42 research outputs found
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Localisation of the Ki-67 antigen within the nucleolus: Evidence for a fibrillarin-deficient region of the dense fibrillar component
The Ki-67 antigen is detected in proliferating cells in all
phases of the cell division cycle. Throughout most of interphase,
the Ki-67 antigen is localised within the nucleolus.
To learn more about the relationship between the Ki-67
antigen and the nucleolus, we have compared the distribution
of Ki-67 antibodies with that of a panel of antibodies
reacting with nucleolar components by confocal laser
scanning microscopy of normal human dermal fibroblasts
in interphase stained in a double indirect immunofluorescence
assay. During early G1, the Ki-67 antigen is
detected at a large number of discrete foci throughout the
nucleoplasm, extending to the nuclear envelope. During Sphase
and G2, the antigen is located in the nucleolus.
Double indirect immunofluorescence studies have revealed
that during early to mid G1 the Ki-67 antigen is associated
with reforming nucleoli within discrete domains which are
distinct from domains containing two of the major
nucleolar antigens fibrillarin and RNA polymerase I.
Within mature nucleoli the Ki-67 antigen is absent from
regions containing RNA polymerase I and displays only
partial co-localisation within domains containing either fibrillarin
or B23/nucleophosmin. Following disruption of
nucleolar structure, induced by treatment of cells with the
drug 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole or
with actinomycin D, the Ki-67 antigen translocates to
nucleoplasmic foci which are associated with neither fibrillarin
nor RNA polymerase I. However, in treated cells
the Ki-67 Ag remains associated with, but not co-localised
to, regions containing B23/nucleophosmin. Our observations
suggest that the Ki-67 antigen associates with a fibrillarin-
deficient region of the dense fibrillar component of
the nucleolus. Integrity of this region is lost following either
nucleolar dispersal or nucleolar segregation
Aging of Hutchinson-Gilford progeria syndrome fibroblasts is characterised by hyperproliferation and increased apoptosis
Hutchinson-Gilford progeria syndrome is a rare genetic disorder that mimics certain aspects of aging prematurely. Recent work has revealed that mutations in the lamin A gene are a cause of the disease. We show here that cellular aging of Hutchinson-Gilford progeria syndrome fibroblasts is characterised by a period of hyperproliferation and terminates with a large increase in the rate of apoptosis. The occurrence of cells with abnormal nuclear morphology reported by others is shown to be a result of cell division since the fraction of these abnormalities increases with cellular age. Similarly, the proportion of cells with an abnormal or absent A-type lamina increases with age. These data provide clues as to the cellular basis for premature aging in HGPS and support the view that cellular senescence and tissue homeostasis are important factors in the normal aging process
Senescent human diploid fibroblasts are able to support DNA synthesis and to express markers associated with proliferation
The characteristic limited reproductive life-span of normal human fibroblasts in culture is due to a
steadily decreasing fraction of cells able to proliferate in the standard rich growth media. We have observed that restricting the growth factor supply to old cells for variable lengths of time in culture increases the fraction of cells that can enter S-phase; although these cells do not go on to divide. Thus, it seems that there is a transient phase between the proliferating state and the irreversibly post-mitotic, senescent state. Perhaps a 'quiescent-G0' state, which
can be maintained in the presence of growth factors, is a stage on the pathway to mortalization and senescence
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Changes in the nuclear distribution of DNA polymerase alpha and PCNA/cyclin during the progress of the cell cycle, in a cell-free extract of Xenopus eggs
The nuclear distribution of DNA polymerase alpha
and PCNA/cyclin in embryonic nuclei has been
investigated, in a cell-free extract of Xenopus eggs
that recapitulates a basic cell-cycle in vitro, by
indirect immunofluorescence microscopy. Both
antigens co-distribute with the chromatin in Sphase
nuclei; however, as DNA replication is completed
and nuclei progress into a G2 state anti-PCNA
fluorescence disappears and anti-DNA polymerase
alpha fluorescence becomes resolved into bright
spots. These spots are initially associated with the
chromatin strands and can be seen to share both
anti-PCNA and anti-DNA polymerase alpha fluorescence,
but as anti-PCNA fluorescence fades the
spots become dissociated from the chromatin and
are redistributed throughout the nucleus until they
are dispersed during nuclear envelope breakdown.
The loss of anti-PCNA fluorescence and displacement
of anti-DNA polymerase alpha fluorescence
from the chromatin can be prevented by inhibiting
DNA synthesis 'with aphidicolin. Under these conditions
both antigens remain associated 'with the
chromatin even after nuclear envelope breakdown
and lamin dispersal. The association of these antigens
with mitotic figures appears to be functional,
as both biotin-11-dUTP and pPJdCTP can be incorporated
efficiently into DNA during the mitotic
period
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Expression of proliferation-dependent antigens during cellular ageing of normal and progeroid human fibroblasts
Normal human fibroblasts display a limited lifespan in
culture, which is due to a steadily decreasing fraction of
cells that are able to proliferate. Using antibodies that react
with antigens present in proliferating cells only, in an
indirect immunofluorescence assay, we have estimated the
fraction of proliferating cells in cultures of normal human
fibroblasts. Furthermore, we have estimated the rate of
decline in the fraction of proliferating cells during the
process of cellular ageing by application of the assay to
normal human fibroblasts throughout their lifespan in
culture. Werner’s Syndrome is an autosomal recessive
disease in which individuals display symptoms of ageing
prematurely. Werner’s Syndrome fibroblasts display a
reduced lifespan in culture compared with normal human
fibroblasts. Like normal human fibroblasts, the growth of
Werner’s Syndrome fibroblasts is characterised by a
decreasing fraction of cells reacting with the proliferationassociated
antibodies throughout their lifespan in culture.
However, the rate of loss of proliferating cells in Werner’s
Syndrome fibroblasts during the process of cellular ageing
is accelerated 5- to 6-fold compared with the rate determined
for normal human fibroblasts
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Internal iamin structures within G1 nuclei of human dermal fibroblasts
The nuclear lamina is a mesh-like network of fibres subjacent
to the inner nuclear membrane that is believed
to be involved in the specific spatial reorganisation of
chromatin after mitosis. To determine how the lamina
might be involved in chromatin reorganisation, we have
performed indirect immunofluorescence studies on quiescent
and proliferating human dermal fibroblasts
(HDF). Two monoclonal antibodies recognising human
lamins A and C and three different fixation methods
were employed. In indirect immunofluorescence studies,
cultures of quiescent cells displayed a uniform perinuclear
distribution of the antibodies. In proliferating cultures
two distinct populations of cells were observed:
one population displayed a typical perinuclear antibody
distribution, while the second population displayed an
unusual pattern consisting of a series of spots and fibres
within the nucleus. By inducing cell-cycle synchrony in
cultures we were able to determine that the unusual
internal distribution of the lamin antibodies was
restricted to cells in G1. Optical sectioning and 3-D
reconstruction of the lamina structures in G1 nuclei was
performed with a confocal laser scanning microscope
(CLSM). This revealed that the internal lamin structures
consisted of small foci and fibres proliferating
throughout the nucleus. These structures were shown to
be closely associated with areas of condensed chromatin
but not nuclear membrane. As cells progress towards S
phase the internal lamin foci disappear
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Weaving a pattern from disparate threads: lamin function in nuclear assembly and DNA replication
The major residual structure that remains associated with
the nuclear envelope following extraction of isolated nuclei
or oocyte germinal vesicles with non-ionic detergents,
nucleases and high salt is the lamina (Fawcett, 1966;
Aaronson and Blobel, 1975; Dwyer and Blobel, 1976). The
nuclear lamina is composed of intermediate filament
proteins, termed lamins (Gerace and Blobel, 1980; Shelton
et al., 1980), which polymerise to form a basket-weave
lattice of fibrils, which covers the entire inner surface of the
nuclear envelope and interlinks nuclear pores (Aebi et al.,
1986; Stewart and Whytock, 1988; Goldberg and Allen,
1992). At mitosis, the nuclear envelope and the lamina both
break down to allow chromosome segregation. As a consequence,
each structure has to be rebuilt during anaphase
and telophase, allowing cells an opportunity to reposition
chromosomes (Heslop-Harrison and Bennett, 1990) and to
reorganise looped chromatin domains (Franke, 1974;
Franke et al., 1981; Hochstrasser et al., 1986), which may
in turn control the use of subsets of genes. Because of the
position that it occupies, its dynamics during mitosis and
the fact that it is an essential component of proliferating
cells, the lamina has been assigned a number of putative
roles both in nuclear metabolism and in nuclear envelope
assembly (Burke and Gerace, 1986; Nigg, 1989). However,
to date there is little clear cut evidence that satisfactorily
explains the function of the lamina in relation to its
structure. In this Commentary we will describe some of the
recent work that addresses this problem and attempt to
provide a unified model for the role of lamins in nuclear
envelope assembly and for the lamina in the initiation of
DNA replication
Farnesyltransferase inhibitor treatment restores chromosome territory positions and active chromosome dynamics in Hutchinson-Gilford progeria syndrome cells
Copyright @ 2011 Mehta et al.; licensee BioMed Central Ltd. This article has been made available through the Brunel Open Access Publishing Fund.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a premature ageing syndrome that affects children leading to premature death, usually from heart infarction or strokes, making this syndrome similar to normative ageing. HGPS is commonly caused by a mutation in the A-type lamin gene, LMNA (G608G). This leads to the expression of an aberrant truncated lamin A protein, progerin. Progerin cannot be processed as wild-type pre-lamin A and remains farnesylated, leading to its aberrant behavior during interphase and mitosis. Farnesyltransferase inhibitors prevent the accumulation of farnesylated progerin, producing a less toxic protein. RESULTS: We have found that in proliferating fibroblasts derived from HGPS patients the nuclear location of interphase chromosomes differs from control proliferating cells and mimics that of control quiescent fibroblasts, with smaller chromosomes toward the nuclear interior and larger chromosomes toward the nuclear periphery. For this study we have treated HGPS fibroblasts with farnesyltransferase inhibitors and analyzed the nuclear location of individual chromosome territories. We have found that after exposure to farnesyltransferase inhibitors mis-localized chromosome territories were restored to a nuclear position akin to chromosomes in proliferating control cells. Furthermore, not only has this treatment afforded chromosomes to be repositioned but has also restored the machinery that controls their rapid movement upon serum removal. This machinery contains nuclear myosin 1β, whose distribution is also restored after farnesyltransferase inhibitor treatment of HGPS cells. CONCLUSIONS: This study not only progresses the understanding of genome behavior in HGPS cells but demonstrates that interphase chromosome movement requires processed lamin A.This work was funded by an ORSAS award and the Brunel Progeria Research Fund
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The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts
The sites of nascent DNA synthesis were compared
with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-11-dUTP. The sites of incorporation of biotin into injected cells
were compared with the distribution of PCNA by
indirect immunofluorescence microscopy and laser
scanning confocal microscopy (LSCM). In common
with other studies, a progression of patterns for both biotin incorporation and PCNA localisation was observed. However, we did not always observe
coincidence in these patterns, the pattern of biotin incorporation often resembling the expected, preceding distribution of PCNA. In nuclei in which the pattern of biotin incorporation appeared to be identical to the distribution of PCNA, LSCM revealed that not all of the sites of PCNA immunofluorescence were incorporating biotin at the same time. Secondly,
nuclei which had been isolated from quiescent
cultures of HDF were innoculated into cell-free
extracts of Xenopus eggs which support DNA replication in vitro. Following innoculation into these extracts DNA replication was initiated in each nucleus. The sites of DNA synthesis were detected by biotin-11-dUTP incorporation and compared with the distribution of PCNA by indirect immunofluorescence. Only a single pattern of biotin incorporation and PCNA distribution was observed. PCNA accumulated
at multiple discrete spots some 15min before any biotin incorporation was observed. When biotin incorporation did occur, LSCM revealed almost complete coincidence between the sites of DNA synthesis and the sites at which PCNA was localised.Brunel Open Access Publishing Fun