Dany Rouillard

The pag gene product, a physiological inhibitor of c-abl tyrosine kinase, is overexpressed in cells entering S phase and by contact with agents inducing oxidative stress

The human pag gene product is an inhibitor of the c‐abl tyrosine kinase and belongs to a new family of proteins. We show here that higher levels of pag gene expression are observed following induction of proliferation and contact with compounds inducing oxidative stress such as diethyl maleate and sodium arsenate. A weaker overexpression is seen in a macrophage cell line using hydrogen peroxide or menadione as inducers. Pag gene expression increases in synchronized cells entering the S phase. This raises the possibility that elevated levels of pag counteract the cytostatic activity of abl. Treatment of growth arrested cells with diethyl maleate and sodium arsenate induces pag gene overexpression, independently of cell proliferation. Thus, enhanced pag gene expression occurs in two cellular events: proliferation and response to oxidative stress.

Regulation of CD26/DPPIV gene expression by interferons and retinoic acid in tumor B cells

Interferons (IFNs α, β and γ) and all trans retinoic acid (RA) have the ability to activate genes with GAS sites. We have found that the promoter of CD26/dipeptidylpeptidase IV (DPPIV) contains a consensus GAS site TTCnnnGAA located at bp-35 to -27, and computer analysis confirmed this sequence to be a putative Stat binding site. Consistent with this finding, we show that IFNs and RA rapidly enhanced CD26 gene and protein expression in chronic B lymphocytic leukemia (B-CLL) cells. Immunoblot analyses revealed that unstimulated B-CLL cells expressed detectable levels of serine/tyrosine-phosphorylated Stat1α, and RA and IFN-γ treatment led to increased levels of tyrosine phosphorylation of Stat1α and its nuclear accumulation. As shown by electrophoretic mobility shift assay, RA and IFN-γ increased the binding of a nuclear protein to the GAS-CD26 element. Shift-Western blotting identified Stat1α as the GAS-CD26 binding factor. Augmented levels of CD26 protein in malignant B cells cultured with IFNs or RA coincided with the enhancement of DPPIV activity. Taken together, our results are in favor of the IFN-/RA-mediated upregulation of CD26/DPPIV in B-CLL through the signaling pathway involving Stat1α and the GAS response element of CD26 promoter.

Correlations between p53-protein accumulation, serum antibodies and gene mutation in colorectal cancer

Only half of colorectal‐cancer patients elicit serum antibodies in response to intratumoral p53‐gene mutations. Our study was designed to compare cellular events (p53‐protein accumulation and gene mutations) with the presence of circulating anti‐p53 antibodies (p53‐Ab). Thirty‐five colorectal‐cancer patients were studied for their intratumoral p53‐protein accumulation and circulating p53‐Ab. Tumour DNA was analyzed for genomic mutations in a sub‐set of 28 patients. In all, 18 tumours (51.4%) were positive by immunohistochemistry, and 17 tumour extracts were shown to contain “mutant” conformation p53 protein, 16 of them being were concordant by both methods. Of the 28 tumours tested by DGGE, 16 contained alterations in p53 exons 5 to 8 (57.1%). Of 12 tumours without detectable mutations, 10 were “mutant”‐conformation‐negative by immunohistochemistry and ELISA. Paradiploid tumors presented more frequently wild‐type p53 genes and were significantly less frequently immunohistochemistry‐ or p53‐Ab‐positive than polyploid tumors. Circulating p53‐Ab were detected in the serum of 11 patients (31%). In 9/11 cases, a gene mutation was found in the corresponding tumour. Three of four mutations in exon 8 and 3/3 mutations in exons 5‐6 were associated with p53‐Ab, in contrast with only 3/9 mutations in exon 7. We found good agreement in the detection of p53‐gene alterations by different methods. However, our data suggest that all gene mutations may not be equivalent in term of immunogenicity. Int. J. Cancer 81:712–718, 1999.

Treatment of multiple sclerosis patients with interferon-beta primes monocyte-derived macrophages for apoptotic cell death.

Although interferon (IFN)‐β has shown a significant clinical benefit in multiple sclerosis (MS), its mechanism of action remains unclear. We found that IFN‐β treatment of patients with MS resulted in a significant increase in apoptotic cell death (measured by annexin V staining and nuclear fragmentation) of monocyte‐derived macrophages, as compared with cells derived from patients before treatment. Stimulation of the cells with IFN‐β in vitro resulted in an even further increase of annexin V binding, as well as increased Fas (CD 95, APO‐1) expression. However, no increased Fas expression, apoptotic monocytes, or monocytopenia were observed upon in vivo treatment. This indicates that IFN‐β does not deliver a death signal to monocytes but rather primes for subsequent macrophage apoptosis upon activation or differentiation.

γ-glutamyl transpeptidase activity mediates NF-κB activation through lipid peroxidation in human leukemia U937 cells

Gamma-glutamyl transpeptidase (GGT) is a key enzyme in the catabolism of glutathione (GSH). Recently, it has been reported that the extracellular cleavage of GSH by GGT induced the production of reactive oxygen species (ROS), suggesting that GGT plays a pro-oxidant role. In the present study, we investigated the nature of the oxidative stress generate by glutathione and GGT and the possibility that this stress affects the activity of NF-κB a prototypical oxidant-stress-responsive transcription factor. We found that, in the presence of iron, a natural substrate of GGT, glutathione induces lipid peroxidation in U937 cells. This induction depends on GGT activity as it is prevented by the Serine/Borate complex, a GGT inhibitor. We found that γ-glutamyl transpeptidase activity induces NF-κB DNA binding activity, an effect which is significantly reduced by the addition of GGT inhibitors (Serine/Borate complex and Acivicin). Moreover, we show that lipid peroxidation is involved in GGT-dependent NF-κB activation since vitamin E, which completely inhibits GGT-induced generation of lipid peroxides, prevents the GGT-dependent NF-κB activation. Finally, inhibition of GGT by either the Serine/Borate complex or by Acivicin resulted in cell apoptosis. This finding suggests that GGT-mediated NF-κB activation plays a role in the control of apoptosis in U937 cells.

Cell cycle arrest by oxaliplatin on cancer cells

Oxaliplatin (L‐OHP) is the only platinum compound to show activity in colorectal cancer. We evaluated the cytotoxicity of L‐OHP on four human cancer cell lines and its influence on the cell cycle, when treated during long exposure (72 h) and different post‐incubation times (24 or 72 h). We used a panel of cell lines: HT29 (colon cancer), MCF7 (breast cancer), Hela (uterine cervix) and A549 (lung adenocarcinoma). Inhibition concentration (IC)50 was assessed by MTT assay. Cell cycle modifications were determined using dual parameter bromodeoxyuridine and propidium iodide. L‐OHP yielded a superior cytotoxicity on HT29 and MCF7 relative to Hela and A549 after treatment, the post‐incubations demonstrate that growth inhibition was irreversible for HT29 and Hela cell lines contrary to MCF7 and A549. The main effects of L‐OHP are G2/M cell cycle arrest and transient S phase delay. Taken together, L‐OHP treatment results on HT29, MCF7 and Hela, are in favor of lengthening the infusion duration to patients during further clinical trials.

gamma-Glutamyl transpeptidase expression in Ewing's sarcoma cells: up-regulation by interferons.

The genetic hallmark of Ewings sarcoma family of tumours (ET) is the presence of the translocation t(11;22)(q24;q12), which creates the ET fusion gene, leading to cellular transformation. Five human gamma-glutamyl transpeptidase (gamma-GT) genes are located near the chromosomal translocation in ET. gamma-GT is a major enzyme involved in glutathione homoeostasis. Five human cell lines representative of primary or metastatic tumours were investigated to study whether gamma-GT alterations could occur at the chromosomal breaks and rearrangements in ET. As shown by enzymic assays and FACS analyses, all ET cell lines consistently expressed a functional gamma-GT which however did not discriminate steps of ET progression. As shown previously [Sancéau, Hiscott, Delattre and Wietzerbin (2000) Oncogene 19, 3372-3383], ET cells respond to the antiproliferative effects of interferons (IFNs) type I (alpha and beta) and to a much less degree to IFN type II (gamma). IFN-alpha and -beta arrested cells in the S-phase of the cell cycle. We found an enhancement of gamma-GT mRNA species with IFN-alpha and -beta by reverse transcriptase-PCR analyses. This is reflected by up-regulation of gamma-GT protein, which coincides with the increase in gamma-GT-specific enzymic activity. Similarly, IFNs up-regulate the levels of gamma-GT in another IFN-responsive B cell line. Whether this up-regulation of gamma-GT by IFNs is of physiological relevance to cell behaviour remains to be studied.

Regulation of the cell cycle by p53 after DNA damage in an amphibian cell line.

In mammalian cells, the p53 protein is a key regulator of the cell cycle following DNA damage. In the present study, we investigated the function of p53 in the A6 amphibian cell line. Using various specific Xenopus p53 monoclonal antibodies, we showed that Xenopus p53 accumulates after DNA damage, including gamma and UV irradiation or treatment with adriamycin. Such accumulation is accompanied by an increase in the apparent molecular weight of the protein. This change was shown to be the result of a phosphorylation event that occurs after DNA damage. Accumulation of Xenopus p53 is parallel to a drastic change in the cell cycle distribution. Brief exposure to adriamycin or gamma irradiation induces reversible growth arrest, whereas long-term exposure to adriamycin leads to apoptosis. Taken together, these results indicate that p53 has a similar behaviour in frog cells and mammalian cells, and that it conserves two activities, cell cycle arrest and apoptosis.

TNFα Potentiates 2‐Methoxyestradiol‐Induced Mitochondrial Death Pathway

Abstract: Ewing sarcoma cells are resistant to TNFα‐induced cell death and this resistance results from the activation of the transcription factor NF‐κB. Here, we investigated whether NF‐κB activation interferes with 2‐Me‐induced cell death signaling in Ewing sarcoma cells and we examined the effect of treatment of these cells with 2‐Me either alone or in combination with TNFα. Our results show that TNFa cooperates with 2‐Me to induce apoptosis in Ewing tumor cells through mitochondrial cell death signaling. These results suggest that the use of TNFα in combination with 2‐Me may be beneficial for Ewing tumor treatment.

Synergistic effect of prolactin on IFN-γ-mediated growth arrest in human monoblastic cells: correlation with the up-regulation of IFN-γ receptor gene expression

Abstract Interferon-γ (IFN-γ) stimulates the development of monocytic features in human myeloid precursors. Because transcriptional regulation by IFN-γ and the pituitary hormone prolactin (PRL) has been described to involve common Jak-STAT pathways, we addressed here the question of whether PRL plays a role in monoblastic (U937) cell growth and macrophage maturation. In contrast to IFN-γ, PRL did not affect U937 cell growth nor induction of differentiation as assessed by the unchanged cell surface expression of maturation markers CD11b and HLA-DR class II. However, PRL in synergy with IFN-γ inhibited, in a time- and dose-dependence, proliferation of U937 cells without influencing their maturation induced by IFN-γ. IFN-γ and PRL both affected the expression of the IFN-γ receptor (IFN-γR) gene by increasing IFN-γR mRNA levels. The rise in IFN-γR transcripts was accompanied by a low but significant release of IL-6 which has previously been shown to stabilize IFN-γR mRNA. Moreover, a transient increase in surface expression of IFN-γR was observed in U937 cells treated by IFN-γ alone or in combination with PRL, whereas no apparent modulation of cell surface IFN-γR was observed in cells treated with PRL. Lastly, PRL did not induce transcriptional activation of IFN-γ inducible IRF-1 and FcγRI genes in U937 cells. Together, our data indicate that IL-6 secretion and increased expression of the IFN-γR gene correlate with U937 cell growth arrest induced by IFN-γ and PRL, probably through a signaling mechanism which does not involve the Stat 1 IRF -1 pathway.