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A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1
Abstract:
Initiation of T-lymphocyte proliferation by mitogen or antigen involves a cascade of gene activation events. Thus, by the time mitogen-activated T cells have reached the G1/S interface, many genes that are transcriptionally silent in G0, like the c-myc, IL-2, IL-2 receptor (IL-2R) and transferrin receptor (TfR) genes, have been transcriptionally activated. To understand the role of the individual genes in the activation process, one must be able to interfere specifically with the expression or function of each particular gene product. In this way, by blocking the IL-2R with an antibody, it has been demonstrated that IL-2/IL-2R interaction is required to induce TfR expression in activated T cells. When the function or expression of intracellular proteins is to be blocked, however, the need to introduce antibodies into the cytoplasm of viable cells, although possible, is a limiting factor. We have taken another approach, namely the exogenous addition to bulk cell cultures of small antisense oligomers. Sequence-specific antisense oligodeoxyribonucleotides have been reported to inhibit intracellular viral replication without interfering with cellular protein synthesis. Similarly, rabbit globin mRNA translation in a cell-free system and in rabbit reticulocytes has been inhibited by oligomers complementary to the globin mRNA initiation codon region. Recently, a pentadecadeoxyribonucleotide complementary to the initiation codon and four downstream codons of human c-myc mRNA was reported to inhibit the proliferation of the human leukaemic cell line HL-60 specifically. We report here that the same c-myc complementary oligonucleotide inhibits mitogen-induced c-myc protein expression in human T lymphocytes and prevents S phase entry. Interestingly, c-myc antisense treatment did not inhibit G0 to G1 traversal as assessed by morphologic blast transformation, transcriptional activation of the IL-2R and TfR genes, or induction of 3H-uridine incorporation.
Insights
Antisense oligomers targeting c-myc mRNA inhibit T-lymphocyte proliferation by blocking S phase entry. This method selectively blocks c-myc protein expression without affecting early activation events like gene transcription.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- T-lymphocyte proliferation involves gene activation, including c-myc, IL-2, and IL-2 receptor (IL-2R).
- Understanding individual gene roles requires specific interference with gene products.
- Antibodies are limited for blocking intracellular proteins; antisense oligomers offer an alternative approach.
Purpose of the Study:
- To investigate the role of c-myc in T-lymphocyte activation.
- To assess the efficacy of antisense oligodeoxynucleotides in inhibiting c-myc expression and T-cell proliferation.
Main Methods:
- Utilized sequence-specific antisense oligodeoxynucleotides complementary to human c-myc mRNA.
- Applied these oligomers to bulk cultures of human T lymphocytes stimulated by mitogen.
- Assessed effects on c-myc protein expression, S phase entry, and early activation markers (blast transformation, IL-2R, TfR gene transcription).
Main Results:
- A c-myc antisense oligonucleotide specifically inhibited mitogen-induced c-myc protein expression in human T lymphocytes.
- This inhibition prevented T cells from entering the S phase of the cell cycle.
- c-myc antisense treatment did not impede G0 to G1 phase transition, including morphological changes or transcriptional activation of IL-2R and TfR genes.
Conclusions:
- Antisense oligomers targeting c-myc effectively inhibit T-lymphocyte proliferation at the G1/S interface.
- c-myc is crucial for progression into S phase but not for initial G0 to G1 activation events.
- Antisense technology provides a specific tool for studying gene function in T-cell activation.