Numerical aberrations of chromosomes 1 and 17 correlate with tumor site in human gastric carcinoma of the diffuse and intestinal types. Fluorescence in situ hybridization analysis on gastric biopsies.

Recent studies predict that tumor aneuploidy plays a direct role in tumor instability. The relationship between interphase cytogenetics, histology, grade, and tumor site was analyzed in 20 primary gastric carcinomas. Using fluorescence in-situ hybridization, the numerical changes of centromeric sequences of chromosomes 1, 3, 10, and 17 were directly analyzed in gastric biopsies. Polysomic copy numbers of chromosomes 1 and 17 were discovered in 63% (10 of 16) and 59% (10 of 17), respectively, of informative cancer cases. Chromosome 3 and 10 signal number changes were found in only 6% (1 of 16) and 13% (1 of 8), respectively, of informative cancer cases. There was a positive correlation between the appearance of polysomic nuclear target sites of chromosomes 1 and 17 (correlation coefficient r = 0.72; p < 0.005). Copy number changes were not significantly related to histologic subtypes of either the Laurén or WHO classifications. However, incidence of cancers having dual polysomic signal number abnormalities for both chromosomes 1 and 17 was significantly correlated to tumor location at the cardia. The data suggests that (i) human gastric cancer appears in two genomic groups that can be reliably diagnosed by fluorescence in-situ hybridization on routine biopsy sections, (ii) numerical aberrations of chromosomes 1, 3, 10, and 17 are largely independent of histologic subtypes, and (iii) polysomic copy number abnormalities of chromosomes 1 and 17 correlate to intragastric tumor site and are highest in cardia cancers, suggesting high tumor instability at this particular location.

Polyploid giant cells provide a survival mechanism for p53 mutant cells after DNA damage.

The relationships between delayed apoptosis, polyploid 'giant' cells and reproductive survivors were studied in p53-mutated lymphoma cells after DNA damage. Following severe genotoxic insult with irradiation or chemotherapy, cells arrest at the G(2)-M cell cycle check-point for up to 5 days before undergoing a few rounds of aberrant mitoses. The cells then enter endoreduplication cycles resulting in the formation of polyploid giant cells. Subsequently the majority of the giant cells die, providing the main source of delayed apoptosis; however, a small proportion survives. Kinetic analyses show a reciprocal relationship between the polyploid cells and the diploid stem line, with the stem line suppressed during polyploid cell formation and restituted after giant cell disintegration. The restituted cell-line behaves with identical kinetics to the parent line, once re-irradiated. When giant cells are isolated and followed in labelling experiments, the clonogenic survivors appear to arise from these cells. These findings imply that an exchange exists between the endocyclic (polyploid) and mitotic (diploid or tetraploid) populations during the restitution period and that giant cells are not always reproductively dead as previously supposed. We propose that the formation of giant cells and their subsequent complex breakdown and subnuclear reorganization may represent an important response of p53-mutated tumours to DNA damaging agents and provide tumours with a mechanism of repair and resistance to such treatments.

Release of mitotic descendants by giant cells from irradiated Burkitt's lymphoma cell line..

Polyploid giant cells are produced as part of the response of p53 mutant Burkitt's lymphoma cell lines to high doses of irradiation. Polyploid giant cells arise by endo-reduplication in the first week after a single 10 Gray dose of irradiation. Within the giant cells a sub-nuclear structure is apparent and within this, sub-nuclear autonomy is evident, as displayed by independent nuclear structure and DNA replication in different parts of the nucleus. The majority of these cells soon die as apoptotic polykaryons. However, approximately 10-20% of giant cells remain viable into the second week after irradiation and begin vigorous extrusion of large degraded chromatin masses. During the second week, the giant cells begin to reconstruct their nuclei into polyploid 'bouquets', where chromosome double-loops are formed. Subsequently, the bouquets return to an interphase state and separate into several secondary nuclei. The individual sub-nuclei then resume DNA synthesis with mitotic divisions and sequester cytoplasmic territories around themselves, giving rise to the secondary cells, which continue mitotic propagation. This process of giant cell formation, reorganization and breakdown appears to provide an additional mechanism for repairing double-strand DNA breaks within tumour cells.

Crystalloid smooth endoplasmic reticulum in the quail uropygial gland.

The occurrence, localization and organization of crystalloid smooth endoplasmic reticulum (SER) membrane aggregates in the male quail uropygial gland was investigated by electron microscopy. The lattice-like structures exhibiting a hexagonal honeycomb pattern are regularly found in the perinuclear region of the fully developed intermediate cell (type II) which is most effective in lipid biosynthesis and constitutes the middle layers of the stratified glandular epithelium undergoing sebaceous transformation. The crystalloids frequently exhibit a rectangular shape and tend to cluster, the latter exceeding 5 microns in length. They are composed of sets of highly ordered and densely packed tubular SER profiles. Diaminobenzidine (DAB) stained peroxisomes exhibit a close spatial relationship to the borders of crystalloids, but the organelles do not participate in the formation of these grid-like structures. The functional significance of the conformational change of the SER organization is not known. Local accumulation of specific lipogenic enzymes within this functional SER domain is discussed.

Clofibrate increases the number of peroxisomes and of lamellar bodies in alveolar cells type II of the rat lung.

The hypolipidemic drug clofibrate, which causes a striking proliferation of hepatic peroxisomes, and the induction of peroxisomal lipid metabolizing enzymes, was tested for its influence on rat lung. Alveolar cells type II of the lung are the major source of the surface-active phospholipid-apoprotein complex. Their surfactant-containing lamellar bodies are part of the pulmonary surfactant system. To test the possible relationship between lung peroxisomes and the phospholipid-rich lamellar bodies in alveolar cells type II, clofibrate was administered to male rats. Drug treatment for 7 days resulted in a 30% (p less than 0.001) increase in the number of lamellar bodies within the type II cells, as estimated by morphometry on semithin sections of the lung. In contrast, the average number of type II cells per area of lung remained unchanged which indicates that type II cell proliferation did not occur. Intraalveolar macrophages were consistently vacuolated and markedly increased in size in the lungs of the treated rats. Peroxisomes (microperoxisomes) were identified cytochemically using the alkaline diaminobenzidine (DAB) method for catalase, a marker enzyme of these organelles. Ultrastructural-morphometric analysis of the lungs showed that clofibrate treatment resulted in a 60% increase in the profile number of DAB-positive peroxisomes (p less than 0.005) in alveolar cells type II which are known to be actively involved in the synthesis of the pulmonary surfactant. The number of mitochondria remained unchanged. A great variation in shape and size of the proliferated peroxisomes was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Two hypolipidemic peroxisome proliferators increase the number of lamellar bodies in alveolar cells type II of the rat lung.

Male rats treated with either clofibrate or nafenopin, two peroxisome proliferating compounds with potent hypolipidemic properties, show identical structural changes in their lungs. In both cases, two types of lung cells were affected by these agents: (i) the alveolar epithelial cells type II and (ii) the intraalveolar macrophages. These lung cells are known to be involved in the metabolism of the pulmonary surfactant which serves to reduce the surface tension within alveoli. The size of the alveolar cells type II was conspicuously increased in the treated lungs. Compared to controls, intraalveolar macrophages apparently were slightly more numerous. Their enlarged cytoplasm was strongly vacuolated. The osmiophilic lamellar bodies within alveolar cells type II represent the intracellular presecretory pulmonary surfactant. Their number per individual alveolar cell type II was estimated by means of light microscopic morphometry on Epon-embedded semithin sections. Compared to control lungs, the number was increased by about 30% in rats treated with clofibrate (11.4 +/- 0.5 lamellar inclusions in the control cells; P less than 0.001). The increase in the number of lamellar bodies per type II cell was close to 60% in animals fed the nafenopin diet. In contrast the frequency of alveolar cells type II, estimated per area of lung tissue, remained unchanged. These results demonstrate that clofibrate and nafenopin, two drugs with hypolipidemic properties, cause identical structural changes in the rodent lung. It is concluded from these data that (i) the morphological changes observed in the surfactant metabolizing cells represent a specific action of hypolipidemic agents at the lungs and (ii) hypolipidemic peroxisome proliferators influence the metabolism of the pulmonary surfactant.

Light microscopic quantitative investigation on the effects of prostaglandin E2 (PGE2) on the lectin binding pattern in rat gastric mucosa injured by ethanol or sodium taurocholate.

We have used quantitative lectin histochemistry to study the cellular binding of peanut agglutinin (PNA) and soybean agglutinin (SBA) at the rat gastric corpus mucosa that has been injured experimentally by either ethanol or sodium-taurocholate and was protected by pretreatment of a cytoprotective dosage of the natural prostaglandin E2 (PGE2). The investigation was carried out in order to get access to possible differences in the amounts of intracellular secretory glycoproteins, associated with the prostaglandin-administration. The stomachs of rats injured by either ethanol or taurocholate revealed significantly reduced intramucosal amounts of glycoproteins with specific affinity for PNA and SBA, whereas in the stomachs of rats equally injured by the necrotizing agents, but pretreated by prostaglandin E2, the amounts of these glycoproteins remained close to the data observed in the control animals. These results give raise to two possible explanations: a) the decrease in the amounts of intramucosal glycoproteins might be due to a shedding of epithelial cells, the extent of which would then be less high in the prostaglandin treated stomachs. b) A second concept would imply influences on the glycoprotein metabolism, suggesting opposite traffics to be induced by the necrotizing agents than by the protective ones. Taken at their face value, our findings make it very likely that i) although prostaglandin-cytoprotectian did occur, this was not associated with a complete prevention from superficial cell loss at the rat gastric corpus mucosa and ii) the cytoprotective effect of prostaglandins might be mediated by influences on the intracellular glycoprotein metabolism, a process which could favour the defensive anti-ulcerogenic factors of the gastric mucosa.

Modification of surfactant metabolizing cells in rat lung by clofibrate, a hypolipidemic peroxisome proliferating agent. Evidence to suggest that clofibrate influences pulmonary surfactant metabolism.

The influence of clofibrate (ethyl-alpha-p-chlorophenoxy-isobutyrate), a hypolipidemic peroxisome proliferating agent, has been tested on the lungs of adult male rats. Drug administration for 7 days caused structural changes in two types of lung cells, both of which are involved in the metabolism of the pulmonary surfactant. By light microscopy the prominent features were the presence of enlarged type II alveolar epithelial cells and foamy intraalveolar macrophages. Compared with controls, type II cells in treated rats apparently contained more numerous surfactant-containing lamellar bodies, as visualized in semi-thin sections of Epon-embedded tissue. This difference was quantified morphometrically by light microscopy: the number of lamellar bodies was estimated as the profile number per individual type II alveolar cell, transsected at its nucleus. Clofibrate administration for 7 days resulted in a significant increase in the number of the lamellar inclusions. In contrast the number of type II alveolar cells per area of lung remained unchanged. There was no evidence of atelectasis or inflammatory infiltration in the drug-treated lungs, a finding confirmed in sections of perfusion-fixed, paraffin-embedded whole lung-lobes. By electron microscopy the lamellar inclusion bodies in the type II alveolar cells in treated rats, apart from being more numerous and sometimes smaller, were morphologically identical to those in controls. The vacuolated alveolar macrophages seen in treated rats also contained various lamellar phospholipid inclusions.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of actin microfilaments in the intracytoplasmic inclusions present in recurring infantile digital fibromatosis (Reye tumor).

The diagnostic intracytoplasmic perinuclear inclusion bodies within the fibroblastic tumor cells of recurring digital fibrous tumor of childhood (Reye tumor) were found to be ultrastructurally composed of condensed microfilaments that were continuous with axially oriented cytoplasmic filament bundles running toward the cell membrane. Immunofluorescence microscopy performed with antibodies raised against vimentin and against actin revealed a strong positive reaction for vimentin in the tumor cell cytoplasm, whereas the spherical inclusion bodies were actin-positive. From these results the following conclusions may be drawn: (I) the mesenchymal origin of the Reye tumor cells is consistent with the identification of vimentin intermediate filaments and (II) the actin-positive spherical inclusion bodies appear to represent pathological aggregations of microfilaments.

Nafenopin-induced rat liver peroxisome proliferation reduces DNA methylation by N-nitrosodimethylamine in vivo.

The hypolipidaemic drug nafenopin (NAF) has been shown to enhance the hepatocarcinogenic effect of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine in rats. We have investigated whether the NAF-induced peroxisome proliferation in hepatocytes interferes with NDMA's metabolism and interaction with DNA. Adult male Wistar rats received a single i.p. injection of [14C]NDMA (2 mg/kg) and were killed 4 h later. DNA was isolated from liver and kidney, hydrolysed in 0.1 N HCl and analysed by Sephasorb chromatography. In rats pre-treated with NAF (0.2% in the diet over a period of 3 weeks), the concentration of N7-methylguanine in hepatic DNA (mumol/mol guanine) was 46% below control values. This is probably due to the greater amount of target DNA, as NAF caused a marked hepatomegaly with a 50% increase in total liver DNA content. Concentrations of N7-methylguanine in kidney DNA were twice as high in NAF-pre-treated animals when compared to control rats. This is unlikely to result from a shift in the metabolism of NDMA from liver to other rat tissues since the time course and extent of the conversion of [14C]NDMA to 14CO2 and 14C-labelled urinary metabolites were identical in NAF-treated and control animals. There was no indication that NAF inhibits the activity of the hepatic O6-alkylguanine-DNA alkyltransferase.

The cytoprotective effect of prostaglandin E2 on taurocholate-induced erosions in gastric mucosa of the rat. Light microscopic, morphometric and scanning electron microscopic investigations.

The morphological changes associated with the cytoprotective effect of prostaglandin E2 (PGE2) following sodium-taurocholate (NaTC) erosive injury in the gastric mucosa were investigated in male rats. A single topic application of NaTC (80 mMol) induced multiple gastric erosions in all animals. Application of 200 micrograms PGE2/kg body weight prior to NaTC treatment led to a significant 90% decrease in the lesion-score in PGE2-protected animals. Light microscopic morphometric studies of the mucus-producing cells in the fundus mucosa were carried out. Within the PGE2-protected animals a significant increase was observed in the length of zones of the mucus-producing cells at the surface and in the foveolae (both PAS-positive and alcian-blue-positive cells). Compared to the NaTC-injured animals, this increase amounted to 8.1% for the PAS-positive, and 6.1% for the alcian-blue-positive zone. Compared to the untreated controls, these values were 4.7% and 3.2% respectively. In the scanning electron microscope we observed a characteristic explosive release of mucus and damage of the cell's surface membranes in the NaTC-treated animals. The PGE2-protected rats showed a predominance of exocytosis of mucus vesicles which formed a characteristic mucus network at the surface membrane. Our investigations suggest that the cytoprotective effect of PGE2 may, in part, be due to an increase in mucus production and to a modification of mucus adherence at the cell membrane.

Time course of peroxisome biogenesis during adaptation to mild hyperthyroidism in rat liver: a morphometric/stereologic study by electron microscopy.

The subcellular reaction pattern of the peroxisome compartment in rat liver cells has been studied after thyroid hormone administration for 1, 3, 5, 8, and 15 days (20 micrograms of 3, 3', 5'-triiodothyronine (T3) per 100 gm. of body weight per day). Mild hyperthyroidism caused by T3 resulted in the enlargement of the peroxisome volume fraction. This was achieved exclusively by an increase in organelle number (31 x 10(9) +/- 1.5 x 10(9) per 1 cu. cm. per cu. cm. of cytoplasm in controls), since the average organelle volume (0.27 +/- 0.02 cu. micrometer in controls) was simultaneously reduced. The remodeling of the peroxisome compartment was, for the most part, completed on day 3 and consisted of a first preliminary doubling of peroxisome numbers after 1 day, followed by a second doubling on day 3. Each doubling was accompanied by a one-third reduction in the average organelle volume. Peroxisome biogenesis due to T3 resulted in (1) the formation of microperoxisomes, consisting of small profiles, lacking the core-structure and (2) an increase of peroxisomes arranged in clusters. Cluster types with two to three profiles appeared from the beginning of biogenesis, whereas clusters with four or more profiles were mostly observed at the end. The latter was taken as an indication of a late phase of adaptation of the peroxisome compartment to hyperthyroidism, following a rapid early phase, mainly expressed on the organellar level and completed for the most on day 3. The parallel response of peroxisomes and mitochondria, as seen by the simultaneous enlargement of the fractional volume of both compartments, is taken as an indication of a close functional relationship of both organelle systems under stimulated cellular respiration.

Centrioles, microtubules and microfilaments in activated mononuclear and multinucleate macrophages from rat peritoneum: electron-microscopic and immunofluorescence microscopic studies.

In male Wistar rats of the BD I strain, mononuclear macrophages and multinucleate giant cells obtained from the peritoneum 1 day to 5 weeks after implantation of coverslips coated with dermoid cyst sebum, were examined by light microscopy and immunofluorescence microscopy, using antibodies specific for actin and tubulin and also by scanning and transmission electron microscopy. In activated mononuclear macrophages, microtubules radiate from the centrioles, situated in the perinuclear area, into the cytoplasm and the major cell processes. Microfilaments form a dense meshwork beneath the plasmalemma. When mononuclear macrophages fuse to form multinucleate giant cells, the initially unordered ("Foreign body") syncytia still reveal the original distribution patterns of centrioles, microtubules and microfilaments similar to those seen in the individual cells. In the ordered (Langhans) multinucleate giant cell all centrioles are accumulated in a main pluricorpuscular central group. Centrioles are the initiating and organising centres in the formation of microtubules. From the centrioles microtubules extend into the entire cytoplasm of the syncytium as a uniformly organised, stellate, radial system. The centrosphere, which is characteristic for ordered multinucleate giant cells, seems free from microfilaments, which form a ring-shaped woven network encircling the nuclei. Depolymerisation and inhibition of microtubules upon exposure to colchicine, indicates that both the organisation of the cytoplasm and the cellular movements depend on the undisturbed coordination of centrioles, microtubules and microfilaments. This applies also to the fusion of mononuclear macrophages to form syncytia, the ordering processes within multinucleate giant cells, and the function of ordered giant cells.