Cell cycle distribution in-vivo of mononuclear blood cells of renal allograft recipients

In 47 healthy blood donors (controls) and 29 renal allograft recipients (patients) the relative contents of RNA and DNA of peripheral blood mononuclear (PBM) cell populations were estimated from the intensities of green and red fluorescences emitted by complexes that form with DNA and RNA, respectively, after staining the cells with the metachromatic dye, acridine orange. Based on the correlated DNA and RNA estimates for large numbers of cells, the percentages and the relative RNA contents of cells in particular compartments of the cell cycle were determined. PBM cell populations of controls contained less than 0.5% proliferating (SG2M) cells with highly variable relative RNA contents. Among controls, neither percentages nor relative RNA contents of SG2M-cells were correlated with percentages or relative RNA contents of G1-cells with an RNA content 2 (2SD) or 3 (3SD) standard deviations above the mean of the entire G0/1-cell population. Unlike controls, PBM cell populations of patients contained significantly higher percentages of SG2M-cells which were significantly correlated with the relative coefficients of variation (rCV) of the F530 histograms of G0/1-cells; the rCV represents the ratio of a patient's CV to a control's CV. Moreover, significant statistical correlations existed between percentages and relative RNA contents of 2SD-, 3SD-, SG2M- and G0/1-cells, suggesting a well-orchestrated progression of cells through the cell cycle. Different pairs of correlated parameters characterized clinically stable, acutely rejecting, and infected patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-color multiparametric method for flow cytometric DNA analysis of carcinomas using staining for cytokeratin and leukocyte-common antigen

Solid tumors contain heterogenous cell populations, resulting in flow cytometric (FCM) DNA quantitations of a mixture of tumor and host cells. Such mixed populations can result in dilution of the tumor cells by the host cells, in difficulty defining the diploid reference mean and in histogram peak overlap, precluding cell-cycle analysis. In this study, epithelial (tumor) cells and contaminating host cells in 100 consecutively accessioned human mammary and colorectal carcinomas were segregated in a multiparametric two-color FCM DNA analysis of intact, ethanol-fixed cells. These two carcinomas and bladder carcinomas contain a cytoskeleton of simple epithelium that is selectively stained with an FITC-labeled monoclonal antibody (MAb) to cytokeratin (CK: CAM 5.2-FITC). This MAb detects the CK 8, CK 18 and CK 19 consistently present in all layers of normal and neoplastic urothelium, colonic epithelium and mammary epithelium. Gating on CK in these tumors enables the nonstaining leukocytes, stromal fibroblasts and endothelial cells to be excluded from DNA analysis. A separate aliquot of each tumor evaluated was labeled with an MAb to leukocyte-common antigen (LCA-FITC) to serve as a patient-specific intrinsic diploid reference standard. Both the CK-labeled and LCA-labeled cells were then dual labeled for DNA with propidium iodide. This method (1) correctly identified the intrinsic diploid (LCA-positive) channel, allowing an accurate definition of normal cell DNA content for calculation of the DNA index; and (2) resulted in an increased sensitivity in the identification of both diploid and abnormal hyperdiploid tumor cell populations. It also (3) limited DNA cell cycle analysis to urothelial, colonic and mammary epithelial cells, the majority of which were neoplastic in carefully selected tumor samples. In addition, this method (4) clarified near-tetraploid populations that overlap the normal nonepithelial G2M region by diminishing the normal G2M peak and accentuating the aneuploid tetraploid G0G1 peak and (5) deconvoluted overlapping histograms composed of normal host and diploid-range or aneuploid tumor cells by gating on tissue-specific markers. This exclusion of host cells in both classes of tumors resulted in more accurate cell-cycle calculations in the former and allowed calculation of the S-phase fractions in the latter.     

Isolation of tetraploid clones with high efficiency from diploid 3Y1 rat fibroblasts treated with sodium butyrate

Summary Sodium butyrate causes proliferation arrest with a G2 (4C) DNA content and induces formation of tetraploid cells upon removal of the inhibitor, in rat 3Y1 diploid fibroblasts. We isolated tetraploid clones from the butyrate-treated 3Y1 cells with high efficiency; among 21 clones randomly isolated, 5 were pure diploid, 7 were mainly tetraploid with a small contaminating diploid population, and 7 were pure tetraploid. Among the pure tetraploid clones, two showed doubled chromosome numbers with slightly broader distributions than that seen in parental 3Y1 cells. Butyrate further induced polyploid formation in the tetraploid cells thus produced, but octaploid cells that resulted could not be maintained for prolongeed, cultivation. We found no difference between the tetraploid and the (parental and parallel isolated) diploid clones in terms of colony-forming ability, proliferation rate, and sensitivity to density-dependent inhibition of proliferation. These results suggest that doubling of chromosome number by itself does not cause a change in proliferation property. The tetraploid clones had lower average saturation densities possibly due to enlargement of cell size represented by higher cellular protein content.     

The DNA content of micronuclei induced in mouse bone marrow by gamma-irradiation: evidence that micronuclei arise from acentric chromosomal fragments.

The DNA contents of 75 micronuclei found in mouse bone marrow 48 h after a 260 R dose of gamma-rays were measured individually and compared to 71 diploid G1 nuclei. The coefficient of variation for the diploid cells was 6.3%. The DNA content of micronuclei varied from 0.5 to 11.1% of the diploid G1 nuclei with a mean of 3.5%. These results are in agreement with the expectations based upon the hypothesis that micronuclei arise from acentric chromosomal fragments produced by random breakage of the mouse genome.     

Intraclonal and interclonal differences of ploidy in transplantable rat rhabdomyosarcoma RA-2 detected using DNA flow cytometry

DNA contents were determined using flow cytometry in cells of 71 clones of the rat transplantable rhabdomyosarcoma passed through more than 70 cycles of selection for increased malignancy. 58 clones (82%) consisted of cells with diploid DNA content, whereas 13 clones (18%) displayed not only diploid, but also near tetraploid cell subpopulations, the latter covering from 20 to 60% of the total number of cells measured. The data obtained show the existence of intra- and interclonal karyotypic heterogeneity in populations of tumor clonogenic cells.     

Flow cytometric analysis of unbalanced control of protein accumulation and DNA synthesis in differentiating mouse myeloid leukemia cells

The protein and DNA contents of mouse myeloid leukemia M1 (clone B24) cells were determined by flow cytometry (FCM) after double fluorescent staining of the cells with fluorescein isothiocyanate and propidium iodide. FCM analysis showed that there was a linear relationship between the DNA and protein contents in logarithmically growing cells, although the protein content showed some variation. B24 cells can be induced to differentiate into macrophage-like cells by treatment with a protein inducer(s) in conditioned medium (CM) of hamster embryo cells. When the cells were treated with various concentrations of CM, cells with a 2C DNA content, G1/0 cells, increased and protein accumulated in these G1/0 cells. The increases in the number of G1/0 cells and in their protein content per cell were proportional to the concentration of CM. Serial analysis of changes in the contents of DNA and protein in differentiating B24 cells showed that DNA synthesis was suppressed by differentiation-induced block of the cell cycle at the G1/0 phase, whereas increase in the protein content was not completely suppressed by block of the cell cycle. These results suggest that unbalanced control of the DNA and protein contents of B24 cells is involved in the mechanisms of the morphological changes during differentiation into macrophages.     

Flow cytometric DNA analysis of renal-cell carcinoma. A study of fine needle aspiration biopsies in comparison with multiple surgical samples

The flow cytometric (FCM) DNA analysis of fine needle aspiration (FNA) biopsy material was compared with the DNA analysis of multiple surgical biopsy material from 44 renal-cell carcinomas. Twenty tumors were heterogeneous with respect to their DNA content. Eleven of the 17 tumors that had both diploid and aneuploid tumor cell clones in the surgical specimens gave a diploid DNA content in the aspiration biopsies; the other 6 cases showed aneuploidy in the aspirate. The fine needle aspirates from 18 homogeneously diploid tumors and 9 tumors with an aneuploid DNA content in all eight surgical samples revealed DNA indices similar to those found in the surgical samples. These findings show that aneuploid clones of renal-cell carcinoma can remain undetected by the use of FNA biopsy as a method for obtaining samples for FCM DNA analysis.     

Retinoblastoma cell cycling

Techniques for the measurement of bromodeoxyuridine (BrdUrd) positive cells generally include either microscopic evaluation of paraffin embedded sections or measurements on cell suspensions using a fluorescent activated cell sorter. The accuracy of these measurements and their correlations can be affected by a number of technical and intrinsic tumor factors. Extrinsic parameters including degree of necrosis and tumor growth fraction are less easily analyzed in BrdUrd stained material. Retinoblastoma tumor cell cycling was prospectively studied in 11 children using in vivo and one child using in vitro BrdUrd. BrdUrd measurements were made by staining cell suspensions or sections of paraffin embedded tumor and analyzing by microscopy. Approximately 14% of viable cells were in the synthesis-phase of the cell cycle. The correlation between BrdUrd in cell suspensions and BrdUrd in paraffin embedded sections did not reach significance (r = 0.48). DNA analysis of these tumors was also performed using flow cytometry. Nine tumors were found to have a normal diploid DNA content, one had a G1 peak below the diploid control, two had a G1 peak above the diploid control, and one had two G1 peaks (a diploid and a hyperdiploid peak). There was no correlation between abnormal DNA content and the percent of cells in synthesis.     

Altered nucleic acid contents of mononuclear cells in blood of renal allograft recipients

The DNA and RNA contents of blood mononuclear cell populations of 29 cadaver renal allograft recipients and 49 blood donors (controls) were estimated by acridine orange flow cytometry (AO FCM) to assess their cell cycle status. All patients received azathioprine and prednisone for immunosuppression. The patients represented three clinical categories: clinically stable patients, those with acute rejections (clinically overt or impending), and those with infections. Three cell cycle compartments were analyzed for percentage (%) and RNA content (R) of cells: G0/1, consisting of all cells with diploid DNA content; 2 S.D., consisting of cells with diploid DNA content and RNA content 2 standard deviations above the mean RNA content of controls; and SG2M, consisting of cells with a DNA content higher than that of G0/1 cells. The relative coefficient of variation (rCV) of the DNA distribution of G0/1 cells was also determined. In such cell cycle evaluations, the means of rCV and SG2M% of stable recipients were significantly different from those of blood donors. Multivariate analysis of the variables of the three categories of patients resulted in the tentative formulation of two simple logistic equations: one that differentiates stable patients from those with impending or overt rejections based on 2SD% and another one that distinguishes infected patients from those with impending or overt rejections based on SG2M% and RG0/1.     

杂种杨无性系的光系统Ⅱ放氧活性、光合色素及叶绿体超微结构对光胁迫的响应

 用氧电极仪、红外CO2气体分析仪及叶绿素荧光仪,结合透射电镜技术对几个杂种杨无性系在光胁迫下的光系统Ⅱ活性、光合色素及叶绿体超微结构进行了测定。随着预处理光强的增加,各无性系叶片的净光合速率和光系统Ⅱ放氧活性明显下降。二倍体无性系B11的光系统Ⅱ最大光化学效率（Fv/Fm）及实际光化学效率［(Fm′-F)/Fm′］高于两个三倍体无性系B346和B342。强光下三倍体无性系B346的非光化学淬灭远高于B342和二倍体无性系B11。叶绿素含量和光合速率没有明显的线性关系,叶绿素a/b含量在自然光胁迫下存在季节变化,受强光胁迫2个三倍体无性系的叶绿素a/b增大。预处理光强PFD超过3 000 μmol photons·m-2·s-1,各无性系的基粒类囊体片层结构遭到破坏,但二倍体无性系的类囊体片层结构受破坏程度较三倍体无性系轻。叶绿体蛋白合成抑制剂（硫酸链霉素,SM）可加剧叶绿体超微结构的破坏。     

A cytophotometric analysis of the structure of hypothalamic cell populations in the frog,Rana temporaria (L.), with special reference to seasonal changes in chromatin status

Summary We used cytophotometry after the Feulgen reaction and UV cytophotometry to measure the DNA content of quiescent cells of the hypothalamic preoptic region (HPR) of adult and juvenile frogs (Rana temporaria) that had been caught in their natural habitat in winter, spring and summer. The histone-to-DNA ratio in cell nuclei was cytophotometrically determined using a combined Feulgen, heparine and alcian-blue staining procedure. The vast majority of HPR cells studied had nuclei with a diploid DNA content. However, we observed great variability in the Feulgen-DNA content of the HPR cell population, which was not detected in the diploid standard (hepatocytes). This heterogeneity in the diploid sample of the HPR cell populations was always greater in prespawning frogs and may have been due to differences in the chromatin arrangement in nuclei. About 1% of cells had a DNA content either ranging between diploid and tetraploid levels (H2C cells) or at the tetraploid level (4C and 2C x 2 cells). The proportion of these cells was not affected by the age of the animals or the annual cycle, thus suggesting that there is no age-related increase in the mean DNA content in the frog HPR. The mean DNA contents of H2C and 4C cells were much higher than those in the standard (hepatocytes). This cannot be simply attributed to the presence of different amounts of nuclear proteins, but rather indicates that at least a certain proportion of the highest DNA contents may be due to a real extra-DNA synthesis.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Isolation and characterization of diploid clones from adult and newborn rat liver cell lines.

A high frequency of diploid and near-diploid clones were developed from cell lines derived from adult and newborn rat liver using micropipettes. There were some differences in morphology, biochemical properties and growth rate between clones. Cloned cells had low levels of tyrosine transaminase activity, glucose-6-phosphatase activity and albumin content. A diploid clone and pseudodiploid clone derived from adult rat liver cell line were positive for alpha-fetoprotein.     

Selection, establishment and characterization of cell lines derived from a chemically-induced rat mammary heterogeneous tumor, by flow cytometry, transmission electron microscopy, and immunohistochemistry

Summary In order to isolate, characterize, and establish culture cell lines with different diagnostic and prognostic significance, derived from multiclonal neoplasms, a ductal infiltrating mammary tumor was induced in rats by 7,12-dimethylbenz[a]anthracene. Clones with different DNA/protein content, being the DI of 1.16, 1.30, and 1.60, respectively, were observed in the primary tumor. Biparametric flow cytometry suggested that the clone at 1.30 is made up of two subpopulations with different protein and slightly different DNA contents. The culture, after a few passages, exhibited the presence of aneuploid cells and the absence of diploid components, demonstrating that only tumor cells survived. The limiting dilution method gave rise to four lines with DI of 1.16, 1.25, 1.30, and 1.50; a mean chromosome number of 45, 46, 47, and 88, respectively; and different morphological and ultrastructural features. These characteristics were stable during the experimental procedure, that is, for about 20 passages. Conversely, the detection of cytoskeletal proteins indicated that the tumor epithelial cells underwent early dedifferentiation into sarcoma-like cells showing markers of stromal cell type and thus exhibiting phenotypic instability in vitro, a feature reported in many advanced human breast cancers in vivo. In conclusion, this cellular model represents the in vivo situation and appears suitable for in vitro studies of tumor cell characteristics and might be used to predict clinical behavior.     

Transformation with specific fragments of adenovirus DNAs. II. Analysis of the viral DNA sequences present in cells transformed with a 7% fragment of adenovirus 5 DNA

Five clones of rat kidney cells transformed by a small restriction endonuclease fragment of adenovirus 5 (Ad5) DNA (fragment HsuI G, which represents the left terminal 7% of the adenovirus genome) were analyzed with respect to the viral DNA sequences present in the cellular DNAs. In these analyses, the kinetics of renaturation of 32P-labeled specific fragments of Ad5 DNA was measured in the presence of a large amount of DNA extracted either from each of the transformed cell lines or from untransformed cells. The fragments were produced by digestion of 32P-labeled adenovirus 5 DNA with endo R.HsuI, or by digestion of 32P-labeled fragment HsuI G of adeno 5 DNA with endo R.HpaI. All five transformed lines were found to contain DNA sequences homologous to 75--80% of Ad5 fragment HsuI G only. Clones II and V contained approximately 48 copies per quantity of diploid cell DNA, clone VI about 35 copies, clone IV 22 copies and clone III 5--10 copies. These results indicate that a viral DNA segment as small as 5.5% of the Ad5 genome, contains sufficient information for the maintenance of transformation.     

Cytogenetic and virological characteristics of the initial line and clones of RH cells with various degrees of sensitivity to the tick-borne encephalitis virus

Virological and cytogenetic characterization of line RH and its two clones (RHk-20 and RHk-13/6), having different sensitivity to the tick-borne encephalitis virus (TBE), has been made. The RHk-13/6-cells are 10,000 times more sensitive to cytopathic effect of the tick-borne encephalitis virus in comparison with the RHk-20-cells. By its sensitivity to the virus the base line is in the intermediate position. Cytogenetic analysis revealed the variability interval decrease as to the chromosome number at cloning, and showed the structural stability of the line and clone karyotypes. The line RH and its clone K-20 have modal number of chromosomes equal to 65; the cell population with the chromosome number equal to 66 dominated in RHk-13/6. The application of methods of chromosome differential (G, C) staining and these for the detection of active nucleolar organizers (NO) permitted identifying the marker chromosomes and determining the difference between the clones and the line as associated with the presence of an additional homologue of chromosome 8 in the sensitive clone.     

Establishment of a diploid reference value for DNA ploidy analysis by image cytometry in mouse cells.

OBJECTIVE: To establish a diploid reference value for DNA ploidy analysis of mouse cells (Mus musculus) by image cytometry using the CAS 200, an analysis system suitable for DNA content studies in human cells. STUDY DESIGN: To establish this standard, we used spleen imprints from 26 normal animals. A minimum of 150 lymphocytes present in each imprint was counted. The mean DNA content (pg/cell) of the G0/G1 peak and the DNA index observed in all samples were statistically analyzed. Cytospins with peritoneal cells from the same animals were then analyzed with this reference DNA value to confirm the diploid range. RESULTS: The DNA diploid reference value was determined by the mean DNA content of all spleen samples, which was 6.42 +/- 0.234 pg/cell, and the diploid range, defined as the diploid value +/- 10%, was 5.78-7.06 pg/cell. All the peritoneal samples showed a DNA diploid histogram, with a mean value for the G0/G1 peak DNA content of 6.742 +/- 0.15. CONCLUSION: The diploid reference value found in this study differs from those reported for other species, including the human being, and should be used in further studies of mouse pathology.     

DNA stainability in aneuploid breast tumors: comparison of four DNA fluorochromes differing in binding properties.

The aim of this study was to evaluate whether or not the differences in chromatin structure between diploid stromal cells or lymphocytes, which are often used as DNA ploidy standard, and aneuploid breast tumor cells can significantly affect the estimates of the DNA index of these tumors. To this end, the DNA content estimates of 34 aneuploid breast tumors, differing in size, degree of differentiation, and presence or absence of estrogen and progesterone receptors and metastases, were compared using four common DNA fluorochromes: DAPI, Hoechst 33342, propidium iodide, and acridine orange. These dyes differ in their mode of interaction with DNA (binding to minor groove or intercalation) and for each of them binding to DNA is restricted to a different degree by nuclear proteins. It was expected, therefore, that if differences in chromatin structure play a role in DNA content estimates, the DNA index of the measured tumors may vary depending on the dye. The cell nuclei were isolated from the tumors using a detergent-based procedure and stained with each of the dyes and the DNA index was estimated using peripheral blood lymphocytes as a DNA content standard. For each of the tumors, the DNA index estimates with all four dyes correlated very well. When the results obtained with individual dyes were compared in pairs, the correlation coefficients (r) of DNA indices were all above 0.96 (correlation at p less than 0.001). The best concordance was seen between specimens stained with Hoechst 33342 and DAPI (r = 0.99), and the least between those stained with Hoechst 33342 and propidium iodide (r = 0.96). The data indicate that DNA content analysis of unfixed nuclei, utilizing the above fluorochromes, is not significantly biased by differences in chromatin structure of the measured cells.     

Isolation and characterization of diploid clones from adult and newborn rat liver cell lines

Summary A high frequency of diploid and near-diploid clones were developed from cell lines derived from adult and newborn rat liver using micropipettes. There were some differences in morphology, biochemical properties and growth rate between clones. Cloned cells had low levels of tyrosine transaminase activity, glucose-6-phosphatase activity and albumin content. A diploid clone and a pseudodiploid clone derived from adult rat liver cell line were positive for α-fetoprotein. This work was supported by a grant for cancer research from the Japanese Ministry of Education.     

Oxidative stress induces the expression of the major histocompatibility complex in murine tumor cells.

The effect of t-butyl hydroperoxide (t-BOOH) on the induction of the Major Histocompatibility Complex (MHC) class I genes has been studied in two cell clones (B9 and G2) of the methylcholanthrene-induced murine fibrosarcoma GR9. These two clones were selected based on their different biological and biochemical behavior specially related to their tumor induction capability when injected into a BALB/c mouse. t-BOOH (0.125 mM) induced the expression of H-2 molecules in both cell clones. In B9 cell clone, in which MHC basal expression is very low or absent, t-BOOH significantly induced H-2Kd, H-2Dd and H-2Ld molecules. In G2 cell clone the expression of MHC class I genes was also enhanced by the xenobiotic, the effect being especially significant on the H-2Ld molecule which is not expressed under basal conditions. H-2 molecules expression was accompanied by the activation of the transactivator factor NF kappa B. These results suggest that oxidative stress may modulate the antigen expression of tumor cells and thus the immune response of the host organism. Basal levels of oxidative parameters, such as anti-oxidant enzymes, malondialdehyde (MDA) and the DNA damaged base 8-hydroxy-2'-deoxyguanosine (8-OHdG), showed differences between the two fibrosarcoma cell clones.     

Interclonal variation in methylation patterns for expressed and non-expressed genes.

A mass culture of human diploid fibroblasts, and eight clones isolated from that mass culture, were examined for methylation patterns in several regions of DNA. Plasmid-inserted cDNA sequences were used as probes for alpha-hCG, beta-globin, A gamma- and G gamma-globin, and beta- and gamma-actin gene regions. Each probe revealed a different clone-specific pattern of DNA methylation, indicating a striking degree of inter-clonal heterogeneity, for those gene regions which are not normally expressed in diploid fibroblasts (alpha-hCG, gamma-globin and beta-globin). Intra-clonal variation was also evident in many instances, implying that heterogeneity could arise de novo in pure cell clones during serial passage. Thus methylation patterns, in particular for repressed genes, appear to be unstably inherited in these cells, and this instability may lead to random derepression in some cell lineages during mitotic growth.