Subclassification of murine T cells by computerized microphotometric analysis

Splenocytes separated by physical means and classified as T cells by immunologic tests and computerized microphotometric analysis are differentiated into subgroups by analysis of the distribution patterns of Feulgen-positive nuclear DNA. In like fashion T cells obtained as purified preparations after separation on a nylon column, and accepted as T cells by micromorphometric analysis were subjected to further computerized morphometric analysis of nuclear DNA to form subgroups of cells. In each case, the number and composition of the detected subgroups were consistent. The classification does not appear to reflect any obvious phases of the cell cycle and is not dependent upon the sex and strain of mice from which the cells were obtained.     

Computerized morphometric differentiation of chromatin changes in splenocytes from normal and calorie-restricted mice

Morphometric image analysis of splenocytes obtained from mice fed calorie-restricted diets for periods of up to three weeks revealed slight but significant changes in nuclear size, nuclear texture and total amounts of stainable Feulgen-positive DNA as compared to age-and-sex-matched control cells. The cellular changes were associated with body weight loss and changes in the distribution of splenic T cells and B cells during the three weeks of dietary restriction. The reduced nuclear area and Feulgen-stained DNA may reflect an altered susceptibility of cells from nutrient-restricted sources toward acid hydrolysis. This in turn may be indicative of a temporarily altered synthesis of subcellular components, as these phenomena seem to lessen by the third week of dietary restriction.     

Localization of HPV-16 DNA sequence in CaSki cells by electron microscopic hybridocytochemistry.

We investigated the HPV-16 DNA sequence in the CaSki cervical carcinoma cell line by electron microscopic hybridocytochemistry using biotinylated HPV-16/18 probes. At the light microscopic level, reaction product of hybridized HPV-16 DNA sequence was not seen in the cytoplasm but appeared as spots or rods randomly distributed in the nuclei. By electron microscopy, reaction product was seen aggregated in several regions in the nuclei. Most of the stained areas did not reveal particular architecture but showed part of the chromatin structure. In other nuclei, reaction product was observed to be associated with strings of loop-like structure, and some stained loops were seen to be connected directly to the nuclear filamentous chromatin structure. The skeletonized images of hybridized HPV-16 DNA in the nuclei were illustrated by computerized image analysis. In conclusion, we have demonstrated the HPV-16 DNA sequence in the nuclei of CaSki cells by electron microscopy. The identification of stained areas localized only in the chromatin suggests an integrated form of HPV-16 DNA sequence in the cells. This method could be used to identify an integrated or episomal form of viral DNA in the virus-containing cells.     

Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells.

The subnuclear distribution of simian virus 40 large T antigen within nuclei of transformed Cos and C6 monkey cells was examined. Cos cells express wild-type T antigen but lack viral sequences required for DNA replication, whereas C6 cells contain a functional viral origin but express a replication-defective mutant T antigen which is unable to bind specifically to viral DNA. Discrete subpopulations of T antigen were isolated from the soluble nucleoplasm, chromatin, and nuclear matrix of both cell lines. Although only a small quantity (2 to 12%) of the total nuclear T antigen from Cos cells was associated with the nuclear matrix, a high proportion (25 to 50%) of C6 T antigen was bound to this structure. Results obtained from lytically infected monkey cells showed that early in infection, before viral replication was initiated, a higher proportion (22%) of T antigen was found associated with the nuclear matrix compared with amounts found associated with this structure later in infection (5 to 8%). These results suggest that an increased association of T antigen with this structure is not correlated with viral replication. T antigen isolated from the C6 nuclear matrix was more highly phosphorylated than was soluble C6 T antigen and was capable of binding to the host p53 protein. C6 DNA contains three mutations: two corresponding to N-terminal changes at amino acid positions 30 and 51 and a third located internally at amino acid position 153. By analysis of the subnuclear distribution of T antigen from rat cells transformed by C6 submutant T antigens, it was determined that one or both of the mutations at the NH2 terminus are responsible for the increased quantity of C6 T antigen associated with the nuclear matrix. These results suggest that neither a functional viral DNA replication origin nor the origin binding property of T antigen is required for association of this protein with the nuclear matrix.     

Isotachophoretic and HPLC determination of nucleotides in rat liver cell nuclei isolated by non-aqueous technique

Rat liver whole cells and cell nuclei were prepared by a non-aqueous technique (glycerol). The nuclear preparations were of different purity as determined by RNA/DNA ratios (0.17-1.60) and accordingly were divided into 3 subgroups (mean values 0.29, 1.04 and 1.48). RNA nucleotides were separated by isotachophoresis and HPLC and calculated per mg DNA. Two of the nuclear subgroups (RNA/DNA = 1.04 and 1.48) had significantly elevated nucleotide values in relation to RNA/DNA. UDP-N-acetylhexosamine/DNA, on the contrary, was reduced in conformity with RNA in the preparations. Our findings may indicate different nucleotide concentrations in different parts of the cell.     

Computerized measurement of the DNA content, areas, and autoradiographic grains of the same nuclei: demonstration that lightly (3H)thymidine-labeled bone marrow cells are predominantly in G0/G1 and G2

A computerized "flying spot" microdensitometer and scanning stage have been combined to measure the cellular DNA content, nuclear areas, and autoradiographic grain areas of the same cells. The slide positions of the Feulgen-stained, (3H)thymidine-labeled cells are mapped with the computerized stage, and nuclear DNA content and areas are then determined by integral absorbance measurements at 588 nm. Following autoradiograph preparation, the cells are relocated and the areas of the autoradiographic grains over each nucleus are measured at a light wavelength (625 nm) and an optical density setting (greater than 0.10) that do not detect the Feulgen stain. The microcomputer calculates the portion of each nucleus covered with autoradiographic grains (grain area proportion, GAP), and it links the GAP value to the DNA content of each nucleus in the computer file for subsequent sorting and analysis. By using this system in a study of mouse bone marrow cells labeled in vivo with (3H)thymidine, we found that all S-phase cells were clearly labeled after 8 or more days of autoradiographic exposure. Prolonged exposures (up to 64 days) led to detection of lightly labeled cells (0.1 less than GAP less than 0.8) with G1/G0 and G2 DNA content.     

Induction of the nuclear protein cyclin in serum-stimulated quiescent 3T3 cells is independent of DNA synthesis

Inhibition of DNA synthesis and cell proliferation of mouse 3T3 cells by aphidicolin did not affect the expression of cyclin, a nuclear protein whose synthesis correlates with cell proliferation, as determined by quantitative two-dimensional gel electrophoresis analysis. Serum stimulation of quiescent 3T3 cells revealed that cyclin synthesis increases shortly before DNA synthesis. Inhibition of DNA synthesis by aphidicolin in serum-stimulated quiescent cells did not affect the increase of cyclin following stimulation. These results demonstrate that cyclin synthesis is not coupled to DNA synthesis and that it is one of the latest events before DNA replication.     

Initiation of DNA synthesis and uptake of T antigen by chick erythrocyte nuclei in hterokaryons with SV40-transformed human cells.

Nonsynchronized and hydroxyurea (HU)-synchronized SV40-transformed human cells (W98VaD) were fused with chick embryo erythrocytes (CE). The uptake of T antigen by CE nuclei was compared with initiation of chick nuclear DNA synthesis. Uptake of T antigen by CE nuclei occurred at about the same time after fusion with asynchronous as with HU-synchronized cells. CE nuclei rapidly became T antigen-positive between 16 h and 28 h after fusion and usually almost all CE nuclei were T antigen-positive by 48 h after fusion. In contrast, initiation of chick nuclear DNA synthesis occurred as a function of time after reversal of the HU block, when the host cell nuclei were also synthesizing DNA. Chick nuclear DNA synthesis occurred in many heterokaryons before the CE nuclei became T antigen-positive by immunofluorescence.     

Characterization of cytoplasmic DNA of mouse-myeloma cells.

Cytoplasmic DNA from mouse myeloma cells comprised between 1% and 2% of the total cellular DNA. Detergent-prepared cytoplasmic lysate consisted mainly of 8-S and 22-S species. While these DNA species were present in the 13000 times g pellet of the detergent-prepared cytoplasmic lysate, only the light DNA species was present in the 13000 times g supernatant fraction. In neutral CsCl gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm3, which is identical to that of nuclear DNA. The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed-circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent-prepared cytoplasmic lysate. It was found that poly (A)-containing mRNA and 28-S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA.     

Synthesis of nuclear acidic proteins in density-inhibited fibroblasts stimulated to proliferate

Previous work from this laboratory (Rovera and Baserga, 1971) has shown that, when density-inhibited WI-38 human diploid fibroblasts are stimulated to proliferate by a change of medium, the synthesis of nuclear acidic proteins increases within 30 minutes after stimulation; several hours before DNA synthesis begins to increase. Similar results have now been obtained with density-inhibited 3T6 mouse fibroblasts, also stimulated by a change of medium. Gel electrophoretic analysis of nuclear acidic proteins in both WI-38 human diploid fibroblasts and 3T6 mouse fibroblasts stimulated to proliferate indicates that the increased synthesis of nuclear acidic proteins is limited to certain classes of proteins while other classes are totally unaffected. The increase in nuclear acidic proteins synthesis is inhibited when WI-38 cells or 3T6 cells are stimulated in the presence of 5-azacytidine (10 μg/ml), a treatment which also inhibits the subsequent stimulation of DNA synthesis. These results, confirming and extending similar findings previously reported in other models of stimulated DNA synthesis, lend further support to the hypothesis that nuclear acidic proteins may play a critical role in the control of DNA synthesis and cell division in mammalian cells.     

The effect of in vitro heat exposure on the recovery of nuclear matrix-bound DNA polymerase alpha activity during the different phases of the cell cycle in synchronized HeLa S3 cells.

HeLa S3 cells were synchronized by a double thymidine block or aphidicolin treatment and the levels of nuclear matrix-bound DNA polymerase alpha activity were then measured using activated calf thymus DNA as template. The nuclear matrix was obtained by 2 M NaCl extraction and DNase I digestion of isolated nuclei incubated at 37 degrees C for 45 min prior to subfractionation. In all phases of the cell cycle 25-30% of nuclear DNA polymerase alpha activity remained matrix-bound, even when cells were in the G1 phase. No dynamic association of DNA polymerase alpha activity with the matrix was seen, at variance with previous results obtained in regenerating rat liver. The variations measured in matrix-bound activity closely followed those detected in isolated nuclei throughout the cell cycle. If nuclei were not heat-stabilized very low levels of DNA polymerase alpha activity were measured in the matrix (1-2% of total nuclear activity). Heat incubation of nuclei failed to produce any enrichment in matrix-associated newly replicated DNA, whereas the sulfhydryl cross-linking chemical sodium tetrathionate did. Therefore the results obtained after the heat stabilization procedure do not completely fit with the model that envisions the nuclear matrix as the active site where eucaryotic DNA replication takes place.     

The HMG Protein T160 Colocalizes with DNA Replication Foci and Is Down-regulated during Cell Differentiation

The high mobility group protein T160, the murine homolog of the human structure-specific recognition protein 1, was first supposed to be involved in the process of V-(D)-J recombination, since it could bind to recombination signal sequence probes. We have recently cloned T160 by using an unrelated DNA probe and shown that it binds to either cruciform or linear DNA with no sequence specificity. In this work, we performed a detailed analysis of T160 expression and immunolocalization. We show that T160 is a phosphoprotein broadly conserved from yeast to mammals, with a high level of expression in all the cell lines tested and in tissues containing a high degree of proliferating cells. Indirect immunofluorescence analysis by confocal laser microscopy revealed that T160 distribution in the cell nucleus is not uniform, and focus-like staining was observed. Cell cycle studies by BrdU incorporation suggest that the appearance of T160 nuclear foci is specific of mid to late S phase. Furthermore, while T160 expression does not change during the cell cycle, it is dramatically down-regulated when cells begin to differentiate, as highlighted in C2C12 myoblasts and myotubes. The disappearance of T160 nuclear staining in multinucleated myotubes is shown. Taken together, these data suggest that its function may be less specific than V-(D)-J recombination and more related to some cellular basic process, such as DNA replication or repair.     

Image analysis of Feulgen-stained NIH/3T3 cells transformed with DNA of 4-nitroquinoline 1-oxide treated human breast epithelial cells.

The nuclear phenotypes of Feulgen-stained NIH/3T3 cells transformed with 4-nitroquinoline 1-oxide (4NQO) treated, human breast epithelial cell (HBEC) DNA were studied by scanning microspectrophotometry and image analysis and compared with data obtained for nontransformed cells and for NIH/3T3 cells under ras oncogene transfecting situations. The Feulgen-DNA content of the individual nuclei (NQ1, NQ2, and NQ3 phenotypes) of the transformed cells was found not to be deeply affected, although presence of chromatin structures resembling double minutes could be verified in part of the metaphases of the transformed cells. On the other hand, the chromatin supraorganization of these cells showed some changes involving increased (NQ2, NQ3) or decreased (NQ1) levels of condensation. The changes in chromatin packing states, however, were of small magnitude compared with those reported for NIH/3T3 cells transfected with a c-H-ras oncogene or an N-ras-containing MCF-7 cell DNA. It was assumed that the transformation of the NIH/3T3 cells is not always necessarily accompanied by high levels of chromatin condensation. The transformation of the NIH/3T3 cells induced by the 4NQO-treated HBEC DNA and particularly the changes in chromatin condensation in these transformed cells could not be attributed merely to a ras activation elicited by the carcinogen. It is suggested that a more complex transforming mechanism is involved, probably owing to the fact that a whole genomic DNA of the 4NQO-treated HBEC has been used for transfection.     

pp32 overexpression induces nuclear pleomorphism in rat prostatic carcinoma cells

Abstract. Nuclear pleomorphism is an important diagnostic factor in tumour pathology. Traditionally, nuclear pleomorphism is evaluated qualitatively or semiquantitatively, often as a component of tumour grade; the molecular basis of nuclear pleomorphism, however, remains unclear. In this study, we investigated the quantitative effects on nuclear morphology of overexpressing pp32, a recently described nuclear phosphoprotein highly expressed in self-renewing and neoplastic cell populations. Assessment of Feulgen-stained transfected and control lines of AT3.1, a rat prostatic carcinoma cell line, using a computerized Cellular Image Analysis System (BD CAS-200) showed that stable overexpression of human pp32 in AT3.1 cells is accompanied by marked increases in the coefficient of variation of nuclear shape, nuclear size and chromatin textures but not in DNA content. In contrast, stable transfection with control vector, with ras , or with bcl-2 failed to affect nuclear morphology. Cell cycle analysis further showed that pp32-related increases in variation of nuclear structure manifested principally in G_1. These studies suggest that pp32 plays a role either directly or indirectly in the control of nuclear shape of G₁ cells.     

Cell fusion of human and mouse cells as a source for new cells retaining human markers. Analysis of DNA content, membrane and cytoplasmic antigen expression.

Flow cytometry and ultrastructural morphometry were used to study some characteristics of cells obtained by fusion with polyethylene-glycol 4000 between mouse fibroblasts 3T3.4E and normal keratinocytes (3T3.4E x NHK) or hand wart human keratinocytes (3T3.4E x HWK), at late passages. The cell cycle and the expression of human beta 2-microglobulin, human EGF-receptors (EGF-r), vimentin were simultaneously studied by flow cytometry. Epithelial CaSki cells, derived from a human uterine carcinoma, expressing high levels of beta 2-microglobulin, EGF-r and vimentin, were used as a positive control. In mouse fibroblasts 3T3.4E only vimentin was expressed whereas in cells derived from fusion, human beta 2-microglobulin, human EGF-r and vimentin were detected. The cell cycle analysis revealed that the peak position of G0/G1 differed with the cells (channel 11 for 3T3.4E cells, 13 for 3T3.4E x HWK and 15 for 3T3.4E x NHK). The area of the cell compartments from each cell type was also different by quantitative ultrastructural morphometry. The hybrid phenotype was maintained in late passages in cells (3T3.4E x NHK) and (3T3.4E x HWK), as shown by the expression of human antigens, differences in DNA contents and nuclear area. Flow cytometry may be a very accurate and precise tool for studying low antigenic expression. The combination of different methods including analysis of DNA content, antigenic expression and ultrastructural morphometry confirmed that 3T3.4E, 3T3.4E x NHK and 3T3.4E x HWK cells are different cell types. These techniques are complementary to cell phenotype analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear protein following heat shock: protein removal kinetics and cell cycle rearrangements

Nuclear protein and DNA content of HeLa cells was determined as a function of time following hyperthermia by staining isolated nuclei with two fluorescent dyes: fluorescein isothiocyanate (FITC) for protein content and propidium iodide (PI) for DNA content. Bivariate FITC and PI histograms were obtained by flow cytometry. Univariate flow cytometric analysis was shown to be inadequate for this study, because some of the nuclear protein changes were due to cell cycle redistribution. Posthyperthermia cell kinetics could be divided into two distinct phases: an early phase characterized by the removal of heat-induced excess nuclear proteins with little or no cell progression through the cell cycle; and a late phase characterized by a redistribution of cells in the cell cycle resulting in an accumulation of cells in G2. The duration of these phases was dependent upon the hyperthermia dose. In the early phase, the rate of removal of excess nuclear protein was found to vary with heating time and temperature for time-temperature combinations which resulted in the same amount of excess nuclear protein. In the late phase, the cells blocked in G2 did not reduce their nuclear protein levels back to control values.