In silico analysis of the transportome in human pancreatic ductal adenocarcinoma.

The altered expression and/or activity of ion channels and transporters (transportome) have been associated with malignant behavior of cancer cells and were proposed to be a hallmark of cancer. However, the impact of altered transportome in epithelial cancers, such as pancreatic ductal adenocarcinoma (PDAC), as well as its pathophysiological consequences, still remains unclear. Here, we report the in silico analysis of 840 transportome genes in PDAC patients' tissues. Our study was focused on the transportome changes and their correlation with functional and behavioral responses in PDAC tumor and stromal compartments. The dysregulated gene expression datasets were filtered using a cut-off of fold-change values ≤-2 or ≥2 (adjusted p value ≤0.05). The dysregulated transportome genes were clearly associated with impaired physiological secretory mechanisms and/or pH regulation, control of cell volume, and cell polarity. Additionally, some down-regulated transportome genes were found to be closely linked to epithelial cell differentiation. Furthermore, the observed decrease in genes coding for calcium and chloride transport might be a mechanism for evasion of apoptosis. In conclusion, the current work provides a comprehensive overview of the altered transportome expression and its association with predicted PDAC malignancy with special focus on the epithelial compartment.

Effects of p38α/ß inhibition on acute lymphoblastic leukemia proliferation and survival in vivo.

P38α/ß has been described as a tumor-suppressor controlling cell cycle checkpoints and senescence in epithelial malignancies. However, p38α/ß also regulates other cellular processes. Here, we describe a role of p38α/ß as a regulator of acute lymphoblastic leukemia (ALL) proliferation and survival in experimental ALL models. We also report first evidence that p38α/ß phosphorylation is associated with the occurrence of relapses in TEL-AML1-positive leukemia. First, in vitro experiments show that p38α/ß signaling is induced in a cyclical manner upon initiation of proliferation and remains activated during log-phase of cell growth. Next, we provide evidence that growth-permissive signals in the bone marrow activate p38α/ß in a novel avian ALL model, in which therapeutic targeting can be tested. We further demonstrate that p38α/ß inhibition by small molecules can suppress leukemic expansion and prolong survival of mice bearing ALL cell lines and primary cells. Knockdown of p38α strongly delays leukemogenesis in mice xenografted with cell lines. Finally, we show that in xenografted TEL-AML1 patients, ex vivo p38α/ß phosphorylation is associated with an inferior long-term relapse-free survival. We propose p38α/ß as a mediator of proliferation and survival in ALL and show first preclinical evidence for p38α/ß inhibition as an adjunct approach to conventional therapies.

c-Rel is a critical mediator of NF-κB-dependent TRAIL resistance of pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies with an overall life expectancy of 6 months despite current therapies. NF-κB signalling has been shown to be critical for this profound cell-autonomous resistance against chemotherapeutic drugs and death receptor-induced apoptosis, but little is known about the role of the c-Rel subunit in solid cancer and PDAC apoptosis control. In the present study, by analysis of genome-wide patterns of c-Rel-dependent gene expression, we were able to establish c-Rel as a critical regulator of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in PDAC. TRAIL-resistant cells exhibited a strong TRAIL-inducible NF-κB activity, whereas TRAIL-sensitive cells displayed only a small increase in NF-κB-binding activity. Transfection with siRNA against c-Rel sensitized the TRAIL-resistant cells in a manner comparable to siRNA targeting the p65/RelA subunit. Gel-shift analysis revealed that c-Rel is part of the TRAIL-inducible NF-κB complex in PDAC. Array analysis identified NFATc2 as a c-Rel target gene among the 12 strongest TRAIL-inducible genes in apoptosis-resistant cells. In line, siRNA targeting c-Rel strongly reduced TRAIL-induced NFATc2 activity in TRAIL-resistant PDAC cells. Furthermore, siRNA targeting NFATc2 sensitized these PDAC cells against TRAIL-induced apoptosis. Finally, TRAIL-induced expression of COX-2 was diminished through siRNA targeting c-Rel or NFATc2 and pharmacologic inhibition of COX-2 with celecoxib or siRNA targeting COX-2, enhanced TRAIL apoptosis. In conclusion, we were able to delineate a novel c-Rel-, NFATc2- and COX-2-dependent antiapoptotic signalling pathway in PDAC with broad clinical implications for pharmaceutical intervention strategies.

Compartmentalization of TNF-related apoptosis-inducing ligand (TRAIL) death receptor functions: emerging role of nuclear TRAIL-R2.

Localized in the plasma membrane, death domain-containing TNF-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induce apoptosis and non-apoptotic signaling when crosslinked by the ligand TRAIL or by agonistic receptor-specific antibodies. Recently, an increasing body of evidence has accumulated that TRAIL receptors are additionally found in noncanonical intracellular locations in a wide range of cell types, preferentially cancer cells. Thus, besides their canonical locations in the plasma membrane and in intracellular membranes of the secretory pathway as well as endosomes and lysosomes, TRAIL receptors may also exist in autophagosomes, in nonmembraneous cytosolic compartment as well as in the nucleus. Such intracellular locations have been mainly regarded as hide-outs for these receptors representing a strategy for cancer cells to resist TRAIL-mediated apoptosis. Recently, a novel function of intracellular TRAIL-R2 has been revealed. When present in the nuclei of tumor cells, TRAIL-R2 inhibits the processing of the primary let-7 miRNA (pri-let-7) via interaction with accessory proteins of the Microprocessor complex. The nuclear TRAIL-R2-driven decrease in mature let-7 enhances the malignancy of cancer cells. This finding represents a new example of nuclear activity of typically plasma membrane-located cytokine and growth factor receptors. Furthermore, this extends the list of nucleic acid targets of the cell surface receptors by pri-miRNA in addition to DNA and mRNA. Here we review the diverse functions of TRAIL-R2 depending on its intracellular localization and we particularly discuss the nuclear TRAIL-R2 (nTRAIL-R2) function in the context of known nuclear activities of other normally plasma membrane-localized receptors.

Mutant PIK3CA licenses TRAIL and CD95L to induce non-apoptotic caspase-8-mediated ROCK activation.

Constitutively active PI3K catalytic subunit alpha (PIK3CA) interfered with apoptosis induction downstream of death receptor-signaling complex formation allowing robust caspase-8 activation without triggering the execution steps of apoptosis. In mutant PIK3CA-expressing cells, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95L stimulated nuclear factor kappaB (NFkappaB) activation, invasion, and transition to an amoeboid-like morphology. NFkappaB activation and adoption of amoeboid shape were inhibited by caspase-8 knockdown or FLIP-S expression, but only the cell morphology alterations required caspase-8 activity. Furthermore, we identified caspase-8-mediated, caspase-3-independent cleavage of the protein kinase rho-associated, coiled-coil containing protein kinase 1 as a novel mechanism for acquiring amoeboid shape and enhanced invasiveness in response to TRAIL and CD95L. Taken together, we provide evidence that mutated PIK3CA converts the 'tumor surveillance' activity of cancer cell-expressed death receptors and caspase-8 toward tumor promotion.

Anti-apoptotic and growth-stimulatory functions of CK1 delta and epsilon in ductal adenocarcinoma of the pancreas are inhibited by IC261 in vitro and in vivo.

BACKGROUND: Pancreatic ductal adenocarcinomas (PDACs) are highly resistant to treatment due to changes in various signalling pathways. CK1 isoforms play important regulatory roles in these pathways. AIMS: We analysed the expression levels of CK1 delta and epsilon (CK1delta/in) in pancreatic tumour cells in order to validate the effects of CK1 inhibition by 3-[2,4,6-(trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) on their proliferation and sensitivity to anti-CD95 and gemcitabine. METHODS: CK1delta/in expression levels were investigated by using western blotting and immunohistochemistry. Cell death was analysed by FACS analysis. Gene expression was assessed by real-time PCR and western blotting. The putative anti-tumoral effects of IC261 were tested in vivo in a subcutaneous mouse xenotransplantation model for pancreatic cancer. RESULTS: We found that CK1delta/in are highly expressed in pancreatic tumour cell lines and in higher graded PDACs. Inhibition of CK1delta/in by IC261 reduced pancreatic tumour cell growth in vitro and in vivo. Moreover, IC261 decreased the expression levels of several anti-apoptotic proteins and sensitised cells to CD95-mediated apoptosis. However, IC261 did not enhance gemcitabine-mediated cell death either in vitro or in vivo. CONCLUSIONS: Targeting CK1 isoforms by IC261 influences both pancreatic tumour cell growth and apoptosis sensitivity in vitro and the growth of induced tumours in vivo, thus providing a promising new strategy for the treatment of pancreatic tumours.

Complementary effects of HDAC inhibitor 4-PB on gap junction communication and cellular export mechanisms support restoration of chemosensitivity of PDAC cells.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease and one of the cancer entities with the lowest life expectancy. Beside surgical therapy, no effective therapeutic options are available yet. Here, we show that 4-phenylbutyrate (4-PB), a known and well-tolerable inhibitor of histone deacetylases (HDAC), induces up to 70% apoptosis in all cell lines tested (Panc 1, T4M-4, COLO 357, BxPc3). In contrast, it leads to cell cycle arrest in only half of the cell lines tested. This drug increases gap junction communication between adjacent T3M-4 cells in a concentration-dependent manner and efficiently inhibits cellular export mechanisms in Panc 1, T4M-4, COLO 357 and BxPc3 cells. Consequently, in combination with gemcitabine 4-PB shows an overadditive effect on induction of apoptosis in BxPc3 and T3M-4 cells (up to 4.5-fold compared to single drug treatment) with accompanied activation of Caspase 8, BH3 interacting domain death agonist (Bid) and poly (ADP-ribose) polymerase family, member 1 (PARP) cleavage. Although the inhibition of the mitogen-activated protein kinase-pathway has no influence on fulminant induction of apoptosis, the inhibition of the JNK-pathway by SP600125 completely abolishes the overadditive effect induced by the combined application of both drugs, firstly reported by this study.

TRAIL promotes metastasis of human pancreatic ductal adenocarcinoma.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted considerable attention for its potential use in tumor therapy, as some recombinant variants of this ligand induce apoptosis in tumor cells without harming most normal cells. Here, we show that TRAIL strongly induces the expression of the proinflammatory cytokines interleukin-8 and monocyte chemoattractant protein 1 and enhances the invasion of apoptosis-resistant pancreatic ductal adenocarcinoma cells in vitro by upregulation of the urokinase-type plasminogen activator expression. Most importantly, we also demonstrate for the first time that TRAIL treatment results in strongly increased distant metastasis of pancreatic tumors in vivo. We orthotopically transplanted human pancreatic ductal adenocarcinoma cells to the pancreata of severe combined immunodeficiency mice and observed a dramatic increase in metastatic spread including a sixfold increase in the volume and fourfold increase in the number of liver metastases upon TRAIL treatment. Our results point to the necessity to carefully evaluate in vivo side effects of TRAIL and to select therapy conditions that not only enhance apoptosis induction but in addition prevent proinvasive and proinflammatory non-apoptotic TRAIL signaling.

Cathepsin D links TNF-induced acid sphingomyelinase to Bid-mediated caspase-9 and -3 activation.

Acidic noncaspase proteases-like cathepsins have been introduced as novel mediators of apoptosis. A clear role for these proteases and the acidic endolysosomal compartment in apoptotic signalling is not yet defined. To understand the role and significance of noncaspases in promoting and mediating cell death, it is important to determine whether an intersection of these proteases and the caspase pathway exists. We recently identified the endolysosomal aspartate protease cathepsin D (CTSD) as a target for the proapoptotic lipid ceramide. Here, we show that tumor necrosis factor (TNF)-induced CTSD activation depends on functional acid sphingomyelinase (A-SMase) expression. Ectopic expression of CTSD in CTSD-deficient fibroblasts results in an enhanced TNF-mediated apoptotic response. Intracellular colocalization of CTSD with the proapoptotic bcl-2 protein family member Bid in HeLa cells, and the ability of CTSD to cleave directly Bid in vitro as well as the lack of Bid activation in cathepsin-deficient fibroblasts indicate that Bid represents a direct downstream target of CTSD. Costaining of CTSD and Bid with Rab5 suggests that the endosomal compartments are the common 'meeting point'. Caspase-9 and -3 activation also was in part dependent on A-SMase and CTSD expression as revealed in the respective deficiency models. Our results link as novel endosomal intermediates the A-SMase and the acid aspartate protease CTSD to the mitochondrial apoptotic TNF pathway.

Multiple and synergistic deregulations of apoptosis-controlling genes in pancreatic carcinoma cells.

Inability to die by apoptosis is one of the reasons for the deregulated growth of tumour cells and the frequently observed failure of chemotherapy. In this study we thought to identify the common and functionally important characteristics responsible for the apoptosis resistance of pancreatic tumour cells. We analysed cell surface expression level of death receptors CD95 and TRAIL-R1-4 as well as the expression profile of sixteen apoptosis-relevant proteins in five pancreatic carcinoma cell lines Capan1, Colo357, PancTuI, Panc89 and Panc1. These data were evaluated in the context of sensitivity towards anti-CD95 and TRAIL-mediated apoptosis. Here we report that except for resistant Panc1 cells, which only marginally expressed CD95, all other cell lines showed comparable levels of CD95 and TRAIL receptors irrespectively of their apoptotic phenotype. Interestingly, we found that the elevated expression of FLIP, Bcl-x(L) and IAP in parallel with a downregulation of FADD and Bid was common for the resistant cell lines. Consequently, stable overexpression of XIAP, Bcl-x(L) or dominant negative FADD in sensitive cells significantly reduced the death receptor mediated apoptosis while the overexpression of Bid rendered the resistant cells sensitive.

FAP-1 in pancreatic cancer cells: functional and mechanistic studies on its inhibitory role in CD95-mediated apoptosis.

In this study we investigated the functional role of FAP-1 as a potential inhibitor of CD95 (Fas, APO-1)-mediated apoptosis in pancreatic cancer cells. Stable transfection of the CD95-sensitive, FAP-1-negative cell line Capan-1 with an FAP-1 cDNA resulted in a strongly decreased sensitivity to CD95-induced apoptosis, as measured by DNA fragmentation and caspase-3 activity. Inhibition of cellular protein tyrosine phosphatases with orthovanadate dose-dependently increased CD95-induced apoptosis in CD95-resistant FAP-1-positive Panc89 and Capan-1-FAP-1 cells almost to the level seen in wild-type Capan-1 cells. Blocking the CD95/FAP-1 interaction in Panc89 cells by cytoplasmic microinjection of a synthetic tripeptide mimicking the C terminus of CD95 resulted in a mean 5.5-fold increase in apoptosis compared to cells that received a control peptide. Using confocal laser scanning microscopy we show that in Panc89 cells FAP-1 is mainly associated with the Golgi complex and with peripheral vesicles. FAP-1 displayed enhanced colocalization with CD95 upon CD95 stimulation in the Golgi complex but not in surface-associated vesicles. This correlated with a decrease in plasma membrane staining for CD95 as determined by FACS analysis. Inhibition of Golgi anterograde transport by brefeldin A abolished the anti-CD95-induced colocalization of FAP-1 and CD95 as well as the decrease in cell-surface-associated CD95. Finally, we demonstrate by immunohistochemistry that FAP-1 is strongly expressed in tumor cells from pancreatic carcinoma tissues. Taken together, these results show that FAP-1 can protect pancreatic carcinoma cells from CD95-mediated apoptosis, probably by preventing anti-CD95-induced translocation of CD95 from intracellular stores to the cell surface.

CD95 and TRAIL receptor-mediated activation of protein kinase C and NF-kappaB contributes to apoptosis resistance in ductal pancreatic adenocarcinoma cells.

The molecular alterations in tumour cells leading to resistance towards apoptosis induced by CD95 and TRAIL-receptors are not fully understood. We report here that the stimulation of the CD95- and TRAIL-resistant human pancreatic adenocarcinoma cell line PancTuI with an agonistic anti-CD95 antibody or TRAIL resulted in activation of protein kinase C and NF-kappaB. Inhibition of protein kinase C by Gö6983 sensitized these cells to apoptotic challenges and strongly diminished activation of NF-kappaB by anti-CD95 and TRAIL. Similarly, inhibition of NF-kappaB by MG132 or by transient transfection with a dominant negative mutant of IkappaBalpha restored the responsiveness of PancTuI cells to both death ligands. In the CD95 and TRAIL-sensitive cell line Colo357 the induction of protein kinase C and NF-kappaB following activation of CD95 and TRAIL-R was very moderate compared with PancTuI cells. However, pre-incubation of these cells with PMA strongly reduced their apoptotic response to anti-CD95 and TRAIL. Taken together, we show that activation of protein kinase C operates directly in a death receptor-dependent manner in PancTuI cells and protect pancreatic tumour cells from anti-CD95 and TRAIL-mediated apoptosis by preventing the loss DeltaPsim and Cytochrome c release as well as by induction of NF-kappaB.

Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis.

In this study we sought to clarify the role of the proapoptotic potential of mitochondria in the death pathway emanating from the TRAIL (APO-2L) and CD95 receptors in pancreatic carcinoma cells. We focused on the role of the Bcl-2 family member Bcl-XL, using three pancreatic carcinoma cell lines as a model system, two of which have high (Panc-1, PancTuI) and one has low (Colo357) Bcl-XL expression. In these cell lines, the expression of Bcl-XL correlated with sensitivity to apoptosis induced by TRAIL or anti-CD95. Flow cytometric analysis revealed cell surface expression of TRAIL-R1 and TRAIL-R2 on PancTuI and Colo357, and TRAIL-R2 on Panc-1 cells. In Colo357 cells retrovirally transduced with Bcl-XL, caspase-8 activation in response to treatment with TRAIL or anti-CD95 antibody was not different from parental cells and EGFP-transfected controls, however, apoptosis was completely suppressed as measured by the mitochondrial transmembrane potential deltapsim, caspase-3 activity (PARP cleavage) and DNA-fragmentation. Inhibition of Bcl-XL function by overexpression of Bax or administration of antisense oligonucleotides against Bcl-XL mRNA resulted in sensitization of Panc-1 cells to TRAIL and PancTuI cells to anti-CD95 antibody-induced cell death. The results show that Bcl-XL can protect pancreatic cancer cells from CD95- and TRAIL-mediated apoptosis. Thus, in these epithelial tumour cells the mitochondrially mediated 'type II' pathway of apoptosis induction is not only operative regarding the CD95 system but also regarding the TRAIL system.

The putative apoptosis inhibitor IEX-1L is a mutant nonspliced variant of p22(PRG1/IEX-1) and is not expressed in vivo.

IEX-1L has been claimed to act as an apoptosis inhibitor involved in NFkappaB-mediated survival in Jurkat cells [Wu et al. (1998) Science 281, 998-1001]. It represents a mutant nonspliced variant of the early response gene p22(PRG1/IEX-1) exhibiting one insertion and two deletions compared to the genomic sequence of p22(PRG1/IEX-1). Direct DNA sequencing of PCR products generated from human genomic DNA only detected the regular genomic sequence of p22(PRG1/IEX-1). No IEX-1L mRNA could be identified by RT-PCR analysis and subsequent DNA sequencing of total, nuclear, or cytoplasmic RNA fractions from PMA-stimulated Jurkat cells. The only functional transcript residing in the cytoplasm is regularly spliced p22(IEX-1/PRG1) mRNA. Substantial amounts of nonmutated nonspliced p22(IEX-1/PRG1) pre-mRNA were identified in the nucleus. Thus, IEX-1L seems to be a mutant variant of p22(IEX-1/PRG1) not existing in vivo. Antiapoptotic effects obviously represent transdominant negative inhibition of endogenous p22(PRG1/IEX-1) in Jurkat cells and several other tumor cell lines.

Human PACAP response gene 1 (p22/PRG1): proliferation-associated expression in pancreatic carcinoma cells.

p22/PACAP response gene-1 (PRG1) is a novel rat early response gene expressed during induction of proliferation and stress response. In humans, a homolog of p22/PRG1, designated IEX-1/DIF-2, exists, yet the exact function of this gene remains elusive. To characterize the expression of p22/PRG1 in human cancers, we analyzed the expression of p22/PRG1 in the human pancreatic carcinoma cell lines 818-4, PT45, and PancTu1. Serum or growth factors, like epidermal growth factor (EGF) and hepatocyte growth factor (HGF), rapidly and transiently induced transcription of p22/PRG1 in these cells, correlating with the mitogenic response. Treatment with TNF-alpha was followed by a rapid increase of p22/PRG1 messenger RNA (mRNA) levels in PT45 and Panc-Tul cells, which proliferate in the presence of TNF-alpha, but not in 818-4 cells, which are growth-inhibited when treated with TNF-alpha. Our findings suggest that human p22/PRG1 is expressed in various pancreatic carcinoma cells as a growth-associated early response gene.

The promoter of human p22/PACAP response gene 1 (PRG1) contains functional binding sites for the p53 tumor suppressor and for NFkappaB.

We describe functional binding sites for the tumor suppressor p53 and for NFkappaB residing in the promoter of the novel human early response gene p22/PRG1 (IEX-1/DIF-2). Gel shift and supershift assays demonstrate binding of p53 and NFkappaB to their corresponding sites in vitro. CAT-reporter gene assays show transactivation of the human p22/PRG1 promoter by p53 in Hep3B cells stably transfected with a temperature-sensitive mutant p53, but not in p53-deficient Hep3B cells. TNF alpha induced NFkappaB dependent transactivation was shown in HepG2 cells or in 818-4 pancreatic cancer cells. These data imply that human p22/PRG1 is a target gene for p53 and NFkappaB involved in growth regulation and stress response.

The proliferation-associated early response gene p22/PRG1 is a novel p53 target gene.

The novel early response gene p22/PRG1 is linked to cell cycle entry and the induction of proliferation in various cell types although its exact function is still unknown. The p22/PRG1 promoter region contains a 20 bp sequence matching the consensus binding motif for the tumor suppressor protein p53. Gel shift assays demonstrated that p53 specifically binds to an oligonucleotide derived from the p53 binding site of the p22/PRG1 promoter. Chloramphenicol acetyltransferase (CAT) reporter gene assays confirmed that this site confers p53-dependent transcriptional activity to the p22/PRG1 promoter. In Hela cells, p22/PRG1 promoter constructs induced CAT expression only when cotransfected with an expression plasmid for wild-type, but not for mutant p53. Similarly, CAT expression was inducible at the permissive (31 degrees C) but not at the non-permissive temperature (39 degrees C) in the rat embryo fibroblast-derived cell line clone-6 that expresses a temperature-sensitive mutant p53. Conversion of this mutant p53 to a functional p53 at the permissive temperature was accompanied by a significant increase of endogenous p22/PRG1 mRNA level in this cell line. Gamma-irradiation of rat splenocytes or doxorubicin-treatment of Hela cells increased p53 levels followed by transcriptional activation of p22/PRG1 and p21/Waf1 in parallel. Our data demonstrate that p22/PRG1 transcription is induced by p53 during p53-dependent cell cycle arrest and apoptosis. Therefore, p22/PRG1 represents a novel target for transcriptional activation by p53.

p22/PACAP response gene 1 (PRG1): a putative target gene for the tumor suppressor p53.

In this study we describe a novel putative p53-responsive gene, designated p22/PACAP response gene 1 (PRG1), recently identified as a proliferation-associated early-response gene in rats. By means of electrophoretic mobility shift assay and CAT-reporter gene assay, we could demonstrate that the p53 binding site residing in the promoter of p22/PRG1 is functional in vitro. Furthermore, in clone 6 cells expression of p22/PRG1 is induced in parallel to p21/Waf1 under conditions permitting mutant p53 to adopt wild-type configuration. An increase of p22/PRG1 transcription was also observed in gamma-irradiated rat splenocytes, which undergo p53-dependent apoptosis. Our findings demonstrate that p22/PRG1 fulfills all essential criteria as a p53 target gene and might be implicated in p53-dependent apoptosis.

Plasticity- and neurodegeneration-linked cyclic-AMP responsive element modulator/inducible cyclic-AMP early repressor messenger RNA expression in the rat brain.

In order to explore the role of CREM (cyclic-AMP responsive element modulator) gene expression in the function of the central nervous system, the gene transcripts were investigated in the rat brain in several conditions linked to increased neuronal activity. Up-regulation of CREM messenger RNA levels in the hippocampus was found to follow intraperitoneal administration of kainate (10 mg/kg). This increase was observed in both the dentate gyrus and hippocampus proper (CA subfields) and reached its maximum at 6 h after the treatment. Intrahippocampal injection of N-methyl-D-aspartate (200 nmol) resulted in elevated CREM messenger RNA expression as well. A similar increase of the messenger RNA abundance was also observed in the retrosplenial cortex after treating the female rats with a high dose (5 mg/kg) of dizocilpine maleate, an N-methyl-D-aspartate receptor antagonist. All these conditions are linked to neuronal excitation and neurodegeneration. However, an increase in CREM messenger RNA accumulation was also observed in the visual cortex after exposure of dark-adapted animals to the light, a procedure linked to neuronal plasticity. In the latter condition, it was found that CREM messenger RNA reached its highest levels at 6 h, i.e. later than the maximal increase of expression of immediate early genes such as c-fos, jun B and zif268, observed 45 min following the onset of visual stimulation. The ICER (inducible cyclic-AMP early repressor) form of CREM messenger RNA was identified to be induced by the light exposure. Finally, it was also found that cycloheximide, an inhibitor of protein synthesis, overinduces CREM/ICER gene expression. Together, these data suggest that CREM/ICER may be responsive to neuronal activation. Furthermore, given that CREM products have been shown previously to down-regulate expression of immediate early genes in vitro, they suggest that ICER may function as a molecular switch involved in down-regulation of immediate early gene expression in the rat brain.