Direct gene transfer into cardiac myocytes in vivo

Recent studies have demonstrated that cardiac and skeletal myocytes share the ability to take up and stably express plasmid DNA injected directly into myocardium or skeletal muscle in vivo. Although this is a relatively inefficient process, with less than 1% of the myocytes expressing the injected recombinant DNA, expression in these cells is stable for periods of at least 6 months. The majority of the injected DNA is maintained in myocytes as an episome and apparently does not undergo DNA replication. The direct DNA injection approach has been used to map cardiac-specific transcriptional regulatory elements in cellular promoter/enhancers. Expression of recombinant proteins in the heart following direct DNA injection also holds promise for the treatment of a variety of acquired and inherited cardiovascular diseases.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29917/1/0000274.pd

Two distinct mechanisms of interleukin-2 gene expression in human T lymphocytes

Interleukin-2 (IL-2) gene regulation was investigated in primary cultures of highly purified human peripheral blood CD28+T cells. Two discrete mechanisms for induction of T-cell proliferation could be distinguished by examining cell cycle progression and the expression of the IL-2 gene. Stimulation of cells by CD3 MoAb induced only transiently expressed, small amounts of IL-2 mRNA that was completely suppressed by cyclosporine. Costimulation of T cells with CD3 MoAb and either CD28 MoAb or PMA, but not calcium ionophore, induced a 50-100-fold increased in IL-2 gene expression and secretion. High levels of IL-2 gene expression could also be achieved by stimulation with calcium ionophore and PMA or CD28 MoAb and PMA, but not by CD28 MoAb plus calcium ionophore. IL-2 gene expression and T-cell proliferation induced by CD3 MoAb plus PMA or calcium ionophore plus PMA were completely suppressible by cyclosporine. In contrast, IL-2 gene expression and T-cell proliferation induced by CD28 MoAb plus PMA were unaffected by cyclosporine. The CD28 signal was dependent on new protein synthesis. Nuclear run-on transcription assays showed that anti-CD28 did not affect lymphokine transcription. A major effect of CD28 stimulation on mRNA stability was shown by studies using actinomycin D; CD28 stimulation substantially increased the half-life of IL-2 and TNF-alpha mRNA. The effects of anti-CD28 stimulation were specific for growth factors, and thus differ from previously described effects of cycloheximide on mRNA stability. These studies suggest the existence of two biochemical pathways for the induction of IL-2 production, one that occurs at the transcriptional level and is mediated by intracellular calcium release and protein kinase C and is cyclosporine-sensitive, and one that acts post-transcriptionally, is mediated by CD28 stimulation, and is cyclosporine-resistant.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27894/1/0000314.pd

Polymorphic markers related to a single Tcrb-V6 gene segment

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46749/1/251_2004_Article_BF00230507.pd

The 4F2 heavy chain gene: a molecular model of inducible gene expression in human T cells

We have utilized the human 4F2 heavy chain (4F2HC) gene as a model system in studies designed to elucidate the molecular events involved in regulating inducible gene expression during normal human T-cell activation. In previous studies we have shown that steady state levels of 4F2HC mRNA are induced 50-60-fold within 6 h of T-cell activation by phytohemagluttinin (PHA) and that the induction of 4F2HC gene expression involves both the protein kinase C and calcium-mediated activation pathways. Despite the fact that the 4F2HC gene is highly regulated in T cells, the 5' upstream region of the 4F2HC gene contains a housekeeping promoter which is G + C rich, lacks TATA or CCAAT sequences, and contains four potential binding sites for the ubiquitous Sp1 transcription factor. The major regulatory elements of the 4F2HC gene do not reside within this 5' upstream region but instead, map to the exon 1-intron 1 region of the gene. The low levels of mature 4F2HC mRNA in resting T cells result from a block to transcription elongation within the exon 1-intron 1 region of the gene rather than promoter inactivity. Phorbol ester stimulation of resting T cells induces 4F2HC gene expression by removing this block to transcription elongation. We now report that in addition to its ability to serve as a transcriptional attenuator, the 4F2HC first intron contains a powerful enhancer element which is active in a wide variety of cell types including malignant human T cells. Full enhancer activity is displayed by a 186 bp fragment of the first intron which contains binding sites for two novel nuclear proteins (NF-4FA and NF-4FB) which flank a consensus binding site for the AP-1 transcription factor. A cDNA encoding the NF-4FB enhancer binding protein has been cloned by screening a lambda gt11 cDNA library with a rabiolabelled oligonucleotide corresponding to the NF-4FB recognition sequence.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27895/1/0000315.pd

DNA fragments of altered electrophoretic mobility in leukemia samples can arise from double-strand DNA breaks at nuclease hypersensitive sites of active genes

Chromosome translocations that disrupt or alter gene function have been implicated in the pathogenesis of a variety of malignancies. Therefore, identification of a translocation breakpoint has become a more important means by which to identify genes involved in cellular transformation. A common site of translocation in myeloid and lymphoid malignancies involves 11q23. One human protooncogene, ETS1, has been localized to this chromosomal segment, and several tumors with 11q23 translocations have been shown to have altered ETS1 DNA migration after restriction enzyme digestion. Two laboratories, however, have recently localized the 11q23 breakpoint region to a small region of DNA telomeric of the CD3 loci, a region at considerable distance from the ETS1 gene locus. Therefore, it is difficult to reconcile the studies that suggest altered migration of fragments associated with ETS1 and lack of a localization of the breakpoint to a region near the ETS1 gene. Recently, in our studies to characterize the promoter/enhancer region of the ETS1 protooncogene, we had the opportunity to analyze DNA from 18 patients with acute leukemia involving chromosome 11q23 aberrations. We were unable to demonstrate rearrangement of the ETS1 gene in this group, thus confirming that the 11q23 breakpoint does not involve ETS1 protooncogene. In one patient, however, a DNA break in the region of the ETS1 promoter was detected reproducibly. This DNA break was mapped to the major DNaseI hypersensitive site in the ETS1 promoter. Mapping from both sides of the break demonstrated that the break must have occurred during processing of the leukemic cells for DNA analysis. Therefore, artifactual DNA breaks can occur at nuclease-hypersensitive sites of active genes. These data suggest that previous reports of chromosomal translocations involving the ETS1 protooncogene may have resulted from DNA breaks at nuclease hypersensitive sites. This mechanism may account for sporadic case reports of altered restriction enzyme fragment migration involving genes that are not ultimately shown to be associated with the chromosome translocation being examined.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30693/1/0000338.pd

T-cell receptor gene rearrangement and expression in human natural killer cells: natural killer activity is not dependent on the rearrangement and expression of T-cell receptor α , β , or γ genes

To test the hypothesis that the T-cell receptor ( Tcr ) λ gene encodes a natural killer (NK) cell receptor molecule, three human NK clones and fresh peripheral blood lymphocytes with NK activity from two patients with a CD16 + lymphocytosis were analyzed for rearrangements and expression of the human Tcr α, β , and λ genes. Two of the clones displayed distinct rearrangements of their Tcr β and λ genes and expressed mature Tcr α, β , and αl RNA. However, one of the clones and both patient samples displayed marked NK activity but failed to rearrange or express any of their Tcr genes. These findings demonstrate that human natural killer activity is not dependent on Tcr λ gene rearrangement and expression. In addition, they confirm previous findings concerning the lack of Tcr α and β gene expression in some natural killer cells. Thus, they suggest the existence of additional NK-specific recognition molecules.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46745/1/251_2004_Article_BF00376117.pd

Polymorphism in a T-cell receptor variable gene is associated with susceptibility to a juvenile rheumatoid arthritis subset

This report demonstrates a T-cell receptor (Tcr) restriction fragment length polymorphism, defined by a Tcrb-V6.1 gene probe and Bgl II restriction enzyme, to be absolutely correlated with allelic variation in the coding sequence of a Tcrb-V6.1 gene. A pair of non-conservative amino acid substitutions distinguish the Tcrb-V6.1 allelic variants. An association of this Tcrb-V6.1 gene allelic variant with one form of juvenile rheumatoid arthritis (JRA) was established in a cohort of 126 patients. The association was observed in patients possessing the HLA-DQA1*0101 gene. Among HLA-DQA*0101 individuals, 19 of 26 patients (73.1%) carried one particular Tcrb-V6.1 gene allele as opposed to 11 of 33 controls (33%; p<0.005). Haplotypes carrying this HLA gene have previously been shown to confer increased risk for progression of arthritis in JRA. This demonstration of a disease-associated Tcrb-V gene allelic variant has not, to our knowledge, been previously reported and supports the contribution of polymorphism in the Tcr variable region genomic repertoire to human autoimmune disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46750/1/251_2004_Article_BF00166831.pd