A Prospective Study on the Usefulness of Initial Voriconazole Dose Adjustment Based on CYP2C19 Gene Polymorphism Analysis.

Genetic polymorphism exists for CYP2C19, a dominant metabolic enzyme of voriconazole (VRCZ), and VRCZ pharmacokinetics has been shown to fluctuate according to the CYP2C19 phenotype. Although dosages for different phenotypes have been recommended in various retrospective studies, few reports have adjusted the initial VRCZ dose based on CYP2C19 phenotype determined prior to administration. In this study, we prospectively evaluated the usefulness of CYP2C19 polymorphism analysis in adjusting the initial VRCZ maintenance dose. The study enrolled 19 patients who underwent analysis of CYP2C19 polymorphism prior to VRCZ administration. Subjects were classified into 3 phenotype subgroups: extensive metabolizer (EM), intermediate metabolizer (IM), and poor metabolizer (PM). The initial VRCZ maintenance doses given twice daily were proposed as follows: approximately 8, 6, and 4 mg/kg/day for EM, IM and PM, respectively, according to previous reports. In EM, the initial maintenance dose was 8.0 ± 0.5 mg/kg/day, and trough level was 6.6 ± 2.4 µg/mL. By contrast, the initial maintenance doses in IM and PM were 5.5 ± 0.7 and 4.1 ± 0.3 mg/kg/day, and the initial trough concentrations were 2.9 ± 1.2 and 2.6 ± 0.4 µg/mL, respectively. The attainment rate of target trough concentration of 1-6 µg/mL was 50% in EM, and was 100% in IM and PM. Determining the initial dose of VRCZ only by phenotype based on CYP2C19 gene polymorphism was found to be challenging. However, decreasing the initial maintenance dose in IM and PM may be important for adjusting the initial trough level to target range.

Comparison of whole-blood tacrolimus concentrations measured by different immunoassay systems.

INTRODUCTION: Different measured values for tacrolimus were obtained with different automated immunoassays. We aimed to examine the differences in the blood tacrolimus concentrations measured by the major immunoassay systems commercially available in Japan. METHODS: Whole-blood samples from 118 patients were assayed by 3 commercial assays: chemiluminescent enzyme immunoassay (CLIA), affinity column-mediated immunoassay (ACMIA), and enzyme-multiplied immunoassay technique (EMIT). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for reference. KEY FINDINGS: The correlation coefficient of immunoassay vs LC-MS/MS was excellent for ACMIA (.83) and CLIA (.81) and good for EMIT (.71). The mean error was negative for ACMIA and positive for CLIA and EMIT. The mean absolute error and root-mean-square error were almost the same for ACMIA and CLIA and lower than those for EMIT. CONCLUSIONS: The ACMIA and CLIA yield considerably better results than the EMIT for monitoring blood tacrolimus concentrations.

Clinical Implications of Plasma N-acetyl-seryl-aspartyl-lysyl-proline Level in Stable Kidney Transplant Recipients.

BACKGROUND: N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is partially eliminated in urine, accumulation of AcSDKP due to chronic renal failure may cause anemia. However, the status of plasma AcSDKP level in stable kidney transplant recipients is unknown although some recipients develop anemia after kidney transplantation. In this study, we investigated the relationship between plasma AcSDKP-like immunoreactive substance (IS) level and clinical characteristics associated with renal anemia in stable kidney transplant recipients. METHODS: Forty Japanese kidney transplant recipients who underwent transplantation more than 90 days prior to the study were included. Morning blood samples were collected and plasma AcSDKP-IS levels were measured using an enzyme immunoassay. RESULTS: A significant correlation was observed between plasma AcSDKP-IS level and creatinine clearance. On the other hand, no significant correlation was observed between plasma AcSDKP-IS level and prolyl oligopeptidase activity, angiotensin II, or erythropoietin level. A significant difference in plasma AcSDKP-IS level was observed between recipients with no renal anemia and those with renal anemia. CONCLUSIONS: These results suggest that plasma AcSDKP level may depend largely on renal function and suggest a possibility that accumulation of AcSDKP may be partially involved in the pathogenesis of renal anemia in stable kidney transplant recipients.

CYP3A5 polymorphism affects the increase in CYP3A activity after living kidney transplantation in patients with end stage renal disease.

AIMS: It has been reported that cytochrome P450 (CYP)3A activity increases significantly in patients with end stage renal disease (ESRD) after kidney transplantation, with wide interindividual variability in the degree of increase. The aim of this study was to evaluate the influence of CYP3A5 polymorphism on the increase in CYP3A activity after living kidney transplantation, by measuring the plasma concentration of 4ß-hydroxycholesterol. METHODS: This prospective study recruited 22 patients with ESRD who underwent a first living kidney allograft transplantation, comprising 12 patients with CYP3A5*1 allele (CYP3A5*1/*1 or *1/*3) and 10 patients without CYP3A5*1 allele (CYP3A5*3/*3). RESULTS: No significant difference in estimated glomerular filtration rate over time was observed between patients with the CYP3A5*1 allele and patients without the CYP3A5*1 allele, suggesting that the degrees of recovery in renal function after living kidney transplantation were similar in the two groups. However, plasma concentrations of 4ß-hydroxycholesterol on days 90 (57.1 ± 13.4 vs. 39.5 ± 10.8 ng ml(-1)) and 180 (55.0 ± 14.5 vs. 42.4 ± 12.6 ng ml(-1)) after living kidney transplantation were significantly higher in the presence of the CYP3A5*1 allele than in the absence of the CYP3A5*1 allele [P = 0.0034 (95% confidence interval of difference 6.55, 28.6) and P = 0.043 (95% confidence interval of difference 0.47, 24.8), respectively], suggesting that CYP3A activity may increase markedly associated with recovery of renal function in patients with the CYP3A5*1 allele. CONCLUSIONS: These findings suggest that the presence of the CYP3A5*1 allele contributes to marked elevation of CYP3A activity associated with recovery of renal function after kidney transplantation.

Association of plasma concentration of 4ß-hydroxycholesterol with CYP3A5 polymorphism and plasma concentration of indoxyl sulfate in stable kidney transplant recipients.

Several studies have shown that renal failure decreases CYP3A activity and that uremic toxins may play a role via transcriptional or translational modifications of cytochrome P450 (P450) enzymes and direct inhibition of P450-mediated metabolism. In this study, we evaluated the relationship between CYP3A activity (using plasma concentration of 4ß-hydroxycholesterol as a biomarker) and clinical characteristics including plasma concentrations of indoxyl sulfate (3-INDS) and indole-3-acetic acid (3-IAA) in stable kidney transplant recipients. Forty-five Japanese kidney transplant recipients who underwent transplantation more than 90 days prior to the study were included. Morning blood samples were collected and plasma concentrations of 4ß-hydroxycholesterol, 3-INDS, and 3-IAA were measured. Plasma concentrations of 4ß-hydroxycholesterol were 57.1 ± 11.2, 42.1 ± 11.8, and 34.5 ± 7.3 ng/ml in recipients with CYP3A5*1/*1 (n = 5), *1/*3 (n = 15), and *3/*3 (n = 25) genotypes, respectively, with significant differences between three genotypes. A significant correlation was observed between plasma concentrations of 4ß-hydroxycholesterol and 3-INDS but not 3-IAA. Multiple regression analysis identified the number of CYP3A5*3 alleles in genotype, plasma concentration of 3-INDS, and body weight as independent variables associated with plasma concentration of 4ß-hydroxycholesterol. In conclusion, these results suggest that CYP3A5 polymorphism and plasma concentration of 3-INDS may account for the interindividual variability of CYP3A activity, and that plasma concentration of 3-INDS may partially explain the gap in CYP3A activity that cannot be explained by genetic contribution in patients with renal failure.

Significant decrease in plasma N-acetyl-seryl-aspartyl-lysyl-proline level in patients with end stage renal disease after kidney transplantation.

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous peptide released from its precursor (thymosin-ß4) by prolyl oligopeptidase. AcSDKP is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. AcSDKP has been shown to be a potent angiogenic factor and to suppress renal fibroblast proliferation. Impairment of renal function has been suggested to have a significant impact on plasma AcSDKP level. The aim of this study was to assess whether improvement of renal function after kidney transplantation has an impact on plasma AcSDKP-like immunoreactive substance (IS) level. Fourteen patients with end stage renal disease (ESRD) who were scheduled to undergo the first kidney allograft transplantation were enrolled. Plasma AcSDKP-IS levels were measured before and 3, 7, 10, 14, 21, 30, 60 and 90 d after kidney transplantation. Plasma AcSDKP-IS level decreased significantly from day 3 after kidney transplantation compared to before kidney transplantation. Creatinine clearance increased significantly from day 7 after kidney transplantation. A significant negative correlation was observed between creatinine clearance and plasma AcSDKP-IS level from before transplantation to 90 d after kidney transplantation. Stepwise multiple regression analysis identified creatinine clearance as the only significant independent factor associated with plasma AcSDKP-IS levels. These results suggest that recovery of kidney function after kidney transplantation may lead to a decrease in plasma AcSDKP level in patients with ESRD, and that plasma AcSDKP level may depend largely on renal function.

Significant increase in plasma 4ß-hydroxycholesterol concentration in patients after kidney transplantation.

Several previous studies have shown that renal failure decreases not only renal elimination but also metabolic clearance of drugs, particularly those metabolized by CYP3A. However, whether recovery of renal function results in recovery of hepatic CYP3A activity remains unknown. In this study, we evaluated the effect of renal function on CYP3A activity after kidney transplantation in patients with end-stage renal disease (ESRD) by measuring the change in CYP3A activity using plasma concentration of 4ß-hydroxycholesterol as a biomarker. The study enrolled 13 patients with ESRD who underwent the first kidney allograft transplantation. Morning blood samples were collected before and 3, 7, 10, 14, 21, 30, 60, 90, 120, 150 and 180 days after kidney transplantation. Plasma concentration of 4ß-hydroxycholesterol was measured using GC-MS. Compared with before kidney transplantation, creatinine clearance increased significantly from day 3 after kidney transplantation and stabilized thereafter. Plasma concentration of 4ß-hydroxycholesterol was elevated significantly on days 90 and 180 after kidney transplantation. In conclusion, this study suggests the recovery of CYP3A activity with improvement in renal function after kidney transplantation in patients with ESRD.

Association of CYP3A5 polymorphisms and parathyroid hormone with blood level of tacrolimus in patients with end-stage renal disease.

Because tacrolimus is predominantly metabolized by CYP3A, the blood concentration/dose (C/D) ratio is affected by CYP3A5 polymorphism. Parathyroid hormone (PTH) expression increases in secondary hyperparathyroidism, which is frequently associated with end-stage renal disease. Recently, PTH has been shown to downregulate CYP3A expression at mRNA level. In this study, we examined the influence of CYP3A5 polymorphism on and association of serum intact-PTH (iPTH) level with blood tacrolimus concentration in patients with end-stage renal disease just before kidney transplantation. Forty-eight patients who satisfied the selection criteria were analyzed. Subjects were classified into two phenotype subgroups: CYP3A5 expressor (CYP3A5*1/*1 and *1/*3; n = 15) and CYP3A5 nonexpressor (CYP3A5*3/*3; n = 33). The blood tacrolimus C/D (per body weight) ratio was significantly lower in CYP3A5 expressors than that in CYP3A5 nonexpressors. A significant positive correlation was found between tacrolimus C/D and iPTH concentrations (r = 0.305, p = 0.035), and the correlation coefficient was higher after excluding 20 patients co-administered CYP3A inhibitor or inducer (r = 0.428, p = 0.023). A multiple logistic regression analysis by stepwise selection identified CYP3A5 polymorphism and serum iPTH level as significant factors associated with tacrolimus C/D. These results may suggest the importance of dose design considering not only the CYP3A5 phenotype but also serum iPTH level when using tacrolimus in patients who undergo renal transplantation.

Hypoxia-response plasmid vector producing bcl-2 shRNA enhances the apoptotic cell death of mouse rectum carcinoma.

Hypoxia-induced gene expression frequently occurs in malignant solid tumors because they often have hypoxic areas in which circulation is compromised due to structurally disorganized blood vessels. Hypoxia-response elements (HREs) are responsible for activating gene transcription in response to hypoxia. In this study, we constructed a hypoxia-response plasmid vector producing short hairpin RNA (shRNA) against B-cell leukemia/lymphoma-2 (bcl-2), an anti-apoptotic factor. The hypoxia-response promoter was made by inserting tandem repeats of HREs upstream of cytomegalovirus (CMV) promoter (HRE-CMV). HRE-CMV shbcl-2 vector consisted of bcl-2 shRNA under the control of HRE-CMV promoter. In hypoxic mouse rectum carcinoma cells (colon-26), the production of bcl-2 shRNA driven by HRE-CMV promoter was approximately 2-fold greater than that driven by CMV promoter. A single intratumoral (i.t.) injection of 40 microg HRE-CMV shbcl-2 to colon-26 tumor-bearing mice caused apoptotic cell death, and repetitive treatment with HRE-CMV shbcl-2 (40 microg/mouse, i.t.) also significantly suppressed the growth of colon-26 tumor cells implanted in mice. Apoptotic and anti-tumor effects were not observed in tumor-bearing mice treated with CMV shbcl-2. These results reveal the ability of HRE-CMV shbcl-2 vector to suppress the expression of bcl-2 in hypoxic tumor cells and suggest the usefulness of our constructed hypoxia-response plasmid vector to treat malignant tumors. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10054FP].

Factors involved in phenoconversion of CYP3A using 4ß-hydroxycholesterol in stable kidney transplant recipients.

BACKGROUND: Phenoconversion is a phenomenon whereby some genotypic extensive metabolizers transiently exhibit drug metabolizing enzyme activity at similar level as that of poor metabolizers. Renal failure is known to decrease CYP3A activity in humans. Indoxyl sulfate, parathyroid hormone (PTH), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) have been reported to cause CYP3A downregulation in renal failure. We measured plasma concentrations of the above compounds in stable kidney transplant recipients, and evaluated their relations with phenoconversion of CYP3A evaluated by plasma concentration of 4ß-hydroxycholesterol, a biomarker of CYP3A activity. Phenoconversion was defined as a genotypic extensive/intermediate metabolizer exhibiting CYP3A activity below the cutoff value that discriminates extensive/intermediate from poor metabolizers. METHODS: Sixty-three Japanese kidney transplant recipients who underwent transplantation more than 180 days prior to the study were included. Morning blood samples were collected, and CYP3A5 polymorphism as well as plasma concentrations of 4ß-hydroxycholesterol, indoxyl sulfate, intact-PTH, IL-6 and TNF-α were determined. RESULTS: Significantly higher plasma 4ß-hydroxycholesterol concentration was observed in recipients with CYP3A5*1 allele (n = 23) compared to those without the allele (n = 40), and the cut-off value was 40.0 ng/mL. Ten recipients with CYP3A5*1 allele exhibited CYP3A activity below 40.0 ng/mL (phenoconversion). Only plasma indoxyl sulfate concentration was significantly higher in recipients with CYP3A phenoconversion compared to those without phenoconversion. CONCLUSIONS: These findings suggest that higher plasma indoxyl sulfate concentration may be involved in CYP3A phenoconversion. Dose adjustment of drugs metabolized by CYP3A may be needed in patients with CYP3A5*1 allele and high blood indoxyl sulfate.

Performance characteristics between TDx®FLx and TBA™-25FR for the therapeutic drug monitoring of methotrexate.

BACKGROUND: High-dose methotrexate (HDMTX) is used in the treatment of certain malignancies, including leptomeningeal metastases, systemic non-Hodgkin lymphoma, acute lymphoblastic leukemia, and osteosarcoma. High circulating levels of methotrexate can cause severe myelosuppression. The present study aimed to examine the differences in plasma MTX concentrations measured by two immunoassay systems currently available in the Japanese market, a TDX/FLX analyzer and a TBA-25FR analyzer. METHODS: A total of 69 plasma samples from 16 patients were assayed by a fluorescence polarization immunoassay technique using a TDx/FLx analyzer (Abbott Diagnostics, Chicago, Illinois, U.S.A.) and a homogeneous enzyme immunoassay technique using a TBA-25FR analyzer (Toshiba Medical Systems, Tokyo, Japan). RESULTS: Assay results were very consistent between the two systems, with good correlation 24 h after the start of treatment (TBA-25FR = 1.06・TDX/FLX, -1.31, r = 0.99), 48 h after the start of treatment (TBA-25FR = 1.00・TDX/FLX, +0.027, r > 0.99), and 72 h after the start of treatment (TBA-25FR = 1.09・TDX/FLX, +0.011, r > 0.99). CONCLUSIONS: The calibration curve spanned one order of magnitude with a linear working range from the lowest to the highest standard. The standard deviations show the excellent reproducibility of repeated measurements at each standard level for both immunoassay systems. However, when using the TBA-25FR, it is necessary to perform measurements in the low-concentration range with care.

Association of metabolic complications with plasma mid-regional pro-adrenomedullin level in stable kidney transplant recipients.

BACKGROUND: Recently, increased plasma mid-regional pro-adrenomedullin (MR-proADM) concentrations have been demonstrated in patients with chronic renal failure. In this study, we attempted to identify significant clinical factors associated with MR-proADM concentration in stable kidney transplant recipients. METHODS: Forty-seven Japanese kidney transplant recipients who underwent transplantation >180days prior to the study were analyzed. To facilitate comparability of anti-hypertensive regimens across recipients taking different drugs, we calculated the treatment intensity score of anti-hypertensive drugs in each recipient. Morning blood samples were collected and plasma MR-proADM concentrations were measured using an enzyme immunoassay. RESULTS: Multiple regression analysis identified treatment intensity score for anti-hypertensive drugs, serum albumin, creatinine clearance and use of lipid-lowering agents as significant independent factors associated with plasma MR-proADM concentration. Adjusted coefficient of determination for this model was 0.46. CONCLUSION: Apart from indicating lowered renal function, plasma MR-proADM concentration may be a useful biomarker for metabolic disorders, especially hypertension and hyperlipidemia, in stable kidney transplant patients.

Activation of cAMP signaling facilitates the morphological maturation of newborn neurons in adult hippocampus.

Previous studies have demonstrated that activation of the cAMP cascade, including the cAMP response element-binding protein (CREB), increases the proliferation and survival of newborn neurons in adult mouse hippocampus. In the present study, we determined whether the cAMP-CREB cascade also influences the morphological maturation of newborn neurons in the subgranular zone of the hippocampus. Rolipram, a selective inhibitor of the cAMP-specific phosphodiesterase type 4, was administered to activate the cAMP cascade, and neuronal morphology was determined by analysis of Golgi-impregnated neurons in the subgranular zone of hippocampus. Rolipram administration significantly increased the number of branch points and length of dendrites relative to vehicle treatment. Increased branch number and length were accompanied by increased levels of phosphorylated CREB, the active form of this transcription factor, in immature neurons. In contrast, the morphology of Golgi-impregnated neurons was not significantly influenced by rolipram treatment in inducible transgenic mice expressing a dominant-negative mutant of CREB in hippocampus. We also tested the influence of cAMP analogs in primary hippocampal cultures and found that activation of the cAMP pathway increased and inhibition of the cAMP cascade decreased the number of branches and length of processes as observed in vivo. These findings indicate that the cAMP-CREB cascade plays an important role in the differentiation and maturation of newborn neurons in hippocampus.

Localization of phosphorylated cAMP response element-binding protein in immature neurons of adult hippocampus.

Neurogenesis continues to occur in the adult hippocampus, although many of the newborn cells degenerate 1-2 weeks after birth. The number and survival of newborn cells are regulated by a variety of environmental stimuli, but very little is known about the intracellular signal transduction pathways that control adult neurogenesis. In the present study, we examine the expression of the phosphorylated cAMP response element-binding protein (pCREB) in immature neurons in adult hippocampus and the role of the cAMP cascade in the survival of new neurons. The results demonstrate that virtually all immature neurons, identified by triple immunohistochemistry for bromodeoxyuridine (BrdU) and polysialic acid-neural cell adhesion molecule (PSA-NCAM), are also positive for pCREB. In addition, upregulation of cAMP (via pharmacological inhibition of cAMP breakdown or by antidepressant treatment) increases the survival of BrdU-positive cells. A possible role for pCREB in the regulation of PSA-NCAM, a marker of immature neurons involved in neuronal remodeling and neurite outgrowth, is supported by cell culture studies demonstrating that the cAMP-CREB pathway regulates the expression of a rate-limiting enzyme responsible for the synthesis of PSA-NCAM. These findings indicate that the cAMP-CREB pathway regulates the survival, and possibly the differentiation and function, of newborn neurons.

Chronic olanzapine or fluoxetine administration increases cell proliferation in hippocampus and prefrontal cortex of adult rat.

BACKGROUND: There has been increasing evidence that atypical antipsychotics are effective in the treatment of mood disorders or for augmenting 5-hydroxytryptamine selective reuptake inhibitors for treatment-resistant depression. METHODS: Upregulation of neurogenesis in the adult hippocampus is a marker of antidepressant activity, and the present study investigated the influence of the atypical antipsychotic drug olanzapine on cell proliferation in the hippocampus of adult rat. The regulation of cell proliferation in the prelimbic cortex of adult rat was also examined. RESULTS: Chronic (21 days) olanzapine administration increased the number of newborn cells in the dentate gyrus of the hippocampus to the same extent as fluoxetine. Olanzapine or fluoxetine treatment also increased the number of proliferating cells in the prelimbic cortex. In contrast, there was no effect of either drug in the subventricular zone or primary motor cortex, and there was a trend for an increase in the striatum. Subchronic (7 days) administration of olanzapine had no effect on cell proliferation in hippocampus or prelimbic cortex, consistent with the time course for the effect of fluoxetine and the therapeutic actions of antidepressant treatment. The combination of olanzapine plus fluoxetine did not result in a greater induction of cell proliferation in either brain region. Analysis of the cell phenotype demonstrated that approximately 20% of the newborn cells in the prelimbic cortex differentiated into endothelial cells but not neurons, in contrast to the dentate gyrus, where most newborn cells differentiated into neurons. CONCLUSIONS: The results demonstrate that antidepressant or atypical antipsychotic medications can increase the proliferation of glia in limbic brain structures, an effect that could reverse the loss of glia that has been observed in depressed patients.

Cortical spreading depression affects Fos expression in the hypothalamic paraventricular nucleus and the cerebral cortex of both hemispheres.

The present experiments were performed to clarify the brain sites whose activity is affected exclusively by cortical spreading depression (CoSD). For this purpose, Fos protein, a product of an immediate early gene, was used as a marker of neuronal activation. Because Fos can be induced by many manipulations such as stress stimuli, we verified CoSD-induced Fos expression by excluding the influence of other factors such as anaesthesia and surgical manipulation. CoSD was induced by applying a KCl solution directly to the dura mater over the cerebral cortex, and Fos expression in the brain was assessed by immunohistochemistry using antibodies against Fos protein. We found that during CoSD, Fos expression was increased specifically in the magnocellular region of the hypothalamic paraventricular nucleus (PVN), as well as in the ipsilateral cortex, whereas reduced Fos expression was observed in both the parvocellular region of the PVN and the whole cortex contralateral to the CoSD site. Consistent with the reduced Fos expression, approximately 40% of neurons in the contralateral cortex revealed a suppression of electrical activity during CoSD. These results suggest that in addition to the ipsilateral cortex, CoSD affects Fos expression exclusively in the PVN and the contralateral cortex.

Paradoxical rise in brainstem PO(2) following umbilical cord occlusion in full-term rat fetuses.

Although the fetus experiences severe hypoxia and ischemia during delivery, the fetal brain is protected from hypoxic-ischemic insults by unknown mechanisms. To investigate this phenomenon, fetal asphyxia was induced in pregnant rats by occlusion of the umbilical cord. Rather than producing cerebral hypoxia, the brainstem PO(2) of at-term fetuses increased following umbilical cord occlusion, while brainstem blood flow was markedly reduced. This paradoxical increase in brainstem PO(2) during asphyxia occurred in the majority of at-term fetuses, less frequently in fetuses 1 day prior to term and did not occur in animals following birth. Because occlusion of the umbilical cord prevents maternal delivery of oxygen to the fetus, we propose that the ability to maintain PO(2) is the result of pre-existing fetal stores of oxygen or from de novo generation of oxygen in the fetal brainstem.

Circadian regulation of mTOR by the ubiquitin pathway in renal cell carcinoma.

Circadian clock systems regulate many biologic functions, including cell division and hormone secretion in mammals. In this study, we explored the effects of circadian control on the pivot cell growth regulatory mTOR, the activity of which is deregulated in tumor cells compared with normal cells. Specifically, we investigated whether the antitumor effect of an mTOR inhibitor could be improved by changing its dosing schedule in RenCa tumor-bearing mice. Active, phosphorylated mTOR displayed a 24-hour rhythm, and levels of total mTOR protein (but not mRNA) also showed a circadian rhythm in RenCa tumor masses. Through investigations of the oscillation mechanism for mTOR expression, we identified the ubiquitination factor Fbxw7 as an mTOR regulator that oscillated in its expression in a manner opposite from mTOR. Fbxw7 transcription was regulated by the circadian regulator D-site-binding protein. Notably, administration of the mTOR inhibitor everolimus during periods of elevated mTOR improved survival in tumor-bearing mice. Our findings demonstrate that the circadian oscillation of mTOR activity is regulated by circadian clock systems, which influence the antitumor effect of mTOR inhibitors.