CDK11 Promotes Cytokine-Induced Apoptosis in Pancreatic Beta Cells Independently of Glucose Concentration and Is Regulated by Inflammation in the NOD Mouse Model.

Background: Pancreatic islets are exposed to strong pro-apoptotic stimuli: inflammation and hyperglycemia, during the progression of the autoimmune diabetes (T1D). We found that the Cdk11(Cyclin Dependent Kinase 11) is downregulated by inflammation in the T1D prone NOD (non-obese diabetic) mouse model. The aim of this study is to determine the role of CDK11 in the pathogenesis of T1D and to assess the hierarchical relationship between CDK11 and Cyclin D3 in beta cell viability, since Cyclin D3, a natural ligand for CDK11, promotes beta cell viability and fitness in front of glucose. Methods: We studied T1D pathogenesis in NOD mice hemideficient for CDK11 (N-HTZ), and, in N-HTZ deficient for Cyclin D3 (K11HTZ-D3KO), in comparison to their respective controls (N-WT and K11WT-D3KO). Moreover, we exposed pancreatic islets to either pro-inflammatory cytokines in the presence of increasing glucose concentrations, or Thapsigargin, an Endoplasmic Reticulum (ER)-stress inducing agent, and assessed apoptotic events. The expression of key ER-stress markers (Chop, Atf4 and Bip) was also determined. Results: N-HTZ mice were significantly protected against T1D, and NS-HTZ pancreatic islets exhibited an impaired sensitivity to cytokine-induced apoptosis, regardless of glucose concentration. However, thapsigargin-induced apoptosis was not altered. Furthermore, CDK11 hemideficiency did not attenuate the exacerbation of T1D caused by Cyclin D3 deficiency. Conclusions: This study is the first to report that CDK11 is repressed in T1D as a protection mechanism against inflammation-induced apoptosis and suggests that CDK11 lies upstream Cyclin D3 signaling. We unveil the CDK11/Cyclin D3 tandem as a new potential intervention target in T1D.

2-Oxaadamant-1-yl Ureas as Soluble Epoxide Hydrolase Inhibitors: In Vivo Evaluation in a Murine Model of Acute Pancreatitis.

In vivo pharmacological inhibition of soluble epoxide hydrolase (sEH) reduces inflammatory diseases, including acute pancreatitis (AP). Adamantyl ureas are very potent sEH inhibitors, but the lipophilicity and metabolism of the adamantane group compromise their overall usefulness. Herein, we report that the replacement of a methylene unit of the adamantane group by an oxygen atom increases the solubility, permeability, and stability of three series of urea-based sEH inhibitors. Most of these oxa-analogues are nanomolar inhibitors of both the human and murine sEH. Molecular dynamics simulations rationalize the molecular basis for their activity and suggest that the presence of the oxygen atom on the adamantane scaffold results in active site rearrangements to establish a weak hydrogen bond. The 2-oxaadamantane 22, which has a good solubility, microsomal stability, and selectivity for sEH, was selected for further in vitro and in vivo studies in models of cerulein-induced AP. Both in prophylactic and treatment studies, 22 diminished the overexpression of inflammatory and endoplasmic reticulum stress markers induced by cerulein and reduced the pancreatic damage.

The BACE1 product sAPPß induces ER stress and inflammation and impairs insulin signaling.

OBJECTIVE: ß-secretase/ß-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a key enzyme involved in Alzheimer's disease that has recently been implicated in insulin-independent glucose uptake in myotubes. However, it is presently unknown whether BACE1 and the product of its activity, soluble APPß (sAPPß), contribute to lipid-induced inflammation and insulin resistance in skeletal muscle cells. MATERIALS/METHODS: Studies were conducted in mouse C2C12 myotubes, skeletal muscle from Bace1-/-mice and mice treated with sAPPß and adipose tissue and plasma from obese and type 2 diabetic patients. RESULTS: We show that BACE1 inhibition or knockdown attenuates palmitate-induced endoplasmic reticulum (ER) stress, inflammation, and insulin resistance and prevents the reduction in Peroxisome Proliferator-Activated Receptor γ Co-activator 1α (PGC-1α) and fatty acid oxidation caused by palmitate in myotubes. The effects of palmitate on ER stress, inflammation, insulin resistance, PGC-1α down-regulation, and fatty acid oxidation were mimicked by soluble APPß in vitro. BACE1 expression was increased in subcutaneous adipose tissue of obese and type 2 diabetic patients and this was accompanied by a decrease in PGC-1α mRNA levels and by an increase in sAPPß plasma levels of obese type 2 diabetic patients compared to obese non-diabetic subjects. Acute sAPPß administration to mice reduced PGC-1α levels and increased inflammation in skeletal muscle and decreased insulin sensitivity. CONCLUSIONS: Collectively, these findings indicate that the BACE1 product sAPPß is a key determinant in ER stress, inflammation and insulin resistance in skeletal muscle and gluconeogenesis in liver.

VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells.

AIM/HYPOTHESIS: Here, our aim was to examine whether VLDL and apolipoprotein (apo) CIII induce endoplasmic reticulum (ER) stress, inflammation and insulin resistance in skeletal muscle. METHODS: Studies were conducted in mouse C2C12 myotubes, isolated skeletal muscle and skeletal muscle from transgenic mice overexpressing apoCIII. RESULTS: C2C12 myotubes exposed to VLDL showed increased levels of ER stress and inflammatory markers whereas peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and AMP-activated protein kinase (AMPK) levels were reduced and the insulin signalling pathway was attenuated. The effects of VLDL were also observed in isolated skeletal muscle incubated with VLDL. The changes caused by VLDL were dependent on extracellular signal-regulated kinase (ERK) 1/2 since they were prevented by the ERK1/2 inhibitor U0126 or by knockdown of this kinase by siRNA transfection. ApoCIII mimicked the effects of VLDL and its effects were also blocked by ERK1/2 inhibition, suggesting that this apolipoprotein was responsible for the effects of VLDL. Skeletal muscle from transgenic mice overexpressing apoCIII showed increased levels of some ER stress and inflammatory markers and increased phosphorylated ERK1/2 levels, whereas PGC-1α levels were reduced, confirming apoCIII effects in vivo. Finally, incubation of myotubes with a neutralising antibody against Toll-like receptor 2 abolished the effects of apoCIII on ER stress, inflammation and insulin resistance, indicating that the effects of apoCIII were mediated by this receptor. CONCLUSIONS/INTERPRETATION: These results imply that elevated VLDL in diabetic states can contribute to the exacerbation of insulin resistance by activating ERK1/2 through Toll-like receptor 2.

Triatoma infestans Calreticulin: Gene Cloning and Expression of a Main Domain That Interacts with the Host Complement System.

Triatoma infestans is an important hematophagous vector of Chagas disease, a neglected chronic illness affecting approximately 6 million people in Latin America. Hematophagous insects possess several molecules in their saliva that counteract host defensive responses. Calreticulin (CRT), a multifunctional protein secreted in saliva, contributes to the feeding process in some insects. Human CRT (HuCRT) and Trypanosoma cruzi CRT (TcCRT) inhibit the classical pathway of complement activation, mainly by interacting through their central S domain with complement component C1. In previous studies, we have detected CRT in salivary gland extracts from T. infestans We have called this molecule TiCRT. Given that the S domain is responsible for C1 binding, we have tested its role in the classical pathway of complement activation in vertebrate blood. We have cloned and characterized the complete nucleotide sequence of CRT from T. infestans, and expressed its S domain. As expected, this S domain binds to human C1 and, as a consequence, it inhibits the classical pathway of complement, at its earliest stage of activation, namely the generation of C4b. Possibly, the presence of TiCRT in the salivary gland represents an evolutionary adaptation in hematophagous insects to control a potential activation of complement proteins, present in the massive blood meal that they ingest, with deleterious consequences at least on the anterior digestive tract of these insects.

Post-Transcriptional Control of LINE-1 Retrotransposition by Cellular Host Factors in Somatic Cells.

Long INterspersed Element-1 (LINE-1 or L1) retrotransposons form the only autonomously active family of transposable elements in humans. They are expressed and mobile in the germline, in embryonic stem cells and in the early embryo, but are silenced in most somatic tissues. Consistently, they play an important role in individual genome variations through insertional mutagenesis and sequence transduction, which occasionally lead to novel genetic diseases. In addition, they are reactivated in nearly half of the human epithelial cancers, contributing to tumor genome dynamics. The L1 element codes for two proteins, ORF1p and ORF2p, which are essential for its mobility. ORF1p is an RNA-binding protein with nucleic acid chaperone activity and ORF2p possesses endonuclease and reverse transcriptase activities. These proteins and the L1 RNA assemble into a ribonucleoprotein particle (L1 RNP), considered as the core of the retrotransposition machinery. The L1 RNP mediates the synthesis of new L1 copies upon cleavage of the target DNA and reverse transcription of the L1 RNA at the target site. The L1 element takes benefit of cellular host factors to complete its life cycle, however several cellular pathways also limit the cellular accumulation of L1 RNPs and their deleterious activities. Here, we review the known cellular host factors and pathways that regulate positively or negatively L1 retrotransposition at post-transcriptional level, in particular by interacting with the L1 machinery or L1 replication intermediates; and how they contribute to control L1 activity in somatic cells.

[In vitro cell response to chemotherapeutic agents, to personalize ovarian cancer treatment: report of two cases]. / Potencial herramienta en la personalización del tratamiento oncológico: casos clínicos.

Our laboratory has implemented an in vitro assay to estimate the response to chemotherapy in ovarian cancer cells pertaining to individual patients. In two selected patients, we determined the correlation between an in vitro assay of cells from suspected ovarian cancer ascites, with the clinical chemotherapy response. Cancer cells isolated from peritoneal fluid with suspected ovarian cancer were tested for cytotoxicity with corresponding chemotherapy regimens. Circulating Cal25 levels and attending physician consultation determined clinical course and response to chemotherapy. The in vitro assay result correlated with Cal25 levels, progression free survival and attending physician evaluation. The assay predicted correctly the failure of two successive chemotherapy regimes in the first patient, while predicting a favorable clinical response in the second subject.

Metformin, at concentrations corresponding to the treatment of diabetes, potentiates the cytotoxic effects of carboplatin in cultures of ovarian cancer cells.

The use of the type 2 diabetics drug metformin has been correlated with enhanced progression-free survival in ovarian cancer. The literature has speculated that this enhancement is due to the high concentration of metformin directly causing cancer cell death. However, this explanation does not fit with clinical data reporting that the women exposed to constant micromolar concentrations of metformin, as present in the treatment of diabetes, respond better to chemotherapy. Herein, our aim was to examine whether micromolar concentrations of metformin alone could bring about cancer cell death and whether micromolar metformin could increase the cytotoxic effect of commonly used chemotherapies in A2780 and SKOV3 cell lines and primary cultured cancer cells isolated from the peritoneal fluid of patients with advanced ovarian cancer. Our results in cell lines demonstrate that no significant loss of viability or change in cell cycle was observed with micromolar metformin alone; however, we observed cytotoxicity with micromolar metformin in combination with chemotherapy at concentrations where the chemotherapy alone produced no loss in viability. We demonstrate that previous exposure and maintenance of metformin in conjunction with carboplatin produces a synergistic enhancement in cytotoxicity of A2780 and SKOV3 cells (55% and 43%, respectively). Furthermore, in 5 (44%) of the 11 ovarian cancer primary cultures, micromolar metformin improved the cytotoxic response to carboplatin but not paclitaxel or doxorubicin. In conclusion, we present data that support the need for a clinical study to evaluate the adjuvant maintenance or prescription of currently approved doses of metformin during the chemotherapeutic treatment of ovarian cancer.

Potencial herramienta en la personalización del tratamiento oncológico: Casos clínicos / In vitro cell response to chemotherapeutic agents, to personalize ovarian cancer treatment: Report of two cases

Our laboratory has implemented an in vitro assay to estimate the response to chemotherapy in ovarian cancer cells pertaining to individual patients. In two selected patients, we determined the correlation between an in vitro assay of cells from suspected ovarian cancer ascites, with the clinical chemotherapy response. Cancer cells isolated from peritoneal fluid with suspected ovarian cancer were tested for cytotoxicity with corresponding chemotherapy regimens. Circulating Cal25 levels and attending physician consultation determined clinical course and response to chemotherapy. The in vitro assay result correlated with Cal25 levels, progression free survival and attending physician evaluation. The assay predicted correctly the failure of two successive chemotherapy regimes in the first patient, while predicting a favorable clinical response in the second subject.

Role of cell cycle re-entry in neurons: a common apoptotic mechanism of neuronal cell death.

Currently, there is no effective treatment for neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Thus, a major focus of neuroscience research is to examine the mechanisms involved in neuronal loss in order to identify potential drug targets. Recent results indicate that DNA damage and re-entry into the cell cycle may constitute a common pathway in apoptosis in neurological diseases. The role of the cell cycle in such disorders is supported by data on the brain of patients who showed an increase in cell-cycle protein expression. Indeed, studies performed in neuronal cell preparations indicate that oxidative stress could be the main mechanism responsible for cell cycle re-entry. DNA damage and repair after oxidative stress may activate the enzyme ataxia telangiectasia mutated, which is a cell-cycle regulator. Once the cell cycle is activated, the increase in the expression of transcription factor E2F-1 could induce neuronal apoptosis. Furthermore, the potential routes involved in E2F-1 induced apoptosis could be p53-dependent or p53-independent. Under this E2F-1 hypothesis of cell death, multiple mitochondria-dependent pathways may be activated, including caspase and caspase-independent signaling cascades. Finally, given that cyclin-dependent kinase inhibitory drugs have neuroprotective and anti-apoptotic effects in experimental models, their potential application for the treatment of neurological disorders should be taken into account.

Antiapoptotic effects of roscovitine on camptothecin-induced DNA damage in neuroblastoma cells.

In the present study dopaminergic neuroblastoma B65 cells were exposed to Camptothecin (CPT) (0.5-10 µM), either alone or in the presence of roscovitine (ROSC). The results show that CPT induces apoptosis through the activation of ataxia telangiectasia mutated (ATM)-induced cell-cycle alteration in neuroblastoma B65 cells. The apoptotic process is mediated through the activation of cystein proteases, namely calpain/caspases. However, whereas a pan-caspase inhibitor, zVADfmk, inhibited CPT-mediated apoptosis, a calpain inhibitor, calpeptin, did not prevent cell death. Interestingly, CPT also induces CDK5 activation and ROSC (25 µM) blocked CDK5, ATM activation and apoptosis (as measured by caspase-3 activation). By contrast, selective inhibition of ATM, by KU55933, and non-selective inhibition, by caffeine, did not prevent CPT-mediated apoptosis. Thus, we conclude that CDK5 is activated in response to DNA damage and that CDK5 inhibition prevents ATM and p53ser15 activation. However, pharmacological inhibition of ATM using KU55933 and caffeine suggests that ATM inhibition by ROSC is not the only mechanism that might explain the anti-apoptotic effects of this drug in this apoptosis model. Our findings have a potential clinical implication, suggesting that combinatory drugs in the treatment of cancer activation should be administered with caution.

Resveratrol inhibits proliferation and promotes apoptosis of neuroblastoma cells: role of sirtuin 1.

Resveratrol prolongs lifespan and prevent cancer formation; however, the mechanisms are not understood. Here we evaluated the cell-cycle inhibition and apoptosis of resveratrol in B65 neuroblastoma cells, and we also studied the effects of resveratrol on the mammalian silent information regulator 2 (SIRT1). Results show that resveratrol reduces cell viability and causes apoptosis at 24 h of treatment. Resveratrol partially blocked cell proliferation, and significantly increased the fraction of cells arrested in the S phase. The role of SIRT1 in cell-cycle effects mediated by resveratrol was studied through changes in the expression of SIRT1 using western blot. Exposure to resveratrol decreased SIRT1 content, concomitant with an increase in the acetylated form of sirtuin substrates p53 and NFκ-ß. Treatment of B65 neuroblastoma cells with resveratrol also reduced the content of the phosphorylated form of AKT. Exposure to the SIRT1 inhibitors nicotinamide and sirtinol altered neither cell viability nor the fraction of apoptotic cells. Furthermore, when cells were exposed simultaneously to resveratrol and nicotinamide or sirtinol, no changes were observed in the fraction of apoptotic cells. Our results show that a decrease in SIRT1 content, caused by exposure to resveratrol, does not appear to be involved in cell-cycle arrest or activation of apoptosis.

Activation of ataxia telangiectasia muted under experimental models and human Parkinson's disease.

In the present study we demonstrated that neurotoxin MPP(+)-induced DNA damage is followed by ataxia telangiectasia muted (ATM) activation either in cerebellar granule cells (CGC) or in B65 cell line. In CGC, the selective ATM inhibitor KU-55933 showed neuroprotective effects against MPP(+)-induced neuronal cell loss and apoptosis, lending support to the key role of ATM in experimental models of Parkinson's disease. Likewise, we showed that knockdown of ATM levels in neuroblastoma B65 cells using an ATM-specific siRNA attenuates the phosphorylation of retinoblastoma protein without affecting other cell-cycle proteins involved in the G(0)/G(1) cell-cycle phase. Moreover, we demonstrated DNA damage, in human brain samples of PD patients. These findings support a model in which MPP(+) leads to ATM activation with a subsequent DNA damage response and activation of pRb. Therefore, this study demonstrates a new link between DNA damage by MPP(+) and cell-cycle re-entry through retinoblastoma protein phosphorylation.

ATM is involved in cell-cycle control through the regulation of retinoblastoma protein phosphorylation.

Ataxia telangiectasia mutated protein (ATM) is a member of the phosphatidylinositol-3 kinase (PI3K) family, which has a role in the cellular response to DNA double-strand breaks (DSBs). In the present study, we evaluated the role of ATM in cell-cycle control in dopaminergic rat neuroblastoma B65 cells. For this purpose, ATM activity was either inhibited pharmacologically with the specific inhibitor KU-55933, or the ATM gene was partially silenced by transfection with small interfering RNA (siRNA). Our data indicate that although ATM inhibition did not affect the cell cycle, both treatments specifically decreased the levels of cyclin A and retinoblastoma protein (pRb), phosphorylated at Ser780. Furthermore, ATM inhibition decreased the active form of p53, which is phosphorylated at Ser15, and also decreased Bax and p21 expression. Using H(2)O(2) as a positive control of DSBs, caused a rapid pRb phosphorylation, this was prevented by KU-55933 and siRNA treatment. Collectively, our data demonstrate how a new molecular network on ATM regulates the cell cycle through the control of pRb phosphorylation. These findings support a new target of ATM.

Effects of MPP+ on the molecular pathways involved in cell cycle control in B65 neuroblastoma cells.

The toxicity caused by cell exposure to 1-methyl-4-phenylpyridinium ion (MPP(+)) is a useful model in the study of Parkinson's disease (PD). However, the exact molecular mechanisms triggered by MPP(+) in cell death are currently unclear. In the present research, we show that exposure to MPP(+) induce the cell death of neuroblastoma-derived dopaminergic B65 cells, which is not reversed by the widely known caspase inhibitor Z-VAD fmk or by calpain inhibition. Likewise, when B65 cells were treated with MPP(+), the DNA damage pathway that involves p53 was activated, and cells were arrested in the G(2)/M phase of the cell cycle. Interestingly, MPP(+) has two effects on the expression of cell cycle-related proteins. It increases the content of cyclins A, E, cdk2 and the phosphorylated form of pRb (serine 780). However, MPP(+) 5mM decreased the expression of cyclin D1, B1 and cdk4. The decrease in the expression of cyclin B1 may be related to the arrest of cells observed in the G(2)/M phase of cell cycle. The increase in S phase cell cycle proteins and retinoblastoma protein phosphorylation was an unexpected result. As the antioxidant trolox attenuated the process of cell loss and changes in the cell cycle, as measured by flow cytometry, we concluded that oxidative stress was involved in the effects of MPP(+) in this cell line. In summary, the present work characterizes the molecular changes involved in damage caused by MPP(+) in B65 cells, and highlights the effects of MPP(+) on molecules involved in the control of cell cycle progression.

Oxidative stress-induced DNA damage and cell cycle regulation in B65 dopaminergic cell line.

Reactive oxygen species and oxidative stress are associated with neuronal cell death in many neurodegenerative conditions. However, the exact molecular mechanisms triggered by oxidative stress in neurodegeneration are still unclear. This study used the B65 rat neuroblastoma cell line as a model to study the molecular events that occur after H(2)O(2) treatment. Treatment of B65 cells with H(2)O(2) rapidly up-regulated the DNA damage pathway involved in double-strand breakage. Subsequently, proteins involved in p53 regulation, such as sirtuin 1 and STAT1, were modified. In addition, H(2)O(2) treatment altered the pattern of cell cycle protein expression. Specifically, a decrease was found in the expression of cyclin D1, cdk4 and surprisingly the levels of cyclin A and the retinoblastoma protein phosphorylated at ser780 were increased. Furthermore, this study shows that pre-treatment of B65 cells with 50 microM trolox confers almost total protection against apoptotic cell death and restores the cell cycle. Likewise, the increase in retinoblastoma phosphorylation was attenuated by KU-55993, a selective ATM inhibitor, and also by trolox. These observations indicate that DNA damage and oxidative stress are responsible for cell cycle regulation. In summary, this study describes the molecular mechanisms involved in cell cycle alterations induced by oxidative stress in B65 cells. These findings highlight the relevance of ATM in the regulation of cell cycle after oxidative stress.

A molecular study of pathways involved in the inhibition of cell proliferation in neuroblastoma B65 cells by the GSK-3 inhibitors lithium and SB-415286.

Pharmacological GSK-3 inhibitors are potential drugs for the treatment of neurodegenerative diseases, cancer and diabetes. We examined the antiproliferative effects of two GSK-3 inhibitors, lithium and SB-415286, on B65 neuroblastoma cell line. Treatment of B65 cells with either drug administered separately caused a decrease in cell proliferation that was associated with G(2)/M cell cycle arrest. Cell-cycle proteins such as cyclins D, E, A, cdk4 and cdk2 were up-regulated. Since lithium and SB-415286-induced G(2)/M arrest we studied changes in the expression of proteins involved in this phase, specifically cyclin B, cdc2 and the phosphorylated form of this protein (tyr15-cdc2). Both drugs increased the expression of tyr15-cdc2, thus inhibiting mitosis. On the other hand, SB-415286 increased the expression of SIRT2, involved in the regulation of proliferation. Moreover, cell-cycle arrest mediated by SB-415286 was accompanied by apoptosis that was not prevented by 100 microM of zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), a pan-caspase inhibitor. Likewise, GSK-3 inhibitors did not affect the mitochondrial release of apoptosis inducing factor (AIF). We conclude that inhibitors of GSK-3 induced cell-cycle arrest, mediated by the phosphorylation of cdc2 and, in the case of SB-415286, SIRT2 expression, which induced apoptosis in a caspase-independent manner.

Neuroprotective effects of SB-415286 on hydrogen peroxide-induced cell death in B65 rat neuroblastoma cells and neurons.

Glycogen synthase kinase-3 (GSK-3) is involved in the pathogenesis of several neurodegenerative diseases. In addition, as oxidative stress has been implicated in all neurodegenerative disorders, the inhibition of both pathways offers a potential strategy for preventing or delaying neurodegeneration. We examined the cytoprotective effects of lithium and SB-415286, two inhibitors of GSK-3, using a rat B65 cell line and also in cerebellar granule cells (CGN). H(2)O(2) decreased the inactive form of GSK-3 (phospho-GSK-3 at Ser9), as measured by immunoblot experiments involving an antibody against the inactive form of the enzyme. Moreover, lithium inhibited this effect. While SB-415286 exerted a protective effect, lithium did not attenuate the toxic effects of H(2)O(2) (1mM). We then examined those mechanisms implicated in the protective effects of SB-415286. When we analyzed reactive oxygen species (ROS) production using the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate in B65 cells, as well as in CGN, we found that SB-415286 strongly reduced DCF fluorescence. Lithium, however, did not exhibit any antioxidant properties. We conclude that the GSK-3 inhibitor SB-415286 has antioxidant properties, which may explain the cytoprotective effects against H(2)O(2) damage. Furthermore, inhibition of GSK-3 activity was not involved in this protective effect.

The antiproliferative activity of melatonin in B65 rat dopaminergic neuroblastoma cells is related to the downregulation of cell cycle-related genes.

A potential application of melatonin is its ability to rescue many cell types from cell death, because of its antioxidant properties. Likewise, recent studies suggest that melatonin may also be used as an anti-tumor drug, due to its anti-proliferative properties in tumor cells when administered at physiologic or pharmacologic doses. In the present study, we investigated the mechanisms involved in the apoptosis induced by acute exposure to melatonin and roscovitine in the rat dopaminergic neuroblastoma B65 cell line. Cell growth studies revealed that, at 24 hr of treatment, roscovitine blocked cell growth and induced apoptosis whereas melatonin delayed cell growth and induced a slight increase in the number of apoptotic nuclei. Melatonin also increased the percentage of cells in the G1-phase of the cell cycle, whereas roscovitine blocked cells in the G2/M-phase. Both compounds significantly downregulated the transcriptional activity of cdk4, while melatonin also downregulated cdk2 and cyclin D1. Taken together, our data show that melatonin at millimolar concentrations inhibits dopaminergic B65 proliferation, induces cell apoptosis, and modulates cell cycle progression by inhibiting the transcriptional activity of cyclins and cdks related to the progression of the G1-phase.