A. Zijno et al., ANALYSIS OF CHROMOSOME SEGREGATION BY MEANS OF FLUORESCENCE IN-SITU HYBRIDIZATION - APPLICATION TO CYTOKINESIS-BLOCKED HUMAN-LYMPHOCYTES, Mutation research, 372(2), 1996, pp. 211-219
The application of methods based on in situ hybridization to centromer
ic regions to cytokinesis-blocked cells provides a convenient way for
the analysis of chromosome segregation in interphase cells. In this wa
y, the reciprocal segregation patterns in daughter nuclei can be visua
lized and most of the problems related to the artefactual loss or gain
of chromosomes which flaw other methods are avoided. In this work, th
e methodology has been applied to human lymphocytes to investigate the
influence of donor age on spontaneous malsegregation rates, the occur
rence of multiple malsegregation events, and the effect of the cytokin
esis-blocking agent cytochalasin B (Cyt B) on spontaneous and induced
chromosome malsegregation. The results obtained with 14 male donors, a
ged 22-57 years, demonstrated a significant (p < 0.001) increase in th
e frequency of micronuclei and X chromosome missegregation (both non-d
isjunction and chromosome loss) with the increasing age of the donors.
Moreover, a similar association was observed with cultures hybridized
with either chromosome 8 or 18 centromere probes, suggesting that the
age-related loss of fidelity in chromosome segregation in vitro may b
e a general trait. The investigation of the distribution of multiple m
alsegregation events in cultured lymphocytes of eight male and nine fe
male donors, with the simultaneous hybridization with pairs of centrom
eric probes (for chromosomes X and 8 or X and 18), demonstrated a larg
e excess of multiple events with respect to that expected by random se
gregation. This fact may highlight the existence of cellular subpopula
tion(s) prone to malsegregate, or indicate that the malsegregation of
one chromosome is able to affect the fidelity of segregation of the ot
her chromosomes. Finally, the possible influence of Cyt B on chemicall
y induced malsegregation has been investigated with the analysis of ch
romosomes X and 8 signals in nuclei of lymphocyte cultures treated wit
h vinblastine (2.5-20 ng/ml) in the presence and absence of 6 mu g/ml
Cyt B. Vinblastine induced a small increase in hyperploidy of either c
hromosome X or 8 at 10 ng/ml in cultures treated with Cyt B. Without C
yt B, a significant increase of hyperploidy was only observed at the h
ighest dose assayed (20 ng/ml). This vinblastine dosage had a severe i
nhibitory effect on cultures treated with Cyt B, where no binucleated
cells were detected. At all doses, a relatively greater mitotic index
was observed in cultures with Cyt B, suggesting a synergistic effect o
f this drug with vinblastine. Most notably, at the two highest vinblas
tine dosages (10 and 20 ng/ml), a large incidence of polyploid nuclei
was observed in cytokinesis-blocked cultures, whereas none or far lowe
r increases of polyploidy were found in the absence of Cyt B. This res
ults provides direct evidence of the potential of Cyt B to indirectly
interfere with chromosome misdistribution induced by a spindle poison,
to be considered before drawing firm conclusions from kinesis-blocked
systems.