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J R Perez

Publications and source records attributed to J R Perez.

9 recordsLinked to original sources
PubMed1994

Sequence-independent induction of Sp1 transcription factor activity by phosphorothioate oligodeoxynucleotides.

Modified analogues of antisense oligodeoxynucleotides (ODNs), particularly phosphorothioates ([S]ODNs), have been extensively used to inhibit gene expression. The potential sequence specificity of antisense oligomers makes them attractive as molecular drugs for human diseases. The use of antisense [S]ODNs to inhibit gene expression has been complicated by frequent nonspecific effects. In this study we show in diverse cell types that [S]ODNs, independent of their base sequence, mediated the induction of an Sp1 nuclear transcription factor. The [S]ODN-mediated Sp1 induction was rapid and was associated with elevated levels of Sp1 protein. This induction was dependent on NF-kappa B activity, since inhibition of NF-kappa B activity abolished the [S]ODN-induced Sp1 activity. [S]ODN-induced Sp1 activity was seen in mouse spleen cells following in vivo administration. Sp1 activity induced by [S]ODNs required the tyrosine kinase pathway and did not have transactivating potential. These results may help to explain some of the non-specific effects often seen with [S]ODNs.

Animals↗
PubMed1994

In vivo toxicological effects of rel A antisense phosphorothioates in CD-1 mice.

To characterize the in vivo toxicity of phosphorothioate antisense oligonucleotides against rel A (p65 subunit of NF-kappa B transcription factor), forty-eight 6-week-old CD-1 mice were split into 4 groups (6/sex/group) receiving vehicle (phosphate-buffered saline) or doses of 50, 100, and 150 mg/kg of rel A antisense oligonucleotides intraperitoneally 3 times weekly for 2 weeks. Clinical signs of toxicity included weakness, and decreased motor activity and food consumption with body weight loss. Mortality occurred in 7 of 12 mice in the 150-mg/kg group and in 2 of 12 mice in the 100-mg/kg group, most of which died within the first 2 to 4 days of treatment. The remaining mice were necropsied on day 15. The major hematological finding was severe dose-dependent thrombocytopenia. The liver enzyme levels were mildly elevated in the serum of mid- and high-dose animals. At necropsy, increased spleen and liver weights were observed in treated mice, some of which also had mild pleural and/or peritoneal effusions. Histopathological examination revealed the likely cause of death to be acute renal failure due to renal cortical or tubular necrosis. Treatment-related changes were also found in the liver, spleen, bone marrow, and several other organs. In summary, the kidney, liver, and bone marrow (megakaryocytic lineage) were identified as the major target organs for toxicity with rel A antisense therapy.

Animals↗
PubMed1994

Regulation of adhesion and growth of fibrosarcoma cells by NF-kappa B RelA involves transforming growth factor beta.

The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion.

Animals↗
PubMed1993

Antisense inhibition of the p65 subunit of NF-kappa B blocks tumorigenicity and causes tumor regression.

The NF-kappa B transcription factor, composed of two proteins, p50 and p65, is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Various cell adhesion molecules have NF-kappa B binding sites and may play an important role in inflammatory response, tumorigenicity, and metastasis. In an earlier study, we demonstrated that adhesion of diverse transformed cells was blocked by antisense inhibition of the p65 subunit of NF-kappa B. Since cell-substratum interactions play an important role in tumorigenicity, we reasoned that antisense p65 could inhibit tumorigenicity. In diverse transformed cell lines, phosphorothioate antisense oligonucleotides to p65 inhibited in vitro growth, reduced soft-agar colony formation, and eliminated the ability of cells to adhere to an extracellular matrix. Stable transfectants of a fibrosarcoma cell line expressing dexamethasone-inducible antisense RNA to p65 showed inhibition of in vitro growth and in vivo tumor development. In response to inducible expression of antisense RNA, a pronounced tumor regression was seen in nude mice. The administration of antisense but not sense p65 oligonucleotides caused a pronounced inhibition of tumorigenicity in nude mice injected with diverse tumor-derived cell lines. Inhibitors of NF-kappa B function may thus be useful in the treatment of cancer.

Animals↗
PubMed1993

A sense phosphorothioate oligonucleotide directed to the initiation codon of transcription factor NF-kappa B p65 causes sequence-specific immune stimulation.

Antisense oligonucleotides have proved effective in achieving targeted inhibition of gene expression. In such experiments, sense oligonucleotides have frequently been used as a control for nonspecific effects, but the results have been variable, raising questions about the reliability of sense oligomers as a control. It is possible that some of the effects of sense oligonucleotides may be specific. We have shown that phosphorothioate antisense oligonucleotides to the p65 subunit of NF-kappa B, a transcription factor, cause a block in cell adhesion. In our efforts to test the efficacy of NF-kappa B p65 oligonucleotides in vivo, we unexpectedly observed that the control p65-sense, but not the p65-antisense, oligonucleotides caused massive splenomegaly in mice. In the current study we demonstrate a sequence-specific stimulation of splenic cell proliferation, both in vivo and in vitro, by treatment with p65-sense oligonucleotides. Cells expanded by this treatment are primarily B-220+, sIg+ B cells. The secretion of immunoglobulins by the p65-sense oligonucleotide-treated splenocytes is also enhanced. In addition, the p65-sense-treated splenocytes, but not several other cell lines, showed an upregulation of NF-kappa B-like activity in the nuclear extracts, an effect not dependent on new protein or RNA synthesis. These results demonstrate that phosphorothioate oligonucleotides can exert sequence-specific effects in vivo, irrespective of sense or antisense orientation.

Animals↗
PubMed1992

Glucocorticoid and retinoid regulation of alpha-2 type I procollagen promoter activity.

Glucocorticoids decrease type I procollagen synthesis by decreasing the steady state levels of procollagen mRNAs and mRNA synthesis. The present studies were undertaken to determine the functional sequences of the pro alpha 2(I) collagen gene required for the glucocorticoid-mediated decrease of type I procollagen mRNA synthesis. Embryonic mouse fibroblasts were stably transfected with the pR40 DNA CAT construct containing the 5' flanking region fragment from -2048 to +54 and the intronic fragment from +418 to +1524 of the mouse alpha 2(I) collagen gene. Dexamethasone treatment of these pR40 transfected fibroblasts resulted in a significant decrease in CAT activity which agrees with the glucocorticoid-mediated decrease of the steady state levels of type I procollagen mRNAs. To determine the possible role of the first intron fragment in the dexamethasone-mediated decrease of CAT activity, pR36, a CAT plasmid containing the first intron fragment and the SV40 early promoter, was transfected into mouse fibroblasts and treated with dexamethasone. No significant decrease in CAT activity was observed. The dexamethasone-mediated response was then localized within the 5' flanking region by preparing a series of constructs containing internal deletions and transfecting these plasmids into mouse fibroblasts. The regions -2048 to -981 and -506 to -351 were required for the dexamethasone response of gene activity. However, the DNA stretch from -981 to -506 was not. Analysis of the DNA sequences of these regions revealed a single GRE at -1023 to -1018 and a modified doublet at -873 to -856.(ABSTRACT TRUNCATED AT 250 WORDS)

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