Katrin Hacke

Regulation of MCP-1 chemokine transcription by p53

BackgroundOur previous studies showed that the expression of the monocyte-chemoattractant protein (MCP)-1, a chemokine, which triggers the infiltration and activation of cells of the monocyte-macrophage lineage, is abrogated in human papillomavirus (HPV)-positive premalignant and malignant cells. In silico analysis of the MCP-1 upstream region proposed a putative p53 binding side about 2.5 kb upstream of the transcriptional start. The aim of this study is to monitor a physiological role of p53 in this process.ResultsThe proposed p53 binding side could be confirmed in vitro by electrophoretic-mobility-shift assays and in vivo by chromatin immunoprecipitation. Moreover, the availability of p53 is apparently important for chemokine regulation, since TNF-α can induce MCP-1 only in human keratinocytes expressing the viral oncoprotein E7, but not in HPV16 E6 positive cells, where p53 becomes degraded. A general physiological role of p53 in MCP-1 regulation was further substantiated in HPV-negative cells harboring a temperature-sensitive mutant of p53 and in Li-Fraumeni cells, carrying a germ-line mutation of p53. In both cases, non-functional p53 leads to diminished MCP-1 transcription upon TNF-α treatment. In addition, siRNA directed against p53 decreased MCP-1 transcription after TNF-α addition, directly confirming a crosstalk between p53 and MCP-1.ConclusionThese data support the concept that p53 inactivation during carcinogenesis also affects immune surveillance by interfering with chemokine expression and in turn communication with cells of the immunological compartment.

The role of tumour necrosis factor-α and tumour necrosis factor receptor signalling in inflammation-associated systemic genotoxicity

Chronic inflammatory diseases are characterised by systemically elevated levels of tumour necrosis factor (TNF)-α, a proinflammatory cytokine with pleiotropic downstream effects. We have previously demonstrated increased genotoxicity in peripheral leukocytes and various tissues in models of intestinal inflammation. In the present study, we asked whether TNF-α is sufficient to induce DNA damage systemically, as observed in intestinal inflammation, and whether tumour necrosis factor receptor (TNFR) signalling would be necessary for the resultant genotoxicity. In the wild-type mice, 500 ng per mouse of TNF-α was sufficient to induce DNA damage to multiple cell types and organs 1-h post-administration. Primary splenic T cells manifested TNF-α-induced DNA damage in the absence of other cell types. Furthermore, TNFR1(-/-)TNFR2(-/-) mice demonstrated decreased systemic DNA damage in a model of intestinal inflammation and after TNF-α injection versus wild-type mice, indicating the necessity of TNFR signalling. Nuclear factor (NF)-κB inhibitors were also able to decrease damage induced by TNF-α injection in wild-type mice. When TNF-α administration was combined with interleukin (IL)-1β, another proinflammatory cytokine, DNA damage persisted for up to 24 h. When combined with IL-10, an anti-inflammatory cytokine, decreased genotoxicity was observed in vivo and in vitro. TNF-α/TNFR-mediated signalling is therefore sufficient and plays a large role in mediating DNA damage to various cell types, subject to modulation by other cytokines and their mediators.

Small-Animal PET/CT for Monitoring the Development and Response to Chemotherapy of Thymic Lymphoma in Trp53−/− Mice

Transgenic mouse models of human cancers represent one of the most promising approaches to elucidate clinically relevant mechanisms of action and provide insights into the treatment efficacy of new antitumor drugs. The use of Trp53 transgenic mice (Trp53 knockout [Trp53−/−] mice) for these kinds of studies is, so far, restricted by limitations in detecting developing tumors and the lack of noninvasive tools for monitoring tumor growth, progression, and treatment response. Methods: We hypothesized that quantitative small-animal PET with 18F-FDG was able to detect the onset and location of tumor development, follow tumor progression, and monitor response to chemotherapy. To test these hypotheses, C57BL/6J Trp53−/− mice underwent longitudinal small-animal PET during lymphoma development and gemcitabine treatment. Trp53 wild-type (Trp53+/+) mice were used as controls, and histology after full necropsy served as the gold standard. Results: In Trp53+/+ mice, the thymic standardized uptake value (SUV) did not exceed 1.0 g/mL, with decreasing 18F-FDG uptake over time. Conversely, all Trp53−/− mice that developed thymic lymphoma showed increasing thymic glucose metabolism, with a mean SUV doubling time of 9.0 wk (range, 6.0–17.5 wk). Using an SUV of 3.0 g/mL as a criterion provided a sensitivity of 78% and a specificity of 100% for the detection of thymic lymphoma. Treatment monitoring with 18F-FDG PET correctly identified all histologic responses and relapses to gemcitabine. Conclusion: 18F-FDG small-animal PET can be used to visualize onset and progression of thymic lymphomas in Trp53−/− mice and monitor response to chemotherapy. Thus, 18F-FDG small-animal PET provides an in vivo means to assess intervention studies in the Trp53 transgenic mouse model.

Genetic modification of mouse bone marrow by lentiviral vector-mediated delivery of hypoxanthine-Guanine phosphoribosyltransferase short hairpin RNA confers chemoprotection against 6-thioguanine cytotoxicity.

We have recently developed a novel and highly efficient strategy that exclusively uses the purine analog 6-thioguanine (6TG) for both pretransplantation conditioning and post-transplantation chemoselection of hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient bone marrow (BM). In a mouse BM transplantation model, combined 6TG preconditioning and in vivo chemoselection consistently achieved >95% engraftment of HPRT-deficient donor BM and long-term reconstitution of histologically and immunophenotypically normal hematopoiesis in both primary and secondary recipients, without significant toxicity and in the absence of any other cytotoxic conditioning regimen. To translate this strategy for combined 6TG conditioning and chemoselection into a clinically feasible approach, it is necessary to develop methods for genetic modification of normal hematopoietic stem cells (HSC) to render them HPRT-deficient and thus 6TG-resistant. Here we investigated a strategy to reduce HPRT expression and thereby confer protection against 6TG myelotoxicity to primary murine BM cells by RNA interference (RNAi). Accordingly, we constructed and validated a lentiviral gene transfer vector expressing short-hairpin RNA (shRNA) that targets the murine HPRT gene. Our results showed that lentiviral vector-mediated delivery of HPRT-targeted shRNA could achieve effective and long-term reduction of HPRT expression. Furthermore, in both an established murine cell line as well as in primary murine BM cells, lentiviral transduction with HPRT-targeted shRNA was associated with enhanced resistance to 6TG cytotoxicity in vitro. Hence this represents a translationally feasible method to genetically engineer HSC for implementation of 6TG-mediated preconditioning and in vivo chemoselection.

Retroviral replicating vector-mediated gene therapy achieves long-term control of tumor recurrence and leads to durable anticancer immunity

Abstract Background. Prodrug-activator gene therapy with Toca 511, a tumor-selective retroviral replicating vector (RRV) encoding yeast cytosine deaminase, is being evaluated in recurrent high-grade glioma patients. Nonlytic retroviral infection leads to permanent integration of RRV into the cancer cell genome, converting infected cancer cell and progeny into stable vector producer cells, enabling ongoing transduction and viral persistence within tumors. Cytosine deaminase in infected tumor cells converts the antifungal prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil, mediating local tumor destruction without significant systemic adverse effects. Methods. Here we investigated mechanisms underlying the therapeutic efficacy of this approach in orthotopic brain tumor models, employing both human glioma xenografts in immunodeficient hosts and syngeneic murine gliomas in immunocompetent hosts. Results. In both models, a single injection of replicating vector followed by prodrug administration achieved long-term survival benefit. In the immunodeficient model, tumors recurred repeatedly, but bioluminescence imaging of tumors enabled tailored scheduling of multicycle prodrug administration, continued control of disease burden, and long-term survival. In the immunocompetent model, complete loss of tumor signal was observed after only 1–2 cycles of prodrug, followed by long-term survival without recurrence for >300 days despite discontinuation of prodrug. Long-term survivors rejected challenge with uninfected glioma cells, indicating immunological responses against native tumor antigens, and immune cell depletion showed a critical role for CD4+ T cells. Conclusion. These results support dual mechanisms of action contributing to the efficacy of RRV-mediated prodrug-activator gene therapy: long-term tumor control by prodrug conversion-mediated cytoreduction, and induction of antitumor immunity.

Effects of 1 GeV/nucleon 56Fe Particles on Longevity, Carcinogenesis and Neuromotor Ability in Atm-Deficient Mice

Abstract As therapeutic uses of high-LET radiation become more prevalent and human space exploration continues to be a focus of NASA, it is important to understand the biological effects of high-LET radiation and the role of genetics in sensitivity to high-LET radiation. To study genetic susceptibility to radiation, we used mice deficient in Atm activity (AtmΔSRI). ATM is important in DNA repair, apoptosis and cell cycle regulation. Although homozygous mutations in ATM are rare, the prevalence of ATM heterozygosity is estimated to be 1% and results in an increased cancer risk. We found that the effects of 1 Gy 1 GeV/nucleon 56Fe particles on life span and tumorigenesis are genotype- and sex-specific. Significant effects of 1 Gy 1 GeV/nucleon 56Fe particles on incidence of non-cancer end points were seen; however, 2 Gy 1 GeV/nucleon 56Fe particles significantly affected neuromotor ability. Our results represent an extensive investigation into the late effects of high-LET radiation exposure in a sex- and genotype-dependent manner and provide a baseline for understanding the long-term risks of high-LET radiation.

P034 When bone marrow transplant recipients become deceased organ donors – A case report

Blood samples for ABO and HLA typing were received from the OPO on a potential deceased donor. The donor medical records indicated AMS due to hemorrhagic stroke, no significant PMH and no recent transfusions. ABO typing was performed by serological agglutination and HLA typing was performed by SSP/melt curve analysis. Results indicated that the donor was blood group O and had HLA typing consistent with the donor’s reported race. The results were reported to the OPO and organ allocation proceeded. Upon review of the UDRAI, a transplant program requested repeat HLA typing on lymph nodes (LN) as the next of kin indicated that the donor received a BMT 15 + years ago for aplastic anemia prior to residing in the USA. The LN results were concordant with the initial blood HLA typing. The repeat indication and testing results were reviewed by the lab general supervisor with the OPO’s AOC since DNA from LN was expected to also have a predominant BMT-donor origin as well. To ensure the correct HLA type of the allocated organs, buccal swab and muscle biopsy samples were requested for repeat testing. Additional HLA typing of these samples confirmed the previous results, indicating that the organ donor most likely received a fully HLA-matched BMT. The confirmed results were reported to the OPO; however, the HLA lab recommended re-allocation as an AB blood group donor since 30–40% of BMTs are ABO incompatible. The OPO reallocated and informed all transplant programs involved that the deceased donor’s tissue ABO typing could not be verified as group O. In conclusion, the HLA lab should be knowledgeable about ABO as it relates to all types of transplantation and provide consultation regarding results and testing limitations. This case also demonstrates the HLA lab can only give proper advice when provided important donor related information. Furthermore, effective communication is vital to managing increasingly complex donors and transplant cases.