Marc Mousli

Down-regulation of nuclear protein ICBP90 by p53/p21Cip1/WAF1-dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition

Checkpoints, which monitor DNA damage and regulate cell cycle progression, ensure genomic integrity and prevent the propagation of transformed cells. DNA damage activates the p53‐dependent checkpoint pathway that induces expression of p21Cip1/WAF1, resulting in cell cycle arrest at G1/S transition by inhibition of cdk activity and DNA replication. ICBP90 was identified as a nuclear protein that binds to the TopoIIα gene promoter and is speculated to be involved in DNA replication. ICBP90 expression is cell cycle regulated in normal cells but stably high throughout cell cycle in various cancer cell lines. We here demonstrate that ICBP90 expression is down‐regulated by the p53/p21Cip1/WAF1‐dependent DNA damage checkpoint signals. The reduction of ICBP90 appeared to be caused by both transcriptional suppression and protein degradation. Adenoviral expression of p21Cip1/WAF1 directly led to ICBP90 reduction in p53−/− HCT116 cells without DNA damage. Furthermore, ICPB90 depletion by RNA interference significantly blocked G1/S transition after DNA damage in HeLa cells. The down‐regulation of ICBP90 is an important mechanism for cell cycle arrest at G1/S transition, which is induced by the activation of a p53/p21Cip1/WAF1‐dependent DNA‐damage checkpoint. Deregulation of ICBP90 may impair the control of G1/S transition during checkpoint activation and lead to genomic instability.

The retinoblastoma gene and its product are targeted by ICBP90: a key mechanism in the G1/S transition during the cell cycle.

The retinoblastoma protein (pRB) is encoded by the RB1 gene whose promoter contains several putative binding sites for ICBP90 (Inverted CCAAT box Binding Protein of 90 kDa), a transcriptional regulator of the topoisomerase IIα gene. ICBP90 has two consensus binding sites for pRB in its primary sequence. Here, we show that pRB and ICBP90 co-immunoprecipitate in cell extracts of proliferating human lung fibroblasts and of proliferating or confluent Jurkat cells. GST pull-down assays and immunocytochemistry, after cell synchronization in late G1 phase, confirmed this interaction. Overexpression of ICBP90 induces downregulation of pRB expression in lung fibroblasts as a result of mRNA decrease. DNA chromatin immunoprecipitation experiment shows that ICBP90 binds to the RB1 gene promoter under its methylated status. Overexpression of ICBP90 increases the S and G2/M phase cell fractions of serum-starved lung fibroblasts as assessed by flow cytometry analysis and increases topoisomerase IIα expression. Together, these results show that ICBP90 regulates pRB at the protein and gene transcription levels, thus favoring the entry into the S phase of the cells. We propose that ICBP90 overexpression, found in cancer cells, is involved in the altered checkpoint controls occurring in cancerogenesis.

Drug discovery targeting epigenetic codes: The great potential of UHRF1, which links DNA methylation and histone modifications, as a drug target in cancers and toxoplasmosis

UHRF1 plays a central role in transferring methylation status from mother cells to daughter cells. Its SRA domain recognizes hemi-methylated DNA that appears in daughter DNA strands during duplication of DNA. UHRF1 recruits DNMT1 to the site and methylates both strands. UHRF1 also binds to HDAC1 and di- and tri-methyl K9 histone H3, ubiquitinates histone H3, and associates with heterochromatin formation, indicating that UHRF1 links histone modifications, DNA methylation, and chromatin structure. UHRF1 is a direct target of E2F1 and promotes G1/S transition. The tumor suppressor p53, which is deficient in 50% of cancers, down-regulates UHRF1 through up-regulation of p21/WAF1 and subsequent deactivation of E2F1. The expression levels of UHRF1 are up-regulated in many cancers, probably partially because of the absence of wild type p53, but it is probably regulated by several other factors. Knockdown of UHRF1 expression in cancer cells suppressed cell growth, suggesting that UHRF1 can be a useful anticancer drug target. Recently, it was revealed that UHRF1 plays important roles not only in carcinogenesis, but also in toxoplasmosis, which is occasionally fatal to people with a weakened immune system, and can cause blindness in the major pathology of ocular toxoplasmosis. Toxoplasma gondii, which causes toxoplasmosis, utilizes UHRF1 to control the cell cycle phase and enhance its proliferation. Thus, knockdown of UHRF1 can be effective at stopping the proliferation of the parasites in infected cells. In this review, we discuss several possible methods that can inhibit the multiple unique functions of UHRF1, which can be utilized for treating cancers and toxoplasmosis.

Cytokine Profiles in Toxoplasmic and Viral Uveitis

BACKGROUND Uveitis is a major cause of visual impairment throughout the world. Analysis of cytokine profiles in aqueous humor specimens may provide insight into the physiopathological processes that underly retinal damage in this context. METHODS Using a multiplex assay, we determined the concentrations of 17 cytokines and chemokines in aqueous humor specimens obtained from patients with ocular toxoplasmosis or viral uveitis and compared these concentrations with those in specimens obtained from patients with noninfectious intermediate uveitis or cataract. RESULTS Five mediators (interleukin [IL]-8, monocyte chemoattractant protein-1, tumor necrosis factor-alpha, IL-4, and IL-10) were detected in >50% of patients in all groups. In contrast, IL-5 and IL-12 were specific for ocular toxoplasmosis, and granulocyte monocyte colony-stimulating factor and IL-1 were specific for viral uveitis; these mediators could present specific markers for diagnostic purposes. Interferon-gamma, IL-6, and macrophage inflammatory protein-1beta were common markers of ocular toxoplasmosis and viral uveitis. IL-17 was a common marker of ocular toxoplasmosis and intermediate uveitis. CONCLUSIONS We found specific cytokine profiles for each type of uveitis, with large interindividual variations and no etiological or clinical correlations. Ocular cytokine mapping contributes to a better understanding of the physiopathology of specific forms of uveitis and provides guidance for new targeted treatment.

Human and rat cutaneous mast cells: involvement of a G protein in the response to peptidergic stimuli

Recent evidence suggests that peptides induce the release of mediators from rat peritoneal mast cell by means of a receptor-independent mechanism, possibly involving an interaction with sialic acid residues at the cell surface followed by the activation of a guanine nucleotide binding protein (G protein). We have now examined the potential involvement of sialic acid residues and of G protein stimulation in the activation of both human and rat cutaneous mast cells by neuropeptide Y, its C-terminal fragments and the wasp venom peptide, mastoparan. Neuropeptide Y-(18-36) was the most effective histamine releaser of the fragments tested, the order of potency being neuropeptide Y-(18-36) > neuropeptide Y-(22-36) > neuropeptide Y-(1-36). This order of potency suggests that the effects of the peptides are not mediated through classical NPY receptors. The hydrolysis of sialic acid residues by neuraminidase and the inhibition of G proteins by benzalkonium chloride or pertussis toxin significantly inhibited the secretory response of cutaneous mast cells to neuropeptide Y-(18-36) and mastoparan. These results demonstrate that the peptidergic pathway described for the activation of peritoneal rat mast cells is also involved in the response of cutaneous human and rat mast cells to peptides.

UHRF1 recruits the histone acetyltransferase Tip60 and controls its expression and activity

Tat-interactive protein, 60kDa (Tip60) is a histone acetyltransferase with specificity toward lysine 5 of histone H2A (H2AK5) and plays multiple roles in chromatin remodeling processes. Co-immunoprecipitation experiments performed on Jurkat cells, showed that Tip60 is present in the same macro-molecular complex as UHRF1 (Ubiquitin-like containing PHD and RING domain 1), DNMT1 (DNA methyltransferase 1), and HDAC1 (histone deacetylase 1). Furthermore, immunocytochemistry experiments confirmed that Tip60 co-localizes with the UHRF1/DNMT1 complex. Although down-regulation of UHRF1 by RNA interference enhanced Tip60 expression, a significant decrease of the level of acetylated H2AK5 was observed. Consistently, we have observed that down-regulation of Tip60 and DNMT1 by RNA interference, dramatically reduced the levels of acetylated H2AK5. Altogether, these results suggest that Tip60 is a novel partner of the epigenetic integration platform interplayed by UHRF1, DNMT1 and HDAC1 involved in the epigenetic code replication.

Structural requirements for neuropeptide Y in mast cell and G protein activation

Incubation of neuropeptide Y or its C-terminal fragments with rat peritoneal mast cells resulted in a dose-dependent histamine release. Fragment 18-36 of neuropeptide Y was the most biologically active peptide. EC25 value on rat mast cells was 7.2 +/- 2.2 nM. Neuropeptide Y was also able to induce a flare response after intradermal injection in humans. The histamine releasing effects of neuropeptide Y related peptides were greatly inhibited by pretreatment of rat mast cells with pertussis toxin or benzalkonium chloride. Neuropeptide Y and C-terminal related peptides also stimulated the GTPase activity of purified heterotrimeric G proteins in a dose-dependent manner from 1 to 50 microM. Binding studies with [125I]neuropeptide Y were unable to provide evidence for the presence of specific binding sites on the surface of mast cells. The alpha helical conformation of neuropeptide Y fragments was studied by measuring the circular dichroism spectra. Neuropeptide Y-(18-36) was the smallest fragment having a strong helical conformation. Our results demonstrate that neuropeptide Y activates mast cells through a non-specific process leading to G protein activation.

Toxoplasma gondii exploits UHRF1 and induces host cell cycle arrest at G2 to enable its proliferation

Toxoplasma gondii is an obligate intracellular parasite that causes severe disease in humans. It is able to infect all nucleated mammalian cells leading to lifelong persistence of the parasite in the host. Here, we studied the effect of T. gondii infection on host cell proliferation and explored the molecular mechanisms involved in host cell cycle progression. We found that T. gondii induced G1/S transition in host cells in the presence of UHRF1, followed by G2 arrest after cyclin B1 downregulation which is probably the major cause of the arrest. Other molecules at the G2/M checkpoint including p53, p21 and Cdk1 were normally regulated. Interestingly, while parasite proliferation was normal in cells that were in the G2 phase, it was suppressed in G1‐arrested cells induced by UHRF1‐siRNA, indicating the importance of the G2 phase via UHRF1‐induced G1/S transition for T. gondii growth.

Increasing role of UHRF1 in the reading and inheritance of the epigenetic code as well as in tumorogenesis.

Epigenetic mechanisms such as DNA methylation and histone posttranslational modifications, allow cells to maintain the phenotype throughout successive mitosis. UHRF1 plays a major role in the inheritance of some epigenetic marks from mother cells to daughter cells due to its particular structural domains. The originality of UHRF1 lies in the fact that it can read epigenetic marks and recruit the enzymes that catalyze the same epigenetic mark. The SRA domain senses the presence of a methylated cytosine on one DNA strand allowing the recruitment of DNMT1, which methylates the cytosine on the newly synthesized DNA. The recently identified tudor domain of UHRF1 senses the presence of methylated histone H3 conducting UHRF1 to recruit histone methyltransferases. Recent studies deciphering the relationships between some of the structural domains of UHRF1 provides new insights on the reading of the epigenetic code over a larger portion of histone tail than usually expected. Furthermore, latest developments highlights that UHRF1 is one of the proteins which is able to directly connect DNA methylation to histone epigenetic marks. This paper reviews the principles how UHRF1 acts as an epigenetic reader and discusses the properties of UHRF1 to be a biomarker as well as a therapeutic target.

Limoniastrum guyonianum aqueous gall extract induces apoptosis in human cervical cancer cells involving p16 INK4A re-expression related to UHRF1 and DNMT1 down-regulation

Several reports have described the potential effects of natural compounds as anti-cancer agents in vitro as well as in vivo. The aim of this study was to evaluate the anti-cancer effect of Limoniastrum guyonianum aqueous gall extract (G extract) and luteolin in the human cervical cancer HeLa cell line, and, if so, to clarify the underlying mechanism. Our results show that G extract and luteolin inhibited cell proliferation and induced G2/M cell cycle arrest in a concentration and time-dependent manner. Both natural products induced programmed cell death as confirmed by the presence of hypodiploid G0/G1 cells. These effects are associated with an up-regulation of the expression of the tumor suppressor gene p16INK4A and a down-regulation of the expression of the anti-apoptotic actor UHRF1 and its main partner DNMT1. Moreover, G extract- and luteolin-induced UHRF1 and DNMT1 down-regulation is accompanied with a global DNA hypomethylation in HeLa cell line. Altogether our results show that G extract mediates its growth inhibitory effects on human cervical cancer HeLa cell line likely via the activation of a p16INK4A -dependent cell cycle checkpoint signalling pathway orchestrated by UHRF1 and DNMT1 down-regulation.