Mitotic reversion in prophase of PTK1 cells induced by argon laser microirradiation

In this paper, we report the effects of laser microirradiation of prophase nucleoli and mitotic chromosomes in cells of female rat kangaroo kidney epithelial cell line PTK1. When the laser power delivered to sample surface was 90-190 mW, irradiation of one of the two nucleoli in the prophase cell did not inhibit the mitotic progress, but resulted in the loss of the irradiated nucleolus in daughter cells. When the laser power was increased to 360-420 mW, either irradiation of the nucleolus or chromosome in midprophase caused a blockage of mitosis at terminal midprophase. The irradiated cells returned morphologically to early prophase. No mitotic reversion occurred in the case of irradiation of chromosomes at late prophase, prometaphase, metaphase, and anaphase. Irradiation of the cytoplasm in prophase cells caused a 50-70 min mitotic delay at prophase. However, the irradiated cells underwent successive mitotic divisions. The mechanism of laser-induced mitotic prophase reversion is discussed.     

Laser microirradiation of kinetochores in mitotic PtK2 cells

Irradiation of the kinetochore region of PtK₂ chromosomes by laser light of 532 nm was used to study the function of the kinetochore region in chromosome movement and to create artificial micronuclei in cells. When the sister kinetochores of a chromosome were irradiated at prometaphase, the affected chromosome detached from the spindle and exhibited no further directed movements for the duration of mitosis. The chromatids of the chromosome remained attached to one another until anaphase, at which point they separated. No poleward movement of the chromatids was observed, and at telophase they passively moved to one of the daughter cells and were enclosed in a micronucleus. The daughter cell containing the micronucleus was then isolated by micromanipulation and followed through subsequent mitoses. At the next mitosis, two chromosomes, each with two chromatids, condensed in the micronucleus. These chromosomes did not attach to the spindle and showed chromatid separation, but no poleward movements at anaphase. They were again enclosed in micronuclei at telophase. The third generation mitosis was similar to the second. Occasionally, both the irradiation-produced and naturally occurring micronuclei exhibited no chromosome condensation at mitosis. Feulgenstained monolayers of PtK₂ cells with naturally occurring micronuclei showed that some micronuclei stain positive for DNA and others do not. This finding raises questions about the fate of chromosomes in a micronucleus.     

Mapping the invisible chromatin transactions of prophase chromosome remodeling

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Mitotic perturbations induced by Nek2 overexpression require interaction with TRF1 in breast cancer cells

NIMA-related kinase 2 (Nek2), a serine–threonine protein kinase, plays a major role in mitotic progression, including timing of mitotic entry, chromatin condensation, spindle organization, and cytokinesis. Nek2 overexpression results in premature centrosome separation, while kinase death Nek2 mutant expression or Nek2-depleted cells lead to centrosome separation failure. In addition, it has been revealed that telomeric repeat binding factor 1 (TRF1) interacts directly with Nek2. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. However, the interactions and correlations between Nek2 and TRF1 are far from clear. Here, we show that mitotic aberrations through Nek2 overexpression are likely to require TRF1. Our results demonstrate that Nek2 directly binds and phosphorylates TRF1 through multiple sites on TRF1. Nek2 overexpression in breast cancer cells, MDA-MB-231 and MCF7, results in increased numbers of centrosomes and multinucleated cells, which leads to cytokinetic failure and aneuploidization. Additionally, TRF1 depletion by siRNA prevents the phenomenon of unaligned chromosomes by Nek2 overexpression during metaphase. Concurrent Nek2 overexpression and TRF1-depleted cells demonstrated ≤ 2 centrosomes per cell, similar to mock plasmid and negative control siRNA-transfected cells. Interestingly, when exogenous TRF1 was added back in Nek2-overexpressed cells with endogenous TRF1 depletion, cells had re-induced cytokinetic failure. Therefore, we propose that TRF1 is required for overexpressed Nek2 to trigger abnormal mitosis and chromosomal instability.     

Increase in IP3 and intracellular Ca2+ induced by phosphate depletion in LLC-PK 1 cells

The mechanisms by which Pi depletion rapidly regulates gene expression and cellular function have not been clarified. Here, we found a rapid increase in intracellular ionized calcium [Ca(2+)](i) by phosphate depletion in LLC-PK(1) cells using confocal microscopy with the green-fluorescence protein based calcium indicator "yellow cameleon 2.1." The increase of [Ca(2+)](i) was observed in the presence or absence of extracellular Ca(2+). At the same time, an approximately twofold increase in intracellular inositol 1,4,5-triphosphate (IP(3)) occurred in response to the acute Pi depletion in the medium. Furthermore, 2-aminoethoxydiphenyl borate completely blocked the [Ca(2+)](i) increase induced by Pi depletion. These results suggest that Pi depletion causes IP(3)-mediated release of Ca(2+) from intracellular Ca(2+) pools and rapidly increases [Ca(2+)](i) in LLC-PK(1) cells.     

TLR4 signaling promotes the expression of VEGF and TGFbeta1 in human prostate epithelial PC3 cells induced by lipopolysaccharide

Chronic inflammation promotes tumor development and progression, and Toll-like receptors (TLRs) may play an important role in this process. In this study, we found that human prostate epithelial PC3 cells constitutively express TLR4 in mRNA and protein level. lipopolysaccharide (LPS) promotes the expression and secretion of immunosuppressive cytokine TGFβ₁ and proangiogenic factor VEGF in human prostate epithelial PC3 cells. We further elucidated that functionally activation of TLR4 is essential for the increased VEGF and TGFβ₁ mRNA expression in the cells. In addition, after LPS stimulation, the increased expression of NF-_KB p65 protein was also detected in human PC3 cells. Our results demonstrate that TLR4 expressed on human PC3 cells is functionally active, and may play important roles in promoting prostate cancer immune escape, survival, progression, and metastasis by inducing immunosuppressive and proangiogenic cytokines.     

PKCdelta and MAPK mediate G(1) arrest induced by PMA in SKBR-3 breast cancer cells

The effects of activating endogenous protein kinase C (PKC) on cell proliferation and the cell cycle were investigated by treating the breast cancer cell line SKBR-3 with phorbol 12-myristate 13 acetate (PMA). This inhibited cell growth in a concentration-dependent manner, causing a marked arrest of cells in G(1). Pre-treatment with GF109203X completely blocked the antiproliferative effect of PMA, and pre-treatment with the PKCdelta inhibitor rottlerin partially blocked it. Infecting SKBR-3 cells with an adenovirus vector containing wild-type PKCdelta, WTPKCdeltaAdV, had similar effects on PMA. Infecting the cells with a dominant-negative PKCdeltaAdV construct blocked the growth inhibition induced by PMA. Downstream of PKC, PMA treatment inhibited extracellular signal-regulated kinase mitogen-activated protein kinase phosphorylation, up-regulated c-jun NH(2)-terminal kinase phosphorylation, and inhibited retinoblastoma (Rb) phosphorylation. These results strongly implicated PKC (mainly PKCdelta) in the G(1) arrest induced by PMA and suggested PKC as a target for breast cancer treatment.     

Vitamin B6 suppresses apoptosis of NM-1 bovine endothelial cells induced by homocysteine and copper

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B₆in vivo. In the present study, we examined effects of vitamin B₆ on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.     

Partial restoration of impaired alpha1-adrenergic responsiveness in parotid cells of aged rats by S-adenosylmethionine treatment

The age related decrease in alpha₁-adrenergic stimulated inositol 1,4,5 trisphosphate (IP₃) production in parotid cells of aged rats can be partially restored by treatment with S-adenosylmethionine (SAM). This effect is completely blocked by S-adenosyl homocysteine (SAH) and occurs in association with an increase in the conversion of phosphatidylethanolamine to phosphatidylcholine and a decrease in membrane viscosity. In contrast, SAM treatment actually inhibits stimulated IP₃ production in cells of young rats. The membrane viscosity of these cells is lower than that of those from aged rats. Although conversion of phosphatidylethanolamine to phosphatidylcholine is enhanced, no further decrease in membrane viscosity is elicited in young cell preparations. These findings suggest that age changes in the membrane environment may result in impaired alpha₁-adrenergic signal transduction and that such alterations may be at least partially reversible by SAM treatment.     

Arachidonic acid metabolism in rat basophilic leukemia (RBL-1) cells

Rat basophilic leukemia (RBL-1) cells metabolized arachidonic acid through more than one enzymatic pathway. The major cyclooxygenase product was prostaglandin (PG) D₂ as established by chromatographic and chemical behavior and the effect on platelet aggregation. PGD₂ formation from exogenous arachidonic acid was inhibited by indomethacin, 1 μg/ml. RBL-1 incubated with exogenous arachidonic acid also formed SRS-A the synthesis of which was not inhibited by indomethacin. However, the SRS-A activity was blocked by the specific receptor antagonist FPL 55712. [¹⁴C]arachidonic acid was effectively incorporated into the phospholipids of RBL-1 cells. Challenge of such prelabelled cells or unlabelled cells with A 23187 caused release of PGD₂, SRS-A and another presently unidentified product. However, with A 23187 as a stimulus, the RBL-1 cyclo-oxygenase could not be blocked by low concentrations of indomethacin. This work further substantiates our earlier findings that SRS-A formed from arachidontic acid is not a cyclooxegenase product.     

Lysophosphatidic acid regulates inflammation-related genes in human endothelial cells through LPA1 and LPA3

Lysophosphatidic acid (LPA) is a low-molecular-weight lysophospholipid (LPL), which regulates endothelial cells participating in inflammation processes via interactions with endothelial differentiation gene (Edg) family G protein-coupled receptors. In this study, we attempted to determine which LPA receptors mediate the inflammatory response in human endothelial cells. Introduction of siRNA against LPA₁ significantly suppressed LPA-induced ICAM-1 mRNA, total protein, and cell surface expressions, and subsequent U937 monocyte adhesion to LPA-treated human umbilical endothelial cells (HUVECs). By knock down of LPA₁ and LPA₃ in HUVECs, LPA-enhanced IL-1β mRNA expression was significantly attenuated. Moreover, LPA₁ and LPA₃ siRNA also inhibited LPA-enhanced IL-1-dependent long-term IL-8 and MCP-1 mRNA expression, and subsequent THP-1 cell chemotaxis toward LPA-treated HUVEC-conditioned media. These results suggest that the expression of LPA-induced inflammatory response genes is mediated by LPA₁ and LPA₃. Our findings suggest the possible utilization of LPA₁ or LPA₃ as drug targets to treat severe inflammation.     

Identification of disulfide bonds and site-specific glycosylation in chicken alpha1-acid glycoprotein by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Recently, we reported the amino acid sequence of chicken alpha1-acid glycoprotein (chicken alpha1-AGP) [Biochem. Biophys. Res. Commun. 295 (2002) 587]. In this study, we located the disulfide bonds and site-specific glycosylation in chicken alpha1-AGP using tryptic digests of carbamidomethylated chicken alpha1-AGP, carbamidomethylated completely deglycosylated chicken alpha1-AGP (cd-alpha1-AGP), and nonreduced denatured cd-alpha1-AGP by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Based on the detection of peptides mlz 3037.4 (amino acid sequences 69-76 plus 161-183) and 3453.3 (amino acid sequences 69-80 plus 161-183), the two disulfide bonds of chicken alpha1-AGP were determined to be located at Cys 6-Cys 146 and Cys 73-Cys 163. The results also showed that Asn 16, 70, 77, and 87 were fully glycosylated and that Asn 62 was partially glycosylated.     

Epithelial to mesenchymal transition (EMT) induced by bleomycin or TFG(b1)/EGF in murine induced pluripotent stem cell-derived alveolar Type II-like cells

Induced pluripotent stem (iPS) cells are derived from reprogrammed somatic cells and are similar to embryonic stem (ES) cells in morphology, gene/protein expression, and pluripotency. In this study, we explored the potential of iPS cells to differentiate into alveolar Type II (ATII)-like epithelial cells. Analysis using quantitative real time polymerase chain reaction and immunofluorescence staining showed that pulmonary surfactant proteins commonly expressed by ATII cells such as surfactant protein A (SPA), surfactant protein B (SPB), and surfactant protein C (SPC) were upregulated in the differentiated cells. Microphilopodia characteristics and lamellar bodies were observed by transmission electron microscopy and lipid deposits were verified by Nile Red and Periodic Acid Schiff staining. C3 complement protein, a specific feature of ATII cells, was present at high levels in culture supernatants demonstrating functionality of these cells in culture. These data show that the differentiated cells generated from iPS cells using a culture method developed previously (Rippon et al., 2006) are ATII-like cells. To further characterize these ATII-like cells, we tested whether they could undergo epithelial to mesenchymal transition (EMT) by exposure to drugs that induce lung fibrosis in mice, such as bleomycin, and the combination of transforming growth factor beta1 (TGF(b1)) and epidermal growth factor (EGF). When the ATII-like cells were exposed to either bleomycin or a TGF(b1)-EGF cocktail, they underwent phenotypic changes including acquisition of a mesenchymal/fibroblastic morphology, upregulation of mesenchymal markers (Col1, Vim, a-Sma, and S100A4), and downregulation of surfactant proteins and E-cadherin. We have shown that ATII-like cells can be derived from skin fibroblasts and that they respond to fibrotic stimuli. These cells provide a valuable tool for screening of agents that can potentially ameliorate or prevent diseases involving lung fibrosis.     

JNK-dependent NFATc1 pathway positively regulates IL-13 gene expression induced by (–)-epigallocatechin-3-gallate in human basophilic KU812 cells

(–)-Epigallocatechin-3-gallate (EGCG) has been reported to possess a wide range of biological and pharmacological properties. In this study, we investigated the effects of EGCG on IL-13 gene expression in human basophilic KU812 cells. The IL-13 mRNA expression level was dose-dependently increased by treatment with EGCG (5–20 μM) for 1 h and additional incubation in a medium for 23 h. EGCG significantly increased the intracellular peroxide level as detected by the peroxide-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate. A pharmacological experiment using catalase and a structure–activity relationship study revealed that the exogenously produced H₂O₂ significantly, but partially, contributed to the IL-13 expression as well as the intracellular oxidative status. Furthermore, EGCG at the concentration required for IL-13 up-regulation activated c-Jun NH₂-terminal kinase (JNK), but not extracellular signal-regulated protein kinase or p38 mitogen-activated protein kinase in KU812 cells. Transfection of a JNK-specific siRNA as well as treatment with a JNK-specific inhibitor, SP600125, significantly reduced the EGCG-induced IL-13 mRNA expression, by 47.1 and 44.6%, respectively. In addition, we observed the nuclear translocation, mRNA up-regulation, and activation of DNA binding with the IL-13 promoter of nuclear factor of activated T cells (NFATc1) in the EGCG-treated cells. These data provide biological evidence that EGCG induces IL-13 mRNA expression via the JNK-dependent NFATc1 pathway in KU812 cells.     

Induction of sister chromatid exchanges and chromosome aberrations in cho cells arrested in the cell cycle by arginine deprivation

Summary Sister chromatid exchanges (SCE) and chromosome aberrations were induced in nondividing CHO cells that had been arrested in their cell cycle by deprivation of the essential amino acid arginine. Cells arrested in arginine-deficient medium (ADM) were treated with one of the mutagenic agents N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or mitomycin C (MMC) and refed with complete medium; the recovering cell population was sampled at various intervals thereafter and mitotic cells analyzed for the presence of chromosome aberrations and SCE. Both chemicals were observed to cause delays in the cell cycle of recovering cells and to induce, chromosome aberrations and SCE at low doses. We have described the variation in the incidence of chromosome aberrations and SCE with respect to sampling time and the number of cell cycles traversed. When ADM-arrested CHO cells were treated with three mutagens at various intervals either before or after release from ADM, it was observed that: (a) UV light induced the greatest number of SCE when applied to cells undergoing DNA synthesis, and SCE yeilds induced by this agent could be reduced by postirradiation incubation in ADM; (b) MNNG induced fewer SCE when applied to cells undergoing DNA synthesis, and SCE yields induced by this agent could not be reduced by posttreatment incubation in ADM for 24 hr. (c) MMC induced the same level irrespective of the time of exposure, and SCE yields induced by this agent could not be reduced by posttreatment incubation in ADM for 24 hr. This work was supported by grants from the British Columbia Foundation for Non-Animal Research (to W. D. M.), and the National Cancer Institute of Canada and the National Research Council of Canada (to H. F. S.). Professor H. F. Stich is a Research Associate of the NCI.     

Sphingosine kinase activity confers resistance to apoptosis by fumonisin B1 in human embryonic kidney (HEK-293) cells

Fumonisin B1 induces cytotoxicity in sensitive cells by inhibiting ceramide synthase due to its structural similarity to the long-chain backbones of sphingolipids. The resulting accumulation of sphingoid bases has been established as a mechanism for fumonisin B1 cytotoxicity. We found that despite the accumulation of sphinganine, human embryonic kidney (HEK-293) cells are resistant to fumonisin B1 toxicity; 25 microM fumonisin B1 exposure for 48 h did not increase apoptosis in these cells, while it did so in sensitive porcine kidney epithelial (LLC-PK1) cells. In this study, DL-threo-dihydrosphingosine, the sphingosine kinase inhibitor (SKI), considerably increased the sensitivity of HEK-293 cells to fumonisin B1. Treatment of these cells with 25 microM fumonisin B1 and 2.5 microM SKI increased apoptosis. Sphingoid bases, sphinganine or sphingosine, added to cell cultures induced apoptosis by themselves and their effects were potentiated by SKI or fumonisin B1. Addition of physiological amounts of sphingosine-1-phosphate prevented the toxic effects induced by SKI inhibition and fumonisin B1. Results indicated that HEK-293 cells are resistant to fumonisin B1 due to rapid formation of sphingosine-1-phosphate that imparts survival properties. Taken together, these findings suggest that sphingoid base metabolism by sphingosine kinase may be a critical event in rendering the HEK-293 cells relatively resistant to fumonisin B1-induced apoptosis.