Triplex DNA in the nucleus: direct binding of triplex-specific antibodies and their effect on transcription, replication and cell growth

Abstract

Jel 318 and Jel 466 are triplex-specific monoclonal antibodies which previously have been shown to bind to cell nuclei and chromosomes by immunofluorescence. Their interaction was further characterized by two methods. First, isolated intact nuclei were encapsulated in agarose. Both antibodies showed significant binding to the nuclei which could be inhibited by adding competing triplex DNA but not by adding Escherichia coli DNA to which the antibodies do not bind. Both triplex-specific antibodies inhibited replication and transcription in the nuclei by about 20%. Secondly, the antibodies were introduced into synchronized myeloma cells by osmotic shock of pynocytic vesicles. Cell-cycle studies showed that the myeloma cells had an S phase of about 10 h and a doubling time of about 20 h. The cells were synchronized with thymidine and both cell growth and cell death were monitored. Introduction of the triplex-specific antibodies caused a marked decrease in cell growth without a significant increase in cell death. The effectiveness of the antibodies was improved by the addition of chloroquine diphosphate which inhibits degradation in the lysosomes. As a control, introduction of an antibody specific for a bacterial protein had little effect. In synchronized cells, inhibition of proliferation reached a maximum at 7 to 13 h after the release from the thymidine block. Thus, cells are most sensitive to the triplex-binding antibodies at the end of S phase and during G2. This result is consistent with the view that triplexes are involved in chromosome condensation/decondensation.