ABSTRACT
The question whether interference with the ubiquitous splicing machinery can lead to cell-type specific perturbation of cellular function is addressed here by T cell specific ablation of the general U5 snRNP assembly factor CD2BP2/U5-52K. This protein defines the family of nuclear GYF domain containing proteins that are ubiquitously expressed in eukaryotes with essential functions ascribed to early embryogenesis and organ function. Abrogating CD2BP2/U5-52K in T cells, allows us to delineate the consequences of splicing machinery interferences for T cell development and function. Increased T cell lymphopenia and T cell death are observed upon depletion of CD2BP2/U5-52K. A substantial increase in exon skipping coincides with the observed defect in the proliferation/differentiation balance in the absence of CD2BP2/U5-52K. Prominently, skipping of exon 7 in Mdm4 is observed, coinciding with upregulation of pro-apoptotic gene expression profiles upon CD2BP2/U5-52K depletion. Furthermore, we observe enhanced sensitivity of naïve T cells compared to memory T cells to changes in CD2BP2/U5-52K levels, indicating that depletion of this general splicing factor leads to modulation of T cell homeostasis. Given the recent structural characterization of the U5 snRNP and the crosslinking mass spectrometry data given here, design of inhibitors of the U5 snRNP conceivably offers new ways to manipulate T cell function in settings of disease.
Subject(s)
Homeostasis , T-Lymphocytes , Animals , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Mice , Apoptosis , Cell Differentiation/immunology , Ribonucleoprotein, U5 Small Nuclear/genetics , Ribonucleoprotein, U5 Small Nuclear/metabolism , Ribonucleoprotein, U5 Small Nuclear/immunology , Cell Proliferation , Lymphopenia/immunology , Lymphopenia/genetics , RNA SplicingABSTRACT
Gene therapy is a new therapeutic approach for the treatment of human cancers. Gene expression systems that can be regulated by drugs have been developed to improve the safety and efficacy of therapeutic transgene delivery. One of the most promising systems is the tetracycline (Tet)-responsive system in the Tet-On configuration. A major problem of the Tet-On system if used in viral vectors is the high basal activity of the Tet response element (TRE) promoter leading to leaky expression of transgenes under uninduced conditions. We therefore evaluated novel TRE promoters for controlling gene expression in an adenovirus vector (AdV) Tet-On system and further investigated them for expression of the pro-apoptotic CD95/Fas ligand (FasL) in human epithelial carcinoma cell line (HeLa) and lung cancer cells. Plasmid-based reporter gene assays showed that modifications within the tetO (7) and minimal immediate early cytomegalovirus promoter (CMV)(min) sequence of the TRE promoter reduced its leakiness and led to a markedly improved regulatability by doxycycline. Among several TRE promoters tested, a new construct (TRE-Tight1) containing modifications of both the tetO (7) sequence and the CMV(min) showed 11-fold reduced leakiness and 1.5-fold increased absolute transgene expression levels after induction, as compared to the original TRE. Under induced conditions, a TRE-Tight1 promoter-dependent AdV expressing the pro-apoptotic CD95L/FasL induced apoptosis and cell lysis in HeLa cells as efficiently as an AdV containing the original TRE promoter. In contrast to the latter, however, the vector with the modified TRE promoter left cells totally unaffected in the absence of the inducer. Stringently regulated induction of apoptosis and cell death by TRE-Tight1-AdV was also demonstrated in three human lung cancer cell lines. These data show that the novel TRE-Tight1 promoter has a high potential for closely controlled and efficient expression of cytotoxic genes in AdV-based anti-cancer approaches.
Subject(s)
Adenoviridae/genetics , Genetic Therapy/methods , Genetic Vectors/genetics , Lung Neoplasms/genetics , Membrane Glycoproteins/genetics , Tumor Necrosis Factors/genetics , Apoptosis/drug effects , Doxycycline/pharmacology , Fas Ligand Protein , Gene Expression Regulation/drug effects , HeLa Cells , Humans , Lung Neoplasms/therapy , Membrane Glycoproteins/drug effects , Promoter Regions, Genetic , Tetracycline/pharmacology , Toxicity Tests , Tumor Cells, CulturedABSTRACT
Restricted replication-competent adenoviruses (RRCAs) using tumor- and tissue-specific promoters (ttsP's) are new tools for cancer gene therapy. In this study we investigated viral and nonviral factors affecting "leakiness" of several ttsP's and their relevance for nonspecific ttsP-dependent RRCA (ttsP-RRCA) replication. The leakiness of the ttsP's in nontarget cells was per se highly variable and correlated with levels of nonspecific ttsP-RRCA replication. Transcriptional regulator elements fused to ttsP's showed variable effects: a hypoxic response element reduced leakiness of an alpha-fetoprotein promoter. In contrast, a mouse tyrosinase enhancer increased leakiness of a tyrosinase promoter, although it was not affected by a human tyrosinase enhancer. Furthermore, leakiness of ttsP's was enhanced by 5'-terminal adenoviral E1A enhancers, and adenoviral E1A-13S was found to be a strong transactivator of ttsP's, leading to "autoactivation" of leaky ttsP-RRCAs. In a proof-of-principle study, ttsP-RRCA replication was shown to be inhibited by a tetracycline-controlled transcriptional silencer via direct ttsP silencing. This opens up the prospect of pharmacological regulation of ttsP-RRCAs. Together, these data indicate that leakiness of ttsP's induced by several factors is a major cause of nonspecific ttsP-RRCA replication. Consideration of these factors may help optimize ttsP-dependent RRCA vectors and may thereby improve their safety.