TPEN, A ZN2+ FE2+ CHELATOR WITH LOW-AFFINITY FOR CA2+, INHIBITS LAMINASSEMBLY, DESTABILIZES NUCLEAR ARCHITECTURE AND MAY INDEPENDENTLY PROTECT NUCLEI FROM APOPTOSIS IN-VITRO/

We used Xenopus egg extracts to examine the effects of TPEN, a chelato r with strong affinities for Zn2+, Fe2+, and Mn2+, on nuclear assembly in vitro. At concentrations above 1 mM, TPEN blocked the assembly of the nuclear lamina and produced nuclei that were profoundly sensitive to stress-induced balloon-like 'shedding' of nuclear membranes away fr om chromatin-associated membranes, TPEN-arrested nuclei were also defe ctive for DNA replication, which could be explained as secondary to th e lack of a lamina. Imaging of TPEN-arrested nuclei by field emission in-lens scanning electron microscopy (FEISEM) revealed clustered, stru cturally-perturbed nuclear pore complexes. TPEN-arrested nuclei were d efective in the accumulation of fluorescent karyophilic proteins. All detectable effects caused by TPEN were downstream of the effects of BA PTA, a Ca2+/Zn2+ chelator that blocks pore complex assembly at two dis tinct early stages, Surprisingly, TPEN-arrested nuclei, but not contro l nuclei, remained active for replication in apoptotic extracts, as as sayed by [P-32]-dCTP incorporation into high molecular weight DNA, sug gesting that TPEN blocks a metal-binding protein(s) required for nucle ar destruction during programmed cell death.