Bivariate cytofluorimetric analysis of DNA and nuclear protein content in plant tissue

Summary Techniques of static biparametric cytofluorimetry were developed to measure DNA and protein fluorescence simultaneously in the same nucleus stained with 4,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate (FITC) fluorochromes. With these cytofluorimetric procedures, we analysed DNA and nuclear protein content in root apices during the first 72 h of pea seed germination. This method allows a more reliable, rapid and less expensive measurement of DNA and proteins than cytophotometry. Nuclear protein content can be considered as a second parameter to define subcompartments of cell cycle phases; it offers the possibility of studying the progression of plant cells through cell cycle and its control in greater detail.Abbreviations DAPI 4,6-diamidino-2-phenylindole - FITC fluorescein isothiocyanate - PI propidium iodide - Tris Tris(hydroxymethyl) aminomethane     

Immunochemical specificity of placental NADPH cytochrome c (P-450) reductase in neoplastic and non-neoplastic human tissue.

NADPH cytochrome c (P-450) reductase was purified from human placental microsomes using a combination of affinity and gel filtration chromatography. Affinity chromatography using agarose-hexane-adenosine 2'5 diphosphate resulted in two protein bands being detected by SDS-PAGE of approximate MwS 68 and 75 kDa. Fractions containing the two proteins were pooled, and then resolved using Sephacryl S-200. Both of the purified proteins displayed enzyme activity, measured by their ability to reduce cytochrome c. The 75 kDa protein obtained was used to immunize three female New Zealand white rabbits. The IgG fraction was partly purified from rabbit sera which suppressed placental microsomal NADPH cytochrome c reductase activity by > 80% using 33% ammonium sulphate. The procured antibody suppressed androstenedione aromatase activity in microsomal preparations of human placental and breast adipose tissue, and NADPH cytochrome c reductase activity in prostate (benign and malignant), MDA-MB-231 breast cancer cells, breast adipose, Hep G2 hepatoma cells and placental microsomal preparations. The extent of NADPH cytochrome c reductase inhibition varied in the order of malignant prostate < benign prostate < MDA < breast adipose < Hep G2 < placenta. The results suggest that human placental NADPH cytochrome c (P-450) reductase shares common antigenic epitopes pertinent to its capability of reducing cytochrome c in all of the above-mentioned tissues. In attempting to associate possible changes in NADPH cytochrome c reductase activity imposed by neoplasia to the obtained immunochemical cross reactivity and enzyme activity results, it was noted that microsomes obtained from MDA cells exhibited enzyme activity significantly less than that of breast adipose microsomes (1.6 and 8.1 nmol/min/mg protein, respectively) and by comparison showed 6% less homology towards the placental antibody. The results obtained for benign and malignant prostate showed no significant difference between the neoplastic states as adjudged by enzyme activity and immunochemical assays.     

Direction of locomotion in clones of non-neoplastic fibroblasts and their neoplastic derivatives

The locomotion of cloned mouse fibroblasts, non-neoplastic and their spontaneously transformed neoplastic derivatives was compared by means of cinephotomicrography. The spontaneous transformants grow as invasive transplantable sarcomas, whereas the non-neoplastic fail to grow as tumors, and do not show the diagnostic characteristics of neoplastic cells in culture; these include certain morphologic alterations, growth in soft agar, and susceptibility to killing by activated macrophages. The non-neoplastic cells tended to maintain the same direction of locomotion in sequential 2.5 h periods, whereas the neoplastic cells did not. Thus, cells in all non-neoplastic lines exhibited a “persisten” walk while cells from the neoplastic lines had a random pattern of locomotion. No relationship between cell density and randomness of locomotion was observed, and the non-neoplastic cells appeared to grow as rapidly as the neoplastic cells. However, the neoplastic cells had higher rates of locomotion possibly associated with their invasive potential in vivo. The deficient amount of lamellar cytoplasm in the neoplastic cells and the high migration rate may account for their random pattern of locomotion.     

Methylation of satellite deoxyribonucleic acid in mouse neoplastic and non-neoplastic cell cultures

Summary Comparisons of nucleic acid methylation between paired neoplastic and non-neoplastic mouse cell lines have shown a striking difference in the deoxyribonucleic acid (DNA) peak eluted from methylated albumin-kieselguhr columns (R. Gantt and V. J. Evans, 1969, Cancer Res. 29: 536–541). Since mouse satellite DNA is relatively highly methylated, its 5-methylcytosine content was compared with mainband DNA in these two paired cell lines to determine whether this might account for the observed differences. The cell DNA was labeled with methyl-labeled methionine and isolated from the cells by repeated neutral cesium chloride isopycnic centrifugation. The satellite DNA strands were then separated in an alkaline cesium chloride gradient. Both the 5-methylcytosine content and the relative amounts of satellite DNA were indistinguishable in the paired cell lines. Further, the results showed that both strands of satellite DNA had virtually equal amounts of 5-methylcytosine, although the heavy strand contains 1.5 times more cytosine than the light strand.     

Susceptibility to neoplastic and non-neoplastic pulmonary diseases in mice: genetic similarities

Chronic inflammation predisposes toward many types of cancer. Chronic bronchitis and asthma, for example, heighten the risk of lung cancer. Exactly which inflammatory mediators (e.g., oxidant species and growth factors) and lung wound repair processes (e.g., proangiogenic factors) enhance pulmonary neoplastic development is not clear. One approach to uncover the most relevant biochemical and physiological pathways is to identify genes underlying susceptibilities to inflammation and to cancer development at the same anatomic site. Mice develop lung adenocarcinomas similar in histology, molecular characteristics, and histogenesis to this most common human lung cancer subtype. Over two dozen loci, called Pas or pulmonary adenoma susceptibility, Par or pulmonary adenoma resistance, and Sluc or susceptibility to lung cancer genes, regulate differential lung tumor susceptibility among inbred mouse strains as assigned by QTL (quantitative trait locus) mapping. Chromosomal sites that determine responsiveness to proinflammatory pneumotoxicants such as ozone (O3), particulates, and hyperoxia have also been mapped in mice. For example, susceptibility QTLs have been identified on chromosomes 17 and 11 for O3-induced inflammation (Inf1, Inf2), O3-induced acute lung injury (Aliq3, Aliq1), and sulfate-associated particulates. Sites within the human and mouse genomes for asthma and COPD phenotypes have also been delineated. It is of great interest that several susceptibility loci for mouse lung neoplasia also contain susceptibility genes for toxicant-induced lung injury and inflammation and are homologous to several human asthma loci. These QTLs are described herein, candidate genes are suggested within these sites, and experimental evidence that inflammation enhances lung tumor development is provided.     

The structure and dynamics of ring chromosomes in human neoplastic and non-neoplastic cells

Acquired ring chromosomes have been found in most types of human neoplasia, with a frequency approaching 10% in malignant mesenchymal tumours. In this study, the composition and dynamics of ring chromosomes were analysed in eight cases of acute myelogenous leukaemia, 17 solid tumours, and five cases with constitutional rings. Chromosomal banding and fluorescence in situ hybridisation were performed to determine the content and the structural heterogeneity of the rings. Telomeric repeats were detected using peptide nucleic acid probes or primed in situ labelling, whereas centromeric activity was evaluated by detection of kinetochore proteins. Mitotic instability was assessed by the frequency of anaphase bridges. The results suggest that human ring chromosomes can be structurally and functionally divided into two categories. In the first of these, size variation is minimal and rearrangement at cell division is uncommon. The majority of such rings contain subtelomeric sequences. Constitutional ring chromosomes and most rings in leukaemias belong to this group, whereas only a few mesenchymal tumours exhibit rings of this type. The second category consists of rings with amplified sequences, primarily from chromosome 12, characteristically occurring in atypical lipomatous tumours and other subtypes of low or borderline malignant mesenchymal neoplasms. Variation in size and number is extensive, and breakage-fusion-bridge events occur at a high frequency. Abnormalities in pericentromeric sequences are common and, in some cases, kinetochores assemble in the absence of alphoid DNA. We conclude that it is not only the ring structure per se or the neoplastic nature of the host cell that determines ring instability, but probably also the functional role of the genes carried in the ring. Received: 24 November 1998 / Accepted: 18 January 1999     

Human colon tissue in organ culture: preservation of normal and neoplastic characteristics

Normal and neoplastic human colon tissue obtained at surgery was used to establish conditions for organ culture. Optimal conditions included an atmosphere of 5% CO₂ and 95% O₂; tissue partially submerged with mucosa at the gas interface; and serum-free medium with 1.5 mM Ca²⁺ and a number of growth supplements. Histological, histochemical, and immunohistochemical features that distinguish normal and neoplastic tissue were preserved over a 2-d period. With normal tissue, this included the presence of elongated crypts with small, densely packed cells at the crypt base and mucin-containing goblet cells in the upper portion. Ki67 staining, for proliferating cells, was confined to the lower third of the crypt, while expression of extracellular calcium-sensing receptor was seen in the upper third and surface epithelium. E-cadherin and β-catenin were expressed throughout the epithelium and confined to the cell surface. In tumor tissue, the same disorganized, abnormal glandular structures seen at time zero were present after 2 d. The majority of cells in these structures were mucin-poor, but occasional goblet cells were seen and mucin staining was present. Ki67 staining was seen throughout the abnormal epithelium and calcium-sensing receptor expression was weak and variable. E-cadherin was seen at the cell surface (similar to normal tissue), but in some places, there was diffuse cytoplasmic staining. Finally, intense cytoplasmic and nuclear β-catenin staining was observed in cultured neoplastic tissue.     

Mechanisms and age-specific characteristics of nuclear DNA replication

Replication mechanisms of nuclear DNA in eucaryotic cells and their changes with the organism aging are discussed. Tge polyrepliconic nature of replicating DNA, discontinuous synthesis of its newly formed chains, enzymic apparatus of replication and regulation of this process are described. Special attention is paid to destabilization of the secondary structure of the replicating molecule. In aging, changes in the DNA replication were found at each level of its regulation, i.e. at synthesis precursors, enzymic replication complex and replicating DNA structure. This may be an indication of the altered regulation of the DNA replication with aging.     

Promoter methylation status of tumor suppressor and tumor-related genes in neoplastic and non-neoplastic gastric epithelia

A number of tumor suppressor and tumor-related genes exhibit promoter hypermethylation with resulting gene silencing in human cancers. In addition, several gene promoters have also been shown to become hypermethylated in non-neoplastic cells during aging. To assess the physiological consequence and clinical significance of gene promoter methylation in gastric epithelia, our laboratory has studied the methylation status of tumor suppressor and tumor-related genes, including APC, DAP-kinase, DCC, E-cadherin, GSTP1, hMLH1, p16, PTEN, RASSF1A, RUNX3 and TSLC1, in neoplastic and non-neoplastic gastric epithelia. The tumor suppressor and tumor-related genes, except APC, were generally unmethylated in non-neoplastic gastric epithelia obtained from younger individuals. The frequencies of methylation increased with age to varying degrees in various genes, although GSTP1 and PTEN methylation was completely absent in both neoplastic and non-neoplastic gastric epithelia. The methylation frequencies in each gene were found to be comparable in neoplastic and non-neoplastic gastric epithelia, except the methylation of RUNX3 and TSLC1, which was mostly cancer-specific (P<0.01). When methylation frequencies were compared between non-neoplastic gastric epithelia from cancer-bearing and non-cancer-bearing stomachs, hMLH1 and p16 methylation was more frequent in those from cancer-bearing stomachs (P<0.01). Promoter methylation in tumor suppressor and tumor-related genes initially occurs in non-neoplastic gastric epithelia, increases with age, and ultimately silences gene function to constitute a field-defect that may predispose tissues to gastric cancer evolution. In clinical applications RUNX3 and TSLC1 methylation may be utilized as molecular diagnostic markers, and hMLH1 and p16 methylation as predictors of malignancy in the stomach.     

Interaction of serum and cell spreading affects the growth of neoplastic and non-neoplastic fibroblasts

Both growth factor availability and cell-to-cell contact have been mechanisms used to explain cell growth regulation at high cell density. Recently Folkman and colleagues have shown that changes in cell shape, rather than cell-to-cell contact, can regulate the growth of fibroblasts. However, in those studies the relation between serum and shape regulation of growth was not studied, nor were neoplastic and non-neoplastic cells compared. In this report we have studied these aspects by varying cell spreading and serum concentration independently for 2 non-neoplastic and 3 neoplastic cell lines. Cell spreading (projected cell area) was controlled by decreasing the adhesiveness of tissue culture plastic plates with poly (hydroxyethyl methacrylate) [poly (HEMA)]. Cell growth was measured as the increase in cell number/day. We have found that more spreading increased net growth of both neoplastic and non-neoplastic cells, while less spreading (toward rounded configuration) depressed growth. There were also quantitative differences between neoplastic and non-neoplastic cells. Neoplastic cells continued to grow under conditions of cell rounding, which completely prevented the growth of their non-neoplastic counterparts. Some neoplastic cells also tended to show little or no increase in net cell number for serum concentrations above 10% as cells became more spread; in contrast, all non-neoplastic cells grew more with increasing concentrations of serum as they became well spread. Thus, in normal cells, it appears that the sensitivity of cells to humoral factors is governed by cell spreading. This interaction between serum and cell shape is less prominent in some neoplastic cells.