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 define genetic determinants of tumor cell resistance to the cytotoxic action of tumor necrosis factor alpha (TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis of available literature data, the striking finding is the association of some differentially expressed genes involved in the phosphatidylinositol-3-kinase/Akt signaling pathway. More notably, we found that the PRNP gene coding for the cellular prion protein (PrP(c)), was 17-fold overexpressed in the 1001 cell line as compared with the MCF7 cell line. This differential expression was confirmed at the cell surface by immunostaining that indicated that PrP(c) is overexpressed at both mRNA and protein levels in the TNF-resistant derivative. Using recombinant adenoviruses expressing the human PrP(c,) our data demonstrate that PrP(c) overexpression converted TNF-sensitive MCF7 cells into TNF-resistant cells, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondria and nuclear condensation.
Subject(s)
Breast Neoplasms/pathology , Cell Death/drug effects , Drug Resistance, Neoplasm , PrPC Proteins/pharmacology , Tumor Necrosis Factor-alpha/toxicity , Cell Line, Tumor , DNA, Complementary/genetics , Enzymes/genetics , Female , Humans , Oligonucleotide Array Sequence Analysis , TransfectionABSTRACT
During the two least decades, the field of tumor immunology has met an expansion of knowledge about the molecular and cellular bases of immune regulation. The identification of cancer antigens has been of critical importance and cancer vaccine is at present a very fast moving field. However, the immunotherapy approaches in cancer are of modest success. This is mainly due to the capacity of tumor cells to escape from immunological detection and to resist to cell mediated cytotoxicity. We will discuss some mechanisms associated with the acquisition of this tumor resistance and the alteration of T cell function and how cancer profiling through genomics approaches may help to reconceptualize immunotherapy strategies.
Subject(s)
Neoplasms/immunology , Tumor Escape/immunology , Apoptosis , Fas Ligand Protein , Humans , Immunologic Surveillance , Lymphocytes, Tumor-Infiltrating/physiology , Membrane Glycoproteins/physiology , NF-kappa B/physiology , Neoplasms/therapy , Perforin , Pore Forming Cytotoxic Proteins , Receptors, Immunologic/physiology , Receptors, KIR , Receptors, Natural Killer Cell , Serine Endopeptidases/physiology , T-Lymphocytes, Cytotoxic/physiology , Tumor Suppressor Protein p53/physiology , fas Receptor/physiologyABSTRACT
The inter-alpha trypsin inhibitor (ITI) family is a group of proteins built up from different combinations of I light chain (ITI-L) and 3 highly homologous heavy chains (ITI-HI, -H2 and -H3). To investigate a potential role of the ITI family chains in cancer and metastasis spreading, we engineered human H460M cell lines expressing both the green fluorescent protein (GFP) and one of these chains. These clones were subcutaneously injected in athymic nude mice, and lung metastasis number and primary tumor weight were determined after 28 days. Expression of the ITI-L chain considerably decreased tumor weight and fluorescent lung metastasis number. ITI-HI and ITI-H3 chain expression induced a significant decrease of metastasis number, whereas no decrease of tumor weight could be detected. In vitro, ITI-L expression significantly decreased chemotaxis and ITI-HI and ITI-H3 expression increased cell attachment. These results argue for the antitumoral or antimetastatic properties of ITI-L, -HI and -H3 chains.
