Alterations in brain leptin signalling in spite of unchanged CSF leptin levels in Alzheimer's disease

Several studies support the relation between leptin and Alzheimer's disease (AD). We show that leptin levels in CSF are unchanged as subjects progress to AD. However, in AD hippocampus, leptin signalling was decreased and leptin localization was shifted, being more abundant in reactive astrocytes and less in neurons. Similar translocation of leptin was found in brains from Tg2576 and apoE4 mice. Moreover, an enhancement of leptin receptors was found in hippocampus of young Tg2576 mice and in primary astrocytes and neurons treated with Aβ_1‐42. In contrast, old Tg2576 mice showed decreased leptin receptors levels. Similar findings to those seen in Tg2576 mice were found in apoE4, but not in apoE3 mice. These results suggest that leptin levels are intact, but leptin signalling is impaired in AD. Thus, Aβ accumulation and apoE4 genotype result in a transient enhancement of leptin signalling that might lead to a leptin resistance state over time.     

补偿生长对异育银鲫IGF-1、IGFBP-1水平及IGF-1、IGF-1RmRNA表达的影响

该实验分析饥饿和恢复投喂对异育银鲫血液IGF-1和IGFBP-1水平和肝脏IGF-1、白肌IGF-1RmRNA表达量的影响。结果显示:饥饿期(14d)血液中IGF-1和IGFBP-1水平逐渐下降,在饥饿第14天均出现显著性降低(P<0.05);恢复投喂后第1天IGF-1迅速恢复到对照组水平,而IGFBP-1水平仍显著低于对照组(P<0.05),随后逐渐升高,直至于恢复投喂第14天后显著高于对照组水平(P<0.05);饥饿期肝脏IGF-1mRNA表达量呈下降趋势,但与对照组无显著性差异(P>0.05);恢复投喂初期(第1、3天),IGF-1mRNA表达量仍继续下降(P<0.05),对营养条件的变化反应滞后,至第7天,表达水平恢复到对照组水平。白肌IGF-1RmRNA表达水平在饥饿第3天出现显著性下降(P<0.05),继续饥饿其水平出现补偿性升高;恢复投喂后第14天IGF-1RmRNA表达量显著高于对照组水平(P<0.05)。该结果揭示恢复投喂期高水平的IGFBP-1含量和IGF-1RmRNA表达量可能通过提高IGF-1的促生长作用参与异育银鲫的补偿生长调节。     

Targeting of the protein interaction site between FAK and IGF-1R

The interaction of focal adhesion kinase (FAK) and insulin-like growth factor-1 receptor (IGF-1R) plays an important role in cancer cell survival. Targeting this interaction with small molecule drugs could be a novel strategy in cancer therapy. By a series of pull-down assays using GST-tagged FAK fragments and His-tagged IGF-1R intracellular fragments, we showed that the FAK-NT2 (a.a. 127-243) domain directly interacts with the N-terminal part of the IGF-1R intracellular domain. Overexpressed FAK-NT2 domain was also shown to co-localize with IGF-1R in pancreatic cells. Computational modeling was used to predict the binding configuration of these two domains and to screen for small molecules binding to the interaction site. This strategy successfully identified a lead compound that disrupts FAK/IGF-1R interaction.     

IGF-1及IGF-1R在胃癌中的表达及意义

目的探讨胰岛素样生长因子1(insulin-like growth factor-1,IGF-1)及胰岛素样生长因子1受体(insulin-like growth factor-1receptor,IGF-1R)在胃癌及癌旁胃黏膜组织中的表达及意义。方法采用免疫组化MaxVision两步法检测80例胃癌和50例癌旁胃黏膜组织中IGF-1及IGF-1R蛋白的表达,并分析两者与胃癌患者临床病理指标间的关系及其相关性。结果 IGF-1及IGF-1R蛋白在胃癌组织中表达阳性率分别为71.25%和75.00%,在癌旁胃黏膜组织中表达阳性率分别为30.00%和24.00%,差异有统计学意义(Z=-4.942,P0.001;Z=-5.688,P0.001)。IGF-1及IGF-1R蛋白的表达与胃癌的组织分化程度、有无淋巴结转移、浸润深度及临床分期相关(Z=-2.067、-2.837,P0.05;Z=-4.117、-3.579,P0.05;Z=-2.885、-2.836,P0.05;Z=-3.286、-3.313,P0.05)。相关性分析显示两者表达呈正相关。结论 IGF-1和IGF-1R蛋白在胃癌组织中呈高表达,两者可能在胃癌的发生、发展过程中具有协同及相互调节的作用。IGF-1和IGF-1R蛋白可能成为胃癌早期诊断、评估胃癌患者预后的重要指标。     

Progesterone treatment decreases traumatic brain injury induced anxiety and is correlated with increased serum IGF-1 levels; prefrontal cortex,amygdala, hippocampus neuron density; and reduced serum corticosterone levels in immature rats

Abstract

Traumatic brain injury (TBI) may cause neuropsychiatric problems, such as anxiety disorder, that have negative effects on cognitive functions and behavior. We investigated the effects of progesterone on traumatic brain injury induced anxiety in 7-day-old rat pups subjected to contusion injury. Progesterone treatment decreased TBI induced anxiety and serum corticosterone levels, and increased serum IGF-1 levels. Moreover, progesterone treatment increased amygdala, prefrontal cortex and hippocampal neuron density. We found a negative correlation between serum corticosterone levels and anxiety tests, and a positive correlation between serum IGF-1 levels and anxiety tests. In addition, progesterone treatment decreased serum corticosterone compared to the controls and sham. Our results indicate that single dose progesterone may be effective for treating anxiety caused by TBI.     

Quantification of the synapses in the hippocampus of aged rats

The synapses in the stratum lacunosum-molecular (str. L-M) of CA1 hippocampal field in 3-month old and 24-month old rats were examined using quantitative ultrastructural methods. No significant difference in the density of synapses and postsynaptic dendritic spines was found between the two age groups. The area of presynaptic terminals and postsynaptic dendritic spines was decreased slightly but significantly in the group of aged as compared to that in the group of young-mature rats. The vesicle number per presynaptic terminal, per area of presynaptic terminals and per volume of neuropil was not changed while the vesicle number per area of synaptic contact zones (SCZ) was increased in the group of aged rats. The mean length, total length and total surface of SCZ were diminished in the group of aged as compared to those in the group of young-mature rats. The same width of the str.radiatum and str.L-M in the two age groups showed that there was no any shrinkage of the neuropil in aged rats. The quantitative alterations in the synapses were accompanied by an increased number of dense and lamellar bodies in presynaptic terminals as well as with a presence of hypertrophic astroglial processes.     

OGG1 mRNA expression and incision activity in rats are higher in foetal tissue than in adult liver tissue while 8-oxo-2'-deoxyguanosine levels are unchanged

This study was set up to investigate the relationships between the formation and removal of DNA damage in form of 8-oxodeoxyguanosine (8-oxodG) in neonatal (day 16 of gestation) as compared to adult rats. The hypothesis addressed was whether the rapidly dividing foetal tissue has an enhanced requirement of DNA repair providing protection against potentially mutagenic DNA damages such as 8-oxodG. The activity of the primary 8-oxodG-repair protein OGG1 was measured by a DNA incision assay and the expression of OGG1 mRNA was measured by Real-Time PCR normalised to 18S rRNA. The tissue level of 8-oxodG was measured by HPLC-ECD. We found a 2-3-fold increased incision activity in the foetal control tissue, together with a 3-15-fold increase in mRNA of OGG1 as compared to liver tissue from adult rats. The levels of 8-oxodG in the foetal tissue were unaltered as compared to the adult groups. To increase the levels of 8-oxodG, the rats received an injection (i.p.) of the hepatotoxin 2-nitropropane. The compound induced significant levels of 8-oxodG in male rat livers 5h after the injection and in the foetuses 24h after the injection, while the female rats showed no increase in 8-oxodG. The incision activity was slightly depressed in both male and female liver tissue and in the foetal tissue 5h after the injection, but significantly increased from 5 to 24h after the injection. However, it did not reach levels significantly above the control levels.In conclusion, this study confirms that foetal tissue has increased levels of OGG1 mRNA and correspondingly an enhanced incision activity on an 8-oxodG substrate in a crude tissue extract.     

Decreased expression and DNA methylation levels of GATAD1 in preeclamptic placentas

Expression of syncytin-1, or the human endogenous retroviral family W member 1 (HERVWE1) in human placental trophoblasts is regulated by DNA methylation. Increased DNA methylation and decreased expression of syncytin-1 have been observed in preeclamptic placentas. The syncytin-1-mediated fusogenic as well as non-fusogenic activities, e.g., cell cycle promotion, anti-apoptosis, and immune suppression, are implicated in the pathogenic changes in preeclamptic placentas. It is noteworthy that in a close vicinity to syncytin-1 there are two genes, peroxisome biogenesis factor 1 (PEX1) and GATA zinc finger domain containing 1 (GATAD1), as well as multiple CpG islands around these genes. In this study we determined if these adjacent genes might, like syncytin-1, subject to epigenetic regulation in preeclamptic placentas. Data from quantitative real-time PCR and Western blotting indicated that while PEX1 expression remained stable, GATAD1 expression was significantly decreased in the third-trimester placentas associated with preeclampsia than those associated with normal pregnancy. Immunohistochemistry detected high GATAD1 expression in trophoblast linage, and confirmed its reduced levels in preeclamptic placentas. However, COBRA and bisulfate sequencing detected decreased DNA methylation in levels in the 3 [prime] region of GATAD1 gene in preeclamptic placentas. The positive correlation between 3 [prime] methylation and GATAD1 expression was confirmed by treatment of choriocarcinoma JAR cells with DNMT inhibitor. These data pointed to a potential role of GATAD1 for the syncytium deficiency often associated with preeclamptic placentas. The sharp contrast of the methylation alterations for the closely positioned GATAD1 and HERVWE1 may provide a useful model for studying the accurate control of DNA methylation as well as their positive and negative impact on gene expression in placental trophoblasts.     

IGF-1R tyrosine kinase expression and dependency in clones of IGF-1R knockout cells (R-)

Insulin-like growth factor 1 receptor (IGF-1R) plays many crucial roles in cancer, like anti-apoptotic activity and necessity for transformation. IGF-1R knockout cells (R-) represent a useful tool for molecular mapping of biological properties of the receptor. R- cells have been shown to be refractory to transformation by viral and cellular oncogenes, highlighting the necessity of this receptor for transformation. Surprisingly, more recent studies have shown that these cells can undergo spontaneous transformation. This observation raises the question as whether R- cells over the years have acquired some properties mimicking those of IGF-1R. Using an IGF-1R inhibitor (cyclolignan PPP) we have identified clones of R- (R-s) that are sensitive to this compound. Since, PPP is closely related to podophyllotoxin, which is an efficient microtubule inhibitor, we first investigated if such a mechanism could explain the sensitivity to PPP. However, highly purified PPP showed no or very slight tubulin binding. Further analysis of R-s revealed expression of a 90 kDa protein being reactive to IGF-1R beta-subunit antibodies. This protein was weakly but constitutively tyrosine phosphorylated and was downregulated by siRNA targeting IGF-1R. This downregulation was paralleled by decreased R-s survival. Taken together, our study suggests that clones of R- express IGF-1R activity and dependency, which in turn may explain that R- can undergo spontaneous transformation.     

Serum IGF-1 levels correlate negatively to liver damage in diabetic rats

Abstract

Diabetes and insulin resistance frequently cause liver damage. Diabetes also causes reduction in liver and blood IGF-1 levels. We investigated the relation between liver damage and IGF-1 levels in diabetic rats. Fourteen Wistar albino rats were divided into control and diabetic groups. Diabetes was induced by streptozotocin. Rats were sacrificed for biochemical and histologic examinations 2 weeks after streptozotocin injection. Serum and liver IGF-1 levels were decreased, liver malondialdehyde (MDA) levels were increased, glutathione peroxidase (GPx) enzymes activities were decreased and serum alanine aminotransferase (ALT) levels were increased in diabetic group. Microscopic examination of liver revealed that normal tissue organization was disrupted in streptozotocin-induced diabetic rats. There was a strongly positive correlation between blood glucose levels and liver injury, and blood and liver IGF-1 levels. There was a strongly negative correlation between blood IGF-1 levels and hepatic injury. Our results suggest that reduction of blood IGF-1 levels correlates with hepatic injury and circulating IGF-1 levels may have predictive value for determining hepatic damage that results from diabetes. In addition, circulating IGF-1 levels are correlated with glutathione levels and the oxidative stress status of diabetic rat liver.     

Low IGF-1 levels are associated with cardiovascular risk factors in haemodialysis patients

Cardiovascular diseases (CVD) constitute a significant risk and may, in part, explain the high morbidity and mortality rates among haemodialysis (HD) patients. Several studies have implicated reduced insulin like growth factor (IGF-1) levels in the development of CVD. However, it is not clear whether IGF-1, and its relationship with other hormones such as leptin, insulin, and growth hormone (GH), as well as anthropometric variables may explain the high incidence of vascular complications in chronic kidney disease (CKD) patients. This study was designed to measure total serum IGF-1, leptin, insulin and GH levels in CKD patients and in age-matched control subjects and to elucidate the relationship between IGF-1 and GH, leptin, and insulin as well as other known aetiological risk factors for CVD including blood pressure, body mass index (BMI), and age. The study consisted of 50 CKD patients [36 M and 14 F; mean age; 41.8 ± 10.3 years) on maintenance haemodialysis and 50 healthy control subjects (36 M and 14 F; mean age 41.6 ± 10.2 years) matched for age and sex. None of the subject among patients and controls reported either smoking or history of diabetes mellitus. The circulating levels of IGF-1 were significantly lower (P < 0.001) in both male and female patients compared to the control subjects. Moreover, IGF-1 was strongly and inversely correlated with both systolic blood pressure (SBP) (r = −0.360; P < 0.01) and diastolic blood pressure (DBP) (r = −0.512; P < 0.001) in the CKD group, and when the two groups were combined SBP (r = −0.396; P < 0.001) and DBP (r = −0.296; P < 0.01). When adjusted for age, the correlation was more significant, however, when adjusted for BMI no significant correlation was observed between IGF-1 and blood pressure. IGF-1 was inversely correlated with age (r = −0.367; P < 0.01) and BMI (r = −0.310; P < 0.05) in the control group, but not the patient group. In controls and patients, respectively, a positive correlation between leptin and BMI (r = 0.358; P < 0.01; r = 0.640, P < 0.001) was observed. The results show that circulating levels of IGF-1 were significantly lower in CKD patients as compared to healthy normal subjects and were inversely correlated with SBP and DBP independent of age, but not BMI indicative of a strong relationship between cardiovascular risk factors and low IGF-1 levels. Although, the data do not clearly indicate low IGF-1 levels as a cause or an effect of these cardiovascular risk factors, they do point to an interesting relationship between low IGF-1 levels and increased cardiovascular risk factors among CKD patients as compared to age-matched healthy control subjects.     

Quantitative succinate dehydrogenase histochemistry in the hippocampus of aged rats

Quantitative histochemical methods (microphotometric kinetic and end-point measurements, and morphometric analyses of reactive areas) were used to investigate the levels of succinate dehydrogenase (SDH) in the hippocampus of young adult (3-6 months old) and aged male rats (24-27 months old). Methodological studies concerning the demonstration of SDH activity, which were performed using hippocampi of young animals, revealed a linear relationship between the reaction time and the amount of reaction product for up to 20 min; kinetic (continuous) and end-point measurements provided the same results. In a number of experiments, it was established that an incubation medium consisting of 100 mM succinate, 10 mM sodium azide, 3 mM nitro blue tetrazolium chloride, 0.25 mM phenazine methosulfate, and 7.5% polyvinylalcohol in 0.05 M Hepes buffer (final pH 7.5) was optimal for quantitative SDH histochemistry in the hippocampus. Comparative quantitative investigations of SDH activity in rat hippocampi showed that, in most regions and layers of the hippocampus of both young and aged rats, the levels of SDH activity increased along the rostrocaudal axis of the hippocampus, i.e., higher levels were present in the caudal than in the rostral pole. In both groups, the highest SDH levels were observed in the molecular layer of the cornu ammonis (CA)-1, the CA-3, and the fascia dentata (middle and outer thirds), most of which are termination fields of the excitatory perforant path arising from the regio ento-rhinalis. Furthermore, in almost all of the investigated layers, the older animals exhibited lower SDH levels than young animals. These differences were statistically significant in the molecular layer of the fascia dentata and in most layers of the CA-3. The lower SDH levels in aged animals are discussed in relation to the reduced capacity for energy metabolism in the aging brain.     

Quantitative succinate dehydrogenase histochemistry in the hippocampus of aged rats

Summary Quantitative histochemical methods (microphotometric kinetic and end-point measurements, and morphometric analyses of reactive areas) were used to investigate the levels of succinate dehydrogenase (SDH) in the hippocampus of young adult (3–6 months old) and aged male rats (24–27 months old). Methodological studies concerning the demonstration of SDH activity, which were performed using hippocampi of young animals, revealed a linear relationship between the reaction time and the amount of reaction product for up to 20 min; kinetic (continuous) and end-point measurements provided the same results. In a number of experiments, it was established that an incubation medium consisting of 100 mM succinate, 10 mM sodium azide, 3 mM nitro blue tetrazolium chloride, 0.25 mM phenazine methosulfate, and 7.5% polyvinylalcohol in 0.05M Hepes buffer (final pH 7.5) was optimal for quantitative SDH histochemistry in the hippocampus. Comparative quantitative investigations of SDH activity in rat hippocampi showed that, in most regions and layers of the hippocampus of both young and aged rats, the levels of SDH activity increased along the rostrocaudal axis of the hippocampus, i.e., higher levels were present in the caudal than in the rostral pole. In both groups, the highest SDH levels were observed in the molecular layer of the cornu ammonis (CA)-1 the CA-3, and the fascia dentata (middle and outer thirds), most of which are termination fields of the excitatory perforant path arising from the regio entorhinalis. Furthermore, in almost all of the investigated layers, the older animals exhibited lower SDH levels than young animals. These differences were statistically significant in the molecular layer of the fascia dentata and in most layers of the CA-3. The lower SDH levels in aged animals are discussed in relation to the reduced capacity for energy metabolism in the aging brain.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the Deutsche Forschungsmeinschaft (Ku 541/2-1)     

Disruption of the protein interaction between FAK and IGF-1R inhibits melanoma tumor growth

FAK (focal adhesion kinase) and IGF-1R (insulin-like growth factor receptor-1) directly interact with each other and thereby activate crucial signaling pathways that benefit cancer cells. Inhibition of FAK and IGF-1R function has been shown to significantly decrease cancer cell proliferation and increase sensitivity to chemotherapy and radiation treatment. As a novel approach in human melanoma, we evaluated the effect of a small-molecule compound that disrupts the protein interaction of FAK and IGF-1R.

Previously, using virtual screening and functional testing, we identified a lead compound (INT2–31) that targets the known FAK-IGF-1R protein interaction site. We studied the ability of this compound to disrupt FAK-IGF-1R protein interactions, inhibit downstream signaling, decrease human melanoma cell proliferation, alter cell cycle progression, induce apoptosis and decrease tumor growth in vivo.

INT2–31 blocked the interaction of FAK and IGF-1R in vitro and in vivo in melanoma cells and tumor xenografts through precluding the activation of IRS-1, leading to reduced phosphorylation of AKT upon IGF-1 stimulation. As a result, INT2–31 significantly inhibited cell proliferation and viability (range 0.05–10 μM). More importantly, 15 mg/kg of INT2–31 given for 21 d via intraperitoneal injection disrupted the interaction of FAK and IGF-1R and effectively decreased phosphorylation of tumor AKT, resulting in significant melanoma tumor regression in vivo.

Our data suggest that the FAK-IGF-1R protein interaction is an important target, and disruption of this interaction with a novel small molecule (INT2–31) has potential anti-neoplastic therapeutic effects in human melanoma.     

Disruption of the protein interaction between FAK and IGF-1R inhibits melanoma tumor growth

FAK (focal adhesion kinase) and IGF-1R (insulin-like growth factor receptor-1) directly interact with each other and thereby activate crucial signaling pathways that benefit cancer cells. Inhibition of FAK and IGF-1R function has been shown to significantly decrease cancer cell proliferation and increase sensitivity to chemotherapy and radiation treatment. As a novel approach in human melanoma, we evaluated the effect of a small-molecule compound that disrupts the protein interaction of FAK and IGF-1R.Previously, using virtual screening and functional testing, we identified a lead compound (INT2–31) that targets the known FAK-IGF-1R protein interaction site. We studied the ability of this compound to disrupt FAK-IGF-1R protein interactions, inhibit downstream signaling, decrease human melanoma cell proliferation, alter cell cycle progression, induce apoptosis and decrease tumor growth in vivo.INT2–31 blocked the interaction of FAK and IGF-1R in vitro and in vivo in melanoma cells and tumor xenografts through precluding the activation of IRS-1, leading to reduced phosphorylation of AKT upon IGF-1 stimulation. As a result, INT2–31 significantly inhibited cell proliferation and viability (range 0.05–10 μM). More importantly, 15 mg/kg of INT2–31 given for 21 d via intraperitoneal injection disrupted the interaction of FAK and IGF-1R and effectively decreased phosphorylation of tumor AKT, resulting in significant melanoma tumor regression in vivo.Our data suggest that the FAK-IGF-1R protein interaction is an important target, and disruption of this interaction with a novel small molecule (INT2–31) has potential anti-neoplastic therapeutic effects in human melanoma.     

Colonizing the world in spite of reduced MHC variation

The major histocompatibility complex (MHC), which harbours the most polymorphic vertebrate genes, plays a critical role in the host-pathogen coevolutionary arms race. However, the extent to which MHC diversity determines disease susceptibility and long-term persistence of populations is currently under debate, as recent studies have demonstrated that low MHC variability does not necessarily hamper population viability. However, these studies typically assayed small and decimated populations in species with restricted distribution, thereby making inferences about the evolutionary potential of these populations difficult. Here, we show that MHC impoverishment has not constrained the ecological radiation and flourishing of falcons (Aves: Falconidae) worldwide. We found two remarkably different patterns of MHC variation within the genus Falco. Whereas MHC variation in kestrels (the basal group within the genus) is very high, falcons exhibit ancestrally low intra- and interspecific MHC variability. This pattern is not due to the inadvertent survey of paralogous genes or pseudogenes. Further, patterns of variation in mitochondrial or other nuclear genes do not indicate a generalized low level of genome-wide variability among falcons. Although a relative contribution of genetic drift cannot be completely ruled out, we propose the falcons went through an evolutionary transition, driven and maintained by natural selection, from primarily highly variable towards low polymorphic and slow-evolving MHC genes with a very specific immune function. This study highlights that the importance of MHC diversity cannot be generalized among vertebrates, and hints at the evolution of compensatory immune mechanisms in falcons to cope with emerging and continuously evolving pathogens.     

Surface L‐type Ca2+ channel expression levels are increased in aged hippocampus

Age‐related increase in L‐type Ca²⁺ channel (LTCC) expression in hippocampal pyramidal neurons has been hypothesized to underlie the increased Ca²⁺ influx and subsequent reduced intrinsic neuronal excitability of these neurons that lead to age‐related cognitive deficits. Here, using specific antibodies against Ca_v1.2 and Ca_v1.3 subunits of LTCCs, we systematically re‐examined the expression of these proteins in the hippocampus from young (3 to 4 month old) and aged (30 to 32 month old) F344xBN rats. Western blot analysis of the total expression levels revealed significant reductions in both Ca_v1.2 and Ca_v1.3 subunits from all three major hippocampal regions of aged rats. Despite the decreases in total expression levels, surface biotinylation experiments revealed significantly higher proportion of expression on the plasma membrane of Ca_v1.2 in the CA1 and CA3 regions and of Ca_v1.3 in the CA3 region from aged rats. Furthermore, the surface biotinylation results were supported by immunohistochemical analysis that revealed significant increases in Ca_v1.2 immunoreactivity in the CA1 and CA3 regions of aged hippocampal pyramidal neurons. In addition, we found a significant increase in the level of phosphorylated Ca_v1.2 on the plasma membrane in the dentate gyrus of aged rats. Taken together, our present findings strongly suggest that age‐related cognitive deficits cannot be attributed to a global change in L‐type channel expression nor to the level of phosphorylation of Ca_v1.2 on the plasma membrane of hippocampal neurons. Rather, increased expression and density of LTCCs on the plasma membrane may underlie the age‐related increase in L‐type Ca²⁺ channel activity in CA1 pyramidal neurons.     

LINE-1 methylation levels in leukocyte DNA and risk of renal cell cancer

Purpose

Leukocyte global DNA methylation levels are currently being considered as biomarkers of cancer susceptibility and have been associated with risk of several cancers. In this study, we aimed to examine the association between long interspersed nuclear elements (LINE-1) methylation levels, as a biomarker of global DNA methylation in blood cell DNA, and renal cell cancer risk.

Experimental Design

LINE-1 methylation of bisulfite-converted genomic DNA isolated from leukocytes was quantified by pyrosequencing measured in triplicate, and averaged across 4 CpG sites. A total of 328 RCC cases and 654 controls frequency-matched(2∶1) on age(±5years), sex and study center, from a large case-control study conducted in Central and Eastern Europe were evaluated.

Results

LINE-1 methylation levels were significantly higher in RCC cases with a median of 81.97% (interquartile range[IQR]: 80.84–83.47) compared to 81.67% (IQR: 80.35–83.03) among controls (p = 0.003, Wilcoxon). Compared to the lowest LINE-1 methylation quartile(Q1), the adjusted ORs for increasing methylation quartiles were as follows: OR(Q2) = 1.84(1.20−2.81), OR(Q3) = 1.72(1.11−2.65) and OR(Q4) = 2.06(1.34−3.17), with a p-trend = 0.004. The association was stronger among current smokers (p-trend<0.001) than former or never smokers (p-interaction = 0.03). To eliminate the possibility of selection bias among controls, the relationship between LINE-1 methylation and smoking was evaluated and confirmed in a case-only analysis, as well.

Conclusions

Higher levels of LINE-1 methylation appear to be positively associated with RCC risk, particularly among current smokers. Further investigations using both post- and pre-diagnostic genomic DNA is warranted to confirm findings and will be necessary to determine whether the observed differences occur prior to, or as a result of carcinogenesis.