ABSTRACT
Metastases are known to be more resistant to therapy than matching primary tumors, in particular they are less prone to apoptosis. In this study we investigated the functional interaction of a CTL clone (LT12) specific for a melanoma TA with the primary tumor (T1) versus its metastatic counterpart (G1). The CTL clone (LT12) was shown to lyse the primary T1 cells more efficiently in a classical cytotoxicity test. This differential susceptibility was not associated with MHC class I down-regulation and conjugate formation but correlated with a differential increase in Ca++ flux in the LT12 CTL when stimulated with the primary versus the metastatic tumor cells. Since LT12 uses perforin/granzyme B to kill its autologous target we analysed perforin and granzyme B mRNA expression in the CTL in the presence of either primary and metastatic melanoma cells. Quantitative PCR analysis showed an increased expression of granzyme B and perforin mRNA levels in LT12 when cocultured in the presence of the primary tumor. However, a similar level of (cytotoxic molecule) degranulation as revealed by CD107 expression was observed when LT12 was stimulated with T1 or G1 cells. These data suggest that the differential susceptibility of primary and metastatic melanoma cells involves at least in part their distinct potential to induce autologous CTL reactivity and the subsequent triggering of granzyme B and perforin in these cells.
Subject(s)
Cytotoxicity, Immunologic , Melanoma/immunology , Skin Neoplasms/immunology , T-Lymphocytes, Cytotoxic/immunology , Aged , Calcium/metabolism , Cell Communication , Cell Degranulation/immunology , Cell Line, Tumor , Clone Cells , Coculture Techniques , Down-Regulation , Granzymes/genetics , Granzymes/metabolism , Humans , Male , Melanoma/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Neoplasm Metastasis , Perforin , Pore Forming Cytotoxic Proteins/genetics , Pore Forming Cytotoxic Proteins/metabolism , RNA, Messenger/metabolism , Skin Neoplasms/metabolism , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
To investigate tumor resistance to T cell lysis, a resistant variant was selected after specific cytolytic T lymphocytes (CTL) selection pressure. Although the resistant variant triggered perforin and granzyme B transcription in specific CTLs, as well as their degranulation, it exhibited a dramatic resistance to cytotoxic T cell killing. It also displayed strong morphological changes with alterations of the actin cytoskeleton. Electron microscopy analysis revealed a loosen interaction between CTLs and the resistant variant despite the formation of apparently normal conjugates. Transcriptional profiling identified a gene expression signature that distinguished sensitive from resistant tumor targets. More notably, we found that actin-related genes ephrin-A1 and scinderin were overexpressed in resistant target. Silencing of these genes using RNA interference resulted in a restoration of normal cell morphology and a significant attenuation of variant resistance to CTL killing. Our present study shows that a shift in cytoskeletal organization can be used, by tumor cells, as a strategy to promote their resistance after CTL selection pressure.
Subject(s)
Actins/metabolism , Polymers/chemistry , T-Lymphocytes, Cytotoxic/immunology , Actins/chemistry , Cell Line, Tumor , Ephrin-A1/chemistry , Gelsolin/metabolism , Gene Silencing , Genetic Variation , Humans , Immunoprecipitation , Microscopy, Confocal , Microscopy, Electron , Neoplasms/metabolism , Oligonucleotide Array Sequence Analysis , Phenotype , Pressure , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes, Cytotoxic/metabolism , Time Factors , Transcription, GeneticABSTRACT
In this study, we have investigated the mechanisms used by wild-type p53 (wtp53) to potentiate tumor cell susceptibility to CTL-mediated cell death. We report that wtp53 restoration in a human lung carcinoma cell line Institut Gustave Roussy (IGR)-Heu, displaying a mutated p53, resulted in up-regulation of Fas/CD95 receptor expression associated with an increase of tumor cell sensitivity to the autologous CTL clone, Heu127. However, when IGR-Heu cells were transfected with Fas cDNA, no potentiation to Heu127-mediated lysis was observed, indicating that induction of CD95 is not sufficient to sensitize target cells to CTL killing. Importantly, our data indicate that the effect of wtp53 on the Fas-mediated pathway involves a degradation of short cellular FLICE inhibitory protein resulting in subsequent caspase 8 activation. Furthermore, we demonstrate that wtp53 restoration also resulted in CTL-induced Bid translocation into mitochondria and a subsequent mitochondrial membrane permeabilization leading to cytochrome c release. These results indicate that tumor cell killing by autologous CTL can be enhanced by targeting degranulation-independent mechanisms via restoration of wtp53, a key determinant of apoptotic machinery regulation.
