Specific Regulation of Gene Expression by Antisense Nucleic Acids: A Summary of Methodologies and Associated Problems

Abstract

Gene therapy based on gene-specific nucleic acids has moved from theory to a practical possibility in a very short time. The new DNA and RNA therapeutic reagents are intended to stop the growth of cancerous cells or the production of viruses. At the practical level, the efficacy of antisense oligomers as therapeutic reagents has been carefully examined in various clinical contexts. For the efficient use of antisense nucleic acids as pharmaceutical agents, a complete analysis of their mechanisms of action is necessary. The use of antisense oligomers always involves the following problems: basepair specificity, stereoisomer specificity, stability and resistance to nucleases of sense-antisense duplexes, permeability of the cell membrane and targeting of the oligomer, safety, and the preparation of large amounts of oligomer. Herein, we review the basic concepts and problems associated with the exploitation of antisense technology. We have identified a new transcription factor triple-helix-binding zinc-finger protein-1 (THZif-1) induced by antisense c-myc RNA in the antisense-transformed HL60 cells. The encoded protein functions as the repressor of c-myc to achieve the reduction of the endogenous expression of c-myc gene. Therefore, the introduction of THZif-1 gene into HL60 cells in conjunction with antisense c-myc oligomers may result in the efficient repression of the expression of the c-myc gene. The molecular features of this factor are herein discussed.

Quantitative Proteomic Identification of MAZ as a Transcriptional Regulator of Muscle-Specific Genes in Skeletal and Cardiac Myocytes

“…MAZi (99.3% amino acid identity to human MAZ) was discovered in human pancreatic islet cells and shown to recognize both single-and double-stranded DNA (65). While THZif-1 has 98.2% amino acid similarity to MAZ, it contains a variant second zinc finger (only 33.3% similar to MAZ) and is capable of binding single-stranded, double-stranded, and triple-helical DNA (35,54). Interestingly, MAZR and MAZi appear to activate, whereas THZif-1 represses, expression of c-myc.…”

Binding of THZif-1, a MAZ-like Zinc Finger Protein to the Nuclease-hypersensitive Element in the Promoter Region of the c-MYC Protooncogene

“…Moreover, the binding avidity of the THZif-1 protein to ss, ds, and ts c-myc NHEs was well correlated with the negative regulation of transcription of the c-MYC gene (60, data not shown) since the THZif-1 protein with a mutant second zinc finger did not have the DNA-binding activity and did not repress transcription of the c-MYC gene (Figs. 1B and 7) (60).…”

“…We purified a DNA-binding protein specific for this NHE from a nuclear extract of AM93-4-12 cells and isolated a recombinant cDNA clone (for THZif-1, triple helix-binding zinc-finger protein-1) of the corresponding gene (60). The deduced THZif-1 is a polypeptide of 253 amino acids with a molecular mass of 27,830 kDa.…”

“…We showed previously that synthesis of the transcription factor THZif-1, a MAZ-like zinc finger protein, can be induced in the nuclei of HL60 cells that have been transformed with antisense c-MYC, and that it serves as a repressor of the endogenous transcription of the c-MYC protooncogene via binding to the NHE element (57)(58)(59)(60). This THZif-1 factor is a zinc finger protein with strong binding affinity for the triple-helical conformation of the NHE of the c-MYC promoter, as well as for the single-stranded and double-stranded NHE (60).…”

“…This THZif-1 factor is a zinc finger protein with strong binding affinity for the triple-helical conformation of the NHE of the c-MYC promoter, as well as for the single-stranded and double-stranded NHE (60). In this report, we compared the relative binding activities of THZif-1 protein to the methylated and unmethylated forms of c-myc NHE.…”