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2.
Br J Cancer ; 109(12): 3084-91, 2013 Dec 10.
Article in English | MEDLINE | ID: mdl-24149177

ABSTRACT

BACKGROUND: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clinical impact on patients' stratification and outcome. METHODS: Specimens were obtained from RMS patients and cell lines, and ALK expression was analysed by quantitative RT-PCR, western blotting, IHC, and copy number analysis. RESULTS: High ALK mRNA expression was detected in the vast majority of PAX3/7-FOXO1-positive tumours, whereas PAX3/7-FOXO1-negative RMS displayed considerably lower amounts of both mRNA and protein. Notably, ALK mRNA distinguished unfavourable PAX3/7-FOXO1-positive tumours from PAX3/7-FOXO1-negative RMS (P<0.0001), and also correlated with larger tumour size (P<0.05) and advanced clinical stage (P<0.01), independently of fusion gene status. High ALK mRNA levels were of prognostic relevance by Cox univariate regression analysis and correlated with increased risk of relapse (P=0.001) and survival (P=0.01), whereas by multivariate analysis elevated ALK mRNA expression resulted a negative prognostic marker when clinical stage was not included. CONCLUSION: Quantitative assessment of ALK mRNA expression helps to improve risk stratification of RMS patients and identifies tumours with adverse biological characteristics and aggressive behaviour.


Subject(s)
RNA, Messenger/biosynthesis , Receptor Protein-Tyrosine Kinases/biosynthesis , Rhabdomyosarcoma/enzymology , Anaplastic Lymphoma Kinase , Cell Line, Tumor , Child , Female , Gene Expression , Humans , Immunohistochemistry , Male , Prognosis , RNA, Messenger/genetics , Receptor Protein-Tyrosine Kinases/genetics , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/pathology , Survival Analysis
3.
Leukemia ; 26(6): 1375-82, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22289917

ABSTRACT

Understanding the mechanisms that control stress-induced apoptosis is critical to explain how tumours respond to treatment, as cancer cells frequently escape drug toxicity by regulating stress response through heat shock protein (HSP) expression. The overexpression of Hsp72, in particular, results in increased incidence of cell transformation, and correlates with poor prognosis in a wide range of cancers. We have shown that Hsp72 assists folding of oncogenic NPM-ALK kinase in anaplastic large-cell lymphomas (ALCLs), but its role in the maintenance of the malignant phenotype remains uncertain. Therefore, we assessed Hsp72 expression in ALCLs, investigating more in detail the mechanisms that regulate its status and activity. We found that Hsp72 is unique among the HSPs involved in tumourigenesis to be overexpressed in ALK(+) tumours and cell lines and to be induced by stress. Different from other HSPs, Hsp72 prevents cell injury, Bax activation and death by apoptosis in ALK(+) cells, acting both upstream and downstream of mitochondria. Conversely, Hsp72 is underexpressed in ALK(-) ALCL cells, and it is unable to protect cells from apoptosis under stress. Moreover, when Hsp72 expression is reduced following NPM-ALK inhibition, lymphoma cells undergo apoptosis, demonstrating the importance of Hsp72 in regulating ALCL stress response and drug sensitivity.


Subject(s)
Apoptosis , HSP72 Heat-Shock Proteins/metabolism , Lymphoma, Large-Cell, Anaplastic/metabolism , Lymphoma, Large-Cell, Anaplastic/pathology , Mitochondria/pathology , Nuclear Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Anaplastic Lymphoma Kinase , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Cell Proliferation , Child , Down-Regulation , Gene Expression Profiling , HSP72 Heat-Shock Proteins/genetics , Humans , Immunoenzyme Techniques , Nuclear Proteins/genetics , Nucleophosmin , Oligonucleotide Array Sequence Analysis , Phosphorylation , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptor Protein-Tyrosine Kinases/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Tissue Array Analysis
6.
Cancer Res ; 61(4): 1671-7, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11245482

ABSTRACT

Beta-catenin undergoes both serine and tyrosine phosphorylation. Serine phosphorylation in the amino terminus targets beta-catenin for proteasome degradation, whereas tyrosine phosphorylation in the COOH terminus influences interaction with E-cadherin. We examined the tyrosine phosphorylation status of beta-catenin in melanoma cells expressing proteasome-resistant beta-catenin, as well as the effects that perturbation of beta-catenin tyrosine phosphorylation had on its association with E-cadherin and on its transcriptional activity. Beta-catenin is tyrosine phosphorylated in three melanoma cell lines and associates with both the ErbB2 receptor tyrosine kinase and the LAR receptor tyrosine phosphatase. Geldanamycin, a drug which destabilizes ErbB2, caused rapid cellular depletion of the kinase and loss of its association with beta-catenin without perturbing either LAR or beta-catenin levels or LAR/beta-catenin association. Geldanamycin also stimulated tyrosine dephosphorylation of beta-catenin and increased beta-catenin/E-cadherin association, resulting in substantially decreased cell motility. Geldanamycin also decreased the nuclear beta-catenin level and inhibited beta-catenin-driven transcription, as assessed using two different beta-catenin-sensitive reporters and the endogenous cyclin D1 gene. These findings were confirmed by transient transfection of two beta-catenin point mutants, Tyr-654Phe and Tyr-654Glu, which, respectively, mimic the dephosphorylated and phosphorylated states of Tyr-654, a tyrosine residue contained within the beta-catenin-ErbB2-binding domain. These data demonstrate that the functional activity of proteasome-resistant beta-catenin is regulated further by geldanamycin-sensitive tyrosine phosphorylation in melanoma cells.


Subject(s)
Antibiotics, Antineoplastic/pharmacology , Cadherins/metabolism , Cysteine Endopeptidases/metabolism , Cytoskeletal Proteins/metabolism , Melanoma/metabolism , Multienzyme Complexes/metabolism , Nerve Tissue Proteins , Quinones/pharmacology , Receptor, ErbB-2/metabolism , Trans-Activators , Benzoquinones , Cell Movement/drug effects , Cysteine Endopeptidases/drug effects , Cysteine Proteinase Inhibitors/pharmacology , Cytoskeletal Proteins/genetics , Humans , Lactams, Macrocyclic , Melanoma/drug therapy , Melanoma/genetics , Melanoma/pathology , Multienzyme Complexes/drug effects , Phosphorylation , Point Mutation , Proteasome Endopeptidase Complex , Protein Tyrosine Phosphatases/metabolism , Receptor-Like Protein Tyrosine Phosphatases, Class 2 , Receptor-Like Protein Tyrosine Phosphatases, Class 4 , Receptors, Cell Surface/metabolism , Transcription, Genetic/drug effects , Transcriptional Activation , Transfection , Tumor Cells, Cultured , Tyrosine/metabolism , beta Catenin
7.
Biochem Pharmacol ; 60(9): 1343-54, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11008128

ABSTRACT

Histones H2A and H2B are known to be reversibly post-translationally modified by ubiquitination. We previously observed in cultured tumor cells that proteasome inhibition stabilizes polyubiquitinated proteins, depletes unconjugated ubiquitin, and thereby promotes the deubiquitination of nucleosomal histones in chromatin. Provocative indirect evidence suggests that histone ubiquitination/deubiquitination cycles alter chromatin structure, which may limit accessibility of DNA repair proteins to damaged sites. In the present study, we focused on the relationship between the ubiquitination status of histone H2A, the structure of chromatin, and the efficiency of nucleotide excision repair (NER) of cisplatin-DNA adducts in human ovarian carcinoma cells exposed to the antitumor drug cisplatin. Pretreating cells with the proteasome inhibitor lactacystin (LC) or N-acetyl-leucyl-leucyl-norleucinal (ALLnL) induced deubiquitination of ubiquitinated histone H2A (uH2A) and concomitantly promoted chromatin condensation, increased the extent of cisplatin-DNA adducts, and diminished NER-dependent repair of cisplatin-DNA lesions, compared with control cells treated with cisplatin alone. Both proteasome inhibitors also prevented the increase in ERCC-1 mRNA expression that occurs in cells exposed to cisplatin. Cells treated with the combination of ALLnL and cisplatin underwent apoptosis, as indicated by caspase-dependent poly(ADP-ribose) polymerase (PARP) cleavage, more quickly than cells treated with either agent alone. Additionally, the combination of ALLnL and cisplatin potently increased p53 levels in cell lysates and stimulated the binding of p53 to chromatin. Together, these observations suggest that proteasome inhibition may be exploited therapeutically for its potential to sensitize ovarian tumor cells to cisplatin.


Subject(s)
Acetylcysteine/analogs & derivatives , Antineoplastic Agents/pharmacology , Apoptosis , Cisplatin/pharmacology , DNA Repair/drug effects , DNA-Binding Proteins , Endonucleases , Enzyme Inhibitors/pharmacology , Leucine/analogs & derivatives , Multienzyme Complexes/antagonists & inhibitors , Acetylcysteine/pharmacology , Cells, Cultured , Chromatin/drug effects , Chromatin/metabolism , Cysteine Endopeptidases , Cytosol/drug effects , Cytosol/metabolism , DNA/chemistry , DNA/drug effects , DNA Adducts , Drug Interactions , Female , Histones/metabolism , Humans , Leucine/pharmacology , Ovarian Neoplasms/pathology , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases , Proteasome Endopeptidase Complex , Protein Biosynthesis , Proteins/genetics , Proteins/metabolism , RNA, Messenger/biosynthesis , Ubiquitins/metabolism
8.
Biochim Biophys Acta ; 1495(3): 308-18, 2000 Feb 28.
Article in English | MEDLINE | ID: mdl-10699468

ABSTRACT

Colon carcinoma and melanoma cells containing either a deletion of the adenomatous polyposis coli tumor suppressor protein (APC) or mutation of the site in beta-catenin phosphorylated by glycogen synthase kinase-3beta (GSK-3beta) display elevated levels of detergent-soluble beta-catenin due to insensitivity of the cytosolic protein to proteasome-dependent degradation. In this study, we have examined the effect of beta-catenin mutation (S37F) or APC loss on the proteasome sensitivity of additional subcellular beta-catenin pools in melanoma cells. In contrast to detergent-soluble beta-catenin, the detergent-insoluble protein remains proteasome-sensitive irrespective of S37F mutation or APC status. This insoluble component appears associated primarily with nuclear cytoskeletal elements. In addition, DNase I treatment solubilized a portion of detergent-insoluble beta-catenin, suggesting that this fraction also contains chromatin-associated protein, and correlating with a proteasome-sensitive elevation in beta-catenin-stimulated reporter activity. Since the detergent-insoluble nuclear component of beta-catenin displays GSK-3beta- and APC-independent proteasome sensitivity, distinct from the soluble nuclear and cytosolic pools of this protein, regulation of beta-catenin proteasome sensitivity and the contribution of this process to beta-catenin function may be more complex than previously appreciated.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cysteine Endopeptidases/metabolism , Cytoskeletal Proteins/metabolism , Multienzyme Complexes/metabolism , Trans-Activators , Adenomatous Polyposis Coli Protein , Cell Nucleus/metabolism , Chromatin/metabolism , Detergents/chemistry , Glycogen Synthase Kinase 3 , Glycogen Synthase Kinases , Humans , Melanoma , Multienzyme Complexes/antagonists & inhibitors , Proteasome Endopeptidase Complex , Solubility , Subcellular Fractions/metabolism , Tumor Cells, Cultured , beta Catenin
9.
Electrophoresis ; 20(2): 418-28, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10197449

ABSTRACT

Ubiquitination of key cellular proteins involved in signal transduction, gene transcription and cell-cycle regulation usually condemns those proteins to proteasomal or lysosomal degradation. Additionally, cycles of reversible ubiquitination regulate the function of certain proteins in a manner analogous to phosphorylation. In this short review we describe the current methodology for measuring ubiquitin and ubiquitination, provide examples which illustrate how various techniques have been used to study protein ubiquination, alert the readers of pitfalls to avoid, and offer guidelines to investigators newly interested in this novel post-translational protein modification.


Subject(s)
Proteins/metabolism , Ubiquitins/metabolism , Animals , Humans
11.
Oncogene ; 16(9): 1131-9, 1998 Mar 05.
Article in English | MEDLINE | ID: mdl-9528855

ABSTRACT

N-myc is a short-lived transcription factor, frequently amplified in human neuroblastomas. The ubiquitin-proteasome system is involved in the degradation of many short-lived cellular proteins and previous studies have shown that ubiquitin-dependent proteolysis is implicated in the turn-over of N-myc in vitro. However, calpain has also been implicated in N-myc degradation in vitro. Here we report that, in vivo, N-myc is a sensitive substrate for the 26S proteasome in N-myc amplified neuroblastoma cells. We observed that inhibition of the 26S proteasome with two inhibitors, ALLnL and lactacystin, led to an elevation of the N-myc protein steady-state and increased N-myc protein polyubiquitination, as revealed by ubiquitin Western blotting. Pulse-chase experiments have shown that the increased N-myc levels resulted from stabilization of the protein. In contrast treatment with several calpain and cathepsin inhibitors failed to block N-myc degradation in vivo. Furthermore, fluorescence microscopy of ALLnL-treated cells localized N-myc exclusively to the nuclear compartment, suggesting the absence of a requirement for transport to the cytoplasm prior to degradation.


Subject(s)
Cysteine Proteinase Inhibitors/pharmacology , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex , Proto-Oncogene Proteins c-myc/metabolism , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Calpain/metabolism , Humans , Lysosomes/enzymology , Neuroblastoma , Substrate Specificity , Tumor Cells, Cultured , Ubiquitins/metabolism
12.
J Biol Chem ; 269(24): 16677-83, 1994 Jun 17.
Article in English | MEDLINE | ID: mdl-8206987

ABSTRACT

The functions of a deoxynucleoside kinase and a deoxynucleotidase can give rise to substrate cycles in which the two enzymes catalyze in opposite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence showed that pyrimidine dNMP cycles occur in cultured cells and participate in the regulation of the size of dNMP pools there by affecting the transport of deoxyribonucleosides across the cell membrane. Here, we apply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catabolic activity of a putative dAMP/deoxyadenosine cycle. A comparison of human CEM lymphoblasts and hamster V79 fibroblasts, including mutant cells lacking kinases for the phosphorylation of deoxyadenosine, shows a much lower deoxyadenosine excretion in CEM cells (0.05% of dATP synthesized by reduction of ADP) as compared with V79 cells (4% of dATP). Mutational loss of deoxycytidine kinase increases these values to 0.3% in CEM cells and to 10% in V79 cells. This strongly suggests the presence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additional loss of adenosine kinase only marginally affects deoxyadenosine excretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in situ activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there.


Subject(s)
Adamantane/analogs & derivatives , Adenosine Kinase/genetics , Deoxyadenosines/metabolism , Deoxycytidine Kinase/genetics , Adamantane/metabolism , Adenine/metabolism , Adenosine/metabolism , Adenosine Kinase/metabolism , Animals , Cell Division , Cell Line , Cricetinae , DNA/biosynthesis , Deoxycytidine/metabolism , Deoxycytidine Kinase/metabolism , Deoxyribonucleotides/metabolism , Fibroblasts/metabolism , Humans , Hypoxanthine , Hypoxanthines/metabolism , Kinetics , Lymphocytes , Thymidine/metabolism , Time Factors , Tritium
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