Janine J. Leffert

Homeostatic control of uridine and the role of uridine phosphorylase: a biological and clinical update

Uridine, a pyrimidine nucleoside essential for the synthesis of RNA and bio-membranes, is a crucial element in the regulation of normal physiological processes as well as pathological states. The biological effects of uridine have been associated with the regulation of the cardio-circulatory system, at the reproduction level, with both peripheral and central nervous system modulation and with the functionality of the respiratory system. Furthermore, uridine plays a role at the clinical level in modulating the cytotoxic effects of fluoropyrimidines in both normal and neoplastic tissues. The concentration of uridine in plasma and tissues is tightly regulated by cellular transport mechanisms and by the activity of uridine phosphorylase (UPase), responsible for the reversible phosphorolysis of uridine to uracil. We have recently completed several studies designed to define the mechanisms regulating UPase expression and better characterize the multiple biological effects of uridine. Immunohistochemical analysis and co-purification studies have revealed the association of UPase with the cytoskeleton and the cellular membrane. The characterization of the promoter region of UPase has indicated a direct regulation of its expression by the tumor suppressor gene p53. The evaluation of human surgical specimens has shown elevated UPase activity in tumor tissue compared to paired normal tissue.

Increased ratio of anti-apoptotic to pro-apoptotic Bcl2 gene-family members in lithium-responders one month after treatment initiation

BackgroundLithium is considered by many as the gold standard medication in the management of bipolar disorder (BD). However, the clinical response to lithium is heterogeneous, and the molecular basis for this difference in response is unknown. In the present study, we sought to determine how the peripheral blood gene expression profiles of patients with bipolar disorder (BD) changed over time following intitiation of treatment with lithium, and whether differences in those profiles over time were related to the clinical response.MethodsIllumina Sentrix Beadchip (Human-6v2) microarrays containing > 48,000 transcript probes were used to measure levels of expression of gene-expression in peripheral blood from 20 depressed subjects with BD prior to and every two weeks during 8 weeks of open-label treatment with lithium.Changes in gene-expression were compared between treatment responders (defined as a decrease in the Hamilton Depression Rating Scale of 50% or more) and non-responders. Pathway analysis was conducted using GeneGO Metacore software.Results127 genes showed a differential response in responders vs. non-responders. Pathway analysis showed that regulation of apoptosis was the most significantly affected pathway among these genes. Closer examination of the time-course of changes among BCL2 related genes showed that in lithium-responders, one month after starting treatment with lithium, several anti-apoptotic genes including Bcl2 and insulin receptor substrate 2 (IRS2) were up-regulated, while pro-apoptotic genes, including BCL2-antagonist/killer 1 (BAK1) and BCL2-associated agonist of cell death (BAD), were down-regulated. In contrast, in lithium non-responders, BCL2 and IRS2 were down-regulated, while BAK1 and BAD up-regulated at the one-month time-point.ConclusionsThese results suggest that differential changes in the balance of pro- and anti- apoptotic gene-expression following treatment with lithium may explain some of the heterogeneity in clinical response in BD patients.

Increased peripheral blood expression of electron transport chain genes in bipolar depression

OBJECTIVE   To identify specific genetic pathways showing altered expression in peripheral blood of depressed subjects with bipolar disorder (BPD). METHODS   Illumina Sentrix BeadChip (Human-6v2) microarrays containing >48,000 transcript probes were used to measure levels of gene expression in peripheral blood from 20 depressed subjects with BPD and in 15 healthy control subjects. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to confirm a subset of these differences. RESULTS   A total of 1,180 genes were differentially expressed between subjects with BPD and healthy controls (fold-change >1.3, false discovery rate-corrected p < 0.05, covaried for age and sex). Of these, 559 genes were up-regulated in BPD subjects and 621 were down-regulated. Surprisingly, there was no difference between medicated (n = 11) and unmedicated (n = 9) subjects with BPD for any of these genes. Pathway analysis using GeneGo MetaCore software showed that the most significantly affected pathway was the mitochondrial electron transport chain (ETC). Of the 85 objects (genes or proteins) in this pathway, 22 were up-regulated and 2 down-regulated in subjects with BPD. qRT-PCR confirmed up-regulation of nuclear encoded ETC genes in complexes I, III, IV, and V and, in addition, demonstrated up-regulation of mitochondrially encoded genes in each of these complexes. CONCLUSION   These results suggest that increased expression of multiple components of the mitochondrial ETC may be a primary deficit in bipolar depression, rather than an effect of medication.

Dose escalation and pharmacokinetic study of irinotecan in combination with paclitaxel in patients with advanced cancer

Purpose: Based on preclinical data demonstrating synergy between camptothecin analogues and taxanes, we determined the maximum tolerated dose (MTD) of irinotecan that could be given in combination with a fixed dose of paclitaxel of 75 mg/m2, when both drugs were delivered on a weekly schedule. The pharmacokinetics of this combination were explored to determine whether the sequence of administration affected the elimination of irinotecan. Methods: For the first cycle patients with advanced cancer were treated with irinotecan given as a 90-min infusion followed immediately by paclitaxel given at a dose of 75 mg/m2 over 1 h. The sequence of drug administration was reversed in subsequent cycles for most patients. Chemotherapy was given weekly for 4 weeks, followed by a 2-week rest. In selected patients, plasma concentrations of irinotecan were determined by high-performance liquid chromatography during the first 24 h of cycle 1 and after the first dose of cycle 2 to determine whether the order of drug administration affected the elimination of irinotecan, or the toxicologic effects of the chemotherapy. Results: A total of 53 cycles were delivered to 21 patients. Reversible neutropenia was dose-limiting. Suppression of the other blood cell elements was modest. There was one partial response in a man with a previously treated cholangiocarcinoma that lasted 26 weeks. Prolonged stabilization of disease (6 months or more) was observed in five of the patients (24%). At the recommended dose of irinotecan (50 mg/m2), transfusions of red cells and platelets were not required. The sequence of drug administration produced no significant differences in the pharmacokinetic parameters of irinotecan or SN-38, which were similar to the values reported when irinotecan is administered alone. The most prominent nonhematologic toxicities were mild diarrhea and fatigue. Conclusions: The recommended dose of irinotecan on this schedule is 50 mg/m2. The sequence of drug administration affects neither the elimination of irinotecan nor the chemotherapy-related toxicity. This combination is well tolerated and causes minimal clinical side effects.

Altered Expression of Cytokine Signaling Pathway Genes in Peripheral Blood Cells of Alcohol Dependent Subjects: Preliminary Findings

BACKGROUND Preclinical and clinical studies have implicated changes in cytokine and innate immune gene-expression in both the development of and end-organ damage resulting from alcohol dependence. However, these changes have not been systematically assessed on the basis of alcohol consumption in human subjects. METHODS Illumina Sentrix Beadchip (Human-6v2) microarrays were used to measure levels of gene-expression in peripheral blood in 3 groups of subjects: those with alcohol dependence (AD, n = 12), heavy drinkers (HD; defined as regular alcohol use over the past year of at least 8 standard drinks/wk for women and at least 15 standard drinks/wk for men, n = 13), and moderate drinkers (MD; defined as up to 7 standard drinks/wk for women and 14 standard drinks/wk for men, n = 17). RESULTS Four hundred and thirty-six genes were differentially expressed among the 3 groups of subjects (false discovery rate corrected p-value < 0.05). Two hundred and ninety-one genes differed between AD and MD subjects, 240 differed between AD and HD subjects, but only 6 differed between HD and MD subjects. Pathway analysis using DAVID and GeneGO Metacore(®) software showed that the most affected pathways were those related to T-cell receptor and Janus kinase-Signal transducer and activator of transcription (JAK-Stat) signaling. CONCLUSIONS These results suggest the transition from heavy alcohol use to dependence is accompanied by changes in the expression of genes involved in regulation of the innate immune response. Such changes may underlie some of the previously described changes in immune function associated with chronic alcohol abuse. Early detection of these changes may allow individuals at high risk for dependence to be identified.

Age-related differences in vincristine toxicity and biodistribution in wild-type and transporter-deficient mice.

The impact of mouse multidrug resistance genes mdrla/b and mrpl on age-related differences in the toxicity and biodistribution of vincristine (VCR) was evaluated in wild-type, mrpl(-/-), mdrla/b(-/-), and combined mdrla/b(-/-), mrpl(-/-) weanling and adult mice given a single IP dose of VCR ranging from 0.0625 to 6 mg/kg. Weanling mice of all four genotypes were more sensitive than adult animals as determined by survival rate, average time of death, and pathologic findings. Wild-type animals were the least sensitive and combined mdrla/b(-/-), mrpl(-/-) mice the most sensitive to VCR toxicity. Mdrla/b(-/-) and mrpl(-/-) genotypes exhibited intermediate sensitivities, with mdrla/b(-/-) mice being more sensitive than mrpl(-/-) animals to the vinca alkaloid. Administration of [3H]VCR to wild-type and mdrla/b(-/-), mrpl(-/-) animals revealed relatively greater accumulation of radioactive VCR equivalents in weanlings over adults in several tissues, with weanling mdrla/b(-/-), mrpl(-/-) lung and heart exhibiting the greatest enhanced accumulation of 26- and 15-fold over adults, respectively. A similar cardiopulmonary differential accumulation of VCR was not observed in wild-type weanlings to adults. Semiquantitative RT-PCR expression analyses of ABC transporter genes in weanling and adult tissues of wild-type and combined mdrla/b(-/-), mrpl(-/-) mice did not reveal major age-related differences in these ABC transporters that would explain the relatively greater toxicity observed in weanling mice. However, the greater cardiopulmonary accumulation of VCR equivalents seen in the combined mdrla/b(-/-), mrpl(-/-) weanlings over that of adults underscores the potential for unique organ and age-related toxicities of this agent in the setting of transporter deficiency.

Altered response to antidepressant treatment in FoxG1 heterozygous knockout mice

Evidence from a variety of sources suggests that structural alterations in the brain, including neurogenesis, may play a role in both the pathogenesis of mood disorders and the mechanism of action of antidepressants. Previous studies have implicated both the transforming growth factor‐beta (TGF‐β), and the phosphatidyl inositol‐3 kinase (PI3K)‐Akt pathways in the neurogenesis‐promoting and behavioral properties of antidepressants. Forkhead box protein G1 (FoxG1) is a major regulator of both of these pathways, and FoxG1 heterozygous null mice (FoxG1+/−) have previously been reported to have deficits in adult hippocampal neurogenesis and behavioral abnormalities including deficits in contextual fear learning. However the role of FoxG1, if any, in the response to antidepressants has not been previously investigated.

Altered transport of folic acid and antifolates through the carrier mediated reduced folate transport system in a human leukemia cell line resistant to (6R) 5,10-dideazatetrahydrofolic acid (DDATHF).

5,10-Dideazatetrahydrofolic acid is a potent antiproliferative agent in vitro and in vivo, arresting or delaying the proliferation of tumor cells in a number of murine and human xenografts models 1,2. In contrast to classical antifolates, (dihydrofolate reductase inhibitors), DDATHF blocks de novo purine biosynthesis as the result of a potent inhibition of both glycinamide ribonucleotide (GAR) and aminoimidazole carboxamide ribonucleotide (AICAR) transformylases.