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L Miraglia

Publications and source records attributed to L Miraglia.

8 recordsLinked to original sources
PubMed2000

Variations in mRNA content have no effect on the potency of antisense oligonucleotides.

A fundamental question with regard to antisense pharmacology is the extent to which RNA content or transcription rate or both affect the potency of antisense drugs. We have addressed this by controlling RNA content and transcription rate using either an exogenous gene expressed after transfection or an endogenous gene induced with a cytokine. We have demonstrated that in both A549 and HeLa cells, varying RNA copy numbers from <1 to >100 copies per cell has no effect on the potency of RNase H-active antisense drugs transfected into cells, nor did variation in transcription rate have an effect on potency. We demonstrate that this is because the number of oligonucleotide molecules per cell is vastly in excess of the RNA copy number. These data further suggest that a significant fraction of cell-associated antisense drug molecules may be unavailable to interact with the target RNA, an observation that is not surprising, as phosphorothioate oligonucleotides interact with many cellular proteins. We suggest that these data may extrapolate to in vivo results.

Dexamethasone↗
PubMed1999

Antisense oligonucleotides to the epidermal growth factor receptor.

Overexpression of the epidermal growth factor receptor (EGFR) has been observed in human breast tumors and is associated with poor prognosis in breast cancer patients. This would suggest that blocking the activity of the EGFR is a logical approach in the treatment of breast cancer. Three 20-mer phosphorothioate oligodeoxynucleotides were designed to target different regions of the human epidermal growth factor receptor (EGFR) mRNA. Several analogs of these oligodeoxynucleotides (the 2'-fluoro analog, the 2'-propoxy analog, and/or the 5-methyl cytosine analog) were also evaluated. We added these compounds to a human ovarian carcinoma cell line (SKOV3) and a human lung carcinoma line (A549), both of which overexpress the EGFR. All of these antisense oligonucleotides inhibited expression of the 10 kb EGFR mRNA (range: 22-97% inhibition) compared to a scrambled control oligonucleotide or an untreated control. Expression of the less prominent 5.6 kb EGFR mRNA band was also inhibited by all but two of the parent oligonucleotides. No inhibition of this 5.6 kb band was found with the control oligonucleotide. The reduction in the expression of EGFR mRNA by the three most potent antisense compounds was accompanied by a significant reduction of EGFR protein (90-98%) and in vitro growth inhibition of SKOV3 cells as compared to the control oligonucleotide.

Base Sequence↗
PubMed1996

Inhibition of growth of human tumor cell lines in nude mice by an antisense of oligonucleotide inhibitor of protein kinase C-alpha expression.

A 20-mer phosphorothioate oligodeoxynucleotide (ISIS 3521) designed to hybridize sequences in the 3'-untranslated region of human protein kinase C-alpha (PKC-alpha) mRNA has been shown to inhibit the expression of PKC-alpha in multiple human cell lines. In human bladder carcinoma (T-24) cells, inhibition of PKC-alpha was both concentration dependent and oligonucleotide sequence specific. ISIS 3521 had a IC50 of 50-100 nM for PKC-alpha mRNA reduction and was without effect on the expression of other members of the PKC family of genes (PKC-eta and zeta). Toxicity studies in mice revealed that the oligodeoxynucleotide was well tolerated at repeat doses of 100 mg/kg i.v. for up to 14 days, with no acute toxicity apparent. The oligodeoxynucleotide was found to also inhibit the growth of three different human tumor cell lines, the T-24 bladder, human lung carcinoma (A549), and Colo 205 colon carcinoma grown in nude mice. The inhibition was dose dependent with ID50 values for the growth inhibition between 0.06 and 0.6 mg/kg daily when given i.v., depending on the cell line examined. Three control phosphorothioate oligodeoxynucleotides not targeting human PKC-alpha were without effect on the growth of the tumors at doses as high as 6 mg/kg. Recovery of ISIS 3521 from tumor tissue and resolution by capillary gel electrophoresis revealed that 24 It after the final dose of oligodeoxynucleotide, intact, full-length 20-mer material was present as well as some apparent exonuclease degradation products (e.g., n-1 and n-2 mers). These studies demonstrate the in vivo antitumor effects of an antisense oligodeoxynucleotide targeting PKC-alpha and suggest that this compound may be of value as a chemotherapeutic agent in the treatment of human cancers.

Animals↗
PubMed1996

Inhibition of interleukin-1 type I receptor expression in human cell-lines by an antisense phosphorothioate oligodeoxynucleotide.

A 20 mer oligodeoxynucleotide designed to hybridize to specific 3'-untranslated sequences in the type I receptor IL-1r mRNA was identified which inhibited the expression of both IL-1r mRNA and protein in human A549 lung carcinoma cells and human dermal fibroblasts. The oligodeoxynucleotide exhibited an IC50 value of approximately 100 nM in both cell lines and reduced IL-1r mRNA expression for up to 48 h. Multiple scrambled control oligonucleotides were without effect on IL-1r mRNA expression. Treatment of A549 cells with this oligodeoxynucleotide (but not scrambled controls) inhibited the IL-1 stimulated expression of the cell adhesion molecule ICAM-1 but was without effect on the TNF-alpha induction of this molecule. This study demonstrates that phosphorothioate oligodeoxynucleotides can selectively inhibit IL-1r expression leading to a reduction in IL-1 dependent gene expression.

Carcinoma↗
PubMed1992

Modulation of eucaryotic initiation factor-4E binding to 5'-capped oligoribonucleotides by modified anti-sense oligonucleotides.

The 5' cap structure of eucaryotic mRNA plays a pivotal role in mRNA metabolism. This report demonstrates that anti-sense oligonucleotides equipped with 3'-overhanging nucleotides modulate the amount of recombinant human eucaryotic initiation factor-4E that binds to a 5'-capped oligoribonucleotide. The degree of inhibition or enhancement of protein binding is dependent upon the number and sequence of overhanging nucleotides. A 45% inhibition of complexation was observed by the addition of one 3'-overhanging guanosine residue. Addition of a second residue (+2/GN) resulted in a higher degree of inhibition, 77-88%. In contrast, addition of one adenosine residue enhanced the formation of the eucaryotic initiation factor-4E-m7GpppRNA complex by 213%. Modulation of protein interactions with the 5'-cap structure is likely to effect several biological events, including pre-mRNA processing, transport of the mRNA from the nucleus to the cytoplasm and translation of the target mRNA. This targeting strategy in anti-sense chemistry may have practical applications in experimental biology and medicine.

Base Sequence↗
PubMed1991

Limited functional equivalence of phylogenetic variation in small nuclear RNA: yeast U2 RNA with altered branchpoint complementarity inhibits splicing and produces a dominant lethal phenotype.

U2 is a highly conserved small nuclear RNA essential for pre-mRNA splicing in mammals and yeast and for trans-splicing in trypanosomes. To test the function of variant U2 RNA structures from different organisms, we conducted phylogenetic exchanges of U2 domains. Replacing nucleotides 1-120 of yeast U2 with the corresponding region of human U2 generates a U2 RNA that is correctly folded and functions in yeast. In contrast, replacement of the branchpoint interaction region of yeast U2 with the corresponding region from trypanosome is dominant lethal. Using a GAL-U2 promoter fusion, we show that the dominant phenotype can be made conditional and that the accumulation of mutant U2 is followed rapidly by inhibition of nuclear pre-mRNA splicing. The results suggest that U2 small nuclear ribonucleoprotein particles normally participate in stable complexes with a limiting splicing factor prior to formation of U2-intron branchpoint base pairs.

Base Sequence↗