Re-modelling of nuclear architecture in quiescent and senescent human fibroblasts.

Spatial organisation of the genome within the nucleus can play a role in maintaining the expressed or silent state of some genes [1]. There are distinct addresses for specific chromosomes, which have different functional characteristics, within the nuclei of dividing populations of human cells [2]. Here, we demonstrate that this level of nuclear architecture is altered in cells that have become either quiescent or senescent. Upon cell cycle exit, a gene-poor human chromosome moves from a location at the nuclear periphery to a more internal site in the nucleus, and changes its associations with nuclear substructures. The chromosome moves back toward the edge of the nucleus at a distinctive time after re-entry into the cell cycle. There is a 2-4 hour period at the beginning of G1 when the spatial organisation of these human chromosomes is established. Lastly, these experiments provide evidence that temporal control of DNA replication can be independent of spatial chromosome organisation. We conclude that the sub-nuclear organisation of chromosomes in quiescent or senescent mammalian somatic cells is fundamentally different from that in proliferating cells and that the spatial organisation of the genome is plastic.

S-phase phosphorylation of lamin B2.

Lamin B2 modification in synchronously dividing populations of human diploid fibroblasts was determined by 2-dimensional gel electrophoresis and [32P]orthophosphate labelling. In quiescent (G0) and G1 cultures of HDF, lamin B2 migrated as 2 spots on 2-dimensional gels. In contrast, in S-phase populations of HDF lamin B2 migrated as a single basic species. The level of lamin B2 phosphorylation was determined after immunoisolation from [32P]orthophosphate labelled cells. The results of these experiments indicated a 2-3-fold increase in the steady state level of lamin B2 phosphorylation in S-phase HDF compared with G0 HDF. Consistent with this evidence, tryptic peptide maps revealed the presence of a phosphopeptide in S-phase lamin B2 which was absent from G0 lamin B2. Since all of the phosphate incorporated into S-phase and G0 lamin B2 was recovered in serine residues we conclude that the S-phase specific phosphopeptide did not represent either of the cdc2 sites associated with entry nuclear lamina breakdown.

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 nucleous. 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 S-phase 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-beta-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.

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.

Association of pKi-67 with satellite DNA of the human genome in early G1 cells.

pKi-67 is a nucleolar antigen that provides a specific marker for proliferating cells. It has been shown previously that pKi-67's distribution varies in a cell cycle-dependent manner: it coats all chromosomes during mitosis, accumulates in nuclear foci during G1 phase (type I distribution) and localizes within nucleoli in late G1 S and G2 phase (type II distribution). Although no function has as yet been ascribed to pKi-67, it has been found associated with centromeres in G1. In the present study the distribution pattern of pKi-67 during G1 in human dermal fibroblasts (HDFs) was analysed in more detail. Synchronization experiments show that in very early G1 cells pKi-67 coincides with virtually all satellite regions analysed, i.e. with centromeric (alpha-satellite), telomeric (minisatellite) and heterochromatic blocks (satellite III) on chromosomes 1 and Y (type Ia distribution). In contrast, later in the G1 phase, a smaller fraction of satellite DNA regions are found collocalized with pKi-67 foci (type Ib distribution). When all pKi-67 becomes localized within nucleoli, even fewer satellite regions remain associated with the pKi-67 staining. However, all centromeric and short arm regions of the acrocentric chromosomes, which are in very close proximity to or even contain the rRNA genes, are collocalized with anti-pKi-67 staining throughout the remaining interphase of the cell cycle. Thus, our data demonstrate that during post-mitotic reformation and nucleogenesis there is a progressive decline in the fraction of specific satellite regions of DNA that remain associated with pKi-67. This may be relevant to nucleolar reformation following mitosis.

Internal lamin 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.

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.

The expression of proliferation-dependent antigens during the lifespan of normal and progeroid human fibroblasts in culture.

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 proliferation-associated 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.

The kinetics of senescence in retinal pigmented epithelial cells: a test for the telomere hypothesis of ageing?

Senescence, or replicative failure, has been reported for a wide variety of human cell types but has seldom been characterized in any detail. The senescence of human fibroblast cultures has been shown to be due to a steadily decreasing percentage of cells able to proliferate in standard media. This paper reports the serial subculture of a strain of adult retinal pigmented epithelial (RPE) cells until replicative failure after approximately 15 population doublings. Measurement of the growth fraction of the RPE cells at each passage using antibodies to the proliferation marker pKi67 demonstrated a rate of decline in the proliferating fraction of 3.66% per population doubling. Similar experiments carried out using a strain of human fibroblasts yielded a decline of approximately 0.88% per population doubling. Thus, individual RPE cells enter senescence significantly faster than control fibroblasts (p < 0.001). At growth arrest the RPE cells retained viability for extended periods but showed elevated endogenous autofluorescence, analogous to observations on post-mitotic human fibroblasts. Taken together these findings suggest that the process of senescence is a common feature of different cell lineages but that the specific rate can differ between them. The significance of these observations for the telomere hypothesis of senescence is discussed.

The gene responsible for Werner syndrome may be a cell division "counting" gene.

Werner syndrome is a rare, autosomal, recessive condition that is frequently studied as a model of some aspects of human aging, although the behavioral changes that are usually associated with old age are only seen very infrequently. A most striking aspect of the phenotype of Werner syndrome, presumably arising from the same gene defect, is a dramatic shortening of the replicative life-span of dermal fibroblasts in vitro. The finite replicative life-span of human cells in vitro is due to the stochastic loss of replicative ability in a continuously increasing fraction of newborn cells at every generation. Normal human fibroblasts achieve approximately 60 population doublings in culture, while Werner syndrome cells usually only achieve approximately 20 population doublings. We describe an analysis of the replicative ability of fibroblasts from Werner syndrome patients and demonstrate that the cells in these cultures usually exit, apparently irreversibly, from the cell cycle at a faster rate than do normal cells, although they mostly start off with a good replicative ability. We propose that the Werner syndrome gene is a "counting" gene controlling the number of times that human cells are able to divide before terminal differentiation.

Cell cycle changes in A-type lamin associations detected in human dermal fibroblasts using monoclonal antibodies.

A new panel of anti-A-type lamin monoclonal antibodies was generated. Epitope mapping was performed by immunoblotting against GST-lamin fusion peptides. Epitopes were mapped to four different regions of human lamin A and three different regions of human lamin C. The distribution of A-type lamins was compared with the distribution of the proliferation marker Ki67 in proliferating and quiescent cultures of human dermal fibroblasts (HDFs) using a double indirect immunofluorescence assay. Antibodies that had been mapped to a region of the lamin C tail stained the nuclear envelope of proliferating and quiescent cells equally brightly. In contrast, antibodies recognizing epitopes in the head domain and rod domain of lamins A and C and the tail domain of lamin A stained the nuclear envelope of quiescent cells strongly but reacted poorly or not at all with the nuclear envelope of proliferating cells. Changes in the level of expression of lamins A and C were not detected in immunoblotting assays. However, epitope masking was revealed, and this occurred by two distinct mechanisms. Epitope masking in the head domain of lamins A and C occurred as a result of protein phosphorylation. Epitope masking in the rod domain of lamins A and C and in the tail domain of lamin A occurred through a physical association between the lamin and chromatin and/or other nuclear proteins. The cell cycle timing of epitope masking was investigated in HDFs that had been restimulated after serum starvation. Extensive epitope masking in restimulated cells only occurred after cells had passed through mitosis. These results are consistent with the hypothesis that rearrangement of A-type lamin filaments, as cells progress from a quiescent to a proliferating state, results in altered lamina associations.

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 15 min 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.

Different kinetics of senescence in human fibroblasts and peritoneal mesothelial cells.

Senescence has been reported for a wide variety of human cell types. In cultures of human fibroblasts the process is due to a percentage of the cells becoming senescent at each passage rather than all the cells entering senescence simultaneously at the end of the life span. By measuring the percentage of fibroblasts which are still cycling at each passage, a rate of decline in the growth fraction, which mirrors the rate of senescence, can be obtained. However, such an analysis has never been undertaken in multiple cell types using the same method to identify cycling cells. It is thus unknown if the rate of senescence is the same or different in cultures of different human cell types. To answer this question the rates of decline in the cycling fractions were simultaneously measured in two cultures of human cells (AGO7086A, peritoneal mesothelial cells; and 2DD, human dermal fibroblasts) which have practically identical in vitro life spans. 2DD fibroblasts showed a rate of decline of 0.89% cycling cells per population doubling when the data obtained were fitted to a simple linear equation. However, AGO7086A gave a decline of approximately 2.2% per population doubling. Thus mesothelial cells enter senescence significantly faster than fibroblasts (P < 0.001). This decline in the growth fraction was accompanied by an increasing fraction of mesothelial cells which retained detectable endogenous beta-galactosidase activity at pH 6. Such activity has previously been shown to be associated with senescent human fibroblasts. These findings suggest that the process of senescence has common features in different cell lineages but that the rate of the process can differ markedly between them.