Magdalena Głowala-Kosińska

Co-treatment with actinomycin D and nutlin-3a (A + N) strongly activates p53. Previously we reported that CHIR-98014 (GSK-3 kinase inhibitor), acting in cells exposed to A + N, prevents activation of TREM2-an innate immunity and p53-regulated gene associated with Alzheimer’s disease. In order to find novel candidate p53-target genes and genes regulated by CHIR-98014, we performed RNA-Seq of control A549 cells and the cells exposed to A + N, A + N with CHIR-98014 or to CHIR-98014. We validated the data for selected genes using RT-PCR and/or Western blotting. Using CRISPR/Cas9 technology we generated p53-deficient cells. These tools enabled us to identify dozens of candidate p53-regulated genes. We confirmed that p53 participates in upregulation of BLNK, APOE and IRF1. BLNK assists in activation of immune cells, APOE codes for apolipoprotein associated with Alzheimer’s disease and IRF1 is activated by interferon gamma and regulates expression of antiviral genes. CHIR-98014 prevented or inhibited the upregulation of a fraction of genes stimulated by A + N. Downregulation of GSK-3 did not mimic the activity of CHIR-98014. Our data generate the hypothesis, that an unidentified kinase inhibited by CHIR-98014, participates in modification of p53 and enables it to activate a subset of its target genes, e.g., the ones associated with innate immunity.

Heat Shock Protein A2 (HSPA2) is a member of the HSPA (HSP70) chaperone family and has a critical role for male fertility. HSPA2 is present in a number of somatic organs. Limited evidence suggests that HSPA2 may be involved in regulating epithelial cell differentiation. HSPA2 also emerged as a cancer-related chaperone; however, no consensus on its functional significance has been reached so far. In this study, we compared the phenotypic effects of HSPA2 deficit in non-transformed human bronchial epithelial cells (HBEC), and in lung, breast, and cervical cancer cells. We used various techniques to inhibit the HSPA2 gene expression in order to examine the impact of HSPA2 deficiency on cell growth, migration, adhesion, and invasion. Our results show that HBEC but not cancer cells are sensitive to HSPA2 deficit. HSPA2 knockdown in HBEC cells impaired their clone-forming ability and adhesiveness. Thus, our results indicate that epithelial cells can rely on a specific activity of HSPA2, but such dependence can be lost in epithelial cells that have undergone malignant transformation.

High-grade serous ovarian carcinoma (HGSOC) is the most frequent histological type of ovarian cancer and the one with worst prognosis. Unfortunately, the majority of established ovarian cancer cell lines which are used in the research have unclear histological origin and probably do not represent HGSOC. Thus, new and reliable models of HGSOC are needed. Ascitic fluid from a patient with recurrent HGSOC was used to establish a stable cancer cell line. Cells were characterized by cytogenetic karyotyping and short tandem repeat (STR) profiling. New generation sequencing was applied to test for hot-spot mutations in 50 cancer-associated genes and fluorescence in situ hybridization (FISH) analysis was used to check for TP53 status. Cells were analyzed for expression of several marker genes/proteins by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS), and immunocytochemistry (ICC). Functional tests were performed to compare OVPA8 cells with five commercially available and frequently used ovarian cancer cell lines: SKOV3, A2780, OVCAR3, ES2, and OAW42. Our newly-established OVPA8 cell line shows morphologic and genetic features consistent with HGSOC, such as epithelial morphology, multiple chromosomal aberrations, TP53 mutation, BRCA1 mutation, and loss of one copy of BRCA2. The OVPA8 line has a stable STR profile. Cells are positive for EpCAM, CK19, and CD44; they have relatively low plating efficiency/ability to form spheroids, a low migration rate, and intermediate invasiveness in matrigel, as compared to other ovarian cancer lines. OVPA8 is sensitive to paclitaxel and resistant to cisplatin. We also tested two FGFR inhibitors; OVPA8 cells were resistant to AZD4547 (AstraZeneca, London, UK), but sensitive to the new inhibitor CPL304-110-01 (Celon Pharma, Łomianki/Kiełpin, Poland). We have established and characterized a novel cell line, OVPA8, which can be a valuable preclinical model for studies on high-grade serous ovarian cancer.

The procedure of autologous peripheral blood stem cell transplantation (autoPBSCT) requires cryopreservation of cells in a mixture containing dimethyl sulfoxide (DMSO). DMSO is necessary to secure cell viability, however, its infusion may be toxic to stem cell recipient. The aim of this study was to prospectively evaluate the impact of DMSO concentration on engraftment after autoPBSCT. One-hundred-fifty patients were randomly assigned to one of three study arms; their leukapheresis products were cryopreserved in 10%, 7.5% or 5% DMSO. The study groups did not differ with regard to the diagnosis (mainly lymphomas and multiple myeloma), age, conditioning regimen, and the number of transplanted hematopoietic stem cells. 143 patients were treated with autoPBSCT. The frequency of adverse effects during and shortly after infusion was the lowest in 5% DMSO arm (p = 0.02 compared to 10% DMSO). 4 patients died due to infection before the engraftment. The median time to leukocyte and neutrophil recovery was 10 days in all study groups (p = 0.36 and p = 0.2). As well, the median day of platelet recovery was the same for all DMSO concentrations and equaled 15 days (p = 0.61). In view of these results, 5% DMSO mixture may be considered a new standard in cryopreservation of hematopoietic stem cells.

HSPA2, a poorly characterized member of the HSPA (HSP70) chaperone family, is a testis‐enriched protein involved in male germ cell differentiation. Previously, we revealed that HSPA2 is present in human stratified epithelia, including epidermis, however the contribution of this protein to epithelial biology remained unknown. Here, we show for the first time that HSPA2 is expressed in basal epidermal keratinocytes, albeit not in keratinocytes exhibiting features attributed to primitive undifferentiated progenitors, and participates in the keratinocyte differentiation process. We found that HSPA2 is dispensable for protection of HaCaT keratinocytes against heat shock‐induced cytotoxicity. We also shown that lentiviral‐mediated shRNA silencing of HSPA2 expression in HaCaT cells caused a set of phenotypic changes characteristic for keratinocytes committed to terminal differentiation such as reduced clonogenic potential, impaired adhesiveness and increased basal and confluency‐induced expression of differentiation markers. Moreover, the fraction of undifferentiated cells that rapidly adhered to collagen IV was less numerous in HSPA2‐deficient cells than in the control. In a 3D reconstructed human epidermis model, HSPA2 deficiency resulted in accelerated development of a filaggrin‐positive layer. Collectively, our results clearly show a link between HSPA2 expression and maintenance of keratinocytes in an undifferentiated state in the basal layer of the epidermis. It seems that HSPA2 could retain keratinocytes from premature entry into the terminal differentiation process. Overall, HSPA2 appears to be necessary for controlling development of properly stratified epidermis and thus for maintenance of skin homeostasis.

The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105⁺CD34^- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation. MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105⁺CD34^- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed. In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105⁺CD34^- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart.