Topoisomerase II is nonfunctional in polyamine-depleted cells

Abstract

The polyamines-putrescine, spermidine, and spermine-are essential for normal cell proliferation. Polyamine depletion affects DNA structure and synthesis. Topoisomerase II (topo II) is also necessary for normal cell proliferation, and it has been shown in vitro that polyamines may affect topo II activity. In order to investigate the effect of polyamine depletion on topo II activity, we treated Chinese hamster ovary cells with either ␣-difluoromethylornithine (DFMO) or 4-amidinoindan-1-one-2Ј-amidinohydrazone (CGP 48664), which are polyamine biosynthesis inhibitors. Treatment with the topo II inhibitor etoposide results in DNA strand breaks only if there is active topo II in the cells. By quantitating DNA strand breaks after etoposide treatment using single cell gel electrophoresis, we were able to estimate intracellular topo II activity. We also quantitated topo II activity in crude nuclear extracts from control and polyamine biosynthesis inhibitor-treated cells. Using single cell gel electrophoresis, we noted a clear decrease in the function of topo II in polyamine biosynthesis inhibitor-treated cells, as compared with untreated control cells. However, the topo II activity in crude nuclear extracts did not differ significantly in control versus polyamine biosynthesis inhibitor-treated cells. Taken together, these results indicate that although the function of topo II in polyamine-depleted cells was impaired, topo II remained functional in an in vitro assay. Using the single cell gel electrophoresis assay, we also found that spermine depletion itself caused DNA strand breaks.

Spermidine/spermine N1-acetyltransferase overexpression in kidney epithelial cells disrupts polyamine homeostasis, leads to DNA damage, and causes G2 arrest

“…Our results support the hypothesis that polyamine depletion is the primary factor that affects DNA integrity and cell proliferation in SSAT-overexpressing cells. Previous studies demonstrating that depletion of SPM leads to DNA strand breaks and that polyamine depletion leads to modification of chromatin and DNA structure, increases susceptibility to damage by genotoxic agents, and interferes with DNA repair support our hypothesis that depletion of SPD and SPM is the primary factor involved in the induction of DNA damage and growth arrest in SSAT-expressing cells (2,45,47,54).…”

“…Polyamine depletion leads to modification of chromatin and DNA structures, increases DNA susceptibility to damage by various genotoxic agents, and interferes with DNA repair (44 -46). The DNA alterations in polyamine-depleted cells may be due to DNA strand breaks as well as inhibition of topoisomerase II function (2). Mammalian cell division also requires the activation of the Raf 3 MEK 3 ERK pathway for G 2 /M progression (31,33,55).…”

Ornithine decarboxylase activity in tumor cell lines correlates with sensitivity to cell death induced by histone deacetylase inhibitors

“…Polyamine-depleted cells were able to take up TSA, block HDAC activity, and induce histone hyperacetylation to the same extent as control cells. Polyamines play a role in higher-order chromatin structure, and polyamine depletion alters chromatin structure in mammalian cells (30,42,43). HDAC inhibitors also induce global reorganization of chromatin structure (44).…”

Caspase activation in etoposide‐treated fibroblasts is correlated to ERK phosphorylation and both events are blocked by polyamine depletion

“…Our results show that an early event triggered by etoposide and leading to phosphorylation of the MAPK members ERKs and JNKs is blocked in polyamine‐depleted cells. Polyamines could be required for signal transduction leading to MAPK activation, or for the activity of topoisomerase II, that is important for etoposide action [42], or could act by affecting the cellular localization of members of the Bcl‐2 family [43]. Another possible model that we hypothesize is correlated to the established role of polyamines in cell proliferation [9].…”