Distinction between the G0 and the late G1 phase of rat cells in vivo and in vitro with the nuclear QDH-fluorescence pattern

The quinacrine dihydrochloride (QDH) staining and the [3H]thymidine incorporation patterns were simultaneously analyzed in nuclei of rat cells from a proliferating (granulation tissue) and a nonproliferating tissue (liver). Nuclei from freshly isolated and cultured cells of the rapidly proliferating subcutaneous granulation tissue showed a cell cycle-related pattern similar to that previously described with growing fibroblast-like cells in vitro. Nuclei of liver cells in smears from biopsies and in histological sections showed a fluorescence pattern similar to that of serum-deprived arrested G0 cells from established cell lines. Treatment of primary cultured rat hepatocytes with phenobarbital altered their degree of chromatin condensation similar to that seen after treatment of rats in vivo. The data indicate that the QDH staining pattern is an early marker, suitable for detecting the cell cycle-promoting activity of chemicals (e.g., of tumor promoters) in nonproliferating cells from various tissues in vivo and in vitro.     

Cell kinetics of irradiated experimental tumors: relationship between the proliferating and the nonproliferating pool.

The role of nonproliferating cells in tumor regeneration has been studied after subcurative doses of low L.E.T. irradiation. Radiation was applied in a single dose at three different levels; 0-47, 0-94 and 1-88 krad. Studies included estimation of the absolute number of cells per tumor, differential cell counts, and autoradiographic determination of kinetic variables, employing transplantable mouse mammary adenocarcinoma DBAH. Quantitative changes of morphologically defined proliferating and nonproliferating cell pools were followed at different time intervals after irradiation. Irradiation resulted in reduction of the number of cells in both pools, with apparent sparing of nonproliferating cells. The regenerative period started with a gradual increase in the number of cells in the proliferating pool, whereas the number of cells in the nonproliferating pool continued to fall in tumors irradiated with 0-94 and 1-88 krad. In the late phase of tumor regrowth, the increasing number of cells in the non proliferating pool corresponded to its replenishment by cell transition from the proliferating pool. In an effort to clarify whether cell transition from the nonproliferating to the proliferating pool may take place during the regrowth of radiation perturbed tumors, cell loss rates from both pools were estimated using experimental data. In addition to cell loses from the tumor as a whole, the 'net loss rate' of the nonproliferating pool reflects the rate of cell transition from the nonproliferating to the proliferating pool, minus the rate of transition in the opposite direction. A similar definition applies to cell loss rates from the proliferating pool. The results showed: (1) high losses in both pools, with excess losses in the proliferating during the early phase after irradiation; (2) in the early stage of regrowth after irradiation, the cell net loss rate f-or the nonproliferating pool increased, in contrast to the behavior of cell loss rate for the proliferating pool and the average cell loss rate for the tumor as a whole; (3) in the late stage of regrowth a decrease in net loss rate for the nonproliferating pool reflects the excess production of nonproliferating cells over control tumors. These results suggest that cell transition from the nonproliferating to the proliferating pool takes place at the beginning of tumor regrowth after subcurative single-dose irradiation.     

Ciliated cells in vitamin A-deprived cultured hamster tracheal epithelium do divide

Summary The pseudostratified tracheal epithelium, composed of a heterogeneous phenotypically varying cell population, was studied with respect to the in vitro cell proliferative activity of differentiated epithelial cells. Ciliated tracheal epithelial cells so far have been considered to be terminally differentiated, nonproliferating cells. Tracheal organ cultures obtained from vitamin A-deprived Syrian Golden hamsters were cultured in a vitamin A-deficient, serum-free, hormone-supplemented medium. In vitamin A-deprived tracheal epithelium treated with physiologically active all-trans retinol and low cigarette-smoke condensate concentrations it is possible to stimulate the cell proliferation of both basal and columnar cells. Therefore, the probability of finding proliferating columnar cells was increased compared with the in vivo and the vitamin A-deprived situation in which cell proliferative activity is relatively low. In the presence of cigarette-smoke condensate in a noncytotoxic concentration, basal, small mucous granule, ciliated, and indifferent tracheal epithelial cells incorporated [methyl-³H]-thymidine into the DNA during the S phase. The finding that ciliated cells were labeled was supported by serial sections showing the same labeled ciliated cell in two section planes separated by 2 to 3 μm, without labeled epithelial cells next to the ciliated cell. Furthermore, a ciliated tracheal epithelial cell incorporating [methyl-³H]thymidine into DNA was also seen in tracheal cultures of vitamin A-deprived hamsters treated with all-trans retinol in a physiologic concentration. The present study was financially supported by the Scientific Advisory Committee on Smoking and Health (Dutch Cigarette Industry Foundation) and the Ministry of Welfare, Health and Cutural Affairs.     

CELL KINETICS OF IRRADIATED EXPERIMENTAL TUMORS: RELATIONSHIP BETWEEN THE PROLIFERATING AND THE NONPROLIFERATING POOL

The role of nonproliferating cells in tumor regeneration has been studied after subcurative doses of low L.E.T. irradiation. Radiation was applied in a single dose at three different levels, 0–47, 0–94 and 1–88 krad. Studies included estimation of the absolute number of cells per tumor, differential cell counts, and autoradiographic determination of kinetic variables, employing transplantable mouse mammary adenocarcinoma DBAH. Quantitative changes of morphologically denned proliferating and non-proliferating cell pools were followed at different time intervals after irradiation. Irradiation resulted in reduction of the number of cells in both pools, with apparent sparing of nonproliferating cells. The regenerative period started with a gradual increase in the number of cells in the proliferating pool, whereas the number of cells in the nonproliferating pool continued to fall in tumors irradiated with 0–94 and 1 -88 krad. In the late phase of tumor regrowth, the increasing number of cells in the non proliferating pool corresponded to its replenishment by cell transition from the proliferating pool. In an effort to clarify whether cell transition from the nonproliferating to the proliferating pool may take place during the regrowth of radiation perturbed tumors, cell loss rates from both pools were estimated using experimental data. In addition to cell losses from the tumor as a whole, the ‘net loss rate’ of the non-proliferating pool reflects the rate of cell transition from the nonproliferating to the proliferating pool, minus the rate of transition in the opposite direction. A similar definition applies to cell loss rates from the proliferating pool. The results showed: (1) high losses in both pools, with excess losses in the proliferating during the early phase after irradiation; (2) in the early stage of regrowth after irradiation, the cell net loss rate for the nonproliferating pool increased, in contrast to the behavior of cell loss rate for the proliferating pool and the average cell loss rate for the tumor as a whole; (3) in the late stage of regrowth a decrease in net loss rate for the nonproliferating pool reflects the excess production of nonproliferating cells over control tumors. These results suggest that cell transition from the nonproliferating to the proliferating pool takes place at the beginning of tumor regrowth after subcurative single-dose irradiation.     

Raman spectroscopy detects biochemical changes due to proliferation in mammalian cell cultures

Raman spectra of cells and nuclei from cultures in the plateau (nonproliferating) and exponential (proliferating) phases of growth were measured and show that Raman spectroscopy can monitor changes due to cell proliferation. A simple fitting routine was developed using a basis set (lipid, protein, DNA, RNA) to estimate the relative amounts of biochemical components in cells and nuclei. Using relative amounts and ratios of biochemical components, reproducible differences can be detected and quantified that are not readily apparent by visual analysis of vibrational bands in the spectra. These differences, due to cell proliferation, can be assigned to specific biochemical changes. They include a decrease in the relative lipid and increases in the relative protein and RNA for both nontumorigenic exponential cells and nuclei, and an increase in the relative RNA for tumorigenic exponential cells. The lipid/RNA ratio decreases for nontumorigenic exponential cells and nuclei and tumorigenic exponential cells. The protein/lipid ratio increases for both tumorigenic and nontumorigenic exponential cells and nuclei. Finally, the lipid/DNA ratio decreases for tumorigenic exponential nuclei. This knowledge will be important for Raman detection of rapidly dividing populations of cancer cells in vivo.     

Selective Staining of Eosinophils and Their Immature Precursors in Tissue Sections and Autoradiographs with Congo Red

The use of Congo red as an elective stain for eosinophilic granulocytes and their precursors in tissue sections and autoradiographs is demonstrated and discussed. The 0.5% alcoholic Congo red solution of Highman, normally used for the detection of amyloid, may also be used with only minor changes. This simple method may aid in the diagnosis of special hematological problems and facilitates the recognition of eosinophil granulocytes as well as proliferating and nonproliferating myelocytes in autoradiographs from paraffin sections.     

Notes on Technic: A Carbonate-Colloidal Iron Staining Reagent

The use of Congo red as an elective stain for eosinophilic granulocytes and their precursors in tissue sections and autoradiographs is demonstrated and discussed. The 0.5% alcoholic Congo red solution of Highman, normally used for the detection of amyloid, may also be used with only minor changes. This simple method may aid in the diagnosis of special hematological problems and facilitates the recognition of eosinophil granulocytes as well as proliferating and nonproliferating myelocytes in autoradiographs from paraffin sections.     

Positive and negative regulation of replication in hybrid cells]

DNA replication blockage in various differentiated cells was investigated on the model of heterokaryons. Two distinct types of DNA synthesis regulation in heterokaryons "differentiated cell + proliferating cell" were revealed: I. Neutrophils and nucleated erythrocytes efficiently prevented the entry of non-malignant proliferating cells nuclei into the S-period but usually failed to substantially inhibit the replication in malignant cells nuclei. Both "mortal" and immortalized proliferating cells activated the DNA synthesis in neutrophil and chicken erythrocyte nuclei. II. Macrophages did not influence the DNA synthesis in the nuclei of non-malignant cells in heterokaryons but drastically inhibited that in the nuclei of malignant cells. Only immortalized cells reactivated DNA synthesis in the nuclei of macrophages. These data show that the mechanisms maintaining differentiated cells in non-proliferating state are not uniform. Nucleated erythrocytes were shown to suppress the duplication of centrioles in partner cells. The possibility of the blockage of DNA replication upon the fusion of two proliferating cells (fibroblast + leukemia cell) was demonstrated for the first time in the present work. The influence of various oncogenes upon the regulation of DNA synthesis in heterokaryons was investigated in detail. New modifications of the methods of cell fusion, enucleation and heterokaryon identification were proposed.     

Discrimination of cell nuclei in early S-phase, mid-to-late S-phase, and G(2)/M-phase by sequential administration of 5-bromo-2'-deoxyuridine and 5-chloro-2'-deoxyuridine.

5-Bromo-2'-deoxyuridine (BrdU) and 5-chloro-2'-deoxyuridine (CldU) were sequentially administered intraperitoneally into mice at 1-hr intervals. After one additional hr, the small intestines were resected, fixed, and embedded in paraffin. In histological sections stained with monoclonal antibody Br-3 reactive to both BrdU and CldU, and CldU antibody reactive only to CldU, three types of staining could be identified in the proliferating zone. Cells with nuclei stained only with Br-3 antibody were estimated to have completed DNA replication during the first 1 hr and were fixed in G(2)/M-phase. Those nuclei were frequently found in apical areas of the simple columnar epithelium of the intestine, whereas other nuclei were located basally in the cells. This observation suggested intracellular movement of cell nuclei in G(2)/M-phase. Identification of cells in early S-phase became possible using these antibodies in combination with DAB and fluorescence stainings. Replication sites in early S-phase nuclei were found to be numerous, whereas in late S-phase they were larger in size and much smaller in number.     

Feasibility of automating the micronucleus assay

The results of a feasibility study on the automation of the micronucleus assay in whole blood cultures of human lymphocytes are reported. The assay requires determination of the number of lymphocytes with micronuclei among the proliferating population. Using an in-house-assembled image analysis system, a prototype software package was developed that addressed two problems: micronuclei identification and discrimination of nonproliferating cells from proliferating lymphocytes (the only ones that can give rise to micronuclei). The results of manual verification of automated micronucleus scoring showed that 70% of all digitized micronuclei were extracted from the images and 90% of them were correctly classified and paired with a parent nucleus by an "affinity function". The discrimination between proliferating and nonproliferating cells was carried out by linear discriminant analysis of simple nuclear features extracted from Feulgen-stained cells. Among the Feulgen-stained nuclei that were identified by autoradiography as proliferating or not, 85% were correctly classified by a six-feature discriminant function.     

Trichrome Staining for Detection of Amyloid

It has been proposed to use trichrome staining of histological sections for the detection of connective tissue fiber and sites for amyloid localization, as well as for increasing color contrast. After incubation in acidin–pepsin solution, sections are dewaxed and successively stained with picrofuchsin according to van Gieson, together with nuclei counterstain with hematoxylin, Congo red, and picroindigocarmine. As a result, the amyloid bound with collagen fibers was stained brick-red, collagen and reticular fibers not bound with amyloid was stained blue-green, and cytoplasm of cells not containing amyloid was stained yellow. Trichrome staining of organs affected by amyloidosis is more informative for the analysis of organs than Congo red stain.     

Polychromatic Stains for Thin Sections of Beta Embedded in Epoxy Resin

Thin sections of leaves and anthers of Beta vulgaris L., fixed in glutaraldehyde-OsO₄ and embedded in epoxy resin, were stained with different stains at pH ranges from 5 to 9 at 50 C to select those that provided polychromatic staining of suitable intensity. The thionin derivatives, Azure B, Toluidine Blue O, and polychrome Methylene Blue provided adequate staining, as did the commercially prepared stain Paragon PS 1301. Azure B stain was superior for sugar beet 0.5μ monitor sections: cytoplasm appeared grey; nuclei, blue-gray; nucleoli, blue; chloroplasts, blue-green; primary walls, blue; and secondary walls, light blue. Choice of one of the stains mentioned probably would depend upon the plant material under study.     

Cell density and proliferation modulate collagen synthesis and procollagen mRNA levels in arterial smooth muscle cells.

Collagen synthesis and procollagen mRNA levels were determined and compared in (1) sparse, rapidly proliferating smooth muscle cells (SMC); (2) postconfluent, density-arrested SMC; and (3) sparse, nonproliferating (mitogen-deprived) rabbit arterial SMC. Collagen synthesis per SMC was decreased by 70% in postconfluent versus proliferating cells. However, relative collagen synthesis, expressed as the percentage of total protein synthesis, increased from 3.7% in sparse cultures to approximately 7% in postconfluent cultures. Slot blot analyses demonstrated that the relative steady state alpha 1(I) and alpha 1(III) procollagen mRNA levels were also increased in postconfluent cultures when compared to sparse cultures. As with collagen synthesis per cell, the mRNA levels per cell for types I and III procollagen in postconfluent cells, determined by densitometry of blots, were likewise approximately half that found in sparse, proliferating cells. In a separate study to determine if cell-cell contact was necessary for eliciting these changes in collagen synthesis, we determined collagen synthesis in mitogen-deprived and proliferating SMC cultures at low density. Mitogen-deprived cultures synthesized only 10% the amount of collagen produced (per cell) by proliferating cultures in 10% fetal bovine serum. Relative collagen synthesis in proliferating and nonproliferating cultures was 5.0 and 8.3%, respectively. These results demonstrate elevated collagen synthesis, per cell, by proliferating cultures compared with nonproliferating cultures, regardless of whether cells were rendered quiescent by density arrest or by mitogen deprivation. Results also suggest a pretranslational mechanism for the regulation of collagen synthesis in rabbit aortic smooth muscle cells.     

The disappearance of a cyclin-like protein and the appearance of statin is correlated with the onset of differentiation during myogenesis in vitro

We have used monoclonal antibodies to statin (S-44) and a cyclin-like protein (S-132) to examine the distribution of these two antigens in proliferating and in nonproliferating populations of cells. We have found that this cyclin-like protein is present in proliferating fibroblasts, whereas statin is absent from these same cell populations; in contrast, in senescent populations of fibroblasts the cyclin-like antigen disappears and statin labeling of nuclei appears. During myogenesis in rat muscle cell cultures, S-132 labeling is present in proliferating myoblasts and disappears after cells fuse and differentiate as multinucleated myotubes. In contrast, statin is absent from proliferating myoblasts, but appears when these cells become postmitotic and begin to differentiate. Similar results were seen during chick myogenesis. We have also found similar results during serum-starvation-induced differentiation in neuroblastoma cells. These results indicate that the cyclin-like protein disappears and statin appears upon commitment to differentiation in vitro, and the presence or the absence of these proteins appears to provide cellular markers for the transition from the proliferative to the nonproliferative state during differentiation.     

Immunofluorescent study of DNA polymerase alpha in rat cells and tissues

An indirect immunofluorescent test based on globulin preparation from a highly specific antiserum against rat liver DNA polymerase alpha was used to direct the enzyme in sections of various tissues of the rat. The immunofluorescent staining was found in cells of the thymus and the wall of intestine crypt, in sparse cells of the intestinal muscular layer, and in cells of the embryo skin epithelium. In sections of liver the intensity of staining and the number of stained cells increased significantly during regeneration. The immunoglobulins did not interact with the cytoplasm and nuclei of skeletal muscle myotubes, with the epithelial cells at the top of intestinal villi, and with erythrocytes. The intracellular localization of the fluorescence observed was of two general types: 1) staining in the region of the nuclear envelope and/or in the cytoplasm; 2) an additional intranuclear staining. The staining of the first type is characteristic of the cells of intact liver and of leyomyocytes. It was also observed in the proliferating cells of thymus and crypt wall, and in cultured myogenic L6 cells. Cells of the embryo skin epithelium, the satellite cells in the skeletal muscle, and about one half of the regenerating liver cells appeared to have the second type of staining. These data serve an indication of possible histotypical differences in in the intracellular localization of DNA polymerase alpha in proliferating cells. It is proposed that the presence of DNA polymerase in resting cells is in association with their ability to respond to the mitogenic stimulus.     

Human Cdc45 is a proliferation-associated antigen

Cell division cycle protein 45 (Cdc45) plays a critical role in DNA replication to ensure that chromosomal DNA is replicated only once per cell cycle. We analysed the expression of human Cdc45 in proliferating and nonproliferating cells. Our findings show that Cdc45 protein is absent from long-term quiescent, terminally differentiated and senescent human cells, although it is present throughout the cell cycle of proliferating cells. Moreover, Cdc45 is much less abundant than the minichromosome maintenance (Mcm) proteins in human cells, supporting the concept that origin binding of Cdc45 is rate limiting for replication initiation. We also show that the Cdc45 protein level is consistently higher in human cancer-derived cells compared with primary human cells. Consequently, tumour tissue is preferentially stained using Cdc45-specific antibodies. Thus, Cdc45 expression is tightly associated with proliferating cell populations and Cdc45 seems to be a promising candidate for a novel proliferation marker in cancer cell biology.     

Differential repair of gamma-ray-induced DNA strand breaks by various cellular subpopulations of mouse jejunal epithelium and bone marrow in vivo

We have examined the induction and repair of gamma-ray-induced DNA strand breaks in different subpopulations of cells in mouse jejunal epithelium and bone marrow using a modification of the alkaline elution methodology whereby different populations of cells are selectively labeled with radioactive DNA precursors. Mice were labeled by intraperitoneal injection with between 0.5 and 2.0 mu Ci/g of [3H]thymidine at various times prior to irradiation with 10 Gy of gamma rays. In the studies with jejunal epithelium, the timing of the injection of the radiolabel relative to the irradiation was varied between 6 and 72 h, depending on the cell population of interest. The DNA damage and repair characteristics representative of both the total cell population and the radiolabeled fraction of these cells were then measured. Little difference was noted in the amount of initial damage induced in these different populations of cells. However, for both the jejunum and bone marrow, cells that incorporated the radiolabel within 6 h after injection (i.e., rapidly proliferating cells) repaired their strand breaks more rapidly than did the remainder of the population. In the case of jejunum, the repair capacity of the radiolabeled cell population progressively diminished as the cells matured and differentiated so that cells that contained the radiolabel 72 h after injection (i.e., mature villus cells) actually repaired their strand breaks more slowly than did the bulk cells.     

Polycyclic aromatic hydrocarbon-DNA adducts determined by semiquantitative immunohistochemistry in human esophageal biopsies taken in 1985

Esophageal endoscopic biopsy samples were obtained in 1985 in Linxian, China, a region with very high esophageal cancer incidence rates, and where ingested food is known to contain substantial amounts of polycyclic aromatic hydrocarbons (PAHs). In this study, the automated cellular imaging system (ACIS) was used for localization and semi-quantitation of PAH-DNA adducts. Fresh tissue sections were cut from archived paraffin blocks and incubated with an antiserum elicited against DNA modified with 7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE). Nuclear PAH-DNA adduct staining was observed in four out of five human samples incubated with the anti-BPDE-DNA. By visual inspection, nuclei in the basal layer of the esophageal epithelium had higher levels of PAH-DNA adducts compared to those found in the adjacent superficial squamous layer. Nuclear PAH-DNA staining was absent in serial sections incubated with either normal rabbit serum or BPDE-DNA-antiserum previously absorbed with the immunogen BPDE-DNA. Semi-quantitative evaluation by ACIS revealed that per nucleus values for PAH-DNA adducts in the basal layer of the esophageal epithelium were 5- to 40-fold higher than those in the adjacent superficial squamous layer (P < 0.0001), using a random effects model). This pilot study demonstrates the presence of PAH-DNA adducts in archived paraffin-embedded endoscopic esophageal biopsy samples that are close to 20 years old, and suggests that an appropriate set of archived samples could be used to prospectively correlate PAH-DNA adduct formation with risk of esophageal cancer development.