Michel Maratrat

The requirement for the p53 proline-rich functional domain for mediation of apoptosis is correlated with specific PIG3 gene transactivation and with transcriptional repression

Wild‐type p53 is a tumor suppressor gene which can activate or repress transcription, as well as induce apoptosis. The human p53 proline‐rich domain localized between amino acids 64 and 92 has been reported to be necessary for efficient growth suppression. This study shows that this property mainly results from impaired apoptotic activity. Although deletion of the proline‐rich domain does not affect transactivation of several promoters, such as WAF1, MDM2 and BAX, it does alter transcriptional repression, reactive oxygen species production and sequence‐specific transactivation of the PIG3 gene, and these are activities which affect apoptosis. Whereas gel retardation assays revealed that this domain did not alter in vitro the specific binding to the p53‐responsive element of PIG3, this domain plays a critical role in transactivation from a synthetic promoter containing this element. To explain this discrepancy, evidence is given for a proline‐rich domain‐mediated cellular activation of p53 DNA binding.

Definition of a p53 transactivation function-deficient mutant and characterization of two independent p53 transactivation subdomains.

The wild-type protein product of the p53 tumor suppressor gene can activate transcription of genes which are involved in mediating either growth arrest, e.g. WAF1 or apoptotis, e.g. BAX and PIG3. Additionally, p53 can repress a variety of promoters, which, in turn, may be responsible for the functional activities exhibited by p53. This study shows that the Q22, S23 double mutation, which is known to inactivate a p53 trans-activation subdomain located within the initial 40 residues of the protein, while abrogating transactivation from the WAF1 promoter, only attenuates apoptosis triggering, transactivation from other p53-responsive promoters and repression of promoters by p53. The Q53, S54 double mutation, which inactivates another p53 transactivation subdomain situated between amino acids 43 and 73 results in attenuation of all of the afore-mentioned p53 activities. In contrast to the Q22, S23 double mutation, this latter mutation set does not alter mdm-2-mediated inhibition and degradation of p53. Finally, mutation of all four residues results in complete abrogation of every p53 activity mentioned above.

Antigenic targets in tienilic acid hepatitis. Both cytochrome P450 2C11 and 2C11-tienilic acid adducts are transported to the plasma membrane of rat hepatocytes and recognized by human sera.

Patients with tienilic acid hepatitis exhibit autoantibodies that recognize unalkylated cytochrome P450 2C9 in humans but recognize 2C11 in rats. Our aim was to determine whether the immune reaction is also directed against neoantigens. Rats were treated with tienilic acid and hepatocytes were isolated. Immunoprecipitation, immunoblotting, and flow cytometry experiments were performed with an anti-tienilic acid or an anti-cytochrome P450 2C11 antibody. Cytochrome P450 2C11 was the main microsomal or plasma membrane protein that was alkylated by tienilic acid. Inhibitors of vesicular transport decreased flow cytometric recognition of both unalkylated and tienilic acid-alkylated cytochrome P450 2C11 on the plasma membrane of cultured hepatocytes. Tienilic acid hepatitis sera that were preadsorbed on microsomes from untreated rats (to remove autoantibodies), poorly recognized untreated hepatocytes in flow cytometry experiments, but better recognized tienilic acid-treated hepatocytes. This recognition was decreased by adsorption with tienilic acid or by preexposure to the anti-tienilic acid or the anti-cytochrome P450 2C11 antibody. We conclude that cytochrome P450 2C11 is alkylated by tienilic acid and follows a vesicular route to the plasma membrane. Tienilic acid hepatitis sera contain antibodies against this tienilic acid adduct, in addition to the previously described anticytochrome P450 autoantibodies.

The Interleukin-2 Receptor Down-regulates the Expression of Cytochrome P450 in Cultured Rat Hepatocytes

BACKGROUND & AIMS Interleukin (IL) 2 is used in advanced cancers, but its effects on cytochrome P450 remain unknown. Other cytokines down-regulate hepatic cytochrome P450, but it is not known whether this involves cytokine receptors. The aim of this study was to determine whether the IL-2 receptor is expressed on hepatocytes and whether its activation by IL-2 depresses cytochrome P450 in cultured rat hepatocytes. METHODS A monoclonal antibody specific for the rat IL-2 receptor alpha chain was used to label the receptor, whereas effects on cytochrome P450 were determined after 24 hours of culture with human recombinant IL-2 (5000 U/mL). RESULTS The presence of the IL-2 receptor in hepatocytes was shown by immunoblots, flow cytometry, and scanning confocal microscopy. IL-2 caused a 46% decrease in total cytochrome P450; a 35%, 35%, 36%, 26%, and 56% decrease in immunoreactive cytochrome P4501A1, 2B, 2C11, 2D1, and 3A, respectively; and a marked decrease in cytochrome P4503A2 and 2C11 messenger RNAs. Addition to the culture medium of the anti-receptor antibody or the tyrosine kinase inhibitor genistein prevented the IL-2-mediated decrease in cytochrome P450. CONCLUSIONS IL-2 down-regulates the expression of cytochrome P450 genes in cultured rat hepatocytes by interacting with its receptor expressed on hepatocytes.

CTS1: a p53-derived chimeric tumor suppressor gene with enhanced in vitro apoptotic properties.

The clinical potential of the p53 tumor suppressor gene is being evaluated currently for gene therapy of cancer. We have built a variant of wild-type p53, chimeric tumor suppressor 1 (CTS1), in which we have replaced the domains that mediate its inactivation. CTS1 presents some very interesting properties: (a) enhanced transcriptional activity; (b) resistance to the inactivation by oncogenic forms of p53; (c) resistance to the inactivation by MDM2; (d) lower sensitivity to E6-induced degradation; (e) ability to suppress cell growth; and (f ) faster induction of apoptosis. Thus, CTS1 is an improved tumor suppressor and an alternative for the treatment of wild-type p53-resistant human tumors by gene therapy.

Effect of tauroursodeoxycholate on actin filament alteration induced by cholestatic agents. A study in isolated rat hepatocyte couplets

The mechanism of the protective effect of ursodeoxycholic acid in cholestatic liver diseases remains unclear. Since there is evidence that alterations in the pericanalicular actin microfilament network play a major role in cholestasis, the aims of this study were (a) to determine the effect of the cholestatic agents, taurolithocholate (TLC) and erythromycin estolate (ERY), on F-actin distribution in isolated rat hepatocyte couplets and (b) to assess the effect of tauroursodeoxycholate (TUDC) and taurocholate on the modifications induced by these two compounds. F-actin was stained with fluorescein-isothiocyanate phalloidin and fluorimetric measurements were performed using a scanning laser cytometer ACAS 570. F-actin distribution was assessed in the couplets by the ratio of the pericanalicular area fluorescence/total couplet fluorescence (CF/TF). At non-cytotoxic concentrations, TLC (50, 100 microM) and ERY (10, 50, 100 microM) induced a significant accumulation of F-actin around the bile canaliculus as indicated by increased fluorescence in the pericanalicular area and by the increased CF/TF ratio compared with the controls. Electron microscopy studies showed significant alterations in bile canaliculi microvilli in couplets treated with 100 microM TLC. Only a few canaliculi showed an increase in pericanalicular microfilaments after treatment with 100 microM ERY. As assessed by scanning laser cytometry, TUDC prevented changes in F-actin distribution when it was added to the medium with taurolithocholate or erythromycin estolate at equimolar concentrations. However, the morphological changes observed by electron microscopy after treatment with TLC were not modified by co-treatment with TUDC. Taurocholate was ineffective. We conclude that (a) abnormalities of pericanalicular F-actin microfilaments occur in two different models of cholestasis, (b) tauroursodeoxycholate prevents the accumulation of pericanalicular F-actin detected by scanning laser cytometry but not the morphological changes of the canaliculus observed by electronic microscopy. Therefore, in these experimental conditions, the protective effect of TUDC appears to be partial.

Flow cytometric detection of erythropoietic cytotoxicity in mouse bone marrow.

Erythroblast proliferation and maturation in bone marrow are the processes leading to the formation of polychromatic erythrocytes (PE) and normochromatic erythrocytes (NE), respectively. PE contain RNA but no DNA, and can therefore be distinguished both from NE (which lack both RNA and DNA) and from nucleated cells (which contain both DNA and RNA). Cytotoxic agents that induce impairment of the maturation process change the PE:NE ratio. We have developed a simple and rapid method of determining the PE:NE ratio, based on flow cytometric analysis of formaldehyde-fixed, acridine orange (AO)-stained cells. The effects of cyclophosphamide (CP), mitomycin C (MMC), and vincristine (VC) were tested and the PE:NE ratio was evaluated over 7 days of treatment. In this study we monitored the kinetics of these compounds and were able to demonstrate both a time- and a dose-dependent effect. We detected a difference between the effects of the alkylating agents tested and those induced by the spindle inhibitor tested. Flow cytometry of fixed bone marrow samples stained with AO provides more information, better and more rapid statistical analysis, than conventional microscopic methods for counting the PE:NE ratio.

A rapid assessment of drugs modifying the DNA methylation pattern: Use of the A4/4 cells

BALMAIN Nicole 1, SCHOEVAERT Damien 2, NASG Georges 2, EL KAK Assem 1, LE GUELLEC Dominique 3 i U.120-INSERM, H6p. R. Debrd, 48 Bd S4rurier, 75019 Paris ; 2 Microseopie quantitative, CHU Bie~tre, 94270 Kremlin.Bic~tre ; 3 Cytologic Mol~culaire, CNRS UPR412, Unioers. Claude Bernard, 69622 Villeurbanne. Q U A N T r r A T I V E IN S I T U I I Y B R I D I Z A T I O N : E X P R E S S I O N O F T I l E T Y P E II C O L L A G E N G E N E IN G R O W T H C A R T I L A G E .