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Aneuploidy is observed in the majority of human cancers and is considered to be causally related to carcinogenesis. Although malignant aneuploid cells are suggested to develop from polyploid cells formed in precancerous lesions, the mechanisms of this process remain elusive. This is partly because no experimental model is available where nontransformed polyploid human cells propagate in vitro. We previously showed that proliferative tetraploid cells can be established from normal human fibroblasts by treatment with the spindle poison demecolcine (DC). However, the limited lifespan of these cells hampered detailed analysis of a link between chromosomal instability and the oncogenic transformation of polyploid cells. Here, we report the establishment of proliferative tetraploid cells from the telomerase-immortalized normal human fibroblast cell line TIG-1. Treatment of immortalized diploid cells with DC for 4 days resulted in proliferation of cells with tetraploid DNA content and near-tetraploid/tetraploid chromosome counts. Established tetraploid cells had functional TP53 despite growing at almost the same rate as diploid cells. The frequency of clonal and sporadic chromosome aberrations in tetraploid cells was higher than in diploid cells and in one experiment, gradually increased with repeated subculture. This study suggests that tetraploid cells established from telomerase-immortalized normal human fibroblasts can be a valuable model for studying chromosomal instability and the oncogenic potential of polyploid cells. © 2016 Wiley Periodicals, Inc.

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

The significant increase in chromosomal instability with aging is well known, but the underlying mechanism is not fully understood. Our earlier studies showed a high frequency of abnormal mitosis, such as mitotic slippage or incomplete mitosis in near-senescent human fibroblasts. This study examined the centrosome aberrations in mitotic and interphase cells from different passages of several strains of human fibroblasts. Analysis by laser scanning cytometry showed increased frequencies of abnormal mitotic cells with supernumerary (>2/cell) centrosomes and misaligned chromosomes in later passages in all strains examined. Numerical centrosome aberrations were prominent in a polyploid subpopulation. In metaphase cells, numerical centrosome aberrations were correlated significantly with chromosome misalignment. Fluorescent in situ hybridization analysis using a centromere-specific probe revealed a correlation between chromosome aneusomy and centrosome over-duplication. These results suggest that abnormal duplication of centrosomes in association with cellular aging may be responsible for the increase in chromosomal instability with aging.

Background/Aims: We previously reported that endothelin (ET)-1 may be involved in the contraction of hepatic sinusoidal endothelial fenestrae (SEF). Rho has emerged as an important regulator of the actin cytoskeleton and consequently cell morphology. To clarify the role of ET receptors [endothelin A receptor (ETAR) and endothelin B receptor (ETBR)] in ET-1-induced defenestration, we studied the size of hepatic SEF under various experimental conditions. Methods: Liver sinusoidal endothelial cells (LSECs) isolated from rat livers by collagenase perfusion were cultured and divided into four groups: control, ET-1 (10−6–10−10 M)-treated, ET-1+selective ETAR antagonist (BQ610)-treated and ET-1+ETBR antagonist (BQ788)-treated groups. SEF morphology was observed by scanning electron microscopy. Protein expressions of ETAR and ETBR, Rho A and phosphorylated myosin light-chain kinase were analyzed by Western blotting. F-actin stress fiber formation was observed by confocal microscopy. Active Rho was measured by Ren's modification. Intracellular free Ca2+ concentration ([Ca2+]i) was measured by fluorescence digital imaging using fura-2 AM by Aqua cosmos. Results: ET-1 induced a reduction in the number and size of SEF. ETAR antagonist pretreatment inhibited defenestration induced by low ET-1 concentrations (10−8–10−10 M), whereas ETBR antagonist pretreatment did not block defenestration at low to high ET-1 concentrations (10−6–10−10 M). F-actin stress fibers, Rho A levels and phosphorylated myosin light-chain kinase levels remained the same in various treatments. Active Rho was not detected in control and various treatments. ET-1 did not increase [Ca2+]i. Western blot showed prominent ETBR but scarce ETAR protein expression in LSECs. Conclusions: The present findings demonstrated that ETBR- and ETAR-induced contractile mechanisms are not involved in ET-1-induced defenestration, and that Rho is also not activated. Therefore, ET-1 induces hepatic defenestration by mechanisms other than receptor-mediated contraction.

To study the role of cell death in the aging process, cell death during spontaneous cellular senescence in vitro was examined with normal human fibroblasts. A small subset of the senescent cells showed aberrant morphology such as remarkable nuclear fragmentation or multiple micronuclei, and such cells often showed positive reactions with antibody to phosphorylated pRb. Cells showing caspase activation and binding of Annexin V, which indicate apoptotic change, increased in the senescent phase in flow cytometry analysis. Propidium iodide-positive cells, however, also increased with passaging. The results suggest that both apoptosis and necrosis are involved in cell death of senescent human fibroblasts.

Caveolin, the principal structural protein in caveolae, is involved in signal transduction. The aim of the present study was to clarify the distribution and ultrastructural localization of caveolin-1 in hepatic sinusoidal endothelial cells (SECs) and hepatic stellate cell (HSCs) by confocal microscopy and the electron immunogold method. Liver tissue sections were prepared from male Wistar rats. SECs and HSCs were isolated from rat livers by collagenase infusion. For immunohistochemistry, liver sections were reacted with anticaveolin-1 antibody. The localization and distribution of caveolin-1 were identified by confocal immunofluorescence. The ultrastructural localization of caveolin-1 on SECs and HSCs was identified by electron microscopy using the immunogold method. Immunohistochemical studies using liver tissues localized caveolin-1 in sinusoidal lining cells, bile canaliculi, portal vein, and hepatic artery. By confocal microscopy, caveolin-1 was mainly demonstrated at the Golgi complex in SECs and HSCs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae (SEF) and vesicles in SECs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of caveolae and vesicles in HSCs. We concluded that caveolin-1 is localized from SEFs to the Golgi complex in SECs and from caveolae to the Golgi complex in HSCs.

Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4 days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2 weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3 days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells.

Centrosome overduplication or amplification has been observed in many human cancers and in premalignant tissue, but the mechanisms leading to such centrosome aberrations are not fully understood. We previously showed that abnormal mitotic cells with supernumerary centrosomes increase with replicative senescence in human fibroblasts, especially in a polyploid subpopulation. This study examines localization of p53 protein at centrosomes in mitotic cells, which is often observed in association with DNA damage response, to investigate a possible association between p53 localization and numerical centrosome aberrations induced by cellular senescence. Cultures at later passages or the 4th day after exposure to H2O2showed increased frequencies of mitotic cells with supernumerary centrosomes, especially in a polyploid subpopulation. Immunohistochemical analysis frequently showed p53-positive foci in mitotic cells, and some were localized at centrosomes. The number of p53-positive foci in mitotic cells and its localization to centrosomes increased with replicative and premature senescence. Supernumerary centrosomes showed higher frequencies of p53 localization compared to normally duplicated centrosomes. Centrosome-associated p53 protein was phosphorylated at Ser15. These data suggest a possible association between localization of p53 protein and numerical centrosome aberrations in replicatively or prematurely senescent cells.

Aberrant nuclear morphology, such as nuclei with irregular shapes or fragmented nuclei, is often observed in senescent cells, but its biological significance is not fully understood. My previous study showed that aberrant nuclear morphology in senescent human fibroblasts is attributable to abnormal mitosis in later passages. In this study, the production of abnormal nuclei in association with premature senescence was investigated. Premature senescence was induced by brief exposure of human fibroblasts to hydrogen peroxide (H(2)O(2)), and mitosis was observed by time-lapse microscopy. In addition, cell cycle and nuclear morphology after exposure to H(2)O(2) were also analyzed using a laser scanning cytometer. Time-lapse analysis revealed that the induction of premature senescence caused abnormal mitoses, such as mitotic slippage or incomplete mitosis, especially in later days after H(2)O(2) exposure and often resulted in abnormal nuclear morphology. Analysis by laser scanning cytometer showed significantly higher frequency of abnormal cells with deformed nuclei and abnormal mitotic cells with misaligned chromosomes in a hypertetraploid subpopulation. These results suggest that unstable hypertetraploid cells, formed in association with H(2)O(2)-induced premature senescence, cause abnormal mitosis that leads to aberrant nuclear morphology.

We have developed an in vitro model for studying vascular injury. After 7-10 days in a three-dimensional collagen gel culture, capillary-like tubes were formed in the collagen gels. We injured these capillary-like tubes with a laser microdissection system or a scrape method with razors and then examined the collagen gel culture by phase contrast and electron microscopy. After laser injury, profuse necrotic cells were observed around the injured capillary-like tubes and within the tubular lumen compared to the razor injury. We then isolated total RNA from these cultures and prepared cDNA for investigations by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Quantitative real time RT-PCR revealed the up-regulation of transcription factor early growth response-1 (Egr-1) after both laser and razor injury, accompanied by the up-regulation of fibroblast growth factor-2 (FGF-2), a proangiogenic factor downstream of Egr-1. The effective laser energy is concentrated on the minute focal spot only. These methods provide a useful in vitro model for studying vascular injury.

Although apoptosis has been shown in vivo to be involved in the aging process, in vitro studies of age-dependent apoptosis are limited. In this study, apoptosis was examined in normal human fibroblasts exposed to H(2)O(2) to induce premature senescence and in spontaneously senescent human fibroblasts. Although apoptosis was not observed for several days after exposure to H(2)O(2), morphological changes indicating apoptosis were evident in about 5% of cells 7 days after exposure to 80microM H(2)O(2), concomitantly with expression of senescent phenotype. The apoptotic changes were preceded by caspase activation in majority of the exposed cells. As well as H(2)O(2)-induced senescent cells, spontaneously senescent human fibroblasts showed apoptotic changes in about 2% of cells and majority of the senescent cells also showed activation of caspases. The results indicate that the apoptosis pathway is activated in H(2)O(2)-induced and spontaneously senescent human fibroblasts in vitro.

DNA of minute specimens taken from formalin-fixed and paraffin-embedded tissue was used as a template for the polymerase chain reaction (PCR) to examine whether the c-Ki-ras gene is activated in atypical bronchioloalveolar cell hyperplasia (ABH) of human lung. The c-Ki-ras gene was successfully amplified on 131 samples from 29 cases of lung adenocarcinoma (87.3% of all samples used as templates) by the nested PCR method. Point mutations at codon 12 of the c-Ki-ras gene could be detected in carcinoma tissue of 6 cases (20.7% of all cases), also detected in ABHs showing moderate to severe atypia of 2 cases. PCR amplification is a useful technique for studying pin-point pathological lesions in a routine paraffin section at the molecular level.

Repeated oral administration of an amine-curing agent for epoxy resin, bis(4-amino-3-methylcyclohexyl)methane, gave rise to severe damage in the choroid plexus of rat brain. The damaged epithelium presented varying degrees of swelling and hydropic vacuolation on light microscopy, and varying numbers of vacuoles and inclusion bodies, frequently with lamellar structure, on transmission electron microscopy. Scanning electron microscopy of the choroid plexus disclosed some irregularity in the size of epithelial cells and occasional loss of microvilli. These changes in the choroid plexus were closely correlated with the dosage of the agent administered and the period of administration. In spite of the severe changes in the choroid plexus, no neurological abnormalities were observed in the animals during the experimental period.