ABSTRACT
INTRODUCTION: Interferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling. RESULTS: We could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment. CONCLUSION: We suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in 'silent' metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Interferon-alpha/physiology , Melanoma/immunology , Skin Neoplasms/immunology , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP-Binding Cassette Transporters/genetics , Adult , Aged , Animals , Antigen Presentation , Humans , Immunotherapy , Interferon-alpha/pharmacology , Janus Kinases , Leukocytes, Mononuclear , Male , Melanoma/drug therapy , Mice, Inbred C57BL , Middle Aged , Neoplasm Transplantation , Phosphorylation , Protein Processing, Post-Translational , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Skin Neoplasms/drug therapy , Up-RegulationABSTRACT
The zona pellucida (ZP) is a glycoprotein matrix surrounding mammalian oocytes. Upon fertilization, ZP hardening prevents sperm from binding to and penetrating the ZP. Here, we report that targeted gene deletion of the liver-derived plasma protein fetuin-B causes premature ZP hardening and, consequently, female infertility. Transplanting fetuin-B-deficient ovaries into wild-type recipients restores fertility, indicating that plasma fetuin-B is necessary and sufficient for fertilization. In vitro fertilization of oocytes from fetuin-B-deficient mice only worked after rendering the ZP penetrable by laser perforation. Mechanistically, fetuin-B sustains fertility by inhibiting ovastacin, a cortical granula protease known to trigger ZP hardening. Thus, plasma fetuin-B is necessary to restrain protease activity and thereby maintain ZP permeability until after gamete fusion. These results also show that premature ZP hardening can cause infertility in mice.
Subject(s)
Fertilization , Fetuin-B/metabolism , Gene Expression Regulation, Developmental , Zona Pellucida/pathology , Animals , Cell Membrane Permeability , Embryo Transfer/methods , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Enzyme Activation , Female , Fertilization in Vitro , Fetuin-B/genetics , Infertility, Female/metabolism , Infertility, Female/pathology , Male , Metalloproteases/antagonists & inhibitors , Metalloproteases/genetics , Metalloproteases/metabolism , Mice , Mice, Inbred C57BL , Oocytes/metabolism , Oocytes/pathology , Ovary/metabolism , Ovary/transplantation , Pregnancy , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Spermatozoa/metabolism , Spermatozoa/physiology , Zona Pellucida/drug effects , Zona Pellucida/metabolismABSTRACT
New biomaterial related reference materials with known genotoxic properties were produced in order to study the sample preparation and in vitro genotoxicity testing of biomaterials. We incorporated genotoxic substances like benzo[a]pyrene into the biomaterial Tecoflex, a polyurethane frequently used for catheters and other applications. We demonstrated that the model compound benzo[a]pyrene is sufficiently extracted by organic solvents, whereas cell culture medium only extracts very limited quantities. By changing the medium several times during extraction the extracted amount was augmented. Using higher amounts of organic solvent in relation to the reference material's surface led to a higher recovery of extracted benzo[a]pyrene. For the in vitro genotoxicity testing using the Mammalian Cell Gene Mutation Test (HPRT test), Mammalian Chromosome Aberration Test, and bacterial umu- and SOS-tests, concentration of extracts is a prerequisite because of the low sensitivity of the test systems. Often cytotoxicity interferes with the evaluation of genotoxic effects. We demonstrated that some recommendations of the ISO 10993-Part 3 and 12,(1),(2) dealing with the biological evaluation of medical devices, seem to be insufficient, and new rules for the in vitro genotoxicity testing of biomaterials have to be established.
