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DNA Topoisomerase I and DNA Topoisomerase II Are Regulated Post‐translationally in Interleukin‐2 Activated HuT 78 Cells
Author(s) -
Foglesong Paul David,
Martinez Adela M.,
Gomez John Eric
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.826.6
Subject(s) - microbiology and biotechnology , topoisomerase , dna , biology , dna replication , gel electrophoresis , transcription (linguistics) , cell culture , aphidicolin , lymphokine , biochemistry , in vitro , genetics , linguistics , philosophy
Interleukin‐2 (IL‐2) is a lymphokine that binds to a high affinity receptor (CD 25) on the surface of human T lymphocytes. Transduction of this signal activates T cells by means of biochemical pathways that result in increased transcription, DNA replication, and, ultimately, cell division. HuT 78, a CD 25 + human T cell lymphoma line, was obtained from ATCC and used in these studies. It was previously shown that the specific activities of both DNA topoisomerase I (Topo I) and DNA topoisomerase II (Topo II) increased 10‐fold to 20‐fold in nuclear extracts prepared from HuT 78 cells within the first 24 hrs. after treatment with IL‐2 (Foglesong, 2006). Those results suggested that both Topo I and Topo II function in transcription and DNA replication in activated human T lymphocytes. The polypeptide composition of the nuclear extracts was analyzed by SDS‐polyacrylamide gel electrophoresis in order to determine whether the observed increases in the specific activities of these enzymes were due to increased synthesis of protein. Aliquots of the nuclear extracts containing 1–5 μg protein were incubated at 100 o C for 2 min. in SDS buffer and electrophoresed at 75 V for 2 hr. on 4 to 20% gradient polyacrylamide gels along with broad range markers (Bio‐Rad). The gels were stained with Coomassie Brilliant Blue. Under these denaturing conditions the relative molecular masses (M r ) for Topo I, Topo IIα, and Topo IIβ are 100, 170, and 180 kDa, respectively. No changes in the abundance of proteins of these sizes were observed in the extracts prepared in the first 24 hrs. following treatment of HuT 78 cells with IL‐2. These results taken together suggest that the activities of Topo I and Topo II are regulated post‐translationally in IL‐2‐activated human T lymphocytes. This work was supported by CA 74388 to PDF.