ABSTRACT
Dendritic cells (DC) are central regulators of immunity. Signal-induced maturation of DCs is assumed to be the starting point for specific immune responses. To further understand this process, we analyzed the alteration of transcript profiles along the time course of CD40 ligand-induced maturation of human myeloid DCs by Affymetrix GeneChip microarrays covering >6800 genes. Besides rediscovery of genes already described as associated with DC maturation proving reliability of the methods used, we identified clusterin as novel maturation marker. Looking across the time course, we observed synchronized kinetics of distinct functional groups of molecules whose temporal coregulation underscores known cellular events during dendritic cell maturation. For example, an early-peaking wave of inflammatory chemokines was followed by a sustained increase of constitutive chemokines and accompanied by slow but continuous induction of survival proteins. After an immediate but transient induction of cytokine-responsive transcripts, there was an increased expression of a group of genes involved in not only the regulation of cytokine effects, but also of transcription in general. Our results demonstrate that microarray studies along time courses combined with real-time PCR not only discover new marker molecules with functional implications, but also dissect the molecular kinetics of biological processes identifying complex pathways of regulation.
Subject(s)
Dendritic Cells/metabolism , Gene Expression Profiling , Genome, Human , 3T3 Cells , Animals , Antigens, CD/analysis , B7-1 Antigen/analysis , B7-2 Antigen , Cell Survival/genetics , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/immunology , Flow Cytometry , Humans , Immunoglobulins/analysis , Membrane Glycoproteins/analysis , Mice , Oligonucleotide Array Sequence Analysis/methods , RNA/genetics , RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Time Factors , Transcription, Genetic , CD83 AntigenABSTRACT
We applied a combined data mining and experimental validation Approach for the discovery of germ cell-specific genes aberrantly expressed in cancer. Six of 21 genes with confirmed germ cell specificity were detected in tumors, indicating that ectopic activation of testis-specific genes in cancer is a frequent phenomenon. Most surprisingly one of the genes represented lactate dehydrogenase C (LDHC), the germ cell-specific member of the lactate dehydrogenase family. LDHC escapes from transcriptional repression, resulting in significant expression levels in virtually all tumor types tested. Moreover, we discovered aberrant splicing of LDHC restricted to cancer cells, resulting in four novel tumor-specific variants displaying structural alterations of the catalytic domain. Expression of LDHC in tumors is neither mediated by gene promotor demethylation, as previously described for other germ cell-specific genes activated in cancer, nor induced by hypoxia as demonstrated for enzymes of the glycolytic pathway. LDHC represents the first lactate dehydrogenase isoform with restriction to tumor cells. In contrast to other LDH isoenzymes, LDHC has a preference for lactate as a substrate. Thus LDHC activation in cancer may provide a metabolic rescue pathway in tumor cells by exploiting lactate for ATP delivery.
Subject(s)
Isoenzymes/biosynthesis , Isoenzymes/genetics , L-Lactate Dehydrogenase/biosynthesis , L-Lactate Dehydrogenase/genetics , Neoplasms/enzymology , Neoplasms/genetics , Alternative Splicing , Databases, Nucleic Acid , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Male , Reverse Transcriptase Polymerase Chain Reaction , Testicular Neoplasms/enzymology , Testicular Neoplasms/genetics , Transcription, GeneticABSTRACT
Cancer/testis-antigens (CTA), a novel and expanding family of immunogenic proteins detected by serological screening of recombinant cDNA expression libraries, encompass promising candidate targets for T-cell based immunotherapy. We screened kryo-preserved tissue of cutaneous T cell lymphoma (CTCL, n=36) such as mycosis fungoides (MF, n=17), pleomorphic cutaneous T-cell lymphoma (n=8) and Sezary's syndrome (SS, n= 11) as well as a non-malignant entity (small plaques parapsoriasis, SPP, n=5), for the expression of CTA by RT-PCR and Northern blot hybridization. From a panel of eleven CTA (MAGE-1, MAGE-C1, MAGE-3, BAGE, GAGE, SSX-1, SSX-2, SSX4, SCP-1, NY-ESO-1 and TS85) (HOM-Tes-85), mRNA expression could be detected for SCP-1 in 8/17 MF and 6/8 pleomorphic CTCL patients but was completely absent in small plaques parapsoriasis. SS patients had a more heterogeneous antigen expression pattern: Gage (1/11), MAGE-1 (3/11), MAGE-3 (6/11), MAGE-C1 (5/11), NY-ESO-1 (7/11) and TS85 (5/11), with expression of MAGE-3 confirmed by immunohistochemistry. CTA could provide defined targets for antigen-based vaccination in a high percentage of cases with CTCL. SCP-1 might serve as an additional diagnostic indicator in early and clinically indistinct lesions suspicious for cutaneous T-cell lymphoma.
