Relationship between cell proliferation, chromatin template activity and accumulation of nuclear proteins

Trypsinization of confluent monolayers of WI-38 cells causes an extensive loss of nuclear proteins. The loss of nuclear proteins is restored only several hours after the cells have been replated at a lower density in 10% serum. When trypsinized fibroblasts are replated at a lower density in 10% serum, there is also a sustained progressive leading to DNA synthesis and cell division. If 0.3% serum is used instead of 10%, there is a modest increase in nuclear template activity, but not accumulation of nuclear proteins and no DNA synthesis or cell division.     

Immunologic similarity of antigens of residual nuclear proteins from various hepatomas

Rabbit antiserum against alkali-insoluble nuclear residual protein of transplantable rat hepatoma 27 reacted with residual proteins during immunodiffusion in agarose gel, provided immunoprecipitation of isolated nuclei and indirect immunofluorescence with Zajdela's rat ascites hepatoma and mouse ascites hepatoma 22a, while the reaction with non-liver tumors--rat Jensen, Yoshida and M 1 sarcomas and mouse Ehrlich ascites carcinoma was negative. Mild indirect immunofluorescence with kidney and spleen sections was eliminated if antiserum was preincubated with acetone powders of these tissues.     

Monoclonal antibodies against nuclear matrix detect nuclear antigens in mammalian, insect and plant cells: an immunofluorescence study

We have applied monoclonal antibodies generated against nuclear matrix in an immunofluorescence study of a variety of plant and animal species. Antibodies P1 and I1 detected antigens in all species examined, including higher and lower plants. Antibodies PI1 and PI2 stained only animal cells, and showed some tissue and/or species-specific variability in staining pattern. The presence of similar nuclear matrix components in such diverse species suggests that nuclear order may be maintained by similar mechanisms in all eukaryotes.     

Specific interactions of three proliferating cell nuclear antigens with replication-related proteins in Aeropyrum pernix

Proliferating cell nuclear antigen (PCNA) is a well-known multifunctional protein involved in eukaryotic and archaeal DNA transactions. The homotrimeric PCNA ring encircles double-stranded DNA within its central hole and tethers many proteins on DNA. Plural genes encoding PCNA-like proteins have been found in the genome sequence of crenarchaeal organisms . We describe here the biochemical properties of the three PCNAs, PCNA1, PCNA2 and PCNA3, from the hyperthermophilic archaeon, Aeropyrum pernix . PCNA2 can form a trimeric structure by itself, and it also forms heterotrimeric structures with PCNA1 and PCNA3. However, neither PCNA1 nor PCNA3 can form homotrimers. The DNA synthesis activity of DNA polymerase I and II, the endonuclease activity of FEN1, and the nick-sealing activity of DNA ligase were stimulated by the complex of PCNA2 and 3 or PCNA1, 2 and 3. These results suggest that the heterotrimeric PCNA at least including PCNA2 and 3 function as the clamp in the replisome. However, PCNA2 is the most abundant in the cells throughout the growth stages among the three PCNAs, and therefore, PCNA2 may perform multitasks by changing complex composition.     

Role of nuclear matrix proteins in the formation of heterochromatin]

Heterochromatin consists mainly of satellite DNAs (stDNA), the most rapidly evolving type of DNA sequences of the eucaryotic genome. On the other hand, stDNA is involved in the formation of the functionally conserved centromere structure. Centromeres and pericentromeric stDNA, are known to be in association with nuclear matrix or chromosome scaffold at all stages of the cell cycle. Several lines of evidence show that attachment of stDNA to the nuclear matrix is specific. The first defined parts of the genes found in association with nuclear matrix/scaffold, MAR/SAR, possess some common features with the stDNA. A number of different mechanisms have previously been implicated in heterochromatin formation and centromere conservation. The role of nuclear matrix proteins, which are able to recognize common structural features of MAR/SAR and stDNA, in constitutive heterochromatin organization is discussed in the current review.     

Regulation of human megakaryocytopoiesis: analysis of proliferation, ploidy and maturation in liquid cultures

A liquid culture technique associated with either double staining and flow cytometry or electron microscopy was used to study human megakaryocytopoiesis. During development from the embryo to the adult, a progressive increase in ploidy classes associated with an enhancement of megakaryocyte (meg) size was observed. Granulocyte-macrophage colony-stimulating factor had no effects on adult marrow cultures. In contrast, interleukin (IL) 3 induced a marked proliferation, but was unable to promote polyploidization. Furthermore, it abrogated the effects on endomitosis of aplastic plasma (AP). This negative effect on polyploidization of IL-3 could be partially dissociated from its effects on proliferation by a delayed addition in culture. AP acted on both proliferation and endoreplication, which was not due to the main hematopoietic growth factors, including IL-6. A synthesis of IL-6 was detected by in situ hybridization in cultured cells including megs which also express receptors for IL-6. These results suggest that terminal meg differentiation may be regulated by an autocrine IL-6 loop, and that megakaryocytopoiesis may be independently regulated at early and late stages of differentiation.     

Autoradiographic study on the synthesis of nuclear proteins

Experiments were carried out on cultures of Chinese hamster fibroblasts. Cells were short-labelled with H³-Iysine or H³-tryptophane. The kinetics of silver grain number over interphase nuclei, metaphases, and individual chromosomes of the first pair were studied. — The grain count distribution over chromosomes of different groups and over sister-chromatids was analyzed. The data presented allow us to conclude that: 1. There is equal distribution of nuclear and chromosomal proteins between daughter nuclei and sister-chromatids, resp., during each mitotic cycle. 2. The synthesis of chromosome proteins, in general, and nonhistone proteins, in particular, takes place at all stages of the cycle. 3. At each stage of the cycle, proteins are incorporated into chromosomes of different groups simultaneously and at equal rate. The rate of this process in S and G2 is two times that in G1.     

Flow cytometric analysis of proliferation associated nuclear antigens using washless staining of unfixed cells.

The expression of different proliferation associated nuclear antigens was analyzed using a washless double-staining method and flow cytometry. It is a simple and rapid two-step procedure which can be performed on low cell numbers. A series of hematopoietic cell lines and fresh lymphoma cells were tested and the methodology was found to be applicable to a number of nuclear antigens (PCNA, Ki-67, p105, MPM-2, fibrillarin). For PCNA, the detectability was dependent on the type of antibody used. The immunofluorescence pattern observed by microscopy was altered for antigens stained by the washless technique in comparison with the pattern obtained with fixed cells. With the washless method, detailed cell cycle analysis could be obtained by dual parameter analysis of PCNA and Ki-67.     

Influence of section thickness, mean nuclear diameter and nuclear crowding on DNA ploidy in histologic sections of melanocytic skin lesions

OBJECTIVE: To determine the influence of section thickness, nuclear diameter (MND) and area percentage of nuclei (a measure of nuclear crowding) on histologic DNA ploidy assessed by image cytometry (ICM) of primary melanocytic skin neoplasms (MSNs). STUDY DESIGN: Initially a feasibility study was performed to determine if comparable DNA ploidy histograms could be obtained from cell disaggregates and tissue sections. Following this, DNA ICM was performed on Feulgen-stained tissue sections (4, 6, 8 and 10 microns thick) from 30 primary MSNs (20 benign, 10 malignant) with nuclear diameters from 5.6 to 8.6 microns. Area percentage of nuclei was assessed in all cases at all section thicknesses. RESULTS: The feasibility study produced comparable results for cytocentrifuge and tissue section preparations. For sectioned MSNs, DNA ploidy histograms from 4-micron sections had a higher coefficient of variation of the 2c peak than those from 6-, 8- and 10-micron sections. Ten-micrometer sections had marked overlapping of nuclei, and only small numbers of cells could be measured, giving inadequate results. MND and area percentage of nuclei did not have an important influence on the results. CONCLUSION: Adequate DNA ploidy profiles can be obtained by DNA ICM on 6- and 8-micron-thick histologic sections of MSNs, provided that a strict measurement protocol is followed.     

Cytofluorimetric study of the glycogen content in hepatocytes of varying ploidy in adult rats]

A combined method, that allows measuring glycogen and DNA contents in one of the same cell, was applied for quantitative determination of these in mono- and binucleate hepatocytes with different ploidy obtained from adult rats. The mean glycogen content was shown to increase proportionally to the genome number within the changes of the hepatocyte ploidy from 2 to 8c.     

Prognostic significance of DNA ploidy and proliferation rate in human astrocytic gliomas

DNA ploidy and the proliferative potential in 75 gliomas were investigated using bromodeoxyuridine labelling index (BrdUrd LI), S-phase fraction (SPF) and argyrophilic nucleolar organizer regions (AgNOR) technique. There were 53 highly malignant (AIII-AIV), and 22 low-grade (AI-AII) gliomas. One fragment of the tumour was fixed in Carnoy's solution for AgNOR test, while the other fragments were used for flow cytometric determination of the labelling index, SPF and DNA ploidy. For the BrdUrdLI, tumour samples from each patient were incubated in vitro for one hour at 37 degrees C with BrdUrd using the high pressure oxygen method. The tumours showed variability in the BrdUrdLI values, SPF and AgNOR counts/cell nucleus. The same percentage of DNA aneuploidy (55%) was found in high-grade as well as in low-grade gliomas. Univariate analysis showed that patients with grade I & II gliomas had significantly higher 3-year survival rate (p = 0.0193) than those with grade III and grade IV gliomas. Also patients with lower proliferation rate of tumours (BrdUrdLI < or =2.3% and AgNOR counts < or =2.6%/cell) had higher 3-year survival rate (p<0.03), which can be helpful in prognosis. Tumour ploidy or SPF had no influence on patients' survival (p = 0.7908). Cox multivariate analysis showed that only patients' age > 45 years and high tumour grade (III and IV) were significant unfavourable prognostic factors in terms of patients' survival.     

Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics

Recent developments in reagent design can address problems in single cells that were not previously approachable. We have attempted to foresee what will become possible, and the sorts of biological problems that become tractable with these novel reagents. We have focused on the novel fluorescent proteins that allow convenient multiplexing, and provide for a time-dependent analysis of events in single cells. Methods for fluorescently labeling specific molecules, including endogenously expressed proteins and mRNA have progressed and are now commonly used in a variety of organisms. Finally, sensitive microscopic methods have become more routine practice. This article emphasizes that the time is right to coordinate these approaches for a new initiative on single cell imaging of biological molecules.