Cyclin D1 targets hexokinase 2 to control aerobic glycolysis in myeloma cells.

Cancer cells are characterized by the Warburg effect, a shift from mitochondrial respiration to oxidative glycolysis. We report here the crucial role of cyclin D1 in promoting this effect in a cyclin-dependent kinase (CDK)4/6-independent manner in multiple myeloma (MM) cells. We show that the cyclin D1 oncoprotein targets hexokinase 2 (HK2), a major glycolysis regulator, through two original molecular mechanisms in the cytoplasmic and nuclear compartments. In the cytoplasm, cyclin D1 binds HK2 at the outer mitochondrial membrane, and in the nucleus, it binds hypoxia-inducible factor-1α (HIF1α), which regulates HK2 gene transcription. We also show that high levels of HK2 expression are correlated with shorter event-free survival (EFS) and overall survival (OS) in MM patients. HK2 may therefore be considered as a possible target for antimyeloma therapy.

Flow cytometry for receptor analysis from ex-vivo brain tissue in adult rat.

BACKGROUND: Flow cytometry allows single-cell analysis of peripheral biological samples and is useful in many fields of research and clinical applications, mainly in hematology, immunology, and oncology. In the neurosciences, the flow cytometry separation method was first applied to stem cell extraction from healthy or cerebral tumour tissue and was more recently tested in order to phenotype brain cells, hippocampal neurogenesis, and to detect prion proteins. However, it remains sparsely applied in quantifying membrane receptors in relation to synaptic plasticity. NEW METHOD: We aimed to optimize a flow cytometric procedure for receptor quantification in neurons and non-neurons. A neural dissociation process, myelin separation, fixation, and membrane permeability procedures were optimized to maximize cell survival and analysis in hippocampal tissue obtained from adult rodents. We then aimed to quantify membrane muscarinic acetylcholine receptors (mAChRs) in rats with and without bilateral vestibular loss (BVL). RESULTS: mAChR's were quantified for neuronal and non-neuronal cells in the hippocampus and striatum following BVL. At day 30 but not at day 7 following BVL, there was a significant increase (P ≤ 0.05) in the percentage of neurons expressing M2/4 mAChRs in both the hippocampus and the striatum. CONCLUSION: Here, we showed that flow cytometry appears to be a reliable method of membrane receptor quantification in ex-vivo brain tissue.

[Interventions to optimize pharmacologic treatment in hospitalized older adults: a systematic review]. / Intervenciones para optimizar el tratamiento farmacológico en ancianos hospitalizados: una revisión sistemática.

OBJECTIVE: To summarise the evidence on interventions aimed at optimising the drug treatment of hospitalised elderly patients. MATERIAL AND METHODS: We conducted a search in the main medical literature databases, selecting prospective studies of hospitalised patients older than 65 years who underwent interventions aimed at optimising drug treatment, decreasing polypharmacy and improving the medication appropriateness, health outcomes and exploitation of the healthcare system. RESULTS: We selected 18 studies whose interventions consisted of medication reviews, detection of predefined drugs as potentially inappropriate for the elderly, counselling from a specialised geriatric team, the use of a computer support system for prescriptions and specific training for the nursing team. Up to 14 studies assessed the medication appropriateness, 13 of which showed an improvement in one or more of the parameters. Seven studies measured the impact of the intervention on polypharmacy, but only one improved the outcomes compared with the control. Seven other studies analysed mortality, but none of them showed a reduction in that rate. Only 1 of 6 studies showed a reduction in the number of hospital readmissions, and 1 of 4 studies showed a reduction in the number of emergency department visits. CONCLUSIONS: Despite the heterogeneity of the analysed interventions and variables, we obtained better results in the process variables (especially in medication appropriateness) than in those that measured health outcomes, which had greater variability.

Senescence-associated secretory phenotype favors the emergence of cancer stem-like cells.

The molecular mechanisms underlying cancer resistance remain elusive. One possible explanation is that cancer stem cells (CSCs) elude drug treatment, emerge and reproduce a tumor. Using multiple myeloma as a paradigm, we showed that cancer stem-like cells (CSLCs) appear after genotoxic stress because of their intrinsic properties. However, these properties do not drive the emergence of the CSLCs. Following genotoxic stress, remaining DNA damages lead to a senescence-associated secretory phenotype (SASP). Senescent cells, which are the non-CSLCs, secrete chemokines contributing to the emergence, maintenance and migration of CSLCs. Downregulation of checkpoint protein 2, a key player of SASP, significantly reduced the emergence of CSLCs. Our results unravel a novel molecular mechanism by which SASP might promote malignancy, underlining the dual role of senescence in tumorigenesis. This mechanism, based on mutual cooperation among tumor cells, illustrates how cancer may relapse; its targeting could represent new therapeutic opportunities.

Cyclin D1 mediates resistance to apoptosis through upregulation of molecular chaperones and consequent redistribution of cell death regulators.

Cyclin D1 is a key regulator of cell proliferation. It also controls other aspects of the cell fate, such as cellular senescence, apoptosis and tumourigenesis. We used B-lymphoid cell lines producing cyclin D1 to investigate the role of this protein in B-cell lymphomas and leukaemias. Constitutive low levels of cyclin D1 had no effect per se on cell proliferation, but conferred resistance to various apoptotic stimuli in B cells. Activation of the pro-apoptotic protein, Bax, was reduced and mitochondrial permeabilization and phosphatidylserine exposure following cytokine withdrawal were delayed in cyclin D1-producing cells. Proteomic analysis showed that the presence of cyclin D1 led to intracellular accumulation of various molecular chaperones. The chaperone, heat shock protein (Hsp)70, bound to both Bax and the mitochondrial apoptosis inducing factor following cytokine withdrawal, and impeded inhibitors of kappaB (IkappaB)-mediated inhibition of nuclear factor-kappaB anti-apoptotic signalling. Impairment of Hsp70 activity--using a pharmacological Hsp inhibitor or transfecting cells with an Hsp70-blocking antibody--restored the cellular response to mitochondrial apoptosis triggering. Thus, constitutive de-novo cyclin D1 production in B cells delays commitment to apoptosis by inducing Hsp70 chaperoning activity on pre- and post-mitochondrial pro-apoptotic factors.

Steep variation in mitochondrial DNA and B chromosomes among natural populations of Eyprepocnemis plorans (Acrididae).

Restriction enzyme analysis of the mitochondrial DNA of Eyprepocnemis plorans with 9 restriction enzymes revealed low variability--only EcoRI revealed any variability with 3 distinguishable digestion patterns, here named types I, II and III. The samples studied were collected from regions where different types of B chromosome exist. The demes from the central area show the B1 type (as a relict of a probably once continuous distribution) and also mtDNA type II. These show a parallel substitution towards the SW neighbouring demes by other Bs and mtDNA type I. However, the latter mtDNA type had a wider distribution and is predominant, or even the only one found in most other samples where other Bs exist. Considering the mtDNA as a marker unlinked with the Bs, some genetic differences should exist among areas defined by the B chromosomes. Our results support the hypothesis that in the central region the B chromosomes and the mtDNA are involved in two different events of substitution, but these do not necessarily occur in the same way.

Upstream mediators of the Fas apoptotic transduction pathway are defective in B-chronic lymphocytic leukemia.

Data concerning the presence and the functionality of Fas receptor in malignant B-cells are controversial. We have analyzed Fas molecules on B-cells from patients with B-chronic lymphocytic leukemia (B-CLL) cells. We observed a large variability, both of percentage of Fas-positive cells and of intensity of Fas level. Fas triggering was inefficient in inducing apoptosis whatever the number of Fas-positive B-cells, the amount of Fas receptors. B-cells were also resistant to etoposide treatment, but able to undergo apoptosis after dexamethasone treatment. We suggest that the Fas apoptotic pathway is altered in B-CLL patients at the initial step(s) of apoptotic machinery.

Ectopic expression of cyclin D1 impairs the proliferation and enhances the apoptosis of a murine lymphoid cell line.

Cyclin D1, a key regulator of the cell cycle, acts as an oncogene when over-expressed in several types of cancer. In some B-chronic lymphoproliferative disorders, the over-expression of cyclin D1 protein is thought to confer a proliferative phenotype. We have generated BaF3 pro-B cell derivatives in which cyclin D1 can be induced rapidly and reversibly in a dose-dependent manner by the hormone muristerone A. When non-expressing clones displayed the same proliferative capacity as the parental cell line, in the sub-clones, a moderate induction of cyclin D1 lengthened the proliferation rate. The over-expression of cyclin D1 had the same effects on cell proliferation but also led ultimately to cell death by apoptosis. The induction of cyclin D1 in growth factor-deprived cells as well as in anticancer drug-treated cells also reinforced the magnitude of apoptosis. Thus, the expression of cyclin D1 in lymphoid cells does not confer a proliferative advantage but rather alters the response of cells towards apoptotic stimuli in a p53-independent manner.

No BCL-2 protein over expression but BCL-2/IgH rearrangements in B cells of patients with persistent polyclonal B-cell lymphocytosis.

INTRODUCTION: Persistent polyclonal B-cell lymphocytosis is a rare hematological disorder, characterized by a chronic, stable and absolute polyclonal lymphocytosis, the presence of binucleated lymphocytes, a polyclonal increase in serum IgM immunoglobulin and clonal cytogenetic abnormalities involving chromosome 3. For explaining the expansion of B-lymphocytes pool in PPBL, an association with cigarette smoking and/or chronic Epstein-Barr virus infection have been suggested but both hypotheses have been ruled out. MATERIALS AND METHODS: We studied the presence of BCL-2/IgH rearrangements in a series of eight PPBL patients (seven females and one male) by a nested polymerase chain reaction (PCR), targeting the Major Breakpoint Region in BCL-2 locus and we explored the BCL-2 protein expression by Western blot. RESULTS: We demonstrated: (a) the constant presence of BCL-2/IgH rearrangements in eight out of eight DNA samples, (b) multiple rearrangements in three out of eight cases and, (c) normal BCL-2 protein expression, as compared to BCL-2 level in B-lymphocytes from healthy population. CONCLUSION: Despite the presence of BCL-2/IgH rearrangements, the accumulation of B lymphocytes in PPBL is not related to an overexpression of BCL-2 protein.

Early and sequential recruitment of apoptotic effectors after focal permanent ischemia in mice.

In experimental models of cerebral ischemia, cells within the damaged territory die by necrosis and by apoptosis that contributes to the expansion of the insult. Apoptotic machinery mobilizes intracellular processes such as induction of Bcl-2 family members, activation of the proteolytic cascade including the caspases, and cleavage of caspase substrates, such as poly(ADP-ribose) polymerase or PARP. Mitochondria play a pivotal role in controlling apoptosis by releasing cytochrome c and modulating redox state, both under the regulation of manganese superoxide dismutase (Mn SOD) via superoxide anion detoxification. The implication and the kinetics of such events in apoptosis induced after focal permanent ischemia in mice remains to be studied. In a paradigm of ischemic insult induced by occlusion of the middle cerebral artery (MCAO) in mice, we showed by immunohistochemistry a constitutive expression of caspase-3 that is enhanced after MCAO in neurons localized within the infarcted zone. As a function of time intervals after MCAO, the cytochrome c amount increased in the cytosolic fraction of ischemic cortical extracts. The kinetics of the release was in concordance with the expression of caspase-3 and the subsequent cleavage of PARP appearing before the internucleosomal fragmentation of DNA, the ultimate step of apoptosis. When the apoptotic markers progressively appeared, no changes of Mn SOD activity or Mn SOD expression were detected after MCAO. We can therefore speculate that the recruitment of Mn SOD did not participate per se in the release of cytochrome c elicited after permanent focal ischemia.

Pharmacological delta1- and delta2-opioid receptor subtypes in the human neuroblastoma cell line SK-N-BE: no evidence for distinct molecular entities.

The two pharmacological delta-opioid receptor subtypes, delta1 and delta2, have been defined on the basis of pharmacological tools but remain to be characterized at the molecular level, since only a single cDNA has been cloned. The present study aimed to investigate the pharmacological properties of delta1- and delta2-opioid subtypes expressed in the human neuroblastoma cell line SK-N-BE and to characterize their putative corresponding mRNAs. Binding experiments using "selective" delta1- and delta2-opioid agonists and antagonists revealed the presence of two binding sites, demonstrating the presence of these delta1-opioid subtypes as they were previously described. The activation of these pharmacological subtypes by the selective agonists induced the incorporation of [alpha-(32)P]azidoanilide-GTP into Galpha(i2)/Galpha(0) subunits with the same efficiency and potency and inhibited adenosine 3', 5'-cyclic monophosphate (cAMP) accumulation with similar efficiency, while their sustained activation for 15 min induced a cross-desensitization. The "selective" delta1 and delta2 antagonists, 7-benzylidenenaltrexone and naltrindole benzofuran, respectively, were found to be as potent in blocking the inhibition of cAMP accumulation induced by both [D-Pen(2,5)]enkephalin and Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH(2). The possibility that delta-opioid subtypes could arise from alternative splicing was ruled out by reverse transcription-polymerase chain reaction (RT-PCR) experiments and the sequencing of PCR products, which revealed the presence of a single transcript encoding for the delta-opioid receptor. Different possibilities which could account for the delta-opioid receptor heterogeneity observed in the SN-N-BE cell line are discussed.

Cyclin D1 expression in patients with multiple myeloma.

INTRODUCTION: Chromosomal abnormalities are detected in 50 to 70% of patients with multiple myeloma (MM). By conventional cytogenetic analysis, a t(11;14)(q13;q32) is observed at a frequency of 3 to 14%. MATERIALS AND METHODS: To demonstrate a cyclin D1 expression in MM patients or MM cell lines, 14 patients with multiple myeloma (MM) and nine human multiple myeloma cell lines (HMCL) were screened by a competitive RT-PCR and/or Northern blot analysis for cyclin D1 expression. Furthermore, we screened 10 MM patients with FISH to demonstrate a relationship between the cyclin D1 expression and the presence of the t(11;14). RESULTS: Five HMCL had a cyclin D1 overexpression: three of them had a t(11;14)(q13;q32) and two had extra copies of chromosome 11. A cyclin D1 expression was found at diagnosis in seven out of 14 untreated MM patients (50%). Out of 14 MM patients, FISH studies were performed in 10 patients. A t(11;14) was detected in three out of 10 patients and extra copies of chromosome 11 were found in two additional patients. CONCLUSION: Cyclin D1 expression is a common event in MM patients (50%) and is associated either with a t(11;14)(q13;q32) or extra copies of chromosome 11. The prognostic role of the cyclin D1 expression and the level of this expression, as compared to other B-cell chronic lymphoproliferative disorders such as mantle cell lymphoma or hairy cell leukemia, remains to be determined in the pathogenesis of multiple myeloma.

Detection by the comet assay of apoptosis induced in lymphoid cell lines after growth factor deprivation.

Dysregulation of apoptosis contributes to various diseases such as neurodegenerative or aging disorders, autoimmune syndromes or cancers. Numerous experimental paradigms have been explored to characterize molecular and cellular modulators of apoptosis. Similarly, numerous techniques have been described for detecting and/or quantifying accurately cells committed to apoptosis. Besides the conventional techniques, we describe in this report that the comet assay, which detects DNA single- and double-strand breaks in situ, at the cellular level, is relevant for the characterization of apoptotic cells. The comet assay is very sensitive and detects DNA fragmentation occurring in the apoptotic process as early as exposure of phosphatidylserine residues on the outer leaflet. Thus the comet assay can be used for the recognition of apoptosis that follows the death signal caused, for example, by genotoxic stress as well as lack of survival signal as in growth factor deprivation.

Transcriptional and post-transcriptional mechanisms induce cyclin-D1 over-expression in B-chronic lymphoproliferative disorders.

Cyclin D1 participates in cell-cycle control, in the progression through the G(1) phase and in the transition from the G(1) to the S phase. The CCND1 locus, located in 11q13, is amplified and cyclin-D1 protein is over-expressed in a wide range of human solid tumors. In some B-lymphoid malignancies, the t(11;14)(q13;q32) translocation joins the Ig heavy-chain locus to the CCND1 locus and leads to cyclin-D1 over-expression. In this study, a series of 127 patients presenting a B-chronic lymphoproliferative disorder (B-CLPD) was analyzed using a competitive RT-PCR designed to detect cyclin-D1-mRNA over-expression. Cyclin-D1 mRNA was expressed in patients with mantle-cell lymphoma (MCL; 10/10), hairy-cell leukemia (HCL; 3/5), B-chronic lymphoid leukemia (B-CLL; 4/111) and B large-cell lymphoma (BLCL; 1/1). Densitometric analysis of RT-PCR products and Western-blot autoradiograms, in addition to cytogenetic data, indicated that activation of the cyclin-D1 gene occurred independently of the t(11;14)(q13;q32) translocation in patients with HCL. Indeed, a normal-sized protein of 36 kDa exhibiting a level incompatible with gene activation by a translocation mechanism was detected in lymphoid cells with a normal karyotype. Moreover, we found a discrepancy between cyclin-D1 mRNA and protein levels in MCL and B-CLL, which suggested that some regulatory mechanisms acting at a post-transcriptional level persist in tumor cells.

Overproduction of Cu/Zn-superoxide dismutase or Bcl-2 prevents the brain mitochondrial respiratory dysfunction induced by glutathione depletion.

Recent work has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Low concentrations of the powerful antioxidant glutathione (GSH) and impaired brain energy metabolism, particularly in the substantia nigra, are key features of Parkinson's disease (PD). The main goal of this study was to better characterize the deleterious effects of brain GSH depletion on mitochondrial function. We depleted GSH in the brains of newborn wild-type (WT) and transgenic (Tg) mice overproducing either human Cu/Zn-superoxide dismutase (h-CuZnSOD) or human Bcl2 (h-Bcl-2), by subcutaneous injection of l-buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. GSH was 97% depleted in brain homogenates and 90% depleted in brain mitochondria for both WT and Tg mice. This depletion of brain GSH led to a decrease in the activity of the GSH-dependent antioxidant enzyme glutathione peroxidase, both in WT and in Tg animals. BSO treatment decreased the activities of respiratory complexes I, II, and IV in the brain homogenates of WT mice. BSO-treated h-CuZnSOD or h-Bcl-2 Tg mice had no respiratory chain deficiencies. Thus, brain GSH depletion leads to the impairment of mitochondrial respiratory chain activity. The protection of mitochondrial respiratory function by overproduction of Bcl-2 may result from a decrease in the generation of reactive oxygen species (ROS) or lipid peroxidation. The protection of mitochondria by overproduction of CuZnSOD is consistent with the involvement of superoxide or superoxide-derived ROS in the mitochondrial dysfunction caused by brain GSH depletion. This study demonstrates that the antioxidant balance is critical for maintenance of brain mitochondrial function, and its disruption may contribute to the pathogenesis of PD.

Reduction of ischemic damage in NGF-transgenic mice: correlation with enhancement of antioxidant enzyme activities.

If permanent focal ischemia is induced by middle cerebral artery occlusion (MCAO), neurons within the infarcted territory die by necrosis and apoptosis (or programmed cell death). We have previously shown, using a mouse strain transgenic (tg) for the nerve growth factor (NGF) gene, that tg mice have consistently smaller infarcted areas than wild-type (wt) animals, correlated with upregulated NGF synthesis and impaired apoptotic cell death. We studied, in wt and tg mice subjected to MCAO, the activities of several antioxidant enzymes and the synthesis of the proteins of the Bcl-2 family. Our results show that the antiapoptotic Bcl-2 protein and glutathione peroxidase are recruited after MCAO. NGF-tg mice also had an intrinsic resistance to oxidative stress because their basal copper zinc superoxide dismutase (SOD) and glutathione transferase activities were high. Additionally, manganese SOD activity increased in NGF-tg mice after MCAO, correlating strongly with the resistance of these mice to apoptosis.

In vitro evaluation of B-CLL cells apoptotic responses to irradiation.

Defective apoptosis is a mechanism which could possibly explain B chronic lymphocytic leukemia (B-CLL) cell accumulation. Differences in evolution and prognosis of B-CLL patients may be due to heterogeneity in apoptotic cell death. We studied the apoptotic response to in vitro gamma radiation of blood mononuclear cells from 18 untreated B-CLL patients. In cells irradiated with 2, 4 or 8 Gy and then cultured for 20 hours, the percentage of trypan blue excluding (viable) cells was not modified (>92%). An apoptotic response to irradiation was detected in the majority of the patients, but the individual percentage of apoptotic cells varied widely (8 to 81% after 8 Gy irradiation) in individual cases. The flow cytometric analysis of nick-end DNA labeling demonstrated a dose effect of irradiation, particularly in patients with an apoptotic response of over 20%. In the future, a valuable clue to the selection of irradiation regimens for B-CLL patients may be the investigation of correlations between in vitro radiation-induced apoptosis and the in vivo response to radiation therapy.

Reduction of cortical infarction and impairment of apoptosis in NGF-transgenic mice subjected to permanent focal ischemia.

The neuroprotective potential of the nerve growth factor (NGF) against permanent ischemic brain damage has been investigated in vivo using NGF-transgenic (tg) mice. The expression of the transgene is driven by part of the promoter of the proto-oncogene c-fos, which belongs to the first set of genes activated after brain ischemic insult. Wild-type (wt) mice and tg mice were subjected to permanent focal ischemia induced by electrocoagulation of the middle cerebral artery. Twenty four hours (h) after the ischemic shock, when compared to wt, tg mice displayed a 40% reduction of the infarcted area, which lasted up to 1 week. However, infarcted brain areas were similar in wt and tg mice within the first hours post-occlusion, indicating that NGF acted to block the progression of neuronal damage. Kinetics of NGF synthesis assessed by ELISA was in good agreement with the observed neuroprotective effect, since NGF content peaked 6 h post-ischemia. This was further correlated with the time-course of c-Fos immunoreactivity, detectable only from 6 h post-ischemia. The neuroprotective effect of NGF involved the impairment of apoptotic cell death, as evidenced by a marked decrease of the number of apoptotic profiles inside the ischemic zone in tg mice. These results underline the potential of c-fos-NGF-tg mice to study in vivo the molecular and cellular mechanisms of the NGF-induced neuroprotective effect against ischemic damage.