Cytoplasmic Redistribution Research Articles

Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a nucleocytoplasmic shuttling protein that regulates gene expression through its action on mRNA metabolism and translation. The cytoplasmic redistribution of hnRNP A1 is a regulated process during viral infection and cellular stress. Here, we show that hnRNP A1 is an internal ribosome entry site (IRES) trans-acting factor that binds specifically to the 5' untranslated region of both the human rhinovirus-2 and the human apoptotic peptidase activating factor 1 (apaf-1) mRNAs, thereby regulating their translation. Furthermore, the cytoplasmic redistribution of hnRNP A1 after rhinovirus infection leads to enhanced rhinovirus IRES-mediated translation, whereas the cytoplasmic relocalization of hnRNP A1 after UVC irradiation limits the UVC-triggered translational activation of the apaf-1 IRES. Therefore, this study provides a direct demonstration that IRESs behave as translational enhancer elements regulated by specific trans-acting mRNA binding proteins in given physiological conditions. Our data highlight a new way to regulate protein synthesis in eukaryotes through the subcellular relocalization of a nuclear mRNA-binding protein.

PAMPs and DAMPs in IBD

An aberrant innate immune response against the enteric microbial flora plays an essential role in the pathogenesis of inflammatory bowel disease (IBD). The innate immune response is driven by myeloid cells through recognition of Pathogen Associated Pattern Molecules (PAMPs). In addition to these “exogenous” danger signals, it has been recently recognized that the “endogenous” danger signals, Damage Associated Molecular Pattern Molecules (DAMPs), can initiate or perpetuate the innate immune response. The prototype DAMP is high mobility group box 1 (HMGB1). While HMGB1 is a chromatin-associated protein and binds to DNA, it is released to extracellular space by cells undergoing necrosis. HMGB1 is also secreted from activated macrophages. Once released extracellulaly, HMGB1 triggers inflammatory signaling pathways in neighboring cells through the receptor for advanced glycation end products (RAGE), as well as toll-like receptor (TLR) 2 and TLR4. The purpose of this study is to elucidate immunologic effects of HMGB1 at the molecular level in macrophages and in vivo in a mouse model of IBD. The murine macrophage cell line, RAW264.7 cells, murine peritoneal and bonemarrow (BM) derived macrophages were stimulated with HMGB1 alone and combination with IFNγ. BM derived macrophages were also stimulated with HMGB1 and various PAMPs in combination. Mucosal HMGB1 in the inflamed colon of IL-10−/− mice was examined by immunohistochemisty. Fecal HMGB-1 levels were determined by a specific ELISA. HMGB1 stimulated IL-12 p40 protein and mRNA expression in RAW264.7 cells, peritoneal and BM derived macrophages. IFNγ in combination with HMGB1 synergistically activated IL-12 p40 expression. Gene expression patterns induced by HMGB1 in macrophages differed from the prototype PAMP, LPS, as HMGB1 did not induce IL-10 expression, while LPS induced IL-10 as well as IL-12 p40. Muramyl-dipeptide (MDP), but not other PAMPs, synergistically en hance IL-12 p40 and TNF expression induced by HMGB1 in BM derived macrophages. In vivo, a marked upregulation of HMGB1 protein was observed in intestinal epithelial cells and lamina propria cells of IL-10−/− mice compared to wild type mice. HMGB1 expression was prominent in the cytoplasm, consistent with the nuclear to cytoplasmic redistribution of HMGB1. Fecal HMGB1 was much more abundant in IL-10−/− mice compared to wild type mice. Fecal HMGB1 levels correlated with histological severity in IL-10−/− mice. HMGB1 stimulates proinflammatory cytokine production from macrophages, likely via distinct signaling pathways from that of PAMPs. HMGB1 specifically synergies with MDP to produce proinflammatory cytokines. HMGB1 is up-regulated in the inflamed colon of IL-10−/− mice and is detected in feces of mice with active colitis suggesting that HMGB1 could play a role in the pathogenesis of IBD.

Nitric oxide controls nuclear export of APE1/Ref-1 through S-nitrosation of Cysteines 93 and 310

Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1, abbreviated as APE1) is a molecule with dual functions in DNA repair and redox regulation of transcription factors. Accumulated work has shown that the biological activities of APE1 are sensitive to oxidative stress; however, whether APE1 functions can be regulated by nitrosative stress remains unknown. In this investigation, we found that S-nitrosoglutathion (GSNO), a nitric oxide donor and also an S-nitrosating agent, effectively stimulated nuclear export of APE1 in a CRM1-independent manner. This nuclear-cytoplasmic translocation was dependent on S-nitrosation modification of APE1, as simultaneous mutation of S-nitrosation target sites Cys93 and Cys310 completely abrogated the cytoplasmic redistribution. The translocation process was reversal and specific, as it could be reversed by reductive reagents, but could not be mimicked by H2O2. In structure, the region aa.64–80 and the beta-strand aa.311–316 in proximity to Cys93 and Cys310 were important for GSNO-induced APE1 relocalization. In addition, a defect of importin-mediated nuclear import pathway was found in the NO-insulted cells, and p50 and HDAC2 were identified as APE1 nuclear export inhibitory proteins. Together, this study may provide a novel molecular mechanism, which links nitrosative stress to APE1-associated physiological and pathological processes.

Altered distribution of heat shock protein 60 (Hsp60) with dysregulated expression of DHX32

DHX32 has an overall similarity to the DHX family of RNA helicases but with a novel helicase domain and nuclear and mitochondrial localizations. The expression of DHX32 is highly regulated during lymphocyte activation and is dysregulated in lymphoid malignancies. In this study, we report our finding of an altered subcellular localization of heat shock protein 60 (Hsp60) in Jurkat-DHX32 cell line in which DHX32 is constitutively expressed. Two-dimensional gel electrophoresis followed by mass spectrometry, electron microscopic immunocytochemistry, and immunoblot analysis showed mainly cytoplasmic localization of Hsp60 in Jurkat-DHX32 cells instead of its mainly mitochondrial localization in control cells. No significant changes were detected in the mitochondrial ultra-structure and the mitochondrial membrane potential activity as a result of dysregulated DHX32 expression. The subcellular distribution of several mitochondrial proteins including cytochrome c, peroxiredoxin 3, manganese superoxide dismutase, Leucine-Rich PentatricoPeptide Repeat Cassette and the 49-kDa subunit of the mitochondrial respiratory Complex I were similar in control and Jurkat-DHX32 cells ruling out non-specific cytoplasmic leakage of mitochondrial proteins. No significant changes in the expression of Hsp60 transcript or total cellular protein were detected. These findings suggest that dysregulated expression of DHX32 might lead to as of yet unknown changes in mitochondrial homeostasis manifested by cytoplasmic redistribution of the molecular chaperon Hsp60.

Molecular Mechanism of Membrane Docking by the Vam7p PX Domain

The Vam7p t-SNARE is an essential component of the vacuole fusion machinery that mediates membrane trafficking and protein sorting in yeast. Vam7p is recruited to vacuoles by its N-terminal PX domain that specifically recognizes PtdIns(3)P in the bilayers, however the precise mechanism of membrane anchoring remains unclear. Here we describe a molecular basis for membrane targeting and penetration by the Vam7p PX domain based on structural and quantitative analysis of its interactions with lipids and micelles. Our results derived from in vitro binding measurements using NMR, monolayer surface tension experiments and mutagenesis reveal a multivalent membrane docking mechanism involving specific PtdIns(3)P recognition that is facilitated by electrostatic interactions and accompanying hydrophobic insertion. Both the hydrophobic and electrostatic components enhance the Vam7p PX domain association with PtdIns(3)P-containing membranes. The inserting Val(70), Leu(71), and Trp(75) residues located next to the PtdIns(3)P binding pocket are surrounded by a basic patch, which is involved in nonspecific electrostatic contacts with acidic lipids, such as PtdSer. Substitution of the insertion residues significantly reduces the binding and penetrating power of the Vam7p PX domain and leads to cytoplasmic redistribution of the EGFP-tagged protein. The affinities of the PX domain for PtdIns(3)P and other lipids reveal a remarkable synergy within the multivalent complex that stably anchors Vam7p at the vacuolar membrane.

Mutations of phosphorylation sites Ser10 and Thr187 of p27Kip1 abolish cytoplasmic redistribution but do not abrogate G0/1 phase arrest in the HepG2 cell line

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 is an important regulator of cell cycle progression as it negatively regulates G0/1 progression and plays a major role in controlling the cell cycle. The screening of the p27Kip1 sequence identified many potential phosphorylation sites. Although Ser10 and Thr187 were shown to be important for p27Kip1 function, the effects of a combined deletion of both sites on p27Kip1 function are still unknown. To investigate the effects of the overexpression of exogenous p27Kip1 protein lacking both the Ser10 and Thr187 sites on subcellular localization, cell cycle, and proliferation, a plasmid was constructed containing mutations of p27Kip1 at Ser10 and Thr187 (S10A/T187A p27), and transfected into the HepG2 cell line with Lipofectamine. Wild-type and mutant p27 plasmids S10A and T187A were transfected separately as control groups. As a result, the proliferation of HepG2 cells was greatly inhibited and cell cycle was arrested in G0/1 phase after exogenous p27Kip1 double-mutant expression. All recombinant p27Kip1 constructs were distributed in the nucleus after synchronization in G0 phase by treatment with leptomycin B. The expressed wild-type and T187A p27Kip1 proteins were translocated from the nucleus into cytoplasm when cells were exposed to 20% serum for 8h, whereas the S10A p27Kip1 and S10A/T187A p27Kip1 proteins remained in the nucleus. FACS profiles and cell growth curves indicated that the Ser10 and Thr187 double mutant has no significant effect on the biological activities of cell cycle control and growth inhibition. Our results suggest that expression of the p27Kip1 double-mutant abolishes its cytoplasmic redistribution but does not abrogate G0/1 phase arrest in the HepG2 cell line.

Effect of a Novel Vacuolar-H+-ATPase Inhibitor on Cell and Tumor Response to Camptothecins

The vacuolar-H(+)-ATPase, functionally expressed in cell membranes, is known to play a relevant role in intracellular pH regulatory mechanisms, because it is implicated in pumping protons into the extracellular environment or in sequestrating excess protons into acidic vacuolar compartments. Because tumor cells exist in a hypoxic microenvironment and produce acidic metabolites, this regulatory mechanism is recognized as a protective function. This study was designed to investigate the effect of NiK-12192 [4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethyl-piperidin-4-yl)-benzamide], an indole derivative identified as an effective inhibitor of vacuolar-H(+)-ATPase, on the cytotoxic activity of two camptothecins, i.e., topotecan and SN-38 (7-ethyl-10-hydroxycamptothecin, the active metabolite of irinotecan). The cellular studies performed in two pairs of human colon carcinoma cell lines, i.e., LoVo and LoVo/DX (overexpressing P-glycoprotein) and HT29 and HT29/Mit (overexpressing breast cancer resistant protein), indicated an enhancement of the antiproliferative effect of camptothecins by concomitant exposure to subtoxic concentrations of NiK-12192. Studies of subcellular distribution indicated that whereas topotecan alone localized mainly in mitochondria and endoplasmic compartment, the simultaneous presence of NiK-12192 caused a cytoplasmic redistribution. In HT29/Mit cells, NiK-12192 reverted the pattern of acidification induced by topotecan. The potentiation of topotecan efficacy by NiK-12192 was documented by an increased efficacy of the combination in both the HT29 tumor xenografts, being more evident in the topotecan-resistant HT29/Mit tumor. In conclusion, the vacuolar-H(+)-ATPase inhibitor NiK-12192 was able to potentiate the cytotoxic/antitumor effects of camptothecins, either in in vitro or in in vivo systems. Such findings support a potential interest for the use of vacuolar-H(+)-ATPase inhibitors in combination therapy to improve camptothecin efficacy.

Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.

Kainate induces rapid redistribution of the actin cytoskeleton in ameboid microglia

Microglia are key mediators of the immune response in the central nervous system (CNS). They are closely related to macrophages and undergo dramatic morphological and functional changes after CNS trauma or excitotoxic lesions. Microglia can be directly stimulated by excitatory neurotransmitters and are known to express many neurotransmitter receptors. The role of these receptors, however, is not clear. This study describes the microglial response to the glutamate receptor agonist kainate (KA) and shows via immunochemistry that the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor subunit GluR1 is present on cultured microglia. In the presence of 100 microM or 1 mM KA, cultured microglia underwent dramatic morphological and cytoskeletal changes as observed by time-lapse photography and quantitative confocal analysis of phalloidin labeling. KA-stimulated microglia showed condensation of cytoplasmic actin filaments, rapid de- and repolymerization, and cytoplasmic redistribution of condensed actin bundles. Rearrangement of actin filaments-thought to be involved in locomotion and phagocytosis and to indicate an increased level of activation (for reviews see Greenberg [ 1995] Trends Cell Biol. 5:93-99; Imai and Kohsaka [ 2002] Glia 40:164-174)-was significantly increased in treated vs. control cultures. Morphological plasticity and membrane ruffling were also seen. These findings suggest direct microglial excitation via glutamate receptor pathways. Thus, neurotransmitter release after brain or spinal cord injury might directly modulate the inflammatory response.

STAT3 NH2-Terminal Acetylation Is Activated by the Hepatic Acute-Phase Response and Required for IL-6 Induction of Angiotensinogen

The signal transducers and activators of transcription (STATs) are cytoplasmic transcription factors mediating acute-phase response (APR) of the human angiotensinogen (hAGT) gene in hepatocytes. The mechanisms of how STAT3 activates target genes are unknown. Here we analyzed the biochemistry of STAT3 activation by interleukin (IL)-6 in hepatocellular carcinoma HepG2 and Balb/C mice. Immunoprecipitation-Western assays and Matrix Assisted Laser Desorption-Time of Flight mass spectrometry determined sites of STAT3 acetylation by the 300-kilodalton target of E1A (p300) co-activator. The subcellular localization of acetylation-deficient STAT3 molecules were studied by microscopic imaging, effects on DNA binding measured by gel shift and chromatin immunoprecipitation (ChIP) assays, and gene transactivation by Northern blot and reporter assays. Two Lys residues at amino acids 49 and 87 in the STAT3 NH2 terminus are acetylated by p300. Lys-to-Arg point mutations (STAT3 K49R/K87R) had no effect on inducible DNA binding, but blocked p300-mediated acetyl(Ac)-STAT3 formation and abrogated IL-6-induced hAGT activation. Although STAT3 K49R/K87R rapidly translocated into the nucleus, it did not bind p300 and had delayed cytoplasmic redistribution. ChIP assays show IL-6-inducible acetylated STAT3 and p300 binding to the native hAGT promoter. Activation of the APR in mice induces nuclear Tyr phosphorylated and acetylated STAT3 in hepatic nuclei. We also observed that STAT3 interacts with histone deacetylases (HDACs), specifically HDAC 1, that down-regulate IL-6-induced hAGT transactivation. IL-6-induced target gene activation requires p300-mediated STAT3 acetylation, and HDACs are involved in the termination of STAT3 action. These studies indicate the acetylation-deacetylation reaction as a novel signaling mechanism controlling the IL-6-STAT3 pathway in the hepatic APR.

The E-Cadherin–β-Catenin Complex and its Implication in Lung Cancer Progression and Prognosis

The E-cadherin-beta-catenin complex plays a pivotal role in epithelial cell-cell adhesion and in the maintenance of differentiated adult epithelia. Perturbation of its expression or function is widely involved in tumor progression and metastasis. Recent years have seen a rapid expansion in the understanding of the biology and the clinical relevance of the E-cadherin adhesion complex in human lung cancer. During human lung cancer progression genomic, transcriptional and post-transcriptional alterations of the E-cadherin-beta-catenin adhesion system are implicated and comprise deletion of the chromosomic region 3p21 that comprise the locus of the gene encoding beta-catenin, transcriptional downregulation of E-cadherin, cytoplasmic redistribution, phosphorylation of both proteins and proteolysis of E-cadherin. E-cadherin-inactivating mutations and oncogenic-activating mutation of beta-catenin are not reported.

Targeting of the FYVE domain to endosomal membranes is regulated by a histidine switch

Specific recognition of phosphatidylinositol 3-phosphate [PtdIns3P] by the FYVE domain targets cytosolic proteins to endosomal membranes during key signaling and trafficking events within eukaryotic cells. Here, we show that this membrane targeting is regulated by the acidic cellular environment. Lowering the cytosolic pH enhances PtdIns3P affinity of the FYVE domain, reinforcing the anchoring of early endosome antigen 1 (EEA1) to endosomal membranes. Reversibly, increasing the pH disrupts phosphoinositide binding and leads to cytoplasmic redistribution of EEA1. pH dependency is due to a pair of conserved His residues, the successive protonation of which is required for PtdIns3P head group recognition as revealed by NMR. Substitution of the His residues abolishes PtdIns3P binding by the FYVE domain in vitro and in vivo. Another PtdIns3P-binding module, the PX domain of Vam7 and p40phox is shown to be pH-independent. This provides the fundamental functional distinction between the two phosphoinositide-recognizing domains. The presented mode of FYVE regulation establishes the unique function of FYVE proteins as low pH sensors of PtdIns3P and reveals the critical role of the histidine switch in targeting of these proteins to endosomal membranes.

Insulin acutely decreases hepatic fatty acid synthase activity

Insulin is viewed as a positive regulator of fatty acid synthesis by increasing fatty acid synthase (FAS) mRNA transcription. We uncover a new mechanism by which insulin acutely reduces hepatic FAS activity by inducing phosphorylation of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and its interaction with FAS. Ceacam1 null mice (Cc1(-/-)) show loss of insulin's ability to acutely decrease hepatic FAS activity. Moreover, adenoviral delivery of wild-type, but not the phosphorylation-defective Ceacam1 mutant, restores the acute effect of insulin on FAS activity in Cc1(-/-) primary hepatocytes. Failure of insulin to acutely reduce hepatic FAS activity in hyperinsulinemic mice, including L-SACC1 transgenics with liver inactivation of CEACAM1, and Ob/Ob obese mice, suggests that the acute effect of insulin on FAS activity depends on the prior insulinemic state. We propose that this mechanism acts to reduce hepatic lipogenesis incurred by insulin pulses during refeeding.

Microsatellite analysis of the adenomatous polyposis coli (APC) gene and immunoexpression of β catenin in nephroblastoma: a study including 83 cases treated with preoperative chemotherapy

Aims: To determine whether microsatellite mutations of the adenomatous polyposis coli (APC) gene have pathological or prognostic significance in nephroblastomas and to correlate APC alterations with β catenin immunoexpression. Methods:...

Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells.

Fatty acid synthase (FAS) activity is a potential therapeutic target to treat cancer and obesity. Here, we have identified a molecular link between FAS and HER2 (erbB-2) oncogene, a marker for poor prognosis that is overexpressed in 30% of breast and ovarian cancers. Pharmacological FAS inhibitors cerulenin and C75 were found to suppress p185(HER2) oncoprotein expression and tyrosine-kinase activity in breast and ovarian HER2 overexpressors. Similarly, p185(HER2) expression was dramatically down-regulated when FAS gene expression was silenced by using the highly sequence-specific mechanism of RNA interference (RNAi). Pharmacological and RNAi-mediated silencing of FAS specifically down-regulated HER2 mRNA and, concomitantly, caused a prominent up-regulation of PEA3, a transcriptional repressor of HER2. A cytoplasmic redistribution of p185(HER2) was associated with marked morphological changes of FAS RNAi-transfected cells, whereas chemical inhibitors of FAS promoted a striking nuclear accumulation of p185(HER2). The simultaneous targeting of FAS and HER2 by chemical FAS inhibitors and the humanized antibody directed against p185(HER2) trastuzumab, respectively, was synergistically cytotoxic toward HER2 overexpressors. Similarly, concurrent RNAi-mediated silencing of FAS and HER2 genes synergistically stimulated apoptotic cell death in HER2 overexpressors. p185(HER2) was synergistically down-regulated after simultaneous inhibition of FAS and HER2 by either pharmacological inhibitors or small interfering RNA. These findings provide evidence of an active role of FAS in cancer evolution by specifically regulating oncogenic proteins closely related to malignant transformation, strongly suggesting that HER2 oncogene may act as the key molecular sensor of energy imbalance after the perturbation of tumor-associated FAS hyperactivity in cancer cells.

Activation of beta-catenin provides proliferative and invasive advantages in c-myc/TGF-alpha hepatocarcinogenesis promoted by phenobarbital.

Previously, we have found that phenobarbital (PB) enhanced cell survival and facilitated tumor growth in our c-myc/transforming growth factor (TGF)-alpha transgenic mouse model of liver cancer. Given that PB selectively promoted initiated cells harboring beta-catenin mutations during chemically induced hepatocarcinogenesis and that Wnt/beta-catenin signaling is involved in both anti-apoptotic and proliferative processes, we now have extended our analysis to investigate whether promotion by PB affects the occurrence of beta-catenin mutations in c-myc/TGF-alpha-driven tumors. The frequency of beta-catenin activation as judged by somatic mutations and/or nuclear localization was significantly increased in hepatocellular carcinomas (HCCs) from c-myc/TGF-alpha mice treated with PB (15/28; 53.6%) as compared with that in control HCCs (2/28; 7.1%). Furthermore, an intact beta-catenin locus was detected in all neoplasms following PB treatment, whereas 57.1% (16/28) of malignant tumors from c-myc/TGF-alpha untreated mice displayed loss of heterozygosity at the beta-catenin locus. Strikingly, in the majority of PB-treated HCCs beta-catenin nuclear localization was limited to small cells with high nuclear/cytoplasmic ratio forming an invasion front (NAinv). beta-Catenin NAinv cells showed cytoplasmic redistribution of E-cadherin associated with intense mucin 1 and matrilysin immunostaining, suggesting their invasive phenotype. All beta-catenin-positive HCCs displayed increased proliferation and tumor size, but no difference in the apoptotic rate when compared with beta-catenin negative tumors. These findings show that PB treatment positively selects for a cell population displaying activation of beta-catenin in c-myc/TGF-alpha HCCs. beta-Catenin activation confers additional growth and invasive advantages in a model of liver cancer already accelerated by synergistic activity of the c-myc and TGF-alpha transgenes.

SMAGP, a new small trans-membrane glycoprotein altered in cancer.

Using the Affymetrix array technology, we previously identified an EST strongly expressed in several metastatic cell lines. In the present study, we cloned the corresponding cDNA that encodes a new glycoprotein composed of 97 amino acids and containing a trans-membrane domain. Therefore, we named it SMAGP for Small trans-Membrane And Glycosylated Protein. SMAGP is strongly conserved during evolution. It is expressed by normal epithelia of various organs, the protein being notably localized to the lateral face of the plasma membrane of cohesive well-polarized epithelial cells. In addition, SMAGP contains binding domains for the protein 4.1 and the PDZ domain of MAGUK proteins. Similar protein features are observed in several cell-surface proteins involved via ternary complexes in intercellular processes leading to cytoskeleton assembly as well as intracellular signalling. Thus, SMAGP might similarly be involved in a scaffolding protein complex, and therefore participate in the epithelium organization or in subsequent functions. Immunohistochemical data obtained using human breast, colon and lung cancer samples sustain this hypothesis since they showed that, in both primary tumours and metastases, reduced expression and/or cytoplasmic redistribution of SMAGP is superimposable with low histological tumour differentiation features, namely a lack of epithelial cell polarity and disorganized tissue phenotype.

HGTSE-1 Expression Stimulates Cytoplasmic Localization of p53

hGTSE-1 (human G(2) and S phase-expressed-1) is a cell cycle-regulated protein mainly localized in the cytoplasm and apparently associated with the microtubules. hGTSE-1 is able to down-regulate levels and activity of the p53 tumor suppressor protein: it binds the C-terminal region of p53 and represses its ability to induce apoptosis after DNA damage. Here we report that, after DNA damage, hGTSE-1 becomes stabilized in a p53-independent way and accumulated in the nucleus. Further characterization of hGTSE-1 localization revealed increased nuclear staining in unstressed cells after treatment with the nuclear export inhibitor leptomycin B, or when a nuclear export signal (NES) located in its C-terminal region was mutated. Finally, we provide evidence that hGTSE-1 ectopic expression, in addition to p53 protein levels down-regulation, is able to enhance cytoplasmic localization of p53. Interestingly, NES-mutated hGTSE-1 accumulates in the nucleus, binds p53 but looses its ability to enhance cytoplasmic redistribution of p53 and to regulate p53 protein levels. Similarly, when wild type hGTSE-1 functions on p53 were analyzed in cells lacking Mdm2, it failed in regulating both p53 localization and protein levels, thus indicating that hGTSE-1 requires an intact NES and functional Mdm2 for the regulation of p53. Our results provide new insights into the mechanism of hGTSE-1 function, whereby its characterized nucleo-cytoplasmic shuttling ability is required to regulate p53.

The cytoplasmic trafficking of DNA topoisomerase IIα correlates with etoposide resistance in human myeloma cells

In this study we have investigated the role of topoisomerase (topo) IIα trafficking in cellular drug resistance. To accomplish this, it was necessary to separate the influence of cell cycle, drug uptake, topo protein levels, and enzyme trafficking on drug sensitivity. Thus, we developed a cell model (called accelerated plateau) using human myeloma H929 cells that reproducibly translocates topo IIα to the cytoplasm. Compared to log-phase cells, the cytoplasmic redistribution of topo IIα in plateau-phase cells correlated with a 10-fold resistance to VP-16 and a 40–60% reduction in the number of drug-induced double-strand DNA breaks. In addition, 7-fold more VP-16 was necessary to achieve 50% topo IIα band depletion, suggesting that there are fewer drug-induced topo–DNA complexes formed in quiescent cells than in log-phase cells. The total cellular amount of topo IIα and topo IIβ protein in log- and plateau-phase cells was similar as determined by Western blot analysis. There was a 25% reduction in S-phase cell number in plateau cells (determined by bromodeoxyuridine (BrdU) incorporation), while there was no significant difference in the equilibrium concentrations of [ 3H]-VP-16 when log cells were compared with plateau cells. Furthermore, the nuclear/cytoplasmic ratio of topo IIα is increased 58-fold in accelerated-plateau H929 cells treated with leptomycin B (LMB) when compared to untreated cells. It appears that the nuclear-cytoplasmic shuttling of topo IIα, which decreases the amount of nuclear target enzyme, is a major mechanism of drug resistance to topo II inhibitors in plateau-phase myeloma cells.

Localization and level of expression of β-catenin in human laryngeal squamous cell carcinoma

Objective We studied the participation of β-catenin in the histologic differentiation of laryngeal squamous cell carcinomas. Study design and setting At the National Institute of Respiratory Diseases, a tertiary referral center, localization and level of expression of β-catenin were compared between normal epithelium (15 cases) and primary tumors in different degrees of differentiation (38 cases), using an immunohistochemical procedure. Results Cell membrane staining of β-catenin was observed in normal epithelium and in well and moderately differentiated carcinomas. Cytoplasmic redistribution was observed in poorly differentiated carcinomas. Loss of β-catenin correlated with tumor dedifferentiation. Conclusion Reduction of cell membrane β-catenin expression correlated with tumor dedifferentiation. Significance Loss of β-catenin may lead to diminishing the strength of the intercellular adhesion system, thereby promoting the invasive phenotype of the squamous cell carcinoma of the larynx.