Re DAPI staining
ZBIGNIEW DARZYNKIEWICZ (darzynk@nymc.edu)
Sun, 30 Jul 1995 11:36:41 -0500
Richard Meister raises the question as to why stainability of cells
treated with hydroxyurea with DAPI is altered.
Only a fraction of DNA in the nucleus is accessible to fluorochromes
(stainable). The size of this fraction varies depending on molecular
structure of the fluorochrome. It also depends very much on chromatin
structure. Binding of any fluorochrome (its accessibility to the
binding sites) is modulated by interactions of DNA with nuclear
proteins, primarily with histones as well as by DNA topology (e.g.
closed loops vs. nicked DNA) (e.g. see Cytometry, 5:355, 1984). In
some cell systems cell differentiation alters DNA stainablility. Also
many drugs interacting with DNA change its stainability. One cannot
always expect, therefore, the stoichiometry in staining of DNA in
situations when cells with different chromatin structure or drug
treated vs. untreated are compared. There ara several possible
reasons why stainability of DNA is changed in cells treated with
hydroxyurea. First, the cells arrested by hydroxyurea are
characterized by growth imbalance, i.e. DNA synthesis is suppressed
while protein and RNA synthesis continues. The protein/DNA or RNA/DNA
ratio may be dramatically increased (e.g see Cytometry 2: 212, 1982).
Some of the synthesized proteins are of nuclear location and their
presence changes chromatin structure and may affect DNA stainability.
In addition, in the presence of hydroxyurea DNA structure also is
changed because, compared with normal DNA replication, there are many
more replication initiation points (nicks), while progression of
replication is halted. The topology of such DNA, thus, is different
than that of the untreated cells. Furthermore, DNA replication is not
entirely stopped in the the presence of hydroxyurea, but is only
markedly suppressed. Thus, depending on cell type, duration of the
hydroxyurea block and the degree of growth imbalance one may expect
significant variability in DNA stainability with many fluorochromes.
Of all fluorochromes, however, DAPI appears to be the least sensitive
to variations in chromatin structure.
Zbigniew Darzynkiewicz
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