Search

Finely tuned decidualization of endometrial stromal fibroblasts into decidual cells is crucial for successful implantation and a healthy pregnancy. Both insulin and androgens are known to modulate decidualization, however, their complex effect on this process has not been fully elucidated. As hyperinsulinemia and hyperandrogenism are associated in clinical conditions, we aimed to investigate the interaction between insulin and androgens on decidualization. Primary human endometrial stromal cells were decidualized in vitro in the presence of insulin and/or androgens (dihydrotestosterone (DHT), testosterone). Gene or protein expressions of decidualization markers were measured, and cells size characteristics were determined. Migration of decidualizing endometrial stromal cells and invasion of HTR‐8/SVneo trophoblast spheroids were assessed. We found that insulin and androgens in combination enhanced the upregulation of several decidualization markers including prolactin, tissue factor, tissue inhibitor of matrix metalloproteinase 3 and connexin‐43, and also interacted in modulating cell size characteristics resulting in enlarged decidualizing cells. However, insulin and DHT together restricted the migration of decidualizing cells and invasion of trophoblast spheroids. Our findings suggest that insulin and androgens interact to potentiate the process of decidualization. On the other hand, inhibited cell migration and trophoblast invasion might negatively impact the function of decidualizing endometrial stromal cells.

Recent studies suggest estradiol (E2)/natural progesterone (P) confers less breast cancer risk compared with conjugated equine estrogens (CEE)/synthetic progestogens. We investigate if differences in the regulation of breast cancer-related gene expression could provide some explanation. This study is a subset of a monocentric, 2-way, open observer-blinded, phase 4 randomized controlled trial on healthy postmenopausal women with climacteric symptoms (ClinicalTrials.gov; EUCTR-2005/001016-51). Study medication was two 28-day cycles of sequential hormone treatment with oral 0.625 mg CEE and 5 mg of oral medroxyprogesterone acetate (MPA) or 1.5 mg E2 as percutaneous gel/day with the addition of 200 mg oral micronized P. MPA and P were added days 15–28/cycle. Material from two core-needle breast biopsies in 15 women in each group was subject to quantitative PCR (Q-PCR). The primary endpoint was a change in breast carcinoma development gene expression. In the first eight consecutive women, RNA was extracted at baseline and after two months of treatment and subjected to microarray for 28856 genes and Ingenuity Pathways Analysis (IPA) to identify risk factor genes. Microarray analysis showed 3272 genes regulated with a fold-change of >±1.4. IPA showed 225 genes belonging to mammary-tumor development function: 198 for CEE/MPA vs. 34 for E2/P. Sixteen genes involved in mammary tumor inclination were subject to Q-PCR, inclining the CEE/MPA group towards an increased risk for breast carcinoma compared to the E2/P group at a very high significance level (p = 3.1 × 10−8, z-score 1.94). The combination of E2/P affected breast cancer-related genes much less than CEE/MPA.

Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors. Funding Agencies|Cancer Research Funds of Radiumhemmet [174283]; Thorsmans Stiftelse for Pre-leukemi Forskning; Gunnar Grimfors Gavofond for Hematologisk Forskning; Cathrine Everts Research Foundation; Lars Hierta Memorial Foundation; Emil Anderson Fund for Medical Research; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2013-08807]; Karolinska Institute Foundation and Funds; Karolinska InstituteKarolinska Institutet

Prokineticin 1 (PROK1) is a key regulator of embryo implantation and placentation, and its dysregulation is associated with pregnancy complications, such as pre‐eclampsia and foetal growth restriction. We have previously shown that insulin strongly enhances the expression of PROK1 in human decidualizing stromal cells. Here, we demonstrate that dihydrotestosterone (DHT), but not testosterone, potentiates insulin to up‐regulate PROK1 in these cells. However, the androgens alone do not influence the expression of PROK1. Our findings suggest that insulin and androgens both are involved in the regulation of PROK1 that could have implications for normal and pathological pregnancies.

Various subsets of bone marrow mesenchymal stromal cells (BM MSCs), including fibroblasts, endothelial, fat and reticular cells, are implicated in the regulation of the hematopoietic microenvironment and the survival of long-lived antibody-secreting cells (ASCs). Nowadays it is widely acknowledged that vaccine-induced protective antibody levels are diminished in adults and children that are treated for hematological cancers. A reason behind this could be damage to the BM MSC niche leading to a diminished pool of ASCs. To this end, we asked whether cell cytotoxic treatment alters the capacity of human BM MSCs to support the survival of ASCs. To investigate how chemotherapy affects soluble factors related to the ASC niche, we profiled a large number of cytokines and chemokines from in vitro-expanded MSCs from healthy donors or children who were undergoing therapy for acute lymphoblastic leukemia (ALL), following exposure to a widely used anthracycline called doxorubicin (Doxo). In addition, we asked if the observed changes in the measured soluble factors after Doxo exposure impacted the ability of the BM niche to support humoral immunity by co-culturing Doxo-exposed BM MSCs with in vitro-differentiated ASCs from healthy blood donors, and selective neutralization of cytokines. Our in vitro results imply that Doxo-induced alterations in BM MSC-derived interleukin 6 (IL-6), CXCL12 and growth and differentiation factor 15 (GDF-15) are not sufficient to disintegrate the support of IgG-producing ASCs by the BM MSC niche, and that serological memory loss may arise during later stages of ALL therapy.

Background Solute carrier family 2 member 1 (SLC2A1; previously known as glucose transporter 1), is the most abundant glucose transporter in human endometrium and is up-regulated during decidualization, whereas high insulin may have a negative impact on this process. The present study aimed to investigate the effect of insulin on the expression of SLC2A1 and glucose uptake in decidualizing human endometrial stromal cells. Methods We induced in vitro decidualization of endometrial stromal cells obtained from regularly menstruating healthy non-obese women. The cells were treated with increasing concentrations of insulin, and the involvement of the transcription factor forkhead box O1 (FOXO1) was evaluated using a FOXO1 inhibitor. SLC2A1 mRNA levels were measured by Real-Time PCR and protein levels were evaluated by immunocytochemistry. Glucose uptake was estimated by an assay quantifying the cellular uptake of radioactive glucose. One-way ANOVA, Dunnett’s multiple comparisons test and paired t-test were used to determine the statistical significance of the results. Results We found that insulin dose-dependently decreased SLC2A1 mRNA levels and decreased protein levels of SLC2A1 in decidualizing human endometrial stromal cells. Transcriptional inactivation of FOXO1 seems to explain at least partly the down-regulation of SLC2A1 by insulin. Glucose uptake increased upon decidualization, whereas insulin treatment resulted in a slight inhibition of the glucose uptake, although not significant for all insulin concentrations. Conclusions These results indicate an impairment of decidualization by high concentrations of insulin. Future studies will determine the clinical significance of our results for endometrial function and decidualization in women with insulin resistance and hyperinsulinemia.

B-cell CLL/lymphoma 6 (BCL6) is a transcriptional master regulator that can repress more than 1200 potential target genes. It exerts oncogenic effects through the inhibition of differentiation, DNA damage sensing and apoptosis in several human hematopoietic malignancies, including multiple myeloma (MM). The multifunctional cytokine interferon γ (IFNγ) exerts pro-apoptotic and anti-proliferative effects on MM cells in vitro, at least partially through the inhibition of the effects of interleukin 6 (IL6), one of the most important growth factor of MM and a strong inducer of BCL6 expression. However, IFNγ was also reported to directly upregulate BCL6 in several cell types. These observations prompted us to analyze the effect of IFNγ on BCL6 expression in MM cells. We discovered that among several myeloma growth/survival factors tested (including IL6, oncostatin M, insulin-like growth factor 1, tumor necrosis factor α and IFNα) IFNγ was the strongest inducer of BCL6 mRNA and protein expression in MM cell lines. IFNγ induced upregulation of BCL6 was dependent on the classical STAT1 signaling pathway, and affected both major BCL6 variants. Interestingly, although IFNα induced stronger STAT1 phosphorylation than IFNγ, it only slightly upregulated BCL6 in MM lines. We proved that IFNα induced BCL6 upregulation was limited by the concomitant activation of STAT5 signaling. We assume that BCL6 upregulation may represent a potentially pro-tumorigenic effect of IFNγ signaling in MM cells.

Prokineticin 1 (PROK1), a hypoxia‐regulated angiogenic factor, has emerged as a crucial regulator of embryo implantation and placentation. Dysregulation of PROK1 has been linked to recurrent pregnancy loss, pre‐eclampsia, foetal growth restriction and preterm birth. These pregnancy complications are common in women with obesity and polycystic ovary syndrome, i.e. conditions associated with insulin resistance and compensatory hyperinsulinaemia. We investigated the effect of insulin on PROK1 expression during in vitro decidualization. Endometrial stromal cells were isolated from six healthy, regularly menstruating women and decidualized in vitro. Insulin induced a significant dose‐dependent up‐regulation of PROK1 on both mRNA and protein level in decidualizing endometrial stromal cells. This up‐regulation was mediated by hypoxia‐inducible factor 1‐alpha (HIF1α) via the phosphatidylinositol 3‐kinase (PI3K) pathway. Furthermore, we demonstrated that PROK1 did not affect the viability, but significantly inhibited the migration of endometrial stromal cells and the migratory and invasive capacity of trophoblast cell lines. This in vitro study provides new insights into the regulation of PROK1 by insulin in human decidualizing endometrial stromal cells, the action of PROK1 on migration of endometrial stromal cells, as well as migration and invasion of trophoblasts. We speculate that hyperinsulinaemia may be involved in the mechanisms by which PROK1 is linked to placenta‐related pregnancy complications.

Endometrial receptivity is crucial for implantation and establishment of a normal pregnancy. The shift from proliferative to receptive endometrium is still far from being understood. In this paper, ...

Endometrial receptivity is crucial for implantation and establishment of a normal pregnancy. The shift from proliferative to receptive endometrium is still far from being understood. In this paper, we comprehensively present the transcriptome of the human endometrium by comparing endometrial biopsies from proliferative phase with consecutive biopsies 7-9 days after ovulation. The results show a clear difference in expression between the two time points using both total and small RNA sequencing. A total of 3,297 messenger RNAs (mRNAs), 516 long noncoding RNAs (lncRNAs), and 102 small noncoding RNAs were identified as statistically differentially expressed between the two time points. We show a thorough description of the change in mRNA between the two time points and display lncRNAs, small nucleolar RNAs, and small nuclear RNAs not previously reported in the healthy human endometrium. In conclusion, this paper reports in detail the shift in RNA expression from the proliferative to receptive endometrium.

B-cell CLL/lymphoma 6 (BCL6) exerts oncogenic effects in several human hematopoietic malignancies including chronic myeloid leukemia (CML), where BCL6 expression was shown to be essential for CML stem cell survival and self-renewal during imatinib mesylate (IM) treatment. As several lines of evidence suggest that interferon γ (IFNγ) production in CML patients might have a central role in the response to tyrosine kinase inhibitor (TKI) therapy, we analyzed if IFNγ modulates BCL6 expression in CML cells. Although separate IFNγ or IM treatment only slightly upregulated BCL6 expression, combined treatment induced remarkable BCL6 upregulation in CML lines and primary human CD34+ CML stem cells. We proved that during combined treatment, inhibition of constitutive signal transducer and activator of transcription (STAT) 5 activation by IM allowed the specific enhancement of the STAT1 dependent, direct upregulation of BCL6 by IFNγ in CML cells. By using colony-forming assay, we found that IFNγ enhanced the ex vivo colony or cluster-forming capacity of human CML stem cells in the absence or presence of IM, respectively. Furthermore, inhibition of the transcriptional repressor function of BCL6 in the presence of IM and IFNγ almost completely blocked the cluster formation of human CML stem cells. On the other hand, by using small interfering RNA knockdown of BCL6, we demonstrated that in an IM-treated CML line the antiapoptotic effect of IFNγ was independent of BCL6 upregulation. We found that IFNγ also upregulated several antiapoptotic members of the BCL2 and BIRC gene families in CML cells, including the long isoform of MCL1, which proved to be essential for the antiapoptotic effect of IFNγ in an IM-treated CML line. Our results suggest that combination of TKIs with BCL6 and MCL1 inhibitors may potentially lead to the complete eradication of CML stem cells.

Type I interferons (IFN) exert multiple effects on both the innate and adaptive immune system in addition to their antiviral and antiproliferative activities. Little is known, however about the direct effects of type I IFNs on germinal center (GC) B cells, the central components of adaptive B cell responses. We used Burkitt's lymphoma (BL) lines, as a model system of normal human GC B cells, to examine the effect of type I IFNs on the expression of BCL-6, the major regulator of the GC reaction. We show that type I IFNs, but not IFNγ, IL-2 and TNFα rapidly down-regulate BCL-6 protein and mRNA expression, in cell lines derived from endemic, but not from sporadic BL. IFNα-induced down-regulation is specific for BCL-6, independent of Epstein-Barr virus and is not accompanied by IRF-4 up-regulation. IFNα-induced BCL-6 mRNA down-regulation does not require de novo protein synthesis and is specifically inhibited by piceatannol. The proteasome inhibitor MG132 non-specifically prevents, while inhibitors of alternate type I IFN signaling pathways do not inhibit IFNα-induced BCL-6 protein downregulation. We validate our results with showing that IFNα rapidly down-regulates BCL-6 mRNA in purified mouse normal GC B cells. Our results identify type I IFNs as the first group of cytokines that can down-regulate BCL-6 expression directly in GC B cells.

Following infection with Epstein–Barr virus (EBV), the virus is carried for life in the memory B-cell compartment in a silent state (latency I/0). These cells do not resemble the proliferating lymphoblastoid cells (LCLs) (latency III) that are generated after infection. It is of fundamental significance to identify how the different EBV expression patterns are established in the latently infected cell. In view of the prompt activatability of CD4 + T cells in primary EBV infection, and their role in B-cell differentiation, we studied the involvement of CD4 + T cells in the regulation of EBV latency. Lymphoblastoid cell lines (LCLs) were cocultured with autologous or allogeneic CD4 + T cells. Activated T cells influenced the expression of two key viral proteins that determine the fate of the infected B cell. EBNA2 was down-regulated, whereas LMP1 was unregulated and the cells proliferated less. This was paralleled by the down-regulation of the latency III promoter (Cp). Experiments performed in the transwell system showed that this change does not require cell contact, but it is mediated by soluble factors. Neutralizing experiments proved that the up-regulation of LMP1 is, to some extent, mediated by IL21, but this cytokine was not responsible for EBNA2 down-regulation. This effect was partly mediated by soluble CD40L. We detected similar regulatory functions of T cells in in vitro-infected lymphocyte populations. In conclusion, our results revealed an additional mechanism by which CD4 + T cells can control the EBV-induced B-cell proliferation.

Kaposi's sarcoma (KS) shows a distinct geographical and ethnic distribution. Genes at both ends of the human herpesvirus 8 (HHV-8) genome have been shown to vary considerably. Seven major molecular subtypes of HHV-8 were defined based on the amino acid sequence of the open reading frame K1 (orf-K1). The aim of the present study was to characterize HHV8 isolates from hospitalized patients in Hungary.A total of 36 archival paraffin-embedded Kaposi's sarcoma tissue samples were collected. Polymerase chain reaction (PCR) was carried out on the extracted DNA, using specific primers for HHV-8. After identifying the presence of HHV-8 by amplification of its orf26 region, the orf-K1 region was amplified, sequenced and used for phylogenetic analysis.From the 36 orf26-positive cases, orf-K1 was amplified and was analyzed successfully in 12 cases. Phylogenetic studies, based on the complete K1 gene/protein sequences, indicate that all strains belong to the A subtype. Specifically, six of them were related to the A1 subgroup, six to the A2 subgroup and three previously reported to the A3 subgroup. Nucleotide sequence data are reported and are available in the Genbank database under accession numbers KF829938-KF829947.

Epstein-Barr viral (EBV) latency-associated promoters Qp, Cp, and LMP1p are crucial for the regulated expression of the EBNA and LMP transcripts in dependence of the latency type. By transient transfection and in vitro binding analyses, many promoter elements and transcription factors have previously been shown to be involved in the activities of these promoters. However, the latency promoters have only partially been examined at the nucleotide level in vivo. Therefore, we undertook a comprehensive analysis of in vivo protein binding and CpG methylation patterns at these promoters in five representative cell lines and correlated the results with the known in vitro binding data and activities of these promoters from previous transfection experiments. Promoter activity inversely correlated with the methylation state of promoters, although Qp was a remarkable exception. Novel protein binding data were obtained for all promoters. For Cp, binding correlated well with promoter activity; for LMP1p and Qp, binding patterns looked similar regardless of promoter activity.

Insulin resistance and compensatory hyperinsulinemia are characteristic features of obesity and polycystic ovary syndrome, and both are associated with reduced fertility and implantation. There is little knowledge about the effect of insulin on the decidualization process and previous findings are contradictory. We investigated the effect of insulin on the regulation of forkhead box protein O1 (FOXO1), one of the most important transcription factors during decidualization. Endometrial stromal cells were isolated from six healthy, regularly menstruating women and decidualized in vitro. Gene expression levels of six putative FOXO1 target genes (including insulin-like growth factor binding protein-1 (IGFBP1) and prolactin (PRL)) were measured with Real-Time PCR following FOXO1 inhibition or insulin treatment. PI3K inhibition was used to identify the possible mechanism behind regulation. Subcellular localization of FOXO1 was analyzed with immunofluorescence. All the genes (IGFBP1, CTGF, INSR, DCN, LEFTY2), except prolactin, were evaluated as FOXO1 target genes in decidualizing stromal cells. Insulin caused a significant dose-dependent inhibition of the verified FOXO1 target genes. It was also demonstrated that insulin regulated FOXO1 target genes by transcriptional inactivation and nuclear export of FOXO1 via PI3K pathway. However, insulin did not inhibit the morphological transformation of endometrial stromal cells via transcriptional inactivation of FOXO1. This study provides new insights on the action of insulin on the endometrium via regulation of FOXO1. It is suggested that hyperinsulinemia results in dysregulation of a high number of FOXO1 controlled genes that may contribute to endometrial dysfunction and reproductive failure. Our findings may illuminate possible reasons for unexplained infertility.

We report that type I interferons (IFNs) upregulate latent membrane protein 1 (LMP-1) expression by direct activation of the ED-L1 promoter in several Epstein-Barr virus (EBV)-carrying Burkitt's lymphoma lines. In EBV-infected primary B cells, IFN-α transiently upregulates LMP-1 mRNA, but not protein levels, followed by downregulation of both, suggesting a novel antiproliferative mechanism of type I IFNs. Furthermore, our results may explain the expression of LMP-1 in memory B cells of systemic lupus erythematosus patients.

We analysed the methylation patterns of CpG dinucleotides in a bidirectional promoter region (LRS, LMP 1 regulatory sequences) of latent Epstein-Barr virus (EBV) genomes using automated fluorescent genomic sequencing after bisulfite-induced modification of DNA. Transcripts for two latent membrane proteins, LMP 1 (a transforming protein) and LMP 2B, are initiated in this region in opposite directions. We found that B cell lines and a clone expressing LMP 1 carried EBV genomes with unmethylated or hypomethylated LRS, while highly methylated CpG dinucleotides were present at each position or at discrete sites and within hypermethylated regions in LMP 1 negative cells. Comparison of high resolution methylation maps suggests that CpG methylation-mediated direct interference with binding of nuclear factors LBF 2, 3, 7, AML1/LBF1, LBF5 and LBF6 or methylation of CpGs within an E-box sequence (where activators as well as repressors can bind) is not the major mechanism in silencing of the LMP 1 promoter. Although a role for CpG methylation within binding sites of Sp1 and 3, ATF/CRE and a sis-inducible factor (SIF) cannot be excluded, hypermethylation of LRS or regions within LRS in LMP 1 negative cells suggests a role for an indirect mechanism, via methylcytosine binding proteins, in silencing of the LMP 1 promoter.