Prior studies demonstrated that ceramide promotes apoptotic cell death in the human myeloid leukemia cell lines HL-60 and U937 (Jarvis, W. D., Kolesnick, R. N., Fornari, F. A., Jr., Traylor, R. S., ...Gewirtz, D. A., and Grant, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 73-77), and that this lethal process is potently suppressed by diglyceride (Jarvis, W. D., Fornari, F. A., Jr., Browning, J. L., Gewirtz, D. A., Kolesnick, R. N., and Grant, S. (1994) J. Biol. Chem. 269, 31685-31692). The present findings document the intrinsic ability of sphingoid bases to induce apoptosis in HL-60 and U937 cells. Exposure to either sphingosine or sphinganine (0. 001 10 microM) for 6 h promoted apoptotic degradation of genomic DNA as indicated by (a) electrophoretic resolution of 50-kilobase pair DNA loop fragments and 0.2-1.2-kilobase pair DNA fragment ladders on agarose gels, and (b) spectrofluorophotometric determination of the formation and release of double-stranded fragments and corresponding loss of integrity of bulk DNA. DNA damage correlated directly with reduced cloning efficiency and was associated with the appearance of apoptotic cytoarchitectural traits. At sublethal concentrations (</=750 nM), however, sphingoid bases synergistically augmented the apoptotic capacity of ceramide (10 microM), producing both a leftward shift in the ceramide concentration-response profile and a pronounced increase in the response to maximally effective levels of ceramide. Thus, sphingosine and sphinganine increased both the potency and efficacy of ceramide. The apoptotic capacity of bacterial sphingomyelinase (50 milliunits/ml) was similarly enhanced by either (a) acute co-exposure to highly selective pharmacological inhibitors of protein kinase C such as calphostin C and chelerythrine or (b) chronic pre-exposure to the non-tumor-promoting protein kinase C activator bryostatin 1, which completely down-modulated total assayable protein kinase C activity. These findings demonstrate that inhibition of protein kinase C by physiological or pharmacological agents potentiates the lethal actions of ceramide in human leukemia cells, providing further support for the emerging concept of a cytoprotective function of the protein kinase C isoenzyme family in the regulation of leukemic cell survival.
We characterized participation of the stress-activated protein kinase (SAPK) cascade in the lethal actions of the cytotoxic lipid messengers ceramide and sphingosine in U937 human monoblastic ...leukemia cells. Acute exposure of U937 cells to either lipid resulted in loss of proliferative capacity, degradation of genomic DNA, and manifestation of apoptotic cytoarchitecture. Ceramide robustly stimulated p46-JNK1/p54-JNK2 activity and increased expression of c-jun mRNA and c-Jun protein; in contrast, sphingosine moderately stimulated p46-JNK1/p54-JNK2 and failed to modify c-jun/c-Jun expression. Dominant-negative blockade of normal c-Jun activity by transfection with the TAM-67 c-Jun NH2-terminal deletion mutant abolished the lethal actions of ceramide but was without effect on those of sphingosine, indicating that ceramide-related apoptosis is directly dependent on activation of c-Jun, whereas sphingosine-induced cell death proceeds via an unrelated downstream mechanism. Characterization of the mitogen-activated protein kinase (MAPK) cascade in these responses revealed a further functional disparity between the two lipids: basal p42-ERK1/ p44-ERK2 activity was gradually reduced by ceramide but immediately and completely suppressed by sphingosine. Moreover, blockade of the MAPK cascade by the aminomethoxyflavone MEK1 inhibitor PD-98059 unexpectedly activated p46-JNK1/p54-JNK2 and induced apoptosis in a manner qualitatively resembling that of sphingosine. Both lipids sharply increased p38-RK activity; selective pharmacological inhibition of p38-RK by the pyridinyl imidazole SB-203580 failed to mitigate the cytotoxicity associated with either ceramide or sphingosine, suggesting that p38-RK is not essential for lipid-induced apoptosis. These findings demonstrate that reciprocal alterations in the SAPK and MAPK cascades are associated with the apoptotic influence of either lipid inasmuch as (i) ceramide-mediated lethality is primarily associated with strong stimulation of SAPK and weak inhibition of MAPK, whereas (ii) sphingosine-mediated lethality is primarily associated with weak stimulation of SAPK and strong inhibition of MAPK. We therefore propose that leukemic cell survival depends on the maintenance of an imbalance of the outputs from the MAPK and SAPK systems such that the dominant basal influence of the MAPK cascade allows sustained proliferation, whereas acute redirection of this balance toward the SAPK cascade initiates apoptotic cell death.
The potential involvement of ceramide-related signaling processes in the induction of apoptosis by tumor necrosis factor α was assessed by multiple biochemical strategies in the human leukemic cell ...lines HL-60 and U937 and the murine fibrosarcoma cell lines L929/LM and WEHI 164/13. Exposure of these cells to tumor necrosis factor α resulted in internucleosomal cleavage of genomic DNA, yielding laddered patterns of oligonucleosomal fragments characteristic of apoptosis when resolved by agarose gel electrophoresis; similar responses were observed after exposure to exogenous sphingomyelinase or synthetic ceramides. Quantitative spectrofluorophotometry demonstrated that these treatments promoted time- and concentration-dependent degradation of DNA, resulting in the formation of and eventual release of small DNA fragments (≤3.0 kb). Corresponding damage to bulk DNA was demonstrated by enhanced-fluorescence alkaline unwinding analysis. DNA fragmentation was not induced by phospholipase C or synthetic diglyceride; in fact, the effects of sphingomyelinase and ceramide were substantially reduced by coexposure to these agents, suggesting opposing roles for diglyceride- and ceramide-mediated signals in the regulation of apoptosis. Phospholipase A2and arachidonic acid failed to promote DNA fragmentation, as did phospholipase D. Characterization of DNA strand breaks by alkaline and neutral elution analyses confirmed that ceramide action was restricted to breakage of mature, double-stranded DNA but not of nascent DNA. The induction of DNA damage was associated with appearance of apoptotic morphology and decreased clonogenicity. These results demonstrate that the ceramide-dependent signaling system selectively induces apoptosis and raise the possibility that ceramide-activated enzymes represent important components in a signaling cascade involved in the regulation of programmed cell death.
The capacity of doxorubicin to inhibit topoisomerase II in the MCF-7 breast tumor cell line is supported by the induction of protein-associated single-strand breaks in DNA, as well as by interference ...with the decatenation activity of nuclear extracts. Doxorubicin also produces non-protein-associated DNA strand breaks (at a supraclinical concentration of 5 microM), which may indicate damage mediated via the generation of free radicals. However, no strand breaks are detected in DNA of MCF-7 cells at the IC50 for doxorubicin (approximately 0.1 microM). At doxorubicin concentrations of 0.05, 0.1, and 0.5 microM, at which growth is inhibited by approximately 15, 50, and 75%, respectively, doxorubicin interferes with radiation-induced unwinding of DNA; doxorubicin also produces a concentration-dependent inhibition of DNA synthesis that corresponds closely to growth inhibition. These studies suggest that DNA strand breaks fail to fully account for the antiproliferative activity of doxorubicin in the MCF-7 breast tumor cell line. Compromised DNA synthesis associated with interference with DNA unwinding may contribute to growth inhibition in MCF-7 cells exposed to doxorubicin.
We recently demonstrated that physiological induction of apoptosis by cytotoxic sphingolipid messengers proceeds via activating protein-1 (AP1)-dependent and AP1-independent mechanisms in U937 human ...monoblastic leukemia cells. Here we examine involvement of the stress-activated protein kinase (SAPK) cascade and AP1 in the initiation of apoptosis in U937 cells by podophyllotoxin-derived inhibitors of topoisomerase II. Induction of apoptotic cell death and DNA damage by treatment of U937 cells with etoposide (100 microM) was associated with phosphorylation and activation of the c-Jun NH(2)-terminal kinase (JNK1) SAPK enzymes p46 and p54-JNK2 and transient increases in expression of the transcription factor c-Jun, a primary JNK substrate. These responses were accompanied by a modest, but sustained, recruitment of the mitogen-activated protein kinases p42-extracellular signal receptor-activated kinase (ERK)1 and p44-extracellular signal receptor-activated kinase 2. The capacity of etoposide to promote double-stranded DNA degradation and cell death was unaffected by manipulations that interfere with SAPK signaling outflow through c-Jun/AP1, including: 1) pharmacological inhibition of AP1 activity by diferuloylmethane and 2) molecular ablation of normal c-Jun function by the Jun dominant-negative mutant TAM-67. Cytotoxicity of the structurally related compound teniposide was similarly unaffected. In parallel trials, the lethal actions of ceramide (but not of sphingosine) were markedly diminished by pretreatment with diferuloylmethane or expression of TAM-67, confirming the effectiveness of these interventions in suppression of SAPK/AP1-dependent apoptosis. The involvement of AP1 in the proapoptotic actions of other inhibitors of topoisomerase II activity was also evaluated. Induction of cell death by the anthracyclines daunorubicin, daunorubicin, and idarubicin was found to be insensitive to pretreatment with diferuloylmethane or expression of TAM-67. Collectively, the present data indicate that induction of apoptosis by etoposide and related inhibitors of topoisomerase II is mediated through a cell death pathway that does not require SAPK-dependent recruitment of AP1. These findings additionally suggest that activation of the SAPK represents a consequence, rather than an underlying cause, of etoposide-induced apoptosis in myeloid leukemia cells.
Abnormal behaviors involving dopaminergic gene polymorphisms often reflect an insufficiency of usual feelings of satisfaction, or Reward Deficiency Syndrome (RDS). RDS results from a dysfunction in ...the "brain reward cascade," a complex interaction among neurotransmitters (primarily dopaminergic and opioidergic). Individuals with a family history of alcoholism or other addictions may be born with a deficiency in the ability to produce or use these neurotransmitters. Exposure to prolonged periods of stress and alcohol or other substances also can lead to a corruption of the brain reward cascade function. We evaluated the potential association of four variants of dopaminergic candidate genes in RDS (dopamine D1 receptor gene DRD1; dopamine D2 receptor gene DRD2; dopamine transporter gene DAT1; dopamine beta-hydroxylase gene DBH).
We genotyped an experimental group of 55 subjects derived from up to five generations of two independent multiple-affected families compared to rigorously screened control subjects (e.g., N = 30 super controls for DRD2 gene polymorphisms). Data related to RDS behaviors were collected on these subjects plus 13 deceased family members.
Among the genotyped family members, the DRD2 Taq1 and the DAT1 10/10 alleles were significantly (at least p < 0.015) more often found in the RDS families vs. controls. The TaqA1 allele occurred in 100% of Family A individuals (N = 32) and 47.8% of Family B subjects (11 of 23). No significant differences were found between the experimental and control positive rates for the other variants.
Although our sample size was limited, and linkage analysis is necessary, the results support the putative role of dopaminergic polymorphisms in RDS behaviors. This study shows the importance of a nonspecific RDS phenotype and informs an understanding of how evaluating single subset behaviors of RDS may lead to spurious results. Utilization of a nonspecific "reward" phenotype may be a paradigm shift in future association and linkage studies involving dopaminergic polymorphisms and other neurotransmitter gene candidates.
Opiate addiction is associated with many adverse health and social harms, fatal overdose, infectious disease transmission, elevated health care costs, public disorder, and crime. Although ...community-based addiction treatment programs continue to reduce the harms of opiate addiction with narcotic substitution therapy such as methadone maintenance, there remains a need to find a substance that not only blocks opiate-type receptors (mu, delta, etc.) but also provides agonistic activity; hence, the impetus arose for the development of a combination of narcotic antagonism and mu receptor agonist therapy. After three decades of extensive research, the federal Drug Abuse Treatment Act 2000 (DATA) opened a window of opportunity for patients with addiction disorders by providing increased access to options for treatment. DATA allows physicians who complete a brief specialty-training course to become certified to prescribe buprenorphine and buprenorphine/naloxone (Subutex, Suboxone) for treatment of patients with opioid dependence. Clinical studies indicate that buprenorphine maintenance is as effective as methadone maintenance in retaining patients in substance abuse treatment and in reducing illicit opioid use. With that stated, we must consider the long-term benefits or potential toxicity attributed to Subutex or Suboxone. We describe a mechanism whereby chronic blockade of opiate receptors, in spite of only partial opiate agonist action, may ultimately block dopaminergic activity causing anti-reward and relapse potential. While the direct comparison is not as yet available, toxicity to buprenorphine can be found in the scientific literature. In considering our cautionary note in this commentary, we are cognizant that, to date, this is what we have available, and until such a time when the real magic bullet is discovered, we will have to endure. However, more than anything else this commentary should at least encourage the development of thoughtful new strategies to target the specific brain regions responsible for relapse prevention.
Executive functions are processes that act in harmony to control behaviors necessary for maintaining focus and achieving outcomes. Executive dysfunction in neuropsychiatric disorders is attributed to ...structural or functional pathology of brain networks involving prefrontal cortex (PFC) and its connections with other brain regions. The PFC receives innervations from different neurons associated with a number of neurotransmitters, especially dopamine (DA). Here we review findings on the contribution of PFC DA to higher-order cognitive and emotional behaviors. We suggest that examination of multifactorial interactions of an individual’s genetic history, along with environmental risk factors, can assist in the characterization of executive functioning for that individual. Based upon the results of genetic studies, we also propose genetic mapping as a probable diagnostic tool serving as a therapeutic adjunct for augmenting executive functioning capabilities. We conclude that preservation of the neurological underpinnings of executive functions requires the integrity of complex neural systems including the influence of specific genes and associated polymorphisms to provide adequate neurotransmission.
Background: It is well established that in both food- and drug-addicted individuals there is "dopamine resistance" associated with the DRD2 gene A1 allele. Based on earlier studies, evidence is ...emerging wherein the potential of utilizing a natural, nonaddicting, safe, putative D2 agonist may play a significant role in the recovery of individuals with reward deficiency syndrome, including those addicted to psychoactive chemicals. Findings: Positive outcomes demonstrated by quantitative electroencephalographic (qEEG) imaging in a randomized, triple-blind, placebo-controlled, crossover study involving oral Synaptose Complex KB220Z™ showed an increase of alpha waves and low beta wave activity in the parietal brain region. Using t statistics, significant differences observed between placebo and Synaptose Complex KB220Z™ consistently occurred in the frontal regions after week 1 and then again after week 2 of analyses (P = 0.03). This is the first report to demonstrate involvement of the prefrontal cortex in the qEEG response to a natural putative D2 agonist (Synaptose Complex KB220Z™), especially evident in dopamine D2 A1 allele subjects. Independently, we have further supported this finding with an additional study of 3 serious polydrug abusers undergoing protracted abstinence who carried the DRD2 A1 allele. Significant qEEG differences were found between those who received 1 dose of placebo compared with those who were administered Synaptose Complex KB220Z™. Synaptose Complex KB220Z™ induced positive regulation of the dysregulated electrical activity of the brain in these addicts. The results are indicative of a phase change from low amplitude or low power in the brain to a more regulated state by increasing an average of 6.169 mV
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across the prefrontal cortical region. In the first experiment we found that while 50% of the subjects carried the DRD2 A1 allele, 100% carried ≥ 1 risk allele. Specifically, based on the proposed addiction risk score for these 14 subjects, 72% had moderate-to-severe addiction risk. Similar findings were obtained by repeating the experiment in 3 additional currently abstinent polydrug abusers carrying the DRD2 A1 allele. Conclusion: This seminal work will provide important information that may ultimately lead to significant improvement in the recovery of individuals with psychostimulant and polydrug abuse problems, specifically those with genetically induced dopamine deficiency. Based on this small sample size, we are proposing that with necessary large populations supporting these initial results, and possibly even additional candidate genes and single nucleotide polymorphisms, we may eventually have the clinical ability to classify severity according to genotype and possession of risk alleles, along with offering a safe, nonaddicting, natural dopaminergic receptor agonist that potentially upregulates instead of downregulates dopaminergic receptors, preferably the D2 subtype.
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