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Biomedical subjects

I M Verma

Publications and source records attributed to I M Verma.

At least 19 recordsLinked to original sources
PubMed1992

Gene therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo.

We have explored the use of primary myoblasts as a somatic tissue for gene therapy of acquired and inherited diseases where systemic delivery of a gene product may have therapeutic effects. Mouse primary myoblasts were infected with replication-defective retroviruses expressing canine factor IX cDNA under the control of a mouse muscle creatine kinase enhancer and human cytomegalovirus promoter. The infected myoblasts were injected into the hindlegs of recipient mice and levels of secreted factor IX protein were monitored in the plasma. We report sustained expression of factor IX protein for over 6 months without any apparent adverse effect on the recipient mice.

Animals↗
PubMed1992

Direct association of pp40/I kappa B beta with rel/NF-kappa B transcription factors: role of ankyrin repeats in the inhibition of DNA binding activity.

To understand the mechanism by which pp40/I kappa B beta inhibits DNA binding activity of the rel/NF-kappa B family of transcription factors, we have investigated the role of ankyrin repeats on the biological function of pp40 by deleting or mutating conserved residues. We show that (i) ankyrin repeats alone are not sufficient to manifest biological activity but require the C-terminal region of the pp40 protein; (ii) four out of the five ankyrin repeats are essential for inhibiting the DNA binding activity; (iii) pp40 mutants that do not inhibit DNA binding of rel protein also do not associate with rel; (iv) although pp40 can associate with the p65 and p50 subunits of NF-kappa B, pp40 inhibits the DNA binding activity of only the p50-p65 heterodimer and the p65 homodimer; and (v) pp40 inhibits the transcription of genes linked to kappa B site; however, mutants that do not affect DNA binding have no effect. We propose that the ankyrin repeats and the C-terminal region of pp40 form a structure that associates with the rel homology domain to inhibit DNA binding activity.

Amino Acid Sequence↗
PubMed1992

I kappa B gamma, a 70 kd protein identical to the C-terminal half of p110 NF-kappa B: a new member of the I kappa B family.

A cDNA corresponding to the 2.6 kb NF-kappa B mRNA species present in a variety of lymphoid cell lines has been molecularly cloned. The deduced 607 amino acid sequence is identical to the sequence of the C-terminal region of 110 kd NF-kappa B protein. A 70 kd protein can be identified in lymphoid cells using antibodies raised against the C-terminal region of p110 NF-kappa B. Comparison of the two-dimensional tryptic peptide maps of the 70 kd protein expressed in cells and the in vitro translated product encoded by the cDNA display extensive homology. The 70 kd protein expressed in bacteria prevents sequence-specific DNA binding of p50-p65 NF-kappa B heterodimer, p50 homodimer, and c-rel. p70 also interferes with transactivation by c-rel and prevents its nuclear translocation. The 70 kd protein, predominantly found in lymphoid cells, is a new member of the I kappa B family of proteins and is referred to as I kappa B gamma.

Amino Acid Sequence↗
PubMed1992

Functional antagonism between c-Jun and MyoD proteins: a direct physical association.

The product of the proto-oncogene Jun inhibits myogenesis. Constitutive expression of Jun in myoblasts interferes with the expression and the function of MyoD protein. In transient transfection assays Jun inhibits transactivation of the MyoD promoter, the muscle creatine kinase enhancer, and a reporter gene linked to MyoD DNA-binding sites. Conversely, MyoD suppresses the transactivation by Jun of genes linked to an AP-1 site. We demonstrate that both in vivo and in vitro MyoD and Jun proteins physically interact. Mutational analysis suggests that this interaction occurs via the leucine zipper domain of Jun and the helix-loop-helix region of MyoD.

Animals↗
PubMed1992

Long-term expression of a retrovirally introduced beta-galactosidase gene in rodent cells implanted in vivo using biodegradable polymer meshes.

Grafts of various types of cells have been performed using bioresorbable polymer matrices. These synthetic fibers are degraded by hydrolysis into normal metabolic intermediates and induce a number of events that are conductive to healing and/or repair, the most important of which may be angiogenesis. The use of biodegradable meshes to deliver genetically altered cells was studied. A beta-galactosidase gene was inserted into Long-Evans rat bone marrow stromal (BMS) cells or fibroblasts derived from C57BL/6J mouse embryos using the retroviral vector LNL-SLX beta gal. Expression was monitored using X-gal staining. X-gal+ cells from monolayer cultures were seeded onto either polyglycolic acid (PGA) or polyglactin (PGL) biodegradable meshes and grown to confluence. Two types of grafts were performed: (1) embryonic C57BL/6J mouse fibroblasts (EMF) into either nude mice or adult C57BL/6J mice, and (2) Long-Evans rat BMS into Long-Evans rats. Beta-Galactosidase activity was found for up to 152 days for EMF in nude mice, 123 days for EMF in adult C57BL/6J mice, and 90 days for grafts of syngeneic BMS cells into Long-Evans rats. Noninfected cells grafted using the same methods did not stain with X-gal.

Animals↗
PubMed1992

Circulating human or canine factor IX from retrovirally transduced primary myoblasts and established myoblast cell lines grafted into murine skeletal muscle.

We have used retroviral vectors to introduce human or canine factor IX cDNAs into cultured primary murine and canine myoblasts and into the established murine myoblast cell line C2C12. In all cases, the stably infected cells produced biologically active factor IX in culture and secreted detectable amounts into the culture medium both before and after differentiation of the cells into myotubes. Myoblasts and differentiated myotubes are therefore capable of performing all the posttranslational modifications of the coagulation factor required for biological activity. We have grafted the genetically modified myoblasts into skeletal muscles of nude mice and have detected stable levels of circulating human factor IX for up to two months after grafting. We propose that grafting genetically modified primary myoblasts or established myoblast cell lines into skeletal muscle may represent a useful approach to gene therapy for a variety of genetic diseases, including intrinsic muscle disease and defects in circulating proteins as in the hemophilias.

Animals↗
PubMed1992

Signal transduction: the nuclear target.

Fos and jun heterodimers activate the transcription of genes containing an AP-1 site. The activity of Fos and Jun proteins is regulated by post-translational modification. The activity of the rel/NF-kappa family of transcriptional factors is regulated by their sequestration in the cytoplasm in association with the inhibitor protein, I kappa B. An ankyrin repeat motif in I kappa B proteins is required for their direct association with rel/NF-kappa B.

Animals↗
PubMed1992

Retroviral vectors for persistent expression in vivo.

Retroviral vectors provide a safe and efficient method of introducing genes of therapeutic interest into dividing cells. The principle limitation of these vectors in the past has been poor gene expression in vivo. This problem has been overcome recently through the use of tissue-specific enhancers in commonly used retroviral vectors. In this review we discuss both the relevant biology and some of the practical applications of retroviral vectors in gene therapy.

Animals↗
PubMed1992

The proto-oncogene bcl-3 encodes an I kappa B protein.

The bcl-3 gene product, overexpressed in chronic lymphocytic leukemia (CLL) patients with the translocation t(14;19), is a member of the I kappa B family. The bcl-3 protein is able to inhibit the DNA binding and trans-activation of authentic NF-kappa B heterodimers p50-p65 and p49-p65, as well as p50 and p49 homodimers. The bcl-3 protein does not inhibit either the DNA-binding activity of the Rel protein or its ability to trans-activate genes linked to the kappa B site. A human 37-kD protein (I kappa B alpha), identified previously as a member of the I kappa B family, is also unable to inhibit DNA-binding activity of the Rel protein. However, unlike bcl-3, the 37-kD (I kappa B alpha) protein has no effect on the DNA-binding activity of p50 or p49 homodimers. Two dimensional phosphotryptic peptide maps of the human bcl-3 and the human 37-kD (I kappa B alpha) proteins reveal that the phosphopeptides from the 37-kD (I kappa B alpha) protein are nested within the bcl-3 protein. Furthermore, bcl-3 antisera immunoprecipitates an in vitro-radiolabeled 37-kD (I kappa B alpha) protein. Proteins of 56 and 38 kD can be identified in HeLa cells stimulated with PMA and immunoprecipitated with bcl-3 antisera. Comparison of tryptic peptide maps of the bcl-3 protein synthesized in vitro, and p56 and p38 from HeLa cells, shows that they are all structurally related. Removal of the amino-terminal sequences of the bcl-3 protein generates a protein that inhibits the DNA binding of the p50-p65 heterodimer but, like the 37-kD (I kappa B alpha) protein, is no longer able to inhibit the binding of the p50 and p49 homodimers with kappa B DNA. We propose that the bcl-3 and 37-kD (I kappa B alpha) proteins are related and are members of the I kappa B family.

3T3 Cells↗
PubMed1992

Transformation by FosB requires a trans-activation domain missing in FosB2 that can be substituted by heterologous activation domains.

Two functionally distinct proteins derived from the FosB gene by alternative splicing have recently been described. FosB protein transforms fibroblasts efficiently, whereas FosB2 protein, a carboxy-terminally truncated form of FosB, does not, despite the fact that both proteins can participate in high-affinity, sequence-specific DNA binding as part of a heterodimeric complex with c-Jun protein. We show here that the functional difference between these proteins is the result of the presence of a potent proline-rich transcriptional activation domain in the carboxy-terminal amino acids unique to FosB. This conclusion is supported by three lines of evidence: (1) Mutations in the carboxy-terminal region of FosB that impair transcriptional activation also reduce transforming potential, despite the fact that DNA binding as part of a complex with c-Jun is not affected; (2) the carboxy-terminal region unique to FosB functions as an activation domain when fused to the DNA-binding domain of GAL4; and (3) transforming potential can be conferred on FosB2 by fusing any of several different well-characterized trans-activation domains. These results identify an additional functional requirement for transformation by Fos proteins and have implications for the mechanism(s) of mitogenic signaling by the AP-1 transcription complex.

Amino Acid Sequence↗
PubMed1992

Alteration of a cyclic AMP-dependent protein kinase phosphorylation site in the c-Fos protein augments its transforming potential.

We have studied the phosphorylation of the nuclear oncoprotein Fos by cyclic AMP-dependent protein kinase (PKA). We demonstrate that the human c-Fos protein, phosphorylated either in vitro with purified PKA or in vivo in JEG3 cells following treatment with forskolin, has similar phosphotryptic peptide maps. Serine 362, which constitutes part of a canonical PKA phosphorylation site (RKGSSS), is phosphorylated both in vivo and in vitro. A mutant of Fos protein in which serine residues 362 to 364 have been altered to alanine residues is not efficiently phosphorylated in vitro. Furthermore, Fos protein in which serines 362 to 364 have been altered to alanine shows increased transforming potential. We propose that phosphorylation of Fos by PKA is an important regulatory step in controlling its activity in normal cell growth and differentiation.

Amino Acid Sequence↗
PubMed1992

Direct gene transfer to the liver with herpes simplex virus type 1 vectors: transient production of physiologically relevant levels of circulating factor IX.

We have used gene transfer vectors derived from a replication-defective mutant of herpes simplex virus type 1 (HSV-1) expressing the hepatitis B virus surface antigen (HBsAg), Escherichia coli beta-galactosidase (beta-gal), or canine factor IX (cFIX) from the immediate early promoter of human cytomegalovirus (hCMV) to infect mouse liver by direct injection or through the portal vein. By either route, high levels of transgene expression were demonstrated by the detection of immunoreactive HBsAg or cFIX in the circulation and by histochemical detection of beta-gal activity in situ. The results were striking in that the serum level of cFIX reached 10% of the normal murine levels. Although the level of transgene expression from the hCMV promoter was transient, a significant number of persistent vectors could be rescued from the livers of recipient mice up to 2 months after inoculation. Replacement of the hCMV promoter with the HSV-1 latency-associated transcript (LAT) promoter resulted in reduced but prolonged expression of both HBsAg and cFIX. The very high level of factor IX expression suggests that clinically useful gene transfer may eventually be feasible through direct vector delivery to the liver.

Animals↗
PubMed1992

Transformation by Jun: requirement for leucine zipper, basic region and transactivation domain and enhancement by Fos.

Mutants in the leucine zipper and basic regions of mouse c-jun were tested for transformation in chicken embryo fibroblast cultures. Reduction or elimination of the ability of Jun to dimerize or to bind to DNA severely decreased transformation. A chicken v-jun gene from which the major transactivation domain was deleted also failed to transform. We conclude that an intact leucine zipper, basic region and transactivation domain are required for Jun-induced oncogenic transformation. Coexpression of chicken c-Fos increased formation of transformed foci by Jun proteins of moderate to low oncogenic potency but had no effect on highly transforming Jun. Chicken c-Fos could also transform chicken embryo fibroblasts on its own, albeit after prolonged culture and at a low efficiency.

Amino Acid Sequence↗
PubMed1991

Retinoic acid is a negative regulator of AP-1-responsive genes.

We present evidence that retinoic acid can down-regulate transcriptional activation by the nuclear protooncogene c-jun. All three members of the retinoic acid receptor (RAR) subfamily (RAR alpha, RAR beta, and RAR gamma) can repress transcriptional induction of the human collagenase gene or a heterologous promoter that contains the collagenase promoter AP-1-binding site. In contrast, the retinoid X receptor fails to repress Jun/AP-1 activity, demonstrating a significant difference between the two regulatory systems through which retinoids exert their transcriptional control. Analysis of RAR alpha mutants in transfection studies reveals that the DNA-binding domain is important for the inhibition of Jun/AP-1 activity, even though the RAR does not bind the collagenase AP-1 site. Rather, gel-retardation assays reveal that bacterially expressed full-length RAR alpha inhibits binding of Jun protein to target DNA. These data suggest that the RAR alpha may form a nonproductive complex with c-Jun and provides a simple mechanisms by which retinoic acid may limit cell growth and possibly malignant progression.

Animals↗
PubMed1991

An alternative spliced form of FosB is a negative regulator of transcriptional activation and transformation by Fos proteins.

Two forms of FosB transcript and their products can be identified in mouse NIH 3T3 cells following serum induction. The larger RNA codes for a 338-amino acid protein, whereas the smaller RNA results from the removal of an additional 140 nucleotides from FosB mRNA by alternative splicing. This alternative splicing event places a stop codon following the "leucine zipper" region and results in a shorter protein (FosB2) of 237 amino acids that lacks 101 amino acids at the carboxyl terminus. FosB2 is able to form heterodimers with c-Jun and bind to an AP-1 site but is not able to activate the transcription of promoters containing AP-1 sites. Furthermore, FosB2 can not only suppress the transcriptional activation by c-Fos and c-Jun of promoters containing an AP-1 site but also interferes with the transforming potential of viral and cellular Fos proteins. We propose that FosB2 protein functions as a trans-negative regulator.

Amino Acid Sequence↗
PubMed1991

Long-term in vivo expression of retrovirus-mediated gene transfer in mouse fibroblast implants.

Toward the goal of gene therapy, we have been attempting to establish model somatic cell systems with the potential of sustained expression of the foreign gene. We report here that long-term expression of foreign genes in mouse embryo fibroblast implants can be achieved if a housekeeping gene promoter is used to drive transcription. Specifically, we have shown that in implants containing a beta-galactosidase gene linked to either an immediate early promoter of cytomegalovirus or a dihydrofolate reductase (DHFR) gene promoter, only the DHFR promoter allows long-term expression. We propose that choice of the promoter manifests significant influence on the long-term expression of genes introduced in fibroblast implants by retroviral vectors.

Animals↗
PubMed1991

c-rel activates but v-rel suppresses transcription from kappa B sites.

We show that the product of the protooncogene c-rel is a constituent of an NF-kappa B-like complex that binds to the kappa B site originally identified in the enhancer of immunoglobulin kappa light chain gene. c-rel protein synthesized in bacteria binds to the kappa B site in a sequence-specific manner. The rel-kappa B complex can be disrupted by incubation with anti-rel antibodies. The rel protein can form oligomers. The c-rel protein can activate transcription from promoters containing kappa B sites; v-rel, on the other hand, suppresses the transcription of genes linked to kappa B sites. Thus, v-rel may interfere with the normal transcriptional machinery of the cell by acting as a dominant negative mutant.

Amino Acid Sequence↗