Erenpreisa et al. 2002

Erenpreisa J, Ivanov A, Cragg M, Selivanova G, Illidge T. 2002. Nuclear envelope-limited chromatin sheets are part of mitotic death. Histochemistry & Cell Biology 117: 243-255.

These authors investigated the structure and formation of nuclear envelope-limited chromatin sheets (ELCS) in two lines of mutant human cells commonly used in cancer studies. ELCS are membranous structures of unknown function; they are flat folds of inner nuclear envelope that project outwards from the nucleus, sometimes as far as the cytoplasm, and enfold chromatin. Larger projections containing chromatin are termed nuclear pockets (NP), and are strongly associated with some types of cancer such as leukaemia in mammals, but are also found less commonly in healthy tissues including spermatogonia.

Cells were treated with irradiation to induce double-stranded breaks and with a microtubule inhibitor to inhibit mitosis. Earlier investigations by these and other authors have developed the concept of “mitotic death”, which is a syndrome of cells including failed mitosis, the uncoupling of mitosis from DNA replication, delayed (but inevitable) apoptosis, and the formation of giant cells with high levels of aneuploidy and often endopolyploidy.

This paper demonstrates that mitotic death can be induced by quite different mechanisms, in this case by the failure of mitosis driven by microtubule depolymerisation and by a large number of double-stranded DNA breaks induced by radiation. In both cases, DNA repair was effected over considerable time, though apparently cells were unable to “repair” their aneuploidy.

Of particular interest to my work in this paper is their investigations of nuclear morphology. Cells were suspended in solution, then treated to prepare them for either nuclear DNA contents measures (by PI-flow cytometry and by Feulgen staining), light microscopy, or electron microscopy. For light microscopy cell- and nuclear-morphology investigation, these authors fixed cells in a 1:1 mixture of ethanol and acetone, hydrolysed the cells (and presumably freed the nuclei) in 0.1N HCl, and washed them in a poorly-described solution that may be either MacIlvain buffer (pH 5) or Toluidene blue stain in MacIlvain buffer. All of this occurred at 4°C. This was followed by a rinse in distilled water, and dehydration of slides carrying mounted cells in “warm tertiary butanol”. I have seen ethanol used as a fixative and preservative agent in histology studies before, but this represents the clearest description I have encountered of the process as applied specifically for investigations of cell nuclear morphology. There are some obvious similarities with our procedure for Feulgen staining, which helps to confirm that ethanol preservation may be reversible for whole-nuclear investigations.