Proteins Marking the Sequence of Genotoxic Signaling from Irradiated Mesenchymal Stromal Cells to CD34+ Cells.

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Separase activity distribution can be a marker of major molecular response and proliferation of CD34+ cells in TKI-treated chronic myeloid leukemia patients.

Separase, a cysteine endopeptidase, is a key player in mitotic sister chromatid separation, replication fork dynamics, and DNA repair. Aberrant expression and/or altered separase proteolytic activity are associated with aneuploidy, tumorigenesis, and disease progression. Since genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML), we have comparatively examined separase proteolytic activity in TKI-treated chronic phase CML. Separase proteolytic activity was analyzed on single cell level in 88 clinical samples and in 14 healthy controls by a flow cytometric assay. In parallel, BCR-ABL1 gene expression and replication fork velocity were measured by qRT-PCR and DNA fiber assays, respectively. The separase activity distribution (SAD) value indicating the occurrence of MNCs with elevated separase proteolytic activity within samples was found to positively correlate with BCR-ABL1 gene expression levels and loss of MMR (relapse) throughout routine BCR-ABL1 monitoring. Analyses of CD34+ cells and MNCs fractionized by flow cytometric cell sorting according to their separase activity levels (H- and L-fractions) revealed that CD34+ cells with elevated separase activity levels (H-fractions) displayed enhanced proliferation/viability when compared with cells with regular (L-fraction) separase activity (mean 3.3-fold, p = 0.0011). BCR-ABL1 gene expression positivity prevailed in MNC H-fractions over L-fractions (42% vs. 8%, respectively). Moreover, expanding CD34+ cells of H-fractions showed decreased replication fork velocity compared with cells of L-fractions (p < 0.0001). Our data suggests an association between high separase activity, residual BCR-ABL1 gene expression, and enhanced proliferative capacity in hematopoietic cells within the leukemic niche of TKI-treated chronic phase CML.

DNA Damage and DNA Damage Response in Chronic Myeloid Leukemia.

DNA damage and alterations in the DNA damage response (DDR) are critical sources of genetic instability that might be involved in BCR-ABL1 kinase-mediated blastic transformation of chronic myeloid leukemia (CML). Here, increased DNA damage is detected by γH2AX foci analysis in peripheral blood mononuclear cells (PBMCs) of de novo untreated chronic phase (CP)-CML patients (n = 5; 2.5 γH2AX foci per PBMC ± 0.5) and blast phase (BP)-CML patients (n = 3; 4.4 γH2AX foci per PBMC ± 0.7) as well as CP-CML patients with loss of major molecular response (MMR) (n = 5; 1.8 γH2AX foci per PBMC ± 0.4) when compared to DNA damage in PBMC of healthy donors (n = 8; 1.0 γH2AX foci per PBMC ± 0.1) and CP-CML patients in deep molecular response or MMR (n = 26; 1.0 γH2AX foci per PBMC ± 0.1). Progressive activation of erroneous non-homologous end joining (NHEJ) repair mechanisms during blastic transformation in CML is indicated by abundant co-localization of γH2AX/53BP1 foci, while a decline of the DDR is suggested by defective expression of (p-)ATM and (p-)CHK2. In summary, our data provide evidence for the accumulation of DNA damage in the course of CML and suggest ongoing DNA damage, erroneous NHEJ repair mechanisms, and alterations in the DDR as critical mediators of blastic transformation in CML.

Modulation of human endogenous retrovirus (HERV) transcription during persistent and de novo HIV-1 infection.

BACKGROUND: The human genome contains multiple LTR elements including human endogenous retroviruses (HERVs) that together account for approximately 8-9% of the genomic DNA. At least 40 different HERV groups have been assigned to three major HERV classes on the basis of their homologies to exogenous retroviruses. Although most HERVs are silenced by a variety of genetic and epigenetic mechanisms, they may be reactivated by environmental stimuli such as exogenous viruses and thus may contribute to pathogenic conditions. The objective of this study was to perform an in-depth analysis of the influence of HIV-1 infection on HERV activity in different cell types. RESULTS: A retrovirus-specific microarray that covers major HERV groups from all three classes was used to analyze HERV transcription patterns in three persistently HIV-1 infected cell lines of different cellular origins and in their uninfected counterparts. All three persistently infected cell lines showed increased transcription of multiple class I and II HERV groups. Up-regulated transcription of five HERV taxa (HERV-E, HERV-T, HERV-K (HML-10) and two ERV9 subgroups) was confirmed by quantitative reverse transcriptase PCR analysis and could be reversed by knock-down of HIV-1 expression with HIV-1-specific siRNAs. Cells infected de novo by HIV-1 showed stronger transcriptional up-regulation of the HERV-K (HML-2) group than persistently infected cells of the same origin. Analysis of transcripts from individual members of this group revealed up-regulation of predominantly two proviral loci (ERVK-7 and ERVK-15) on chromosomes 1q22 and 7q34 in persistently infected KE37.1 cells, as well as in de novo HIV-1 infected LC5 cells, while only one single HML-2 locus (ERV-K6) on chromosome 7p22.1 was activated in persistently infected LC5 cells. CONCLUSIONS: Our results demonstrate that HIV-1 can alter HERV transcription patterns of infected cells and indicate a correlation between activation of HERV elements and the level of HIV-1 production. Moreover, our results suggest that the effects of HIV-1 on HERV activity may be far more extensive and complex than anticipated from initial studies with clinical material.

Impact of unbalanced minor route versus major route karyotypes at diagnosis on prognosis of CML.

Major route additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukaemia (CML) indicate an increased risk of progression and shorter survival. Since major route ACA are almost always unbalanced, it is unclear whether other unbalanced ACA at diagnosis also confer an unfavourable prognosis. On the basis of 1348 Philadelphia chromosome-positive chronic phase patients of the randomized CML study IV, we examined the impact of unbalanced minor route ACA at diagnosis versus major route ACA on prognosis. At diagnosis, 1175 patients (87.2 %) had a translocation t(9;22)(q34;q11) and 74 (5.5 %) a variant translocation t(v;22) only, while a loss of the Y chromosome (-Y) was present in addition in 44 (3.3 %), balanced or unbalanced minor route ACA each in 17 (1.3 %) and major route ACA in 21 (1.6 %) cases. Patients with unbalanced minor route ACA had no significantly different cumulative incidences of complete cytogenetic remission or major molecular remission and no significantly different progression-free survival (PFS) or overall survival (OS) than patients with t(9;22), t(v;22), -Y and balanced minor route karyotypes. In contrast, patients with major route ACA had a shorter OS and PFS than all other groups (all pairwise comparisons to each of the other groups: p ≤ 0.015). Five-year survival probabilities were for t(9;22) 91.4 % (95 % CI 89.5-93.1), t(v; 22) 87 % (77.2-94.3), -Y 89.0 % (76.7-97.0), balanced 100 %, unbalanced minor route 92.3 % (72.4-100) and major route 52.2 % (28.2-75.5). We conclude that only major route, but not balanced or unbalanced minor route ACA at diagnosis, has a negative impact on prognosis of CML.

Identification and functional characterization of imatinib-sensitive DTD1-PDGFRB and CCDC88C-PDGFRB fusion genes in eosinophilia-associated myeloid/lymphoid neoplasms.

Eosinophilia-associated myeloid neoplasms with rearrangement of chromosome bands 5q31-33 are frequently associated with PDGFRB fusion genes, which are exquisitely sensitive to treatment with imatinib. In search for novel fusion partners of PDGFRB, we analyzed three cases with translocation t(5;20)(q33;p11), t(5;14)(q33;q32), and t(5;17;14)(q33;q11;q32) by 5'-rapid amplification of cDNA ends polymerase chain reaction (5'-RACE-PCR) and DNA-based long-distance inverse PCR (LDI-PCR) with primers derived from PDGFRB. LDI-PCR revealed a fusion between CCDC88C exon 25 and PDGFRB exon 11 in the case with t(5;17;14)(q33;q11;q32) while 5'-RACE-PCR identified fusions between CCDC88C exon 10 and PDGFRB exon 12 and between DTD1 exon 4 and PDGFRB exon 12 in the cases with t(5;14)(q33;q32) and t(5;20)(q33;p11), respectively. The PDGFRB tyrosine-kinase domain is predicted to be retained in all three fusion proteins. The partner proteins contained coiled-coil domains or other domains, which putatively lead to constitutive activation of the PDGFRB fusion protein. In vitro functional analyses confirmed transforming activity and imatinib-sensitivity of the fusion proteins. All three patients achieved rapid and durable complete hematologic remissions on imatinib.

Centrosome aberrations in bone marrow cells from patients with myelodysplastic syndromes correlate with chromosomal instability.

Centrosomes play important roles in the maintenance of genetic stability and centrosomal aberrations are a hallmark of cancer. Deregulation of centriole duplication leads to supernumerary centrosomes, sister chromatid missegregation and could result in chromosomal instability (CIN) and aneuploidy. CIN is a common feature in at least 45% of patients with myelodysplastic syndromes (MDS). Therefore, we sought to investigate the centrosomal status and its role for development of CIN in bone marrow (BM) cells of MDS patients. BM cells of 34 MDS patients were examined cytogenetically. Furthermore, cells were immunostained with a centrosome-specific antibody to pericentrin to analyze the centrosomal status. Umbilical cord blood specimens and BM cells of healthy persons (n = 11 and n = 4) served as controls. In addition, the protein expression of the protease separase responsible for genetic stability was examined by western blot analysis. Centrosome abnormalities were detected in 10% (range, 4-17%) of cells of MDS samples, but in only 2% (range, 0-4%) of cells of healthy controls. Normal karyotypes were found in control cells and in BM cells of 16/34 MDS patients. The incidence of centrosomal alterations was higher in BM cells of patients with cytogenetic alterations (mean, 12%) compared to BM cells of patients without cytogenetic changes (mean, 7%). Our results indicate that centrosome alterations are a common and early detectable feature in MDS patients and may contribute to the acquisition of chromosomal aberrations. We assume that centrosome defects could be involved in disease progression and may serve as a future prognostic marker.

SNP array analysis of acute promyelocytic leukemia may be of prognostic relevance and identifies a potential high risk group with recurrent deletions on chromosomal subband 1q31.3.

To search for new copy number alterations (CNAs) in acute promyelocytic leukemia (APL), we analyzed DNA from leukemic blasts of 93 acute promyelocytic leukemia (APL) patients with Genome-Wide SNP 6.0 arrays (SNP-A). We identified 259 CNAs consisting of 170 heterozygous deletions, 82 amplifications, and 7 regions of copy number neutral loss of heterozygosity. One of the most common CNAs was a deletion on chromosomal subband 1q31.3 in 13 of 93 (14%) patients encompassing the coding regions for the microRNAs mir181a1/b1. In multivariable analysis with the covariates age, white blood cell count, platelet count, and FLT3-ITD/FLT3 D835 mutations we found that after adjustment for patients' age (P<0.0001), patients with 2 or more CNAs detected by SNP-A had a higher risk of death (hazard ratio=5.942, P=0.0015) than patients with 0 or 1 CNA. Deletions of 1q31.3 were associated with a higher number of CNAs (median 2 vs. 8, P<0.0001) and were a strong independent prognostic factor for an increased risk of relapse (hazard ratio=28.9, P=0.0031). This study presents a comprehensive assessment of new CNAs as pathomechanistically relevant targets and possible prognostic factors which could refine risk stratification of APL.

Reverse Transcription Can Critically Impact the Diagnostic Outcome of BCR::ABL1 Quantitative Real-Time RT-PCR.

Reverse transcriptases (RT) are essential tools in BCR::ABL1 fusion transcript monitoring in chronic myeloid leukemia (CML). The RT type and cDNA priming method may impair the stoichiometry of cDNA synthesis, thereby potentially introducing a bias in BCR::ABL1 qRT-PCR data. Using the Acrometrix™ BCR::ABL1 reference panel and 37 clinical specimens, we have comparatively investigated the performance of the RTs MLV and SuperScript IV with random hexamer vs. target-specific priming. Quantitative RT-PCR results identified the priming type and RT type as major factors for diagnostic data variation, mainly due to the different efficacies of processing BCR::ABL1 low-copy-numbers (<50) compared to GUSB or ABL1 high-copy targets. The impairment of SuperScript IV in processing low- and high-copy-number RNA targets equally was not reflected by the diagnostically relevant Log (BCR::ABL1/GUSB%) values. Therefore, the correct representation of housekeeping and BCR::ABL1 target genes should have priority when aiming at as high a number of housekeeping gene copies as possible. Our data suggest that for improving BCR::ABL1 assay sensitivity, increased RNA/cDNA amounts and the use of distinct RT/priming combinations are advantageous. However, for inter-laboratory harmonization, the proper conversion factor according to the CML international standard (IS) has to be reevaluated each time the grade of RT is changed.

Gene Expression Pattern of ESPL1, PTTG1 and PTTG1IP Can Potentially Predict Response to TKI First-Line Treatment of Patients with Newly Diagnosed CML.

The achievement of major molecular response (MMR, BCR::ABL1 ≤ 0.1% IS) within the first year of treatment with tyrosine kinase inhibitors (TKI) is a milestone in the therapeutic management of patients with newly diagnosed chronic myeloid leukemia (CML). We analyzed the predictive value of gene expression levels of ESPL1/Separase, PTTG1/Securin and PTTG1IP/Securin interacting protein for MMR achievement within 12 months. Relative expression levels (normalized to GUSB) of ESPL1, PTTG1 and PTTG1IP in white blood cells of patients (responders n = 46, non-responders n = 51) at the time of diagnosis were comparatively analyzed by qRT-PCR. 3D scatter plot analysis combined with a distance analysis performed with respect to a commonly calculated centroid center resulted in a trend to larger distances for non-responders compared to the responder cohort (p = 0.0187). Logistic regression and analysis of maximum likelihood estimates revealed a positive correlation of distance (cut-off) with non-achieving MMR within 12 months (p = 0.0388, odds ratio 1.479, 95%CI: 1.020 to 2.143). Thus, 10% of the tested non-responders (cut-off ≥ 5.9) could have been predicted already at the time of diagnosis. Future scoring of ESPL1, PTTG1 and PTTG1IP transcript levels may be a helpful tool in risk stratification of CML patients before initiation of TKI first = line treatment.

Development of novel PCR assays to detect azole resistance-mediating mutations of the Aspergillus fumigatus cyp51A gene in primary clinical samples from neutropenic patients.

The increasing incidence of azole resistance in Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised/hematological patients emphasizes the need to improve the detection of resistance-mediating cyp51A gene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of the Aspergillus fumigatus cyp51A gene to establish PCR assays with consecutive DNA sequence analysis to detect and identify the A. fumigatus cyp51A tandem repeat (TR) mutation in the promoter region and the L98H and M220 alterations directly in clinical samples. After testing of the sensitivity and specificity of the assays using serially diluted A. fumigatus and human DNA, A. fumigatus cyp51A gene fragments of about 150 bp potentially carrying the mutations were amplified directly from primary clinical samples and subsequently DNA sequenced. The determined sensitivities of the PCR assays were 600 fg, 6 pg, and 4 pg of A. fumigatus DNA for the TR, L98H, and M220 mutations, respectively. There was no cross-reactivity with human genomic DNA detectable. Sequencing of the PCR amplicons for A. fumigatus wild-type DNA confirmed the cyp51A wild-type sequence, and PCR products from one azole-resistant A. fumigatus isolate showed the L98H and TR mutations. The second azole-resistant isolate revealed an M220T alteration. We consider our assay to be of high epidemiological and clinical relevance to detect azole resistance and to optimize antifungal therapy in patients with IA.

A new aberrantly spliced BCR-ABL1 transcript variant (e13a1) identified in routine monitoring using different quantitative reverse transcription polymerase chain reaction techniques in a patient with chronic myeloid leukemia.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of BCR-ABL1 transcript level is an essential part of routine disease monitoring in patients with chronic myeloid leukemia. One patient sample (e13a2 transcript detected by nested PCR) attracted attention by revealing an aberrantly spliced BCR-ABL1 transcript variant e13a1. The last 38 base pairs (bp) of BCR exon 13 were replaced by a 37 bp insertion of the ABL1 intron 1-2/exon 1 sequence. The rare aberrant BCR-ABL1 fusion transcript can cause discrepancies in molecular diagnostics. This scenario highlights the importance of an individual characterization of the BCR-ABL1 fusion sequence in case of unclear qRT-PCR results.

Evidence for Recombinant GRP78, CALR, PDIA3 and GPI as Mediators of Genetic Instability in Human CD34+ Cells.

Soluble factors released from irradiated human mesenchymal stromal cells (MSC) may induce genetic instability in human CD34+ cells, potentially mediating hematologic disorders. Recently, we identified four key proteins in the secretome of X-ray-irradiated MSC, among them three endoplasmic reticulum proteins, the 78 kDa glucose-related protein (GRP78), calreticulin (CALR), and protein disulfide-isomerase A3 (PDIA3), as well as the glycolytic enzyme glucose-6-phosphate isomerase (GPI). Here, we demonstrate that exposition of CD34+ cells to recombinant GRP78, CALR, PDIA3 and GPI induces substantial genetic instability. Increased numbers of γH2AX foci (p < 0.0001), centrosome anomalies (p = 0.1000) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells upon incubation with these factors. Specifically, γH2AX foci were found to be induced 4−5-fold in response to any individual of the four factors, and centrosome anomalies by 3−4 fold compared to control medium, which contained none of the recombinant proteins. Aberrant metaphases, not seen in the context of control medium, were detected to a similar extent than centrosome anomalies across the four factors. Notably, the strongest effects were observed when all four factors were collectively provided. In summary, our data suggest that specific components of the secretome from irradiated MSC act as mediators of genetic instability in CD34+ cells, thereby possibly contributing to the pathogenesis of radiation-induced hematologic disorders beyond direct radiation-evoked DNA strand breaks.

Genotoxic Bystander Signals from Irradiated Human Mesenchymal Stromal Cells Mainly Localize in the 10-100 kDa Fraction of Conditioned Medium.

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) < 10 kDa, (II) 10-100 kDa and (III) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10-100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ∨ 3.8 ± 0.3 aberrant metaphases per CD34+ cell sample; mean ± SEM) when compared to (I) < 10 kDa (0.19 ± 0.01 ∨ 0.3 ± 0.2) or (III) > 100 kDa fractions (0.23 ± 0.04 ∨ 0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ∨ 0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10-100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.

Detection of centrosome aberrations in disease-unrelated cells from patients with tumor treated with tyrosine kinase inhibitors.

OBJECTIVES: Tyrosine kinase inhibitors (TKIs) target various pathways associated with proliferation of aberrant clones in malignant diseases. Despite good response and acceptable tolerability, little is known concerning long-term toxicity. Furthermore, the influence of these inhibitors on disease-unrelated cells is not investigated yet. METHODS: Centrosome aberrations are hallmarks of various cancers. We sought to evaluate the effect of TKIs on centrosomes of disease-unrelated cells. We examined cells of the oral mucosa (OM) and fibroblasts of patients with chronic myeloid leukemia (CML) treated with dasatinib and bosutinib. Results were compared with data from patients with CML treated with imatinib or nilotinib and with data from patients suffering from renal and hepatocellular carcinomas (RCC/HCC) treated with sorafenib or sunitinib. Cells of healthy donors served as controls. RESULTS: OM cells (n = 12) and fibroblasts (n = 7) of patients with CML treated with dasatinib and OM cells of three patients with CML treated with bosutinib showed centrosomal alterations (mean, 14%) compared with 16 (10 OM and 6 fibroblasts) controls (mean, 3%). OM cells of five patients with CML and one patient with systemic mastocytosis treated with imatinib or nilotinib and of eight patients with RCC or HCC treated with sorafenib or sunitinib showed centrosome defects in a mean of 15%. CONCLUSIONS: Our data have shown that TKI treatment of tumor patients may influence centrosomes in disease-unrelated cells or tissues. This may be important with regard to various observed side effects.

Correction: High-risk additional chromosomal abnormalities at low blast counts herald death by CML.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High-risk additional chromosomal abnormalities at low blast counts herald death by CML.

Blast crisis is one of the remaining challenges in chronic myeloid leukemia (CML). Whether additional chromosomal abnormalities (ACAs) enable an earlier recognition of imminent blastic proliferation and a timelier change of treatment is unknown. One thousand five hundred and ten imatinib-treated patients with Philadelphia-chromosome-positive (Ph+) CML randomized in CML-study IV were analyzed for ACA/Ph+ and blast increase. By impact on survival, ACAs were grouped into high risk (+8, +Ph, i(17q), +17, +19, +21, 3q26.2, 11q23, -7/7q abnormalities; complex) and low risk (all other). The presence of high- and low-risk ACAs was linked to six cohorts with different blast levels (1%, 5%, 10%, 15%, 20%, and 30%) in a Cox model. One hundred and twenty-three patients displayed ACA/Ph+ (8.1%), 91 were high risk. At low blast levels (1-15%), high-risk ACA showed an increased hazard to die compared to no ACA (ratios: 3.65 in blood; 6.12 in marrow) in contrast to low-risk ACA. No effect was observed at blast levels of 20-30%. Sixty-three patients with high-risk ACA (69%) died (n = 37) or were alive after progression or progression-related transplantation (n = 26). High-risk ACA at low blast counts identify end-phase CML earlier than current diagnostic systems. Mortality was lower with earlier treatment. Cytogenetic monitoring is indicated when signs of progression surface or response to therapy is unsatisfactory.

Variable transcriptional activity of endogenous retroviruses in human breast cancer.

Human endogenous retroviruses (HERVs) account for up to 9% of the human genome and include more than 800 elements related to betaretroviruses. While mouse mammary tumor virus (MMTV) is the accepted etiological agent of mammary tumors in mice, the role of retroviral elements in human breast cancer remains elusive. Here, we performed a comprehensive microarray-based analysis of overall retroviral transcriptional activities in 46 mammary gland tissue specimens representing pairs of nonmalignant and tumor samples from 23 patients. An analysis of nonmalignant tissue samples revealed a distinct, mammary gland-specific HERV expression profile that consists of 18 constitutively active HERV taxa. For corresponding tumor samples, a general trend toward lower levels of HERV transcription was observed, suggesting common regulatory mechanisms. In various subsets of patients, however, increased transcript levels of single class I HERV families (HERV-T, HERV-E, and HERV-F) and several class II families, including HML-6, were detected. An analysis of transcribed HML-6 sequences revealed either the activation of some or the increased activity of several proviral loci. No evidence for MMTV or human MMTV-like virus transcripts was found, indicating that transcriptionally active, MMTV analogous, exogenous viruses were not present in the breast cancer samples analyzed.

Human Endogenous Retroviruses: Residues of Ancient Times Are Differentially Expressed in Crohn's Disease.

BACKGROUND: Eight percent of the human genome consists of human endogenous retroviruses (HERV). These genetic elements are remnants of ancient retroviral germ-line infections. Altered HERV expression is associated with several chronic inflammatory diseases. A physiological role of the HERV-derived proteins syncytin-1 and -2 has been described for the integrity of the human placental cell layer in terms of maintaining feto-maternal tolerance. The aim of this project was to investigate HERV expression in Crohn's disease (CD) with a further focus on syncytins in the gut. MATERIAL AND METHODS: Seventy-four ileal and colonic tissue samples of CD patients and healthy controls have been investigated for mRNA expression of major HERV groups by a comprehensive microarray screening. The most prominent differences have been validated by qRT-PCR. Immunohistochemistry (IHC), Western Blot (WB) and qRT-PCR were performed for syncytin-1 and -2. RESULTS: HERV microarray screening revealed a distinct expression profile in ileal and colonic tissue, as well as differential expression in CD compared to healthy controls. qRT-PCR validated differential expression of at least 3 HERV-groups in CD. qRT-PCR, IHC and WB showed a tissue-dependent diminished epithelial expression of syncytins in inflamed CD. CONCLUSION: For the first time, HERV expression has been comprehensively studied in the gut. Between CD and healthy controls we could show a tissue dependent differential HERV expression profile. Notably, we could show that syncytin-1 and -2 are expressed in the epithelial layer in ileal and colonic tissue samples, whereas their diminished tissue-dependent expression in inflamed CD might modulate inflammatory processes at the gut barrier.

Antileukemic Efficacy in Vitro of Talazoparib and APE1 Inhibitor III Combined with Decitabine in Myeloid Malignancies.

Malignant hematopoietic cells of myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemias (CMML) and acute myeloid leukemias (AML) may be vulnerable to inhibition of poly(ADP ribose) polymerase 1/2 (PARP1/2) and apurinic/apyrimidinic endonuclease 1 (APE1). PARP1/2 and APE1 are critical enzymes involved in single-strand break repair and base excision repair, respectively. Here, we investigated the cytotoxic efficacy of talazoparib and APE1 inhibitor III, inhibitors of PARP1/2 and APE1, in primary CD34+ MDS/CMML cell samples (n = 8; 4 MDS and 4 CMML) and in primary CD34+ or CD34- AML cell samples (n = 18) in comparison to healthy CD34+ donor cell samples (n = 8). Strikingly, talazoparib and APE1 inhibitor III demonstrated critical antileukemic efficacy in selected MDS/CMML and AML cell samples. Low doses of talazoparib and APE1 inhibitor III further increased the cytotoxic efficacy of decitabine in MDS/CMML and AML cells. Moreover, low doses of APE1 inhibitor III increased the cytotoxic efficacy of talazoparib in MDS/CMML and AML cells. In summary, talazoparib and APE1 inhibitor III demonstrated substantial antileukemic efficacy as single agents, in combination with decitabine, and combined with each other. Hence, our findings support further investigation of these agents in sophisticated clinical trials.