Engagement of SIRPα Inhibits Growth and Induces Programmed Cell Death in Acute Myeloid Leukemia Cells

Background

Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRPα) on macrophages. Although AML cells express SIRPα, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRPα in acute myeloid leukemia.

Design and Methods

We analyzed the expression of SIRPα, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRPα on two low SIRPα expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRPα expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs.

Results

By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRPα is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0–M3. Interestingly, AML patients with high SIRPα expression had a poor prognosis. Our results also showed that SIRPα is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRPα with an agonistic antibody in the cells stably expressing chimeric SIRPα, led to inhibition of growth and induction of programmed cell death. Finally, the SIRPα-derived signaling synergized with the activity of established antileukemic drugs.

Conclusions

Our data indicate that triggering of SIRPα has antileukemic effect and may function as a potential therapeutic target in AML.     

Distinct Patterns of DNA Damage Response and Apoptosis Correlate with Jak/Stat and PI3Kinase Response Profiles in Human Acute Myelogenous Leukemia

Background

Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in intracellular signaling responses. Here SCNP was used to characterize Acute Myeloid Leukemia (AML) disease subtypes based on survival, DNA damage response and apoptosis pathways.

Methodology and Principal Findings

Thirty four diagnostic non-M3 AML samples from patients with known clinical outcome were treated with a panel of myeloid growth factors and cytokines, as well as with apoptosis-inducing agents. Analysis of induced Jak/Stat and PI3K pathway responses in blasts from individual patient samples identified subgroups with distinct signaling profiles that were not seen in the absence of a modulator. In vitro exposure of patient samples to etoposide, a DNA damaging agent, revealed three distinct “DNA damage response (DDR)/apoptosis” profiles: 1) AML blasts with a defective DDR and failure to undergo apoptosis; 2) AML blasts with proficient DDR and failure to undergo apoptosis; 3) AML blasts with proficiency in both DDR and apoptosis pathways. Notably, AML samples from clinical responders fell within the “DDR/apoptosis” proficient profile and, as well, had low PI3K and Jak/Stat signaling responses. In contrast, samples from clinical non responders had variable signaling profiles often with in vitro apoptotic failure and elevated PI3K pathway activity. Individual patient samples often harbored multiple, distinct, leukemia-associated cell populations identifiable by their surface marker expression, functional performance of signaling pathway in the face of cytokine or growth factor stimulation, as well as their response to apoptosis-inducing agents.

Conclusions and Significance

Characterizing and tracking changes in intracellular pathway profiles in cell subpopulations both at baseline and under therapeutic pressure will likely have important clinical applications, potentially informing the selection of beneficial targeted agents, used either alone or in combination with chemotherapy.     

Integrated epigenetics of human breast cancer: synoptic investigation of targeted genes, microRNAs and proteins upon demethylation treatment

Background

The contribution of aberrant DNA methylation in silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations are reversible, it became of interest to determine the effects of the 5-aza-2′-deoxycytidine (DAC) demethylation therapy in breast cancer at different molecular levels.

Methods and Findings

Here we investigate a synoptic model to predict complete DAC treatment effects at the level of genes, microRNAs and proteins for several human breast cancer lines. The present study assessed an effective treatment dosage based on the cell viability, cytotoxicity, apoptosis and methylation assays for the investigated cell lines. A highly aggressive and a non-aggressive cell line were investigated using omics approaches such as MALDI-TOF MS, mRNA- and microRNA expression arrays, 2-D gel electrophoresis and LC-MS-MS. Complete molecular profiles including the biological interaction and possible early and late systematic stable or transient effects of the methylation inhibition were determined. Beside the activation of several epigenetically suppressed TSGs, we also showed significant dysregulation of some important oncogenes, oncomiRs and oncosuppressors miRNAs as well as drug tolerance genes/miRNAs/proteins.

Conclusions

In the present study, the results denote some new molecular DAC targets and pathways based on the chemical modification of DNA methylation in breast cancer. The outlined approach might prove to be useful as an epigenetic treatment model also for other human solid tumors in the management of cancer patients.     

Aquaporin-1 Translocation and Degradation Mediates the Water Transportation Mechanism of Acetazolamide

Background

Diuretic agents are widely used on the treatment of water retention related diseases, among which acetazolamide (AZA) acts originally as a carbonic anhydrase (CA) inhibitor. Aquaporin-1 (AQP1) being located in renal proximal tubules is required for urine concentration. Previously our lab has reported AZA putatively modulated AQP1. Aim of this study is to testify our hypothesis that regulating AQP1 may mediate diuretic effect of AZA.

Methodology/Principal Findings

For in vivo study, we utilized Sprague Dawley rats, as well as AQP1 knock-out (AQP1^−/−) mice to examine urine volume, and human kidney-2 (HK-2) cell line was used for in vitro mechanism study. In our present study we found that AZA decreased CAs activity initially but the activity gradually recovered. Contrarily, diuretic effect was consistently significant. AQP1 protein expression was significantly decreased on day 7 and 14. By utilizing AQP1^−/− mice, we found diuretic effect of AZA was cancelled on day 14, while urine volume continuously increased in wild-type mice. Surface plasmon resonance (SPR) results indicated AQP1 was physiologically bound by myosin heavy chain (MHC), immunoprecipitation and immunofluorescence results confirmed this protein interaction. In vitro study results proved AZA facilitated AQP1 translocation onto cell membrane by promoting interaction with MHC, dependent on ERK/ myosin light chain kinase (MLCK) pathway activation. MHC inhibitor BDM and ERK inhibitor U0126 both abolished above effect of AZA. Eventually AZA induced AQP1 ubiquitination, while proteasome inhibitor MG132 reversed AZA''s down-regulating effect upon AQP1.

Conclusions/Significance

Our results identified AZA exerted diuretic effect through an innovative mechanism by regulating AQP1 and verified its inhibitory mechanism was via promoting MHC-dependent translocation onto cell membrane and then ubiquitin mediated degradation, implicating a novel mechanism and target for diuretic agent discovering.     

Bioluminescence-based high-throughput screen identifies pharmacological agents that target neurotransmitter signaling in small cell lung carcinoma

Background

Frontline treatment of small cell lung carcinoma (SCLC) relies heavily on chemotherapeutic agents and radiation therapy. Though SCLC patients respond well to initial cycles of chemotherapy, they eventually develop resistance. Identification of novel therapies against SCLC is therefore imperative.

Methods and Findings

We have designed a bioluminescence-based cell viability assay for high-throughput screening of anti-SCLC agents. The assay was first validated via standard pharmacological agents and RNA interference using two human SCLC cell lines. We then utilized the assay in a high-throughput screen using the LOPAC¹²⁸⁰ compound library. The screening identified several drugs that target classic cancer signaling pathways as well as neuroendocrine markers in SCLC. In particular, perturbation of dopaminergic and serotonergic signaling inhibits SCLC cell viability.

Conclusions

The convergence of our pharmacological data with key SCLC pathway components reiterates the importance of neurotransmitter signaling in SCLC etiology and points to possible leads for drug development.     

Redox-Sensitive Induction of Src/PI3-kinase/Akt and MAPKs Pathways Activate eNOS in Response to EPA:DHA 6:1

Aims

Omega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have vasoprotective effects, in part, by stimulating the endothelial formation of nitric oxide (NO). This study determined the role of the EPA:DHA ratio and amount, and characterized the mechanism leading to endothelial NO synthase (eNOS) activation.

Methods and Results

EPA:DHA 6∶1 and 9∶1 caused significantly greater endothelium-dependent relaxations in porcine coronary artery rings than EPA:DHA 3∶1, 1∶1, 1∶3, 1∶6, 1∶9, EPA and DHA alone, and EPA:DHA 6∶1 with a reduced EPA + DHA amount, which were inhibited by an eNOS inhibitor. Relaxations to EPA:DHA 6∶1 were insensitive to cyclooxygenase inhibition, and reduced by inhibitors of either oxidative stress, Src kinase, PI3-kinase, p38 MAPK, MEK, or JNK. EPA:DHA 6∶1 induced phosphorylation of Src, Akt, p38 MAPK, ERK, JNK and eNOS; these effects were inhibited by MnTMPyP. EPA:DHA 6∶1 induced the endothelial formation of ROS in coronary artery sections as assessed by dihydroethidium, and of superoxide anions and hydrogen peroxide in cultured endothelial cells as assessed by electron spin resonance with the spin probe CMH, and the Amplex Red based assay, respectively.

Conclusion

Omega-3 fatty acids cause endothelium-dependent NO-mediated relaxations in coronary artery rings, which are dependent on the EPA:DHA ratio and amount, and involve an intracellular activation of the redox-sensitive PI3-kinase/Akt and MAPKs pathways to activate eNOS.     

Mechanisms of resistance to decitabine in the myelodysplastic syndrome

Purpose

The DNA methylation inhibitor 5-aza-2′-deoxycytidine (DAC) is approved for the treatment of myelodysplastic syndromes (MDS), but resistance to DAC develops during treatment and mechanisms of resistance remain unknown. Therefore, we investigated mechanisms of primary and secondary resistance to DAC in MDS.

Patients and Methods

We performed Quantitative Real-Time PCR to examine expression of genes related to DAC metabolism prior to therapy in 32 responders and non-responders with MDS as well as 14 patients who achieved a complete remission and subsequently relapsed while on therapy (secondary resistance). We then performed quantitative methylation analyses by bisulfite pyrosequencing of 10 genes as well as Methylated CpG Island Amplification Microarray (MCAM) analysis of global methylation in secondary resistance.

Results

Most genes showed no differences by response, but the CDA/DCK ratio was 3 fold higher in non-responders than responders (P<.05), suggesting that this could be a mechanism of primary resistance. There were no significant differences at relapse in DAC metabolism genes, and no DCK mutations were detected. Global methylation measured by the LINE1 assay was lower at relapse than at diagnosis (P<.05). On average, the methylation of 10 genes was lower at relapse (16.1%) compared to diagnosis (18.1%) (P<.05).MCAM analysis showed decreased methylation of an average of 4.5% (range 0.6%–9.7%) of the genes at relapse. By contrast, new cytogenetic changes were found in 20% of patients.

Conclusion

Pharmacological mechanisms are involved in primary resistance to DAC, whereas hypomethylation does not prevent a relapse for patients with DAC treatment.     

Antigenic Differences between AS03 Adjuvanted Influenza A (H1N1) Pandemic Vaccines: Implications for Pandemrix-Associated Narcolepsy Risk

Background

Narcolepsy results from immune-mediated destruction of hypocretin secreting neurons in hypothalamus, however the triggers and disease mechanisms are poorly understood. Vaccine-attributable risk of narcolepsy reported so far with the AS03 adjuvanted H1N1 vaccination Pandemrix has been manifold compared to the AS03 adjuvanted Arepanrix, which contained differently produced H1N1 viral antigen preparation. Hence, antigenic differences and antibody response to these vaccines were investigated.

Methods and Findings

Increased circulating IgG-antibody levels to Pandemrix H1N1 antigen were found in 47 children with Pandemrix-associated narcolepsy when compared to 57 healthy children vaccinated with Pandemrix. H1N1 antigen of Arepanrix inhibited poorly these antibodies indicating antigenic difference between Arepanrix and Pandemrix. High-resolution gel electrophoresis quantitation and mass spectrometry identification analyses revealed higher amounts of structurally altered viral nucleoprotein (NP) in Pandemrix. Increased antibody levels to hemagglutinin (HA) and NP, particularly to detergent treated NP, was seen in narcolepsy. Higher levels of antibodies to NP were found in children with DQB1*06∶02 risk allele and in DQB1*06∶02 transgenic mice immunized with Pandemrix when compared to controls.

Conclusions

This work identified 1) higher amounts of structurally altered viral NP in Pandemrix than in Arepanrix, 2) detergent-induced antigenic changes of viral NP, that are recognized by antibodies from children with narcolepsy, and 3) increased antibody response to NP in association of DQB1*06∶02 risk allele of narcolepsy. These findings provide a link between Pandemrix and narcolepsy. Although detailed mechanisms of Pandemrix in narcolepsy remain elusive, our results move the focus from adjuvant(s) onto the H1N1 viral proteins.     

Smac Mimetic SM-164 Potentiates APO2L/TRAIL- and Doxorubicin-Mediated Anticancer Activity in Human Hepatocellular Carcinoma Cells

Background

The members of inhibitor of apoptosis proteins (IAPs) family are key negative regulators of apoptosis. Overexpression of IAPs are found in hepatocellular carcinoma (HCC), and can contribute to chemotherapy resistance and recurrence of HCC. Small-molecule Second mitochondria-derived activator of caspases (Smac) mimetics have recently emerged as novel anticancer drugs through targeting IAPs. The specific aims of this study were to 1) examine the anticancer activity of Smac mimetics as a single agent and in combination with chemotherapy in HCC cells, and 2) investigate the mechanism of anticancer action of Smac mimetics.

Methods

Four HCC cell lines, including SMMC-7721, BEL-7402, HepG2 and Hep3B, and 12 primary HCC cells were used in this study. Smac mimetic SM-164 was used to treat HCC cells. Cell viability, cell death induction and clonal formation assays were used to evaluate the anticancer activity. Western blotting analysis and a pancaspase inhibitor were used to investigate the mechanisms.

Results

Although SM-164 induced complete cIAP-1 degradation, it displayed weak inhibitory effects on the viability of HCC cells. Nevertheless, SM-164 considerably potentiated Apo2 ligand or TNF-related apoptosis-inducing ligand (APO2L/TRAIL)- and Doxorubicin-mediated anticancer activity in HCC cells. Mechanistic studies demonstrated that SM-164 in combination with chemotherapeutic agents resulted in enhanced activation of caspases-9, -3 and cleavage of poly ADP-ribose polymerase (PARP), and also led to decreased AKT activation.

Conclusions

Smac mimetics can enhance chemotherapeutic-mediated anticancer activity by enhancing apoptosis signaling and suppressing survival signaling in HCC cells. This study suggests Smac mimetics are potential therapeutic agents for HCC.     

Reducing the Dietary Omega-6:Omega-3 Utilizing α-Linolenic Acid; Not a Sufficient Therapy for Attenuating High-Fat-Diet-Induced Obesity Development Nor Related Detrimental Metabolic and Adipose Tissue Inflammatory Outcomes

Aims

To examine the effect of manipulating the omega-6:omega-3 (1∶1, 5∶1, 10∶1, and 20∶1) utilizing only α-linolenic and linoleic acid within a clinically-relevant high-fat diet (HFD) composed of up to seven sources of fat and designed to be similar to the standard American diet (MUFA∶PUFA of 2∶1, 12% and 40% of calories from saturated and total fat, respectively) on body composition, macrophage polarization, inflammation, and metabolic dysfunction in mice.

Methods

Diets were administered for 20 weeks. Body composition and metabolism (HOMA index and lipid profile) were examined monthly. GC-MS was utilized to determine the eicosapentaenoic acid (EPA):arachidonic acid (AA) and the docosahexaenoic acid (DHA):AA in AT phospholipids. Adipose tissue (AT) mRNA expression of chemokines (MCP-1, Fetuin-A, CXCL14), marker genes for M1 and M2 macrophages (CD11c and CD206, respectively) and inflammatory markers (TNF-α, IL-6, IL-1β, TLR-2, TLR-4, IL-10, GPR120) were measured along with activation of NFκB, JNK, and STAT-3. Macrophage infiltration into AT was examined using F4/80 immunohistochemistry.

Results

Any therapeutic benefit produced by reducing the omega-6:omega-3 was evident only when comparing the 1∶1 to 20∶1 HFD; the 1∶1 HFD resulted in a lower TC:HDL-C and decreased AT CXCL14 gene expression and AT macrophage infiltration, which was linked to a higher EPA:AA and DHA:AA in AT phospholipids. However, despite these effects, and independent of the omega-6:omega-3, all HFDs, in general, led to similar levels of adiposity, insulin resistance, and AT inflammation.

Conclusion

Reducing the omega-6:omega-3 using α-linolenic acid is not an effective therapy for attenuating obesity and type II diabetes mellitus development.     

Cell Type-Dependent Induction of DNA Damage by 1800 MHz Radiofrequency Electromagnetic Fields Does Not Result in Significant Cellular Dysfunctions

Background

Although IARC clarifies radiofrequency electromagnetic fields (RF-EMF) as possible human carcinogen, the debate on its health impact continues due to the inconsistent results. Genotoxic effect has been considered as a golden standard to determine if an environmental factor is a carcinogen, but the currently available data for RF-EMF remain controversial. As an environmental stimulus, the effect of RF-EMF on cellular DNA may be subtle. Therefore, more sensitive method and systematic research strategy are warranted to evaluate its genotoxicity.

Objectives

To determine whether RF-EMF does induce DNA damage and if the effect is cell-type dependent by adopting a more sensitive method γH2AX foci formation; and to investigate the biological consequences if RF-EMF does increase γH2AX foci formation.

Methods

Six different types of cells were intermittently exposed to GSM 1800 MHz RF-EMF at a specific absorption rate of 3.0 W/kg for 1 h or 24 h, then subjected to immunostaining with anti-γH2AX antibody. The biological consequences in γH2AX-elevated cell type were further explored with comet and TUNEL assays, flow cytometry, and cell growth assay.

Results

Exposure to RF-EMF for 24 h significantly induced γH2AX foci formation in Chinese hamster lung cells and Human skin fibroblasts (HSFs), but not the other cells. However, RF-EMF-elevated γH2AX foci formation in HSF cells did not result in detectable DNA fragmentation, sustainable cell cycle arrest, cell proliferation or viability change. RF-EMF exposure slightly but not significantly increased the cellular ROS level.

Conclusions

RF-EMF induces DNA damage in a cell type-dependent manner, but the elevated γH2AX foci formation in HSF cells does not result in significant cellular dysfunctions.     

Evaluating the Impact of Flexible Alcohol Trading Hours on Violence: An Interrupted Time Series Analysis

Background

On November 24^th 2005, the Government of England and Wales removed regulatory restrictions on the times at which licensed premises could sell alcohol. This study tests availability theory by treating the implementation of Licensing Act (2003) as a natural experiment in alcohol policy.

Methods

An interrupted time series design was employed to estimate the Act’s immediate and delayed impact on violence in the City of Manchester (Population 464,200). We collected police recorded rates of violence, robbery, and total crime between the 1st of February 2004 and the 31st of December 2007. Events were aggregated by week, yielding a total of 204 observations (95 pre-, and 109 post-intervention). Secondary analysis examined changes in daily patterns of violence. Pre- and post-intervention events were separated into four three-hour segments 18∶00–20∶59, 21∶00–23.59, 00∶00–02∶59, 03∶00–05∶59.

Results

Analysis found no evidence that the Licensing Act (2003) affected the overall volume of violence. However, analyses of night-time violence found a gradual and permanent shift of weekend violence into later parts of the night. The results estimated an initial increase of 27.5% between 03∶00 to 06∶00 (ω = 0.2433, 95% CI = 0.06, 0.42), which increased to 36% by the end of the study period (δ = −0.897, 95% CI = −1.02, −0.77).

Conclusions

This study found no evidence that a national policy increasing the physical availability of alcohol affected the overall volume of violence. There was, however, evidence suggesting that the policy may be associated with changes to patterns of violence in the early morning (3 a.m. to 6 a.m.).     

Inhibition of histone deacetylases 1 and 6 enhances cytarabine-induced apoptosis in pediatric acute myeloid leukemia cells

Background

Pediatric acute myeloid leukemia (AML) remains a challenging disease to treat even with intensified cytarabine-based chemotherapy. Histone deacetylases (HDACs) have been reported to be promising therapeutic targets for treating AML. However, HDAC family members that are involved in chemotherapy sensitivities remain unknown. In this study, we sought to identify members of the HDAC family that are involved in cytarabine sensitivities, and to select the optimal HDACI that is most efficacious when combined with cytarabine for treating children with AML.

Methodology

Expression profiles of classes I, II, and IV HDACs in 4 pediatric AML cell lines were determined by Western blotting. Inhibition of class I HDACs by different HDACIs was measured post immnunoprecipitation. Individual down-regulation of HDACs in pediatric AML cells was performed with lentiviral shRNA. The effects of cytarabine and HDACIs on apoptosis were determined by flow cytometry analysis.

Results

Treatments with structurally diverse HDACIs and HDAC shRNA knockdown experiments revealed that down-regulation of both HDACs 1 and 6 is critical in enhancing cytarabine-induced apoptosis in pediatric AML, at least partly mediated by Bim. However, down-regulation of HDAC2 may negatively impact cytarabine sensitivities in the disease. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced cytarabine-induced apoptosis.

Conclusion

Our results further confirm that HDACs are bona fide therapeutic targets for treating pediatric AML and suggest that pan-HDACIs may be more beneficial than isoform-specific drugs.     

Brain networks for integrative rhythm formation

Background

Performance of externally paced rhythmic movements requires brain and behavioral integration of sensory stimuli with motor commands. The underlying brain mechanisms to elaborate beat-synchronized rhythm and polyrhythms that musicians readily perform may differ. Given known roles in perceiving time and repetitive movements, we hypothesized that basal ganglia and cerebellar structures would have greater activation for polyrhythms than for on-the-beat rhythms.

Methodology/Principal Findings

Using functional MRI methods, we investigated brain networks for performing rhythmic movements paced by auditory cues. Musically trained participants performed rhythmic movements at 2 and 3 Hz either at a 1∶1 on-the-beat or with a 3∶2 or a 2∶3 stimulus-movement structure. Due to their prior musical experience, participants performed the 3∶2 or 2∶3 rhythmic movements automatically. Both the isorhythmic 1∶1 and the polyrhythmic 3∶2 or 2∶3 movements yielded the expected activation in contralateral primary motor cortex and related motor areas and ipsilateral cerebellum. Direct comparison of functional MRI signals obtained during 3∶2 or 2∶3 and on-the-beat rhythms indicated activation differences bilaterally in the supplementary motor area, ipsilaterally in the supramarginal gyrus and caudate-putamen and contralaterally in the cerebellum.

Conclusions/Significance

The activated brain areas suggest the existence of an interconnected brain network specific for complex sensory-motor rhythmic integration that might have specificity for elaboration of musical abilities.     

Integration of SNP and mRNA Arrays with MicroRNA Profiling Reveals That MiR-370 Is Upregulated and Targets NF1 in Acute Myeloid Leukemia

Background

Deregulated miRNA expression plays a crucial role in carcinogenesis. Recent studies show different mechanisms leading to miRNA deregulation in cancer; however, alterations affecting miRNAs by DNA copy number variations (CNV) remain poorly studied.

Results

Our integrative analysis including data from high resolution SNPs arrays, mRNA expression arrays, and miRNAs expression profiles in 16 myeloid cell lines highlights that CNV are alternative mechanisms to deregulate the expression of miRNAs in acute myeloid leukemia (AML), and represent a novel approach to identify novel candidate genes involved in AML. We found association between the expression levels of 19 miRNAs and CNVs affecting their loci. Functional analysis showed that NF1 is a direct target of miR-370, and that overexpression of miR-370 has similar effects that NF1 inactivation, increasing proliferation and colony formation in AML cells. Moreover, real time RT-PCR showed that NF1 downregulation is a recurrent event in AML (30.8%), and western blot analysis confirmed this result. MiR-370 overexpression and deletions affecting the NF1 locus were identified as alternative mechanisms to downregulate NF1.

Conclusions

NF1 downregulation is a common event in AML, and both deletions in the NF1 locus and overexpression of miR-370 are alternative mechanisms to downregulate NF1 in this disease. Our results suggest a leukemogenic role of miR-370 through NF1 downregulation in AML cells. Since NF1 deficiency leads to RAS activation, patients with AML and overexpression of miR-370 may potentially benefit from additional treatment with either RAS or mTOR inhibitors.     

The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer

Background

Irinotecan (SN38) and oxaliplatin are chemotherapeutic agents used in the treatment of colorectal cancer. However, the frequent development of resistance to these drugs represents a considerable challenge in the clinic. Alus as retrotransposons comprise 11% of the human genome. Genomic toxicity induced by carcinogens or drugs can reactivate Alus by altering DNA methylation. Whether or not reactivation of Alus occurs in SN38 and oxaliplatin resistance remains unknown.

Results

We applied reduced representation bisulfite sequencing (RRBS) to investigate the DNA methylome in SN38 or oxaliplatin resistant colorectal cancer cell line models. Moreover, we extended the RRBS analysis to tumor tissue from 14 patients with colorectal cancer who either did or did not benefit from capecitabine + oxaliplatin treatment. For the clinical samples, we applied a concept of ‘DNA methylation entropy’ to estimate the diversity of DNA methylation states of the identified resistance phenotype-associated methylation loci observed in the cell line models. We identified different loci being characteristic for the different resistant cell lines. Interestingly, 53% of the identified loci were Alu sequences- especially the Alu Y subfamily. Furthermore, we identified an enrichment of Alu Y sequences that likely results from increased integration of new copies of Alu Y sequence in the drug-resistant cell lines. In the clinical samples, SOX1 and other SOX gene family members were shown to display variable DNA methylation states in their gene regions. The Alu Y sequences showed remarkable variation in DNA methylation states across the clinical samples.

Conclusion

Our findings imply a crucial role of Alu Y in colorectal cancer drug resistance. Our study underscores the complexity of colorectal cancer aggravated by mobility of Alu elements and stresses the importance of personalized strategies, using a systematic and dynamic view, for effective cancer therapy.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1552-y) contains supplementary material, which is available to authorized users.     

Real-World Efficiency of Pharmacogenetic Screening for Carbamazepine-Induced Severe Cutaneous Adverse Reactions

Objectives

We evaluated the cost and efficiency of routine HLA-B*15∶02 screening to prevent carbamazepine-induced Stevens-Johnson syndrome and toxic epidermal necrolysis (CBZ-SJS/TEN) in Hong Kong.

Methods

Data were extracted from patients who commenced CBZ as the first-ever AED treatment or tested for HLA-B*15∶02 allele in three years before policy implementation (pre-policy: 16 September 2005 to 15 September 2008) and three years after (post-policy: 16 September 2008 to 15 September 2011). Using published unit costs, we estimated the cost of screening by comparing the costs to prevent and treat CBZ-SJS/TEN. We compared the number of person-tests needed and the cost to prevent resultant death with cancer screening programs.

Results

The number of screening tests needed to prevent one case of CBZ-SJS/TEN was 442, and to prevent one resultant death was 1,474 to 8,840. The screening cost was $332 per person, of which 42% was attributed to an additional consultation to review result and prescribe appropriate medication. HLA-B*15∶02 screening expended $146,749 to prevent a case of CBZ-SJS/TEN, and $489,386– $2,934,986 to prevent a resultant death. The corresponding numbers of tests and costs for mammography and Pap smear to prevent death due to breast and cervical cancers were 7,150 and 7,000, and $614,900 and $273,000, respectively. Comparing to the SJS/TEN treatment cost, HLA-B*15∶02 screening would become cost saving if a point-of-care test of less than $37 was available.

Conclusions

HLA-B*15∶02 screening is as efficient as mammography and Pap smear in preventing death. Development of point-of-care testing will vastly improve efficiency.     

Mycophenolic acid counteracts B cell proliferation and plasmablast formation in patients with systemic lupus erythematosus

Introduction

Clinical trials revealed a high efficacy of mycophenolate mofetil (MMF) in inducing and maintaining remission in patients with class III-V-lupus nephritis. Also extrarenal manifestations respond to MMF treatment. However, few attempts have been undertaken to delineate its mechanism of action in systemic lupus erythematosus (SLE) a disease characterized by enhanced B cell activation.

Methods

Clinical and paraclinical parameters of 107 patients with SLE were recorded consecutively and analyzed retrospectively. Patients were divided into treatment groups (MMF: n = 39, azathioprine (AZA) n = 30 and controls without immunosuppressive therapy n = 38). To further delineate the effect of mycophenolic acid (MPA) on naive and memory B cells in vitro assays were performed.

Results

Although patients taking AZA flared more frequently than patients on MMF or controls, the analysis of clinical parameters did not reveal significant differences. However, profound differences in paraclinical parameters were found. B cell frequencies and numbers were significantly higher in patients taking MMF compared to those on AZA but lower numbers and frequencies of plasmablasts were detected compared to AZA-treated patients or controls. Notably, MMF treatment was associated with a significantly higher frequency and number of transitional B cells as well as naive B cells compared to AZA treatment. Differences in T cell subsets were not significant. MPA abrogated in vitro proliferation of purified B cells completely but had only moderate impact on B cell survival.

Conclusions

The thorough inhibition of B cell activation and plasma cell formation by MMF might explain the favorable outcomes of previous clinical trials in patients with SLE, since enhanced B cell proliferation is a hallmark of this disease.