Die Rolle des Tumorsuppressors p53 für die TNF-alpha-vermittelte Induktion des MCP-1-Gens

Das Immunsystem besteht aus hochspezialisierten Zellen und Molekülen, die in einer komplexen Beziehung miteinander agieren, um Pathogene zu detektieren und zu eliminieren. Chemotaktische Zytokine, so genannte Chemokine sind entscheidend an der Immunantwort beteiligt und bekannt für ihre Effekte bei der Anlockung und Aktivierung spezifischer Gruppen von Leukozyten. Das "Monocyte-Chemoattractant-Protein-1" (MCP-1/CCL2) ist ein wichtiges Mitglied der Chemokin-Familie und löst die Infiltration von Monozyten/Makrophagen aus. Die Expression von MCP-1 tritt bei verschiedenen Krankheiten, die durch den Einstrom von Monozyten charakterisiert sind, auf. Es gibt eindeutige biologische und genetische Hinweise, dass MCP-1 eine entscheidende Rolle in der Pathogenese von Krankheiten, wie der Arteriosklerose, der Rheumatischen Arthritis und der Multiplen Sklerose, spielt. Zahlreiche Zellen, darunter Monozyten, Fibroblasten, vaskuläre Endothelzellen und glatte Muskelzellen produzieren MCP-1 in vitro als Antwort auf verschiedene proinflammatorische Stimuli, wie Lipopolysaccharid (LPS), Interleukin-1 (IL-1) und Tumor-Nekrose-Faktor-alpha (TNF-alpha). Das Gen, welches für MCP-1 codiert, ist ein "immediate-early" Gen. Die Induktion des MCP-1-Gens durch TNF-alpha wird auf der Ebene der Initiation der Transkription hauptsächlich durch einen Enhancer, der sich 2,2 bis 2,7 bp "upstream" zum Transkriptionsstart befindet, reguliert. Durch eine bioinformatische Analyse dieser 5'-Enhancer-Region wurde eine mögliche Bindungsstelle für p53 identifiziert. Ziel dieser Arbeit war es, zu untersuchen, ob der Tumorsuppressor p53 in der durch TNF-alpha vermittelten Induktion von MCP-1 als Transkriptionsfaktor beteiligt ist. Transiente Transfektionen der Glioblastoma-Zelllinie A172 mit siRNA gerichtet gegen p53 Transkripte reduzierten die Induktion von MCP-1 durch TNF-alpha signifikant. Zusätzlich wurden Hep3B Zellen, stabil transfiziert mit temperatursensitiven p53val135, als Modell verwendet: bei 32°C, wenn p53val135 wtp53 Aktivität besitzt, wurde durch die Stimulation mit TNF-alpha die Transkription von MCP-1 induziert. Im Gegensatz dazu, wurde durch TNF-alpha bei 37°C und inaktivem p53val135 das MCP 1-Gen nicht aktiviert. Die physikalische Interaktion von p53 mit dem MCP-1 Enhancer konnte tatsächlich in p53 DNA Bindungsstudien (EMSA) gezeigt werden. Chromatin Immunopräzipitations- (ChIP) Assays ergaben, dass p53 nach der Stimulation mit TNF-alpha auch in vivo mit der identifizierten p53 Bindungsstelle interagiert. Dieses weist darauf hin, dass unter der Beteiligung des TNF-alpha Signaltransduktionsweg die Bindung von p53 an den MCP-1 Enhancer erfolgt. Daher liefert diese Arbeit nicht nur eine neue Perspektive, wie der Tumorsuppressor p53 an der Immunantwort durch die Modulation der Chemokin-Expression beteiligt ist, sondern liefert zusätzlich auch ein nützliches Modell, um die Vernetzung zwischen p53 und dem TNF-alpha Signaltransduktionsweg im Detail zu verstehen

Regulation of MCP-1 chemokine transcription by p53

<p>Abstract</p> <p>Background</p> <p>Our 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. <it>In silico </it>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.</p> <p>Results</p> <p>The proposed p53 binding side could be confirmed <it>in vitro </it>by electrophoretic-mobility-shift assays and <it>in vivo </it>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.</p> <p>Conclusion</p> <p>These 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.</p

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

BackgroundProdrug-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.MethodsHere 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.ResultsIn 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 &gt;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.ConclusionThese 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

STEM-24. THERAPEUTIC APPROACH OF STEM CELL CARRYING RETROVIRAL REPLICATING VECTORS IN HUMAN GLIOMA MODEL

Abstract Toca 511, an improved retroviral replicating vectors (RRVs) expressing a codon-optimized cytosine deaminase, has shown highly promising evidence of therapeutic benefit in preclinical and clinical studies for gene therapy of glioma. In the present study, we engineered human mesenchymal stem cells (MSC) as tumor-homing cellular carriers that produce and release RRV, and evaluated the effect of this mode of virus delivery on the time course of intratumoral RRV dissemination. Human MSC isolates were engineered to produce RRV (MSC-RRV). Cytotoxicity assays confirmed efficient prodrug activator function in U-87 glioma cells transduced with vectors produced from MSC-RRV. To evaluate intratumoral migration activity and tumor-homing migration activity in vivo, individual MSC isolates were injected either directly into human glioma xenografts, or into the contralateral brain hemisphere. MSC-RRV isolates showing the highest levels of intratumoral migration activity were selected, and the efficiency of intratumoral dissemination and tumoricidal activity achieved by these isolates was compared against injection of RRV virus preparations in subcutaneous glioma models. Compared to virus injection, MSC-RRV achieved 1.6x-higher levels of tumor transduction (p&lt; 0.05), and 2x-reduced tumor growth (p=0.018) at earlier time points. In short-term survival studies using lower doses of MSC-RRV cells vs. RRV virus injected 14 days post-establishment of intracranial U-87 gliomas, a small but statistically significant prolongation of median survival was seen with intracranial tumors treated with MSC-RRV as compared to RRV (26 vs. 20 days, p&lt; 0.05) after only a single cycle of 5-FC prodrug. Thus, MSC can be employed as mobile tumor-homing RRV-producer cells, which release RRV as they migrate to tumor foci in vivo and actively penetrate into individual tumor masses, resulting in more rapid tumor transduction and earlier therapeutic efficacy, which may be advantageous particularly for multi-focal and metastatic disease. This study was funded by the California Institute for Regenerative Medicine (TR2-01791)

Protocol for German trial of Acyclovir and corticosteroids in Herpes-simplex-virus-encephalitis (GACHE): a multicenter, multinational, randomized, double-blind, placebo-controlled German, Austrian and Dutch trial [ISRCTN45122933]

Background The treatment of Herpes-simplex-virus-encephalitis (HSVE) remains a major unsolved problem in Neurology. Current gold standard for therapy is acyclovir, a drug that inhibits viral replication. Despite antiviral treatment, mortality remains up to 15%, less than 20% of patients are able to go back to work, and the majority of patients suffer from severe disability. This is a discouraging, unsatisfactory situation for treating physicians, the disabled patients and their families, and constitutes an enormous burden to the public health services. The information obtained from experimental animal research and from recent retrospective clinical observations, indicates that a substantial benefit in outcome can be expected in patients with HSVE who are treated with adjuvant dexamethasone. But currently there is no available evidence to support the routine use of adjuvant corticosteroid treatment in HSVE. A randomized multicenter trial is the only useful instrument to address this question. Design GACHE is a multicenter, randomized, double-blind, placebo-controlled, parallel group clinical trial of treatment with acyclovir and adjuvant dexamethasone, as compared with acyclovir and placebo in adults with HSVE. The statistical design will be that of a 3-stage-group sequential trial with potential sample size adaptation in the last stage. Conclusion 372 patients with proven HSVE (positive HSV-DNA-PCR), aged 18 up to 85 years; with focal neurological signs no longer than 5 days prior to admission, and who give informed consent will be recruited from Departments of Neurology of academic medical centers in Germany, Austria and The Netherlands. Sample size will potentially be extended after the second interim analysis up to a maximum of 450 patients. Trial Registration Current Controlled Trials ISRCTN4512293

Regulation of MCP-1 chemokine transcription by p53. Mol Cancer 9

Abstract Background: Our previous studies showed that the expression of the monocyte

Effects of 1 GeV/nucleon ⁵⁶Fe particles on longevity, carcinogenesis and neuromotor ability in Atm-deficient mice

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

Renal Transplant Patients Biopsied for Cause and Tested for C4d, DSA, and IgG Subclasses and C1q: Which Humoral Markers Improve Diagnosis and Outcomes?

The association between donor specific antibodies (DSA) and renal transplant rejection has been generally established, but there are cases when a DSA is present without rejection. We examined 73 renal transplant recipients biopsied for transplant dysfunction with DSA test results available: 23 patients diffusely positive for C4d (C4d+), 25 patients focally positive for C4d, and 25 patients negative for C4d (C4d−). We performed C1q and IgG subclass testing in our DSA+ and C4d+ patient group. Graft outcomes were determined for the C4d+ group. All 23 C4d+ patients had IgG DSA with an average of 12,500 MFI (cumulative DSA MFI). The C4d− patients had average DSA less than 500 MFI. Among the patients with C4d+ biopsies, 100% had IgG DSA, 70% had C1q+ DSA, and 83% had complement fixing IgG subclass antibodies. Interestingly, IgG4 was seen in 10 of the 23 recipients’ sera, but always along with complement fixing IgG1, and we have previously seen excellent function in patients when IgG4 DSA exists alone. Cumulative DSA above 10,000 MFI were associated with C4d deposition and complement fixation. There was no significant correlation between graft loss and C1q positivity, and IgG subclass analysis seemed to be a better correlate for complement fixing antibodies in the C4d+ patient group