribution of basic amino acids in average H1 sequences, however, does not allow the assignment of a specific element as a classical NLS. J. Cell. Biochem. 64:573–578. © 1997 Wiley-Liss, Inc.     

Factors controlling the loss of immunoreactive somatic histone H1 from blastomere nuclei in oocyte cytoplasm: a potential marker of nuclear reprogramming

Nuclei of differentiated cells can acquire totipotency following transfer into the cytoplasm of oocytes. While the molecular basis of this nuclear reprogramming remains unknown, the developmental potential of nuclear-transfer embryos is influenced by the cell-cycle stage of both donor and recipient. As somatic H1 becomes immunologically undetectable on bovine embryonic nuclei following transfer into ooplasm and reappears during development of the reconstructed embryo, suggesting that it may act as a marker of nuclear reprogramming, we investigated the link between cell-cycle state and depletion of immunoreactive H1 following nuclear transplantation. Blastomere nuclei at M-, G1-, or G2-phase were introduced into ooplasts at metaphase II, telophase II, or interphase, and the reconstructed embryos were processed for immunofluorescent detection of somatic histone H1. Immunoreactivity was lost more quickly from donor nuclei at metaphase than at G1 or G2. Regardless of the stage of the donor nucleus, immunoreactivity was lost most rapidly when the recipient cytoplast was at metaphase and most slowly when the recipient was at interphase. When the recipient oocyte was not enucleated, however, immunoreactive H1 remained in the donor nucleus. The phosphorylation inhibitors 6-DMAP, roscovitine, and H89 inhibited the depletion of immunoreactive H1 from G2, but not G1, donor nuclei. In addition, immunoreactive H1 was depleted from mouse blastomere nuclei following transfer into bovine oocytes. Finally, expression of the developmentally regulated gene, eIF-1A, but not of Gapdh, was extinguished in metaphase recipients but not in interphase recipients. These results indicate that evolutionarily conserved cell-cycle-regulated activities, nuclear elements, and phosphorylation-linked events participate in the depletion of immunoreactive histone H1 from blastomere nuclei transferred in oocyte cytoplasm and that this is linked to changes in gene expression in the transferred nucleus.     

Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins

Previously, we found that anti-DDDED antibodies strongly inhibited in vivo nuclear transport of nuclear proteins and that these antibodies recognized a protein of 69 kD (p69) from rat liver nuclear envelopes that showed specific binding activities to the nuclear location sequences (NLSs) of nucleoplasmin and SV-40 large T-antigen. Here we identified this protein as the 70-kD heat shock cognate protein (hsc70) based on its mass, isoelectric point, cellular localization, and partial amino acid sequences. Competition studies indicated that the recombinant hsc70 expressed in Escherichia coli binds to transport competent SV-40 T-antigen NLS more strongly than to the point mutated transport incompetent mutant NLS. To investigate the possible involvement of hsc70 in nuclear transport, we examined the effect of anti-hsc70 rabbit antibodies on the nuclear accumulation of karyophilic proteins. When injected into the cytoplasm of tissue culture cells, anti-hsc70 strongly inhibited the nuclear import of nucleoplasmin, SV-40 T-antigen NLS bearing BSA and histone H1. In contrast, anti-hsc70 IgG did not prevent the diffusion of lysozyme or 17.4-kD FITC-dextran into the nuclei. After injection of these antibodies, cells continued RNA synthesis and were viable. These results indicate that hsc70 interacts with NLS-containing proteins in the cytoplasm before their nuclear import.     

In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins

An in vitro system was developed that provides a quick microscopic assay for nuclear transport. The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the amount of accumulation observed (up to 17-fold). Selective accumulation of fluorescent nucleoplasmin is observed microscopically within 30 min with rat liver nuclei, Xenopus embryonic nuclei, regrown Xenopus sperm nuclei, or nuclei reconstituted in vitro from bacteriophage lambda DNA. This transport requires the signal domain of nucleoplasmin. Furthermore, the ability of nuclei to accumulate nucleoplasmin directly correlates with their ability to exclude the fluorescent non-nuclear proteins, FITC-immunoglobulin and phycoerythrin. An active transport model would predict that nuclear transport be temperature- and energy- dependent and that inhibition of transport by either low temperature or energy depletion would be reversible. Both predictions were confirmed in our system. Nucleoplasmin accumulation increases with temperature, while the protein is completely excluded at 0 degrees C. The effects of low temperature are reversible. As found for 125I-labeled nucleoplasmin (Newmeyer, D. D., J. M. Lucocq, T. R. Burglin, and E. M. De Robertis, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:501-510), transport of fluorescent nucleoplasmin is inhibited by ATP depletion. This effect is reversed by later ATP addition. Under ATP-depleted conditions non- nuclear proteins continue to be excluded. These results argue for a direct role of ATP in transport rather than for a simple role in preserving envelope integrity. In a first step towards defining the minimum requirements for a transport medium, egg extracts were depleted of membrane vesicles. Membrane-depleted extracts neither support transport nor maintain the integrity of the nuclear envelope.     

Role of nuclear pore complex in simian virus 40 nuclear targeting.

Cytoplasmically injected simian virus 40 (SV40) virions enter the nucleus through nuclear pore complexes (NPCs) and can express large T antigen shortly thereafter (J. Clever, M. Yamada, and H. Kasamatsu, Proc. Natl. Acad. Sci. USA 88:7333-7337, 1991). The nuclear import of the protein components of introduced SV40 was reversibly arrested by chilling and energy depletion, corroborating our previous observation that the nuclear entry of injected SV40 is blocked in the presence of wheat germ agglutinin and an antinucleoporin monoclonal antibody (mAb414), general inhibitors of NPC-mediated import. The nuclear accumulation of virion protein components and large T antigen in nonpermissive NIH 3T3 cells was similar to that in the permissive host, indicating that the ability to use NPCs as a route of nuclear entry appears to be a general property of the injected virus. Injected virions were capable of completing their lytic cycle and forming plaques in permissive cells. During the early phase of SV40 infection, the cytoplasmic injection of mAb414 effectively blocked nuclear T-antigen accumulation for up to 8 h of infection but had very little effect after 12 h of infection. The time-dependent interference with nuclear T-antigen accumulation by the antinucleoporin antibody is consistent with the hypothesis that the infecting virions enter the nucleus through NPCs. The interference study also suggests that the early phase of infection consists of at least two steps: a step for virion cell entry and intracytoplasmic trafficking and a step for virion nuclear entry followed by large-T-antigen gene expression and subsequent nuclear localization of the gene product. Virions were visualized as electron-dense particles in ultrathin sections of samples in which transport was permitted or arrested. In the former cells, electron-dense particles were predominantly observed in the nucleus. The virions were distributed randomly and nonuniformly in the nucleoplasm but were not observed in heterochromatin or in nucleoli. In the latter cells, the electron-dense particles were seen intersecting the nuclear envelope, near the inner nuclear membrane, and in NPCs. In tangential cross sections of NPCs, which appeared as donut-shaped structures, a spherical electron-dense particle was observed in the center of the structure. Immunoelectron microscopy revealed that NPCs were selectively decorated with 5-nm colloidal gold particles-anti-Vp1 immunoglobulin G at the cytoplasmic entrance to and in NPCs, confirming that the morphologically observed electron-dense particles in NPCs contain the viral structural protein. These results support the hypothesis that the nuclear import of SV40 is catalyzed through NPCs by an active transport mechanism that is similar to that of other karyophiles.     

Purification of N-terminally truncated histone H2A-monoubiquitin conjugates from leukemic cell nuclei: probable proteolytic products of ubiquitinated H2A

To gain insight into the significance of nuclear ubiquitinated proteins, two serial extracts prepared from various leukemic cells were analysed by western blotting with anti-ubiquitin antibody. Two previously unidentified ubiquitinated proteins with molecular masses of 10 and 17 kDa were found in 8 M urea-soluble extracts, obtained from Tris-buffer-insoluble materials, of acute myeloid leukemia OCI/AML 1a cells and the cells from the leukemia patients. Both proteins were successfully purified from the OCI/AML 1a cells and identified as monoubiquitin-truncated H2A conjugates, the 10 kDa ubiquitinated H2A(115-129) and the 17 kDa ubiquitinated H2A(54-129), suggesting that both proteins were produced by limited proteolysis of an intact form (23 kDa) of ubiquitinated H2A(1-129). The 17 kDa protein as well as the 23 kDa ubiquitinated histone H2A were localised in chromatin fractions of the OCI/AML cells and released by high concentrations of salt in a micrococcal nuclease-sensitive manner, suggesting their association with chromatin. In contrast, the 10 kDa protein remained insoluble even when the nuclei were treated with nuclease under high salt concentrations, presumably due to binding to the nuclear matrix. An antibody recognising H2A(70-81) also detected the 17 kDa protein in anti-ubiquitin immunoprecipitates obtained from the OCI/AML cell nuclei. In addition, the 17 kDa protein levels in THP-1 cells were transiently increased, concomitant with a decrease in the 23 kDa ubiquitinated H2A, by treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce differentiation. This is the first report of probable proteolytic products of ubiquitinated H2A, which might have a role in nuclear functions.     

Fulvestrant (ICI 182,780)-dependent interacting proteins mediate immobilization and degradation of estrogen receptor-alpha

The antiestrogen fulvestrant (ICI 182,780) causes immobilization of estrogen receptor-alpha (ERalpha) in the nuclear matrix accompanied by rapid degradation by the ubiquitin-proteasome pathway. In this study we tested the hypothesis that fulvestrant induces specific nuclear matrix protein-ERalpha interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)-ERalpha-activating function-2 (AF2) fusion protein was used to isolate and purify receptor-interacting proteins in cell lysates prepared from human MCF-7 breast cancer cells. After SDS-PAGE and gel excision, mass spectrometry was used to identify two major ERalpha-interacting proteins, cytokeratins 8 and 18 (CK8.CK18). We determined, using ERalpha-activating function-2 mutants, that helix 12 (H12) of ERalpha, but not its F domain, is essential for fulvestrant-induced ERalpha-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ERalpha immobilization/degradation, transient transfection assays were performed using wild type ERalpha,ERalpha with a mutated H12, and ERalpha with a deleted F domain. Of those, only the ERalpha H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ERalpha degradation in CK8.CK18-positive human breast cancer cells, and CK8 and CK18 depletion by small interference RNAs partially blocked fulvestrant-induced receptor degradation. Furthermore, fulvestrant-induced ERalpha degradation was not observed in CK8 or CK18-negative cancer cells, suggesting that these two intermediate filament proteins are necessary for fulvestrant-induced receptor turnover. Using an ERalpha-green fluorescent protein construct in fluorescence microscopy revealed that fulvestrant-induced cytoplasmic localization of newly synthesized receptor is mediated by its interaction with CK8 and CK18. In summary, this study provides the first direct evidence linking ERalpha immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ERalpha to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ERalpha turnover.     

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.     

Drug modulation of chromosomal protein subtypes during specific phases of the submaxillary cell cycle

The H1 subtype proteins a, b, c, d, e, and 1 degree and the high mobility group (HMG) proteins 14 and 17 were extracted from submaxillary gland (SMG) nuclei treated in vivo and sorted from specific phases of the cell cycle, and analyzed by gel electrophoresis. Significant differences in these proteins were noted between quiescent (GO) phase nuclei and proliferating nuclei in both stained and autoradiographic gels. Stimulation of the SMG into cell division with DL-isoproterenol-HCl alone or in conjunction with sodium phenobarbital (PB) provided a system for the analysis of drug effects in stained, 3H lysine, and 32P pulse-labeled nuclear proteins obtained from different phases of the cell cycle.     

Somatic histone H1 microinjected into fertilized mouse eggs is transported into the pronuclei but does not disrupt subsequent preimplantation development

We injected somatic subtypes of histone H1 into newly fertilized mouse eggs, which do not naturally contain this chromosomal protein, and examined the fate of the injected protein and its effect on preimplantation development of recipient eggs. Rhodamine-labelled H1 injected into the cytoplasm of 53 eggs was transported into the pronuclei in 51 cases, and this nuclear accumulation could be detected within 15 min of injection. Unlabelled histone H1, which was detected using immunofluorescence, was also transported following microinjection to the pronuclei, where it colocalized with the chromatin and remained associated with the nuclei following cleavage to the two-cell stage. Nuclear accumulation of injected H1 was inhibited when injected eggs were incubated in the presence of drugs that prevent mitochondrial electron transport or glycolysis, which indicates that nuclear transport occurs through an energy-dependent process, as previously observed in tissue culture cells. To determine whether the presence of somatic H1 in early embryonic nuclei would influence subsequent development, fertilized eggs were injected with an approximately physiological quantity (1–5 pg) of somatic H1 or, as controls, with another small basic protein, cytochrome c. Fifty-three eggs were injected with cytochrome c, of which 51 divided to the two-cell stage, and 32 (60%) reached the blastocyst stage, after 5 days in culture. One hundred and eleven eggs were injected with somatic H1, of which 95 divided to the two-cell stage, and 53 (48%) reached the blastocyst stage, after 5 days in culture. The two groups did not differ statistically (X², P > 0.1) with respect to the fraction of injected embryos that developed to the blastocyst stage. These results show that, although mouse embryos lack the somatic subtypes of histone H1 until the four-cell stage of development, they are able to progress through preimplantation development when these subtypes are present beginning at the one-cell stage. This may imply that the distinctive chromatin composition that characterizes early embryos of a variety of species is not essential for early development in mammals. © 1996 Wiley-Liss, Inc.     

Intracellular pH regulation by Na(+)/H(+) exchange requires phosphatidylinositol 4,5-bisphosphate

The carrier-mediated, electroneutral exchange of Na(+) for H(+) across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na(+)/H(+) exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP(2), in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP(2)-binding motifs. Fusion proteins encoding these motifs bound PIP(2) in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP(2)-binding domains had greatly reduced transport capability, implying that association with PIP(2) is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP(2).     

Core histones and linker histones are imported into the nucleus by different pathways

Histones are the major structural proteins in eukaryotic chromosomes. This group of small very basic proteins consists of the H1 linker histones and the core histones H2A, H2B, H3 and H4. Despite their small size, the nuclear import of histones occurs by an active transport mechanism and not simply by diffusion. Histones contain several nuclear localisation signals (NLS) that can be subdivided into two different types of signal structures. We have previously shown that H1 histones are transported by a heterodimeric import receptor complex consisting of importin beta and importin 7, and we now describe the receptors required for the import of the core histones. Competition experiments using the in vitro transport assay indicate that the import pathway of the core histones differs from that of the linker histones and of nuclear proteins with classical NLS. In vitro binding assays show that each of the import receptors importin beta, importin 5, importin 7 and transportin, has the capacity to bind to any of the four core histones. Reconstitution experiments with recombinant factors indicate that each of these factors can independently serve as an import receptor for each of the core histones.     

Reversible nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase upon serum depletion

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) functions as a glycolytic enzyme within the cytoplasm, but beside its metabolic function it is involved in early steps of apoptosis, which trigger the translocation of GAPDH into the nucleus. As apoptosis can be induced by serum withdrawal, which otherwise causes cell cycle arrest, the linkage between serum deprivation, cell cycle and nuclear transport of GAPDH has been investigated. The intracellular distribution of GAPDH was monitored by confocal laser scanning microscopy of either immuno-stained NIH 3T3 fibroblasts or of cells overexpressing GFP-tagged GAPDH. Serum withdrawal led to an accumulation of GAPDH in the nucleus. In contrast to investigations published so far, this nuclear translocation was a reversible process: cytoplasmic location of endogenous GAPDH or of GFP-GAPDH could be recovered upon serum addition to arrested cells and was not inhibited by cycloheximide treatment. In addition, the nuclear import upon serum depletion had no influence neither on the catalytic activity nor on the expression level of GAPDH. The nuclear export of GFP-GAPDH in serum-deprived cells could be stimulated by serum or directly by the growth factors EGF or PDGE The transport process is not regulated via an initiation of cell cycle arrest, as olomoucine, which causes G1-arrest neither stimulated nuclear accumulation nor prevented nuclear export after serum addition to serum-depleted cultures. Moreover, SV40-transformed 3T3 cells transported GAPDH into the nucleus upon serum deprivation, though the expression of the viral large T-antigen enabled growth factor-independent cell proliferation in this cell line. The recruitment of GAPDH to the cytoplasm upon serum stimulation of arrested cells was not impaired by the inhibition of the MAPK signalling pathway with PD 098059. However, further analysis of the growth factor signalling pathway with specific inhibitors revealed that nuclear export was prevented by LY 294002, an inhibitor of the PI-3 kinase. PI3K links the growth factor signalling pathway with cell death via the repression of an apoptotic inducer. Thus, the nuclear accumulation of GAPDH upon growth factor depletion is a reversible process not related directly to cell cycle and likely triggered by survival signals.     

Selective protection of nuclear thioredoxin-1 and glutathione redox systems against oxidation during glucose and glutamine deficiency in human colonic epithelial cells

Little is known about the relative sensitivities of antioxidant systems in nuclei, mitochondria, and cytoplasm. The present study examined the oxidation of the thiol-dependent antioxidant systems in these subcellular compartments under conditions of limited energy supply of human colonic epithelial HT-29 cells induced by depletion of glucose (Glc) and glutamine (Gln) from the culture medium. Increased oxidation of dichlorofluoroscein (DCF) indicated an increased level of reactive oxygen species (ROS). Redox Western blot analysis showed oxidation of cytosolic thioredoxin-1 (Trx1) and mitochondrial thioredoxin-2 (Trx2) by 24 h, but little oxidation of nuclear Trx1. The Trx1 substrate, redox factor-1 (Ref-1), was also oxidized in cytosol but was reduced in nuclei. Protein S-glutathionylation (PrSSG), expressed as a ratio of protein thiol (PrSH), was also increased in the cytosol, while nuclear PrSSG/PrSH was not. Taken together, the data show that oxidative stress induced by depletion of Glc and Gln affects the redox states of proteins in the cytoplasm and mitochondria more than those in the nucleus. These results indicate that the nuclear compartment has better protection against oxidative stress than cytoplasm or mitochondria. These results further suggest that energy and/or substrate supply may contribute to sensitivity of mitochondrial and cytoplasmic systems to oxidative damage.