Chantal Degraef

Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues

Abstract Paraffin sections from animal or human tissues fixed in different fixatives were submitted to immunostaining with the mouse monoclonal antibody 19A2, developed by Ogata et al. (1987a) against cyclin/PCNA. Detection of the bound antibody was performed by the indirect method with biotinylated sheep antibody and streptavidin‐biotin‐peroxidase complexes. No, or faint, nuclear staining was seen in material fixed in ethanol, Bouin, Bouin‐Hollande, Carnoy or formaldehyde, whereas readily detectable immunocytochemical reaction was constantly observed over nuclei of methanol‐fixed tissues. Hydrolysis with 2 N HCl prior to immunocytochemistry (as currently performed to render incorporated BrdU accessible to antibodies) somewhat improved the results with Bouin or Carnoy and markedly augmented the intensity of the peroxidase reactions in formaldehyde and in methanol‐fixed tissues.

Comparison of methods based on annexin‐V binding, DNA content or TUNEL for evaluating cell death in HL‐60 and adherent MCF‐7 cells

Abstract. HL‐60 and MCF‐7 cells were treated with 0.15 μM camptothecin (CPT) or with the solvent dimethylsulfoxide (DMSO) for the controls, for 2, 3 and 4 h or for 24, 48 and 72 h, respectively. The apoptotic index (AI) was then evaluated in parallel by the following flow cytometric methods: (1) double staining of unfixed cells with fluoresceinated annexin V and propidium iodide (PI), this after detachment by trypsinization in the case of MCF‐7 cultures; (2) prefixation in 70% ethanol, extraction of degraded, low molecular weight DNA with 0.2 M phosphatecitrate buffer and analysis of the DNA content stained with PI; (3) TUNEL, i.e. labelling of DNA strand breaks with biotin‐dUTP, followed by standing with streptavidin‐fluorescein and counterstaining with PI. In HL‐60 cells, the three methods gave similar results for the AI (3‐4% in the controls and at 2 h of CPT treatment, and 35‐43% at 3 and 4 h after CPT). This indicates that CPT‐induced membrane alteration and DNA fragmentation occurred concomitantly in those cells. For MCF‐7 cells, CPT‐induced apoptosis developed more slowly, the AI, whether based on annexin V or on DNA content, remained unchanged at 24 h, then was increasing to 8% at 48 h and to 25% at 72 h of treatment. In these cells, the TUNEL index did not increase prior to 72 h and the increase was minor (up to 9% vs. 2‐3% in the controls) at 72 h of the treatment. This indicates that in MCF‐7 cells DNA strand breaks cannot be effectively labelled, which may be due to inaccessibility of 3′‐OH ends in the breaks to exogenous terminal deoxynucleotidyl transferase. The mechanism of endonucleolytic DNA fragmentation thus may be different, depending on the cell type.

Hepatocyte proliferative activity in human liver cirrhosis

BACKGROUND/AIMS The objective of this study was to validate, with an independent prospective cohort of patients, our previous data indicating that the proliferating cell nuclear antigen-labeling index (PCNA-LI) reflects the liver functional reserve in human cirrhosis and might have prognostic significance for patient survival. We also examined how this proliferative index is related to the expression of transforming growth factor beta1 (TGFbeta1) as a possible correlate of hepatocyte proliferative activity. METHODS The present group (n=70 patients) was similar in composition to our previous group regarding age, sex and severity of liver cirrhosis. PCNA and TGFbeta1 immunostaining were analyzed on methanol-fixed, paraffin-embedded liver biopsies. RESULTS Our data show that PCNA-LI declined significantly with worsening Child class and was negatively correlated with the Pugh score. Twenty-five patients died and 10 underwent liver transplantation during the observation period. Liver function, hepatic venous pressure gradient and hepatocyte PCNA-LI were significantly different in survivors and non-survivors. At a mean follow-up of 356 days, the patients with a PCNA-LI higher than 4.4% (the previously determined best cut-off value) had a significantly higher probability of survival than those with a PCNA-LI < or = 4.4% (0.87 vs 0.48, p=0.0009). TGFbeta1 expression in liver parenchyma correlated negatively with PCNA-LI, suggesting that this cytokine could be involved in the impaired regeneration observed in worsened liver cirrhosis. CONCLUSIONS This prospective study strengthens our previous observation that, in cirrhosis, hepatocyte proliferative activity, as evaluated by the PCNA-LI, provides information on liver functional reserve as well as on the patients prognosis.

A novel partner for D-type cyclins: protein kinase A-anchoring protein AKAP95

Using a yeast interaction screen to search for proteins that interact with cyclin D3 in thyroid gland, we identified the cAMP-dependent AKAP95 (protein kinase A-anchoring protein 95). AKAP95 is a scaffolding protein that primarily co-fractionates with the nuclear matrix, whereas a minor fraction associates with chromatin in interphase cells. In co-transfected Chinese-hamster ovary cells, AKAP95 strongly interacted with the three D-type cyclins, but not with CDK4 (cyclin-dependent kinase 4) or with p27kip1. CDK4 displaced the interaction between cyclin D3 and AKAP95, suggesting that AKAP95 could not be the elusive bridging adaptor between D-type cyclins and CDK4 or play a role in the regulation of cyclin D3-CDK4 activity. Interaction between endogenous AKAP95 and cyclin D3 or cyclin D1 was detected in canine thyrocytes, human fibroblasts and NIH-3T3 cells. As both AKAP95 and cyclins D were recently reported to associate with minichromosome maintenance proteins [Eide, Tasken, Carlson, Williams, Jahnsen, Tasken and Collas (2003) J. Biol. Chem. 278, 26750-26756; Gladden and Diehl (2003) J. Biol. Chem. 278, 9754-9760], we hypothesize that the interaction between AKAP95 and D-type cyclins might serve to facilitate the emerging regulatory role of cyclin D-CDK4 in the formation of the prereplication complex at the DNA replication origins.

The docking properties of SHIP2 influence both JIP1 tyrosine phosphorylation and JNK activity

SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is an ubiquitously expressed phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase which contains various motifs susceptible to mediate protein-protein interaction. In cell models, evidence has been provided that SHIP2 plays a role in insulin and growth factor signaling, cytoskeletal organization, cell adhesion and migration. Herein we describe the c-Jun NH2-terminal kinase (JNK)-interacting protein 1 (JIP1) as a new protein partner of SHIP2. The interaction between SHIP2 and JIP1 was confirmed in both overexpression systems and native cells. Without modifying the association of JIP1 with the MAPKs in the scaffold complex and with no apparent change of Akt phosphorylation, SHIP2 positively modulated the MLK3/JIP1-mediated JNK1 activation. Moreover, SHIP2 positively regulated the tyrosine phosphorylation of JIP1. This up-regulation was prevented by inhibitors of the Src family and Abl kinases, PP2 and Glivec. The effects of SHIP2 on JNK activity and JIP1 tyrosine phosphorylation were independent of the SHIP2 phosphoinositide 5-phosphatase activity, as similar results were obtained when using a SHIP2 catalytic inactive mutant instead of wild-type SHIP2. Together, these data suggest that by its docking properties, SHIP2 can modulate JIP1-mediated JNK pathway signaling.

G1/S Cyclins Interact with Regulatory Subunit of PKA via A-Kinase Anchoring Protein, AKAP95

The mechanisms by which cyclins promote mammalian cell cycle progression havebeen a topic of intense investigation over the last decade. We previously described aninteraction between D-type cyclins and A-kinase anchoring protein, AKAP95. Here, wedemonstrate that AKAP95 can also bind cyclin E1. Association between AKAP95 andcyclins is displaced by CDKs. We show that these G1/S cyclins can interact with RIIsubunit of PKAα through AKAP95. The presence of alternate complexes cyclin-CDKand cyclin D/E-AKAP95-PKA.RIIα suggest different roles of G1/S cyclins and a widerbiological importance of these interactions in cells.

SHIP2 (SH2 Domain-containing Inositol Phosphatase 2) SH2 Domain Negatively Controls SHIP2 Monoubiquitination in Response to Epidermal Growth Factor

The SH2 domain containing inositol 5-phosphatase SHIP2 contains several interacting domains that are important for scaffolding properties. We and others have previously reported that SHIP2 interacts with the E3 ubiquitin ligase c-Cbl. Here, we identified human SHIP2 monoubiquitination on lysine 315. SHIP2 could also be polyubiquitinated but was not degraded by the 26 S proteasome. Furthermore, we identified a ubiquitin-interacting motif at the C-terminal end of SHIP2 that confers ubiquitin binding capacity. However, this ubiquitin-interacting motif is dispensable for its monoubiquitination. We showed that neither c-Cbl nor Nedd4-1 play the role of ubiquitin ligase for SHIP2. Strikingly, monoubiquitination of the ΔSH2-SHIP2 mutant (lacking the N-terminal SH2 domain) is strongly increased, suggesting an intrinsic inhibitory effect of the SHIP2 SH2 domain on its monoubiquitination. Moreover, SHIP2 monoubiquitination was increased upon 30 min of epidermal growth factor stimulation. This correlates with the loss of interaction between the SHIP2 SH2 domain and c-Cbl. In this model, c-Cbl could mask the monoubiquitination site and thereby prevent SHIP2 monoubiquitination. The present study thus reveals an unexpected and novel role of SHIP2 SH2 domain in the regulation of its newly identified monoubiquitination.

Chronic alcohol exposure sensitizes mice to galactosamine-induced liver injury through enhanced keratinocyte chemoattractant and defective IL-10 production

BACKGROUND/AIMS Alcohol sensitizes the liver to several injuries. The mechanisms leading to this sensitization are poorly defined. In the present study, we developed a mouse model of chronic exposure to alcohol vapours that sensitize mice to galactosamine (GAL) liver injury. METHODS C57BL/6 mice were exposed to ethanol vapours for 10 days. Liver injury was induced by intraperitoneal injection of GAL (1 g/kg) and mice were killed 24 h later. RESULTS GAL challenge after ethanol pre-treatment significantly raised serum alanine aminotransaminase (ALT) levels and enhanced liver inflammation when compared with the controls (GAL alone). Serum keratinocyte chemoattractant (KC) and monocyte chemoattractant protein-1 (MCP-1) levels were significantly increased in the GAL+ethanol group. On the contrary, serum interleukin 10 (IL-10) levels were lower than in controls. Anti-KC, anti-tumour necrosis factor alpha antibodies and intestinal decontamination significantly protected mice from liver injury. In GAL+ethanol-treated mice, IL-10 treatment reduced ALT release, KC and MCP-1 serum and hepatic mRNA levels, and improved liver inflammation. CONCLUSIONS Enhancement of GAL-induced liver injury by ethanol is associated with an imbalance between proinflammatory cytokines and the anti-inflammatory cytokine IL-10 and depends on gut bacterial flora.

S phase duration measurement by combined PCNA/cyclin immunostaining and radioautography after a single pulse-labelling with 3H-thymidine

A method for measuring S phase duration is described and evaluated that combines single pulse labelling with 3H-thymidine (TdR), detected by radioautography, and proliferating cell nuclear antigen (PCNA)/cyclin immunostaining to replace the second pulse labelling of the classical double-labelling method. Conditions were set up in which nuclei showing one or both types of label were readily distinguished, hence allowing to verify that cell fluxes in and out of S phase were equal. S phase durations thus measured in different tissues of the mouse were concordant with those obtained by the double 3H-TdR labelling or from labelled mitoses curves. Our method might be used with archived samples of methanol-fixed cells or tissues, singly labelled with 3H-TdR or with bromodeoxyuridine.

Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced

Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.