HTLV-I Tax Increases Genetic Instability by Inducing DNA Double Strand Breaks during DNA Replication and Switching Repair to NHEJ

Background

Appropriate responses to damaged DNA are indispensible for preserving genome stability and preventing cancer. Tumor viruses often target DNA repair machinery to achieve transformation. The Human T-cell leukemia virus type I (HTLV-I) is the only known transforming human retrovirus and the etiological agent of Adult T-cell Leukemia (ATLL). Although HTLV-I-transformed leukemic cells have numerous genetic lesions, the precise role of the viral tax gene in this process is not fully understood.

Results

Our results show a novel function of HTLV-I oncoprotein Tax as an inducer of genomic DNA double strand breaks (DDSB) during DNA replication. We also found that Tax acts as a potent inhibitor of homologous recombination (HR) DNA repair through the activation of the NF-kB pathway. These results were confirmed using HTLV-I molecular clones expressing Tax at physiological levels in a natural context. We further found that HTLV-I- and Tax-transformed cells are not more susceptible to DNA damaging agents and repair DNA lesions at a rate similar to that of normal cells. Finally, we demonstrated that during S phase, Tax-associated DDSB are preferentially repaired using the error-prone non-homologous end joining (NHEJ) pathway.

Conclusions

This study provides new insights in Tax effects on DNA repair and genome instability. Although it may not be self sufficient, the creation of DNA breaks and subsequent abnormal use of the non-conservative NHEJ DNA repair during the S phase in HTLV-I-infected Tax-expressing cells may cooperate with other factors to increase genetic and genome instability and favor transformation.     

Human T Cell Leukemia Virus Reactivation with Progression of Adult T-Cell Leukemia-Lymphoma

Background

Human T-cell leukemia virus-associated adult T-cell leukemia-lymphoma (ATLL) has a very poor prognosis, despite trials of a variety of different treatment regimens. Virus expression has been reported to be limited or absent when ATLL is diagnosed, and this has suggested that secondary genetic or epigenetic changes are important in disease pathogenesis.

Methods and Findings

We prospectively investigated combination chemotherapy followed by antiretroviral therapy for this disorder. Nineteen patients were prospectively enrolled between 2002 and 2006 at five medical centers in a phase II clinical trial of infusional chemotherapy with etoposide, doxorubicin, and vincristine, daily prednisone, and bolus cyclophosphamide (EPOCH) given for two to six cycles until maximal clinical response, and followed by antiviral therapy with daily zidovudine, lamivudine, and alpha interferon-2a for up to one year. Seven patients were on study for less than one month due to progressive disease or chemotherapy toxicity. Eleven patients achieved an objective response with median duration of response of thirteen months, and two complete remissions. During chemotherapy induction, viral RNA expression increased (median 190-fold), and virus replication occurred, coincident with development of disease progression.

Conclusions

EPOCH chemotherapy followed by antiretroviral therapy is an active therapeutic regimen for adult T-cell leukemia-lymphoma, but viral reactivation during induction chemotherapy may contribute to treatment failure. Alternative therapies are sorely needed in this disease that simultaneously prevent virus expression, and are cytocidal for malignant cells.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00041327","term_id":"NCT00041327"}}NCT00041327     

Notch ankyrin repeat domain variation influences leukemogenesis and Myc transactivation

Background

The functional interchangeability of mammalian Notch receptors (Notch1-4) in normal and pathophysiologic contexts such as cancer is unsettled. We used complementary in vivo, cell-based and structural analyses to compare the abilities of activated Notch1-4 to support T cell development, induce T cell acute lymphoblastic leukemia/lymphoma (T-ALL), and maintain T-ALL cell growth and survival.

Principal Findings

We find that the activated intracellular domains of Notch1-4 (ICN1-4) all support T cell development in mice and thymic organ culture. However, unlike ICN1-3, ICN4 fails to induce T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) and is unable to rescue the growth of Notch1-dependent T-ALL cell lines. The ICN4 phenotype is mimicked by weak gain-of-function forms of Notch1, suggesting that it stems from a failure to transactivate one or more critical target genes above a necessary threshold. Experiments with chimeric receptors demonstrate that the Notch ankyrin repeat domains differ in their leukemogenic potential, and that this difference correlates with activation of Myc, a direct Notch target that has an important role in Notch-associated T-ALL.

Conclusions/Significance

We conclude that the leukemogenic potentials of Notch receptors vary, and that this functional difference stems in part from divergence among the highly conserved ankyrin repeats, which influence the transactivation of specific target genes involved in leukemogenesis.     

Genome Wide Association Study of Fetal Hemoglobin in Sickle Cell Anemia in Tanzania

Background

Fetal hemoglobin (HbF) is an important modulator of sickle cell disease (SCD). HbF has previously been shown to be affected by variants at three loci on chromosomes 2, 6 and 11, but it is likely that additional loci remain to be discovered.

Methods and Findings

We conducted a genome-wide association study (GWAS) in 1,213 SCA (HbSS/HbSβ⁰) patients in Tanzania. Genotyping was done with Illumina Omni2.5 array and imputation using 1000 Genomes Phase I release data. Association with HbF was analysed using a linear mixed model to control for complex population structure within our study. We successfully replicated known associations for HbF near BCL11A and the HBS1L-MYB intergenic polymorphisms (HMIP), including multiple independent effects near BCL11A, consistent with previous reports. We observed eight additional associations with P<10⁻⁶. These associations could not be replicated in a SCA population in the UK.

Conclusions

This is the largest GWAS study in SCA in Africa. We have confirmed known associations and identified new genetic associations with HbF that require further replication in SCA populations in Africa.     

Modulation of peripheral T-cell function by interleukin-7 in rheumatoid arthritis

Introduction

Interleukin-7 (IL-7) is a cytokine essential for T-cell lymphopoiesis, survival and polarization with an emerging role in autoimmunity. We previously demonstrated reduced levels of circulating IL-7 in rheumatoid arthritis (RA), although high amounts are expressed in joints, suggesting differences between systemic and synovial effects. We observed healthy levels of IL-7 in 48% of RA patients in clinical remission (CR) and aimed to investigate the consequences of IL-7 deficiency on T-cell responses.

Methods

We used RA patients with active disease and in CR presenting various levels of IL-7, to investigate its modulatory effects on T cells by analysing responses to phyto-haemagglutinin (PHA), expression of polarization or survival factors, or suppression by regulatory T cells (Tregs).

Results

IL-7 levels were normal (>10 pg/ml) in 48% of RA patients in CR. Amongst 63 CR patients followed up for 18 months, lack of IL-7 recovery was observed in 13 out of 15 (86%) patients experiencing relapse but only 11 out of 48 (23%) of those who did not (P = 0.0002). Binary regressions showed high significance for below normal IL-7 levels for self-reported maternal family history of arthritis (odds ratio (OR): 7.66, P = 0.006) and a trend for smoking (OR: 3.33, P = 0.068) with no further demographic or clinical associations. Serum IL-7 correlated with restored CD4⁺T-cell response to PHA (rho = 0.879); this was not related to an increase in T-cell proliferation capacity or expression of survival factors B-cell lymphoma 2 (BCL2) and BCL2-associated protein X (BAX). Expression of Th1 polarization factor (TBET) was also dependent on exposure to IL-7 in vivo (rho = 0.600). In contrast CD25^highTregs’ response to PHA was not affected by in vivo IL-7, but their suppression capabilities were related to circulating IL-7 (rho = 0.589). Co-stimulation with IL-7 (mimicking the joint environment) increased responsiveness of CD4⁺T-cells to PHA, lowering the ability of CD25^highTregs to suppress them.

Conclusions

Our data demonstrate that IL-7 has a critical role in modulating T-cell function in vivo, possibly explaining opposing effects observed systemically and in the joint. Lack of IL-7 recovery in CR by maintaining a suppressed immune system may be a determinant factor in the occurrence of relapse.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0511-3) contains supplementary material, which is available to authorized users.     

The Diagnostic Value of DNA Methylation in Leukemia: A Systematic Review and Meta-Analysis

Background

Accumulating evidence supports a role of DNA methylation in the pathogenesis of leukemia. The aim of our study was to evaluate the potential genes with aberrant DNA methylation in the prediction of leukemia risk by a comprehensive meta-analysis of the published data.

Methods

A series of meta-analyses were done among the eligible studies that were harvested after a careful filtration of the searching results from PubMed literature database. Mantel-Haenszel odds ratios and 95% confidence intervals were computed for each methylation event assuming the appropriate model.

Results

A total of 535 publications were initially retrieved from PubMed literature database. After a three-step filtration, we harvested 41 case-control articles that studied the role of gene methylation in the prediction of leukemia risk. Among the involving 30 genes, 20 genes were shown to be aberrantly methylated in the leukemia patients. A further subgroup meta-analysis by subtype of leukemia showed that CDKN2A, CDKN2B, ID4 genes were significantly hypermethylated in acute myeloid leukemia.

Conclusions

Our meta-analyses identified strong associations between a number of genes with aberrant DNA methylation and leukemia. Further studies should be required to confirm the results in the future.     

BCL10GFP fusion protein as a substrate for analysis of determinants required for Mucosa-Associated Lymphoid Tissue 1 (MALT1)-mediated cleavage

Background

MALT1 belongs to a family of paracaspase and modulates NF-κB signaling pathways through its scaffolding function and proteolytic activity. MALT1 cleaves protein substrates after a positively charged Arginine residue. BCL10, a 233 amino acids polypeptide, is identified as one of the MALT1 proteolytic substrates. MALT1 cleaves BCL10 at the C-terminal end of Arg228. A mere 5 amino acids difference between the substrate and the proteolytic product made it difficult to tell whether the cleavage event took place by using a simple western blot analysis. Here, BCL10GFP was constructed and utilized to examine the specificity and domain determinants for MALT1 cleavage in cells.

Methods

Various BCL10GFP constructs were transfected into HEK293T cell with MALT1 construct by using calcium phosphate-DNA precipitation method. Lysates of transfectants were resolved by SDS/PAGE and analyzed by western blot analysis.

Results

BCL10GFP was proteolytically processed by MALT1 as BCL10. The integrity of caspase recruitment domain (CARD) and MALT1-interacting domain on BCL10 were required for MALT1 proteolytic activity. Besides the invariant P1 cleavage site Arg228, P4 Leu225 played a role in defining BCL10 as a good substrate for MALT1.

Conclusions

We offered a way of monitoring the catalytic activity of MALT1 in HEK293T cells using BCL10GFP as a substrate. BCL10GFP can be utilized as a convenient tool for studying the determinants for efficient MALT1 cleavage in HEK293T cells     

HBV-related hepatocellular carcinoma susceptibility gene KIF1B is not associated with development of chronic hepatitis B

Background

A recent genome-wide association study has identified a new susceptibility locus, kinesin family member 1B gene (KIF1B), strongly associated with progression from chronic hepatitis B (CHB) to hepatitis B virus-related hepatocellular carcinoma (HCC) in Chinese population, this study was carried out to explore the role of the genetic variants in KIF1B in the development of chronic hepatitis B.

Methodology/Principal Findings

Three KIF1B polymorphisms (rs8019, rs17401924, and rs17401966) were selected and genotyped in 473 CHB patients and 580 controls with no history of CHB. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression model. None of these three SNPs showed association with CHBs after adjusting for age and gender. Equivalence-based method analysis confirmed the absence of association. In the further haplotype analysis, three common haplotypes were observed in this study population, but no significant effect was also found for haplotypes in the progression to CHB.

Conclusions/Significance

This study showed the new locus identified for HCC, KIF1B, was not associated with progression to CHB, implying distinct genetic susceptibility factor contributes to the progression from hepatitis B virus infection to HCC. Nevertheless, further comprehensive analyses are warranted to dissect the mechanism.     

Suppression of miR-221 inhibits glioma cells proliferation and invasion via targeting SEMA3B

Background

Gliomas are the most common primary tumors in the central nervous system. Due to complicated signaling pathways involved in glioma progression, effective targets for treatment and biomarkers for prognosis prediction are still scant.

Results

In this study we revealed that a new microRNA (miR), the miR-221, was highly expressed in the glioma cells, and suppression of miR-221 resulted in decreased cellular proliferation, migration, and invasion in glioma cells. Mechanistic experiments validated that miR-221 participates in regulating glioma cells proliferation and invasion via suppression of a direct target gene, the Semaphorin 3B (SEMA3B). The rescue experiment with miR-221 and SEMA3B both knockdown results in significant reversion of miR-221 induced phenotypes.

Conclusion

Taken together, our findings highlight an unappreciated role for miR-221 and SEMA3B in glioma.     

Candidate Sequence Variants and Fetal Hemoglobin in Children with Sickle Cell Disease Treated with Hydroxyurea

Background

Fetal hemoglobin level is a heritable complex trait that strongly correlates swith the clinical severity of sickle cell disease. Only few genetic loci have been identified as robustly associated with fetal hemoglobin in patients with sickle cell disease, primarily adults. The sole approved pharmacologic therapy for this disease is hydroxyurea, with effects largely attributable to induction of fetal hemoglobin.

Methodology/Principal Findings

In a multi-site observational analysis of children with sickle cell disease, candidate single nucleotide polymorphisms associated with baseline fetal hemoglobin levels in adult sickle cell disease were examined in children at baseline and induced by hydroxyurea therapy. For baseline levels, single marker analysis demonstrated significant association with BCL11A and the beta and epsilon globin loci (HBB and HBE, respectively), with an additive attributable variance from these loci of 23%. Among a subset of children on hydroxyurea, baseline fetal hemoglobin levels explained 33% of the variance in induced levels. The variant in HBE accounted for an additional 13% of the variance in induced levels, while variants in the HBB and BCL11A loci did not contribute beyond baseline levels.

Conclusions/Significance

These findings clarify the overlap between baseline and hydroxyurea-induced fetal hemoglobin levels in pediatric disease. Studies assessing influences of specific sequence variants in these and other genetic loci in larger populations and in unusual hydroxyurea responders are needed to further understand the maintenance and therapeutic induction of fetal hemoglobin in pediatric sickle cell disease.     

Antitumoral effects of cyclin-dependent kinases inhibitors CR8 and MR4 on chronic myeloid leukemia cell lines

Background

Although Imatinib mesylate has revolutionized the treatment of chronic myeloid leukemia, some patients develop resistance with progression of leukemia. Alternative or additional targeting of signalling pathways deregulated in Bcr-Abl-driven chronic myeloid leukemia may provide a feasible option for improving clinical response and overcoming resistance.

Results

In this study, we investigate ability of CR8 isomers (R-CR8 and S-CR8) and MR4, three derivatives of the cyclin-dependent kinases (CDKs) inhibitor Roscovitine, to exert anti-leukemic activities against chronic myeloid leukemia in vitro and then, we decipher their mechanisms of action. We show that these CDKs inhibitors are potent inducers of growth arrest and apoptosis of both Imatinib-sensitive and –resistant chronic myeloid leukemia cell lines. CR8 and MR4 induce dose-dependent apoptosis through mitochondrial pathway and further caspases 8/10 and 9 activation via down-regulation of short-lived survival and anti-apoptotic factors Mcl-1, XIAP and survivin which are strongly implicated in survival of Bcr-Abl transformed cells.

Conclusions

These results suggest that CDK inhibitors may constitute a complementary approach to treat chronic myeloid leukemia.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0163-x) contains supplementary material, which is available to authorized users.     

Propyl-2-(8-(3,4-Difluorobenzyl)-2′,5′-Dioxo-8-Azaspiro[Bicyclo[3.2.1] Octane-3,4′-Imidazolidine]-1′-yl) Acetate Induces Apoptosis in Human Leukemia Cells through Mitochondrial Pathway following Cell Cycle Arrest

Background

Due to the functional defects in apoptosis signaling molecules or deficient activation of apoptosis pathways, leukemia has become an aggressive disease with poor prognosis. Although the majority of leukemia patients initially respond to chemotherapy, relapse is still the leading cause of death. Hence targeting apoptosis pathway would be a promising strategy for the improved treatment of leukemia. Hydantoin derivatives possess a wide range of important biological and pharmacological properties including anticancer properties. Here we investigated the antileukemic activity and mechanism of action of one of the potent azaspiro hydantoin derivative, (ASHD).

Materials and Methods

To investigate the antileukemic efficacy of ASHD, we have used MTT assay, cell cycle analysis by FACS, tritiated thymidine incorporation assay, Annexin V staining, JC1 staining and western blot analysis.

Results

Results showed that ASHD was approximately 3-fold more potent than the parent compounds in inducing cytotoxicity. Tritiated thymidine assay in conjunction with cell cycle analysis suggests that ASHD inhibited the growth of leukemic cells. The limited effect of ASHD on cell viability of normal cells indicated that it may be specifically directed to cancer cells. Translocation of phosphatidyl serine, activation of caspase 3, caspase 9, PARP, alteration in the ratio of BCL2/BAD protein expression as well as the loss of mitochondrial membrane potential suggests activation of the intrinsic pathway of apoptosis.

Conclusion

These results could facilitate the future development of novel hydantoin derivatives as chemotherapeutic agents for leukemia.     

Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells

Background

We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin β _B-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A.

Methodology

hGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin β _B-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey''s test.

Principal Findings

GDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin β _B-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin β _B-subunit mRNA and inhibin B production were attenuated by adding FST.

Conclusion

GDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin β _B-subunit mRNA expression and inhibin B production in hGL cells.