Selection of novel mediators of E2F1-induced apoptosis through retroviral expression of an antisense cDNA library

The E2F1 transcription factor is an essential mediator of p53-dependent and p53-independent apoptosis as part of an anti-tumour safeguard mechanism. In this study, a functional so-called technical knockout (TKO) approach was applied to Saos-2ERE2F1 cells that conditionally activate E2F1 by the addition of 4-hydroxytamoxifen to search for p53-independent pro-apoptotic E2F1 targets. The approach was based on random inactivation of genes after retroviral transfer of an antisense cDNA library enriched of E2F1-induced genes, followed by the selection of Saos-2ERE2F1 cells that survive in the presence of the apoptotic stimulus. We identified 13 novel E2F1 target genes encoding proteins of known cellular function, including apoptosis and RNA binding. FACS analysis revealed that E2F1-induced apoptosis was significantly attenuated in cell clones containing the antisense cDNA fragments of these genes, demonstrating their participation in E2F1 death pathways. Moreover, inactivation of the target genes resulted in a clear increase of cell viability (>80%) in response to E2F1 activation compared with controls (∼30%). Four genes showed an increase in expression intensity in the presence of cycloheximide, suggesting a direct effect of E2F1 on gene transcription, whereas one gene was identified as an indirect target. Our data provide new insight in the regulation of E2F1-induced apoptosis

Immune Responses to RHAMM in Patients with Acute Myeloid Leukemia after Chemotherapy and Allogeneic Stem Cell Transplantation

Leukemic blasts overexpress immunogenic antigens, so-called leukemia-associated antigens like the receptor for hyaluronan acid-mediated motility (RHAMM). Persistent RHAMM expression and decreasing CD8+ T-cell responses to RHAMM in the framework of allogeneic stem cell transplantation or chemotherapy alone might indicate the immune escape of leukemia cells. In the present study, we analyzed the expression of RHAMM in 48 patients suffering from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Furthermore, we correlated transcripts with the clinical course of the disease before and after treatment. Real-time quantitative reverse transcriptase polymerase chain reaction was performed from RNA of peripheral blood mononuclear cells. T cell responses against RHAMM were assessed by tetramer staining (flow cytometry) and enzyme-linked immunospot (ELISPOT) assays. Results were correlated with the clinical outcome of patients. The results of the present study showed that almost 60% of the patients were RHAMM positive; specific T-cells recognizing RHAMM could be detected, but they were nonfunctional in terms of interferon gamma or granzyme B release as demonstrated by ELISPOT assays. Immunotherapies like peptide vaccination or adoptive transfer of RHAMM-specific T cells might improve the immune response and the outcome of AML/MDS patients

Actionable perturbations of damage responses by TCL1/ATM and epigenetic lesions form the basis of T-PLL

T-cell prolymphocytic leukemia (T-PLL) is a rare and poor-prognostic mature T-cell malignancy. Here we integrated large-scale profiling data of alterations in gene expression, allelic copy number (CN), and nucleotide sequences in 111 well-characterized patients. Besides prominent signatures of T-cell activation and prevalent clonal variants, we also identify novel hot-spots for CN variability, fusion molecules, alternative transcripts, and progression-associated dynamics. The overall lesional spectrum of T-PLL is mainly annotated to axes of DNA damage responses, T-cell receptor/cytokine signaling, and histone modulation. We formulate a multi-dimensional model of T-PLL pathogenesis centered around a unique combination of TCL1 overexpression with damaging ATM aberrations as initiating core lesions. The effects imposed by TCL1 cooperate with compromised ATM toward a leukemogenic phenotype of impaired DNA damage processing. Dysfunctional ATM appears inefficient in alleviating elevated redox burdens and telomere attrition and in evoking a p53-dependent apoptotic response to genotoxic insults. As non-genotoxic strategies, synergistic combinations of p53 reactivators and deacetylase inhibitors reinstate such cell death execution.Peer reviewe

Adenovirus-mediated TA-p73β gene transfer increases chemosensitivity of human malignant melanomas

Malignant melanoma is the most aggressive form of skin cancer and has proven to be highly resistant to conventional chemotherapy. Intriguingly, the p53 tumor suppressor, a main mediator of chemoresistance in other tumor types, is rarely mutated in melanoma. However, we have previously shown that anti-apoptotic isoforms of p73 (ΔTA-p73), another member of the p53 family, are overexpressed in metastatic melanomas. ΔTA-p73 can oppose the pro-apoptotic functions of p53 and full length p73, and thus it could contribute to melanoma chemoresistance. In this study, we use an efficient adenoviral-based gene transfer approach to introduce a transcriptionally active form of p73 (TA-p73β) in melanoma cells, with the objective of overcoming drug resistance. Interestingly, TA-p73β significantly sensitized 5 out of 7 aggressive melanoma cell lines to the standard therapeutic agents adriamycin and cisplatin. More importantly, TA-p73β displayed a synergistic effect in vivo allowing adriamycin or cisplatin to block melanoma cell growth in mouse xenograft models ( p < 0.05). In summary, our data show that Ad-mediated TA-p73β gene expression can markedly sensitize a subset of melanoma cell lines to adriamycin and cisplatin in vitro and in vivo , suggesting a new chemosensitization strategy for malignant melanomas.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44369/1/10495_2006_Article_3407.pd

Herausforderungen und Chancen bei der Beratung und Behandlung mehrsprachiger Familien mit CI-versorgten Kindern

Hintergrund: Die Themen Migration und Behinderung erhalten in Politik und Gesellschaft immer mehr Aufmerksamkeit - u. a. ausgelöst durch die erste Flüchtlingswelle im Jahr 2015. Jedoch geht aus dem 11. Integrationsbericht der Beauftragten der Bundesregierung für Migration, Flüchtlinge und Integration 2016 hervor, dass weiterhin ein großer Forschungs- und Handlungsbedarf bezogen auf Menschen mit Migrationshintergrund und Behinderung besteht. Kulturelle Unterschiede, sprachliche Barrieren und ein eher geringes Inanspruchnahmeverhalten von Gesundheitsleistungen sind dabei wichtige zu untersuchende Faktoren. Dies gilt auch für Kinder aus mehrsprachigen Familien, die mit einem Cochlea-Implantat (CI) versorgt werden.Viele Studien, die sich dem Thema Mehrsprachigkeit bei CI-versorgten Kindern widmen, adressieren die Sprach- und Kommunikationsentwicklung der Kinder. Zunehmend wird auch die Sicht der Fachleute auf den Umgang mit sprachlichen und kulturellen Unterschieden in der Förderung und Bildung von Kindern mit Hörstörungen untersucht. Bisher gibt es jedoch keine Studien, die sich im speziellen mit den Behandlungs- und Beratungssettings in der Folgetherapie bei mehrsprachigen Familien mit CI-versorgten Kindern befassen. Es lässt sich annehmen, dass aufgrund von Sprachbarrieren und kultureller Identität Herausforderungen entstehen und es deshalb kreativer und flexibler Lösungsansätze bedarf.Ziel der Studie ist, Herausforderungen und Chancen in Beratungs- und Behandlungssettings aufzuzeigen, die sich in CI-Zentren, z. B. aufgrund von möglichen Kommunikations- und Sprachbarrieren sowie kulturellen Einflüssen zwischen Eltern und beratender/behandelnder Person, ergeben. Im Fokus stehen der Umgang mit den Herausforderungen und mögliche Handlungsbedarfe, wie beispielsweise der Umgang mit Verständigungsproblemen oder die Anpassung standardisierter Testverfahren.Material und Methoden: In diesem Forschungsprojekt werden leitfadengestützte problemzentrierte Interviews durchgeführt. Befragt werden drei bis fünf Expert*innen aus den Bereichen Sprachtherapie und Pädagogik in fachzertifizierten CI-Zentren in NRW. Zur Transkription und Analyse des Interviews wird die Software MAXQDA verwendet. Die Interviews werden mittels der qualitativen Inhaltsanalyse nach Mayring analysiert.Ergebnisse: Die Durchführung der Interviews erfolgt zwischen Mitte Mai und Mitte Juni 2023. Auf der DGPP Jahrestagung werden die Studienergebnisse vorgestellt und diskutiert

Adenoviral p53 gene transfer inhibits human Tenon’s capsule fibroblast proliferation

Background/aim: Although antiproliferative drugs have been used successfully to prevent scarring after filtration surgery in patients with glaucoma, complications associated with their use (such as hypotony or endophthalmitis) energise the search for an alternative treatment. Single application of β radiation leads to long term growth arrest and expression of p53 in human Tenon’s capsule fibroblasts (hTf). The authors assume that the activation of p53 is one of the cellular triggers. Their aim was to analyse the effect of p53 overexpression on hTf and to determine which pathways are involved. Methods: A recombinant adenoviral vector (rAd.p53) containing transgenes encoding for human p53 and green fluorescent protein (GFP) was used to induce overexpression of p53 in hTF and a control vector (rAd.GFP). Transgene expression was detected by western blot (p53 and p21(WAF-1/Cip1)). Cell proliferation and viability were investigated using cell counts, 5′-bromodeoxyuridine incorporation (BrdU assay) and tetrazolium reduction (MTT assay). Results: Infection of hTf with rAd.p53 resulted in significant inhibition of cell proliferation, DNA synthesis, and metabolic activity in vitro. Western blot showed increased levels of p53 and p21(WAF-1/Cip1) in rAd.p53 infected cells, but not in rAd.GFP and uninfected cells. Apoptosis was excluded with flow cytometry. Conclusions: Adenoviral p53 gene transfer leads to significant growth inhibition in hTf. P53 induces p21(WAF-1/Cip1) expression and does not cause apoptosis in hTf in vitro. p53 as an antiproliferative drug has the potential to replace mitomycin C and 5-fluorouracil in glaucoma surgery