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Biomedical subjects

J R Perez

Publications and source records attributed to J R Perez.

13 recordsLinked to original sources
PubMed1995

Differentiation of immortal cells inhibits telomerase activity.

Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation-inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, down-regulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity.

Animals↗
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↗
PubMed1993

Evidence for differential functions of the p50 and p65 subunits of NF-kappa B with a cell adhesion model.

The p50 and p65 subunits of NF-kappa B represent two members of a gene family that shares considerable homology to the rel oncogene. Proteins encoded by these genes form homo- and heterodimers which recognize a common DNA sequence motif. Recent data have suggested that homodimers of individual subunits of NF-kappa B can selectively activate gene expression in vitro. To explore this possibility in a more physiological manner, murine embryonic stem (ES) cells were treated with phosphorothio antisense oligonucleotides to either p50 or p65. Within 5 h after exposure to phosphorothio antisense p65 oligonucleotides, cells exhibited dramatic alterations in adhesion properties. Similar findings were obtained in a stable cell line that expressed a dexamethasone-inducible antisense mRNA to p65. Although antisense oligonucleotides raised against both p50 and p65 elicited a significant reduction in their respective mRNAs, only the cells treated with antisense p50 maintained a normal morphology. However, 6 days following removal of leukemia-inhibiting factor, a growth factor which suppresses embryonic stem cell differentiation, adhesion properties of cells treated with the antisense p50 oligonucleotides were markedly affected. The ability of the individual antisense oligonucleotides to elicit differential effects on cell adhesion, a property dependent upon the stage of differentiation, suggests that the p50 and p65 subunits of NF-kappa B regulate gene expression either as homodimers or as heterodimers with other rel family members. Furthermore, the finding that reduction in p65 expression alone had profound effects on cell adhesion properties indicates that p65 plays an important role in nonstimulated cells and cannot exist solely complexed with the cytosolic inhibitory protein I kappa B.

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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PubMedDate not supplied

A DNA motif present in alpha V integrin promoter exhibits dual binding preference to distinct transcription factors.

Antisense inhibition of the RelA subunit but not the NFKB1 subunit of NK-kappa B transcription factor results in a block of cellular adhesion and inhibition of tumor cell growth in vitro and in vivo. Studies aimed at dissecting the molecular mechanism of antisense relA action led to our identification of a kappa B-like motif present in aV integrin promoter. The alpha V/kappa B motif is closely related to RelA/c-Rel-binding sequences, such as 65-2 and TF-1. However, unlike these two kappa Blike motifs, the alpha V/kappa B motif detected a nuclear Sp1 activity distinct from kappa B activity, which was subsequently confirmed to be derived from Sp1. In comparison to the conventional GC box-containing Sp1 motif, the alpha V/kappa B motif also binds in vitro to c-Rel and RelA but not to NFKB1. Antisense inhibition of RelA inhibited the alpha V/kappa B activity. Direct in vivo competition of alpha V/kappa B-binding activity by a decoy approach also resulted in inhibition of alpha V/kappa B activity in intact cells. A variant of the alpha V/kappa B motif was found to retain the dual ability to detect Sp1 and the NF-kappa B complex in the nuclear and cytoplasmic extracts. Such dual interacting ability of a DNA motif offers yet another way of gene regulation in vivo and hence can affect cellular growth. Our results identify alpha V integrin as one of the molecular targets for relA/NF-kappa B and may explain growth inhibition by antisense relA.

Animals↗
PubMedDate not supplied

Transcription factor decoy approach to decipher the role of NF-kappa B in oncogenesis.

Antisense inhibition of the RelA subunit of NF-kappa B transcription factor (but not the NFKB1 subunit) causes pronounced inhibition of tumor cell growth in vitro and in vivo. Inhibition of either subunit, however, results in inhibition of the heterodimeric NF-kappa B complex in antisense-treated cells. Either of the subunits of NF-kappa B can form homo- or heterodimers with other members of the Rel oncogene family. In an effort to decipher the role of homo- vs heterodimeric NNF-kappa B in regulating tumor cell growth, we have used a decoy approach to trap these complexes in vivo. Using double-stranded phosphorothioates as a direct in vivo competitor for homo- vs heterodimeric NF-kappa B, we demonstrate that decoys more specific to RelA inhibit growth tumor cell growth in vitro. We demonstrate that RelA, either as a homodimer or a heterodimer with some other members of the Rel family and not the classical NF-kappa B (RelA/NFKB1), is involved in the differential growth control of tumor cells. Our results indicate that such transcription factor decoys can be a non-antisense tool to study the function of DNA-binding transcription factors.

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