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PubMed2003

Wild type p53 gene causes reorganization of cytoskeleton and, therefore, the impaired deformability and difficult migration of murine erythroleukemia cells.

We studied the role of p53 gene in the biophysics and biology in murine erythroleukemia cell line (MEL), with the goal of understanding the influence of this tumor suppressor gene on the deformability and metastasis of tumor cells. Experiments were performed on MEL and p53-transfected MEL (MEL-M with mutant p53 gene and MEL-W with wild-type p53 gene). The cell growth curves indicated that the over-expression of wild-type p53 gene significantly suppressed the growth of MEL, with G(0)-G(1) arrest and apoptosis shown by flow cytometric assays. Confocal laser scanning microscopy revealed that the MEL-W had a more compact organization of the F-Actin cytoskeleton than MEL and MEL-M. Fluorescence polarization measurement indicated a higher membrane fluidity of MEL-W than the other two groups. Fourier transform infrared spectroscopy (FT-IR) showed changes in the composition and/or structure of membrane lipids in MEL-W, with decreases in secondary structures of proteins such as alpha-helix, turns and bends and random coil, in comparison to MEL and MEL-M. The osmotic fragility curves indicated that MEL-W was more fragile and micropipette experiments showed that they had increased elasticity and reduced deformability in comparison to MEL and MEL-M. The adhesion assay with the use of the flow chamber revealed a lower adhesion rate of MEL-W to endothelial cells at high shear stress. The present study on the molecular biology with biophysics of MEL cells contributes to our knowledge on the tumor suppressor gene p53.

Actins↗
PubMed2004

Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.

BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single-pixel basis and a mathematical procedure called "linear unmixing" of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed. RESULTS: In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight-color immunofluorescence protocol for tissue sections was successfully established. Moreover, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated. CONCLUSIONS: This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET.

Flow Cytometry↗
PubMed2005

LPS-induced ROS generation and changes in glutathione level and their relation to the maturation of human monocyte-derived dendritic cells.

Lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) generation and the concomitant decline in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were demonstrated in human monocyte-derived dendritic cells (DC). Further, their relation to the maturation of DC, characterized by the production of cytokines, up-regulation of cell surface molecules and allo-stimulatory capacity, was examined. The LPS-induced ROS generation was demonstrated using electron paramagnetic resonance spectroscopy in intact cells, and was also confirmed using laser scanning confocal microscopy. The GSH/GSSG was assesed using a glutathione assay kit. When the DC were treated with alpha-phenyl-tert-butylnitrone, the ROS generation was attenuated, but the declined GSH/GSSG was not attenuated, and only cytokine production was suppressed among the above-mentioned maturation characteristics. When the DC were treated with glutathione monoethyl ester, both the ROS generation and the declined GSH/GSSG were attenuated, and the maturation characteristics were all suppressed. These findings suggest that the LPS-induced ROS generation and the concomitant decline in GSH/GSSG occur in human monocyte-derived DC and that the former is involved in cytokine production, while the latter is involved in the up-regulation of cell surface molecules and allo-stimulatory capacity. Since the cytokine production and the allo-stimulatory capacity of DC play an important role in inflammatory and immune responses, differential regulation of the ROS generation and the declined GSH/GSSG may be useful as therapeutic tools in diseases where both responses become entangled, such as sepsis and graft-versus-host disease.

Antigens, Surface↗
PubMed2006

Mutual information-based feature selection in studying perturbation of dendritic structure caused by TSC2 inactivation.

In this study, the effect of protein Tuberous sclerosis 2 (TSC2) on the dendritic spine density and length was demonstrated by using TSC2-RNAinactivation. In addition, the role of rapamycin, an antagonist of the molecular target of rapamycin, in the morphological changes of spine caused by TSC2 silencing was investigated. The features were extracted from highresolution three-dimensional image stacks collected by two-photon laser scanning microscopy of green fluorescing pyramidal cells expressing TSC2-RNA interference (RNAi), or TSC2-RNAi and rapamycin treatment in rat hippocampal slice cultures. We proposed to apply the lognormal distribution method for feature extraction. The extracted features of three cases under investigation, namely, (1) green-fluorescent protein GFP vs TSC2-RNAi, (2) GFP vs TSC2-RNAi and rapamycin, and (3) TSC2-RNAi vs TSC2-RNAi and rapamycin, were analyzed by mutual information-based feature selection and evaluated by three classifiers, K-nearest neighbor, Perceptron, and two-layer neural networks. The results showed that both the spine density and length have significant morphological changes after TSC2-RNAi treatment. However, rapamycin treatment could reverse the effect of TSC2-RNAi on spine length but not on spine density. These results are consistent with the results reported in the scientific literature. Finally, we explored the application of pattern recognition method in a small sample with richer feature properties, namely bootstrap mutual information estimation and a mutual information- based feature selection method.

Algorithms↗
PubMed2003

Colocalization analysis of sialomucins CD34 and CD164.

Flow cytometric protocols are employed to identify and characterize hemopoietic stem/progenitor populations before transplantation. Cell surface antigens, including CD34, are employed in this process and widely used in harvest protocols, which largely ignores the potential functional role of such antigens. Transmembrane glycoprotein sialomucins, including CD34 and CD164, have been implicated in cell-to-cell interactions and activation. CD164, also expressed on early hemopoietic populations, was reported to have a possible function facilitating CD34(+) cells to adhere to bone marrow stroma. In this study, we employed high-definition laser-scanning confocal microscopy to investigate CD34 and CD164 surface co-localization patterns on bone marrow and cord blood cells and to compare the expression patterns using a three-dimensional computer-generated method developed in house. Differential interference microscopy analysis revealed bone marrow membrane activity was higher than the corresponding cord blood counterpart, perhaps indicating the marrow microenvironmental nature. Fluorescence analysis of CD34 and CD164 antigens showed both were expressed first in a halo-like pattern and second in antigen-dense pockets. Three-dimensional computer analyses further revealed that this pocketing corresponded to dense crest-like surface structures appearing to rise from the point of adherence on the slide. Further, it was found that CD34 and CD164 display strong colocalization patterns on cells expressing both antigens. The dual nature of the CD34 and CD164 antigens discovered here lends further evidence to the previous literature implicating a strong functional link between these two sialomucins, which should be considered in the transplantation arena and in the function of such sialomucins as negative regulators of cell proliferation.

Antigens, CD34↗
PubMedDate not supplied

Induction of apoptosis in neoplastic cells by anthracycline antitumor drugs: nuclear and cytoplasmic triggering?

In spite of extensive investigation, the mechanism for cell cytotoxicity of the anthracycline antitumor drug adriamycin (ADR) has not yet been completely understood but the nature of the cytotoxic effects of this drug is generally related to its interaction with nuclear components, such as DNA and topoisomerase II. In a previous paper, we studied, using Confocal Laser Scanning Microscopy (CLSM), the localization of ADR and its glutathione (GSH)-conjugate (ADRIGLU), obtained by the anaerobic reaction of the parent anthracycline with reduced GSH, in drug-sensitive and in multidrug resistant (MDR) cells. In all drug-sensitive lines used, ADR was mostly located in the nuclei, while its GSH-conjugate was found only in the cytoplasm, predominantly in the Golgi region. In this study we examined the morphological changes induced by ADR or its GSH-conjugated adduct (ADRIGLU) treatments in TVM-A12 (clone 2) melanoma and K562 erythroleukemia human cell lines, correlated to programmed cell death (apoptosis). We observed that ADR-induced apoptosis in both cell lines tested after 5 h treatment: CLSM and Scanning Electron Microscopy (SEM) showed cell shrinkage, fragmentation and condensation of nuclear chromatin, cell surface blebbing and cytoplasmic vacuolization. On the contrary, ADRIGLU-induced fragmentation and condensation of nuclear chromatin, typical of apoptosis, only after 48-72 h treatment. Cytoflourimetric assay by propidium iodide staining confirmed the data obtained by CLSM and SEM. Our data suggest that apoptosis activation by anthracycline antitumor drugs is induced not only by direct interaction with nuclear components but also with cytoplasmic compartments.

Antibiotics, Antineoplastic↗
PubMed1998

Functional imaging of mitochondria in saponin-permeabilized mice muscle fibers.

Confocal laser-scanning and digital fluorescence imaging microscopy were used to quantify the mitochondrial autofluorescence changes of NAD(P)H and flavoproteins in unfixed saponin-permeabilized myofibers from mice quadriceps muscle tissue. Addition of mitochondrial substrates, ADP, or cyanide led to redox state changes of the mitochondrial NAD system. These changes were detected by ratio imaging of the autofluorescence intensities of fluorescent flavoproteins and NAD(P)H, showing inverse fluorescence behavior. The flavoprotein signal was colocalized with the potentiometric mitochondria-specific dye dimethylaminostyryl pyridyl methyl iodide (DASPMI), or with MitoTrackerTM Green FM, a constitutive marker for mitochondria. Within individual myofibers we detected topological mitochondrial subsets with distinct flavoprotein autofluorescence levels, equally responding to induced rate changes of the oxidative phosphorylation. The flavoprotein autofluorescence levels of these subsets differed by a factor of four. This heterogeneity was substantiated by flow-cytometric analysis of flavoprotein and DASPMI fluorescence changes of individual mitochondria isolated from mice skeletal muscle. Our data provide direct evidence that mitochondria in single myofibers are distinct subsets at the level of an intrinsic fluorescent marker of the mitochondrial NAD-redox system. Under the present experimental conditions these subsets show similar functional responses.

Animals↗
PubMedDate not supplied

Diet restriction impairs extracellular signal-regulated kinase activation of peritoneal exudative cells after N-formyl-methionyl-leucyl-phenylalanine stimulation in a murine peritonitis model.

BACKGROUND: Phosphorylation of extracellular signal-regulated kinase (ERK) enhances various inflammatory responses in immune cells. It is unknown whether dysfunction of immune cells during malnutrition is attributed to derangement of ERK activation. METHODS: Male Institute of Cancer Research (ICR) mice received chow (146 g/kg per day, ad libitum or 36.5 g/kg per day, diet-restricted) for 7 days. Mice (n = 55) were given 6.5 mg/kg of an ERK inhibitor (PD98059) or vehicle intraperitoneally (IP), at 2 hours before cecal ligation and puncture (CLP). Survival was observed up to 60 hours. Detection of phosphorylated ERK (pERK) in the peritoneal exudative cells (PECs) was done as follows. In a separate study, PECs were harvested by peritoneal lavage 2 hours after an IP injection of 1% glycogen. PECs were incubated with or without 100 nmol/L N-formyl-methionyl-leucyl-phenylalanine (fMLP) for 1 minute. PEC ERK activation was detected with Western blot analysis (n = 38), by densitometric quantification, and with a laser scanning cytometer (LSC; n = 13). Subpopulations of PECs were determined by Wright-Giemsa staining. Unstimulated pERK expression was normalized to 100% for Western blot analysis. RESULTS: Diet restriction reduced survival after CLP compared with the ad libitum mice. ERK inhibition showed no effect on survival in diet-restricted mice but reduced survival in ad libitum mice. There were no differences in subpopulations of PECs 2 hours after glycogen injection between the groups. Western blot analysis revealed that fMLP stimulation significantly increased the phosphorylation of ERK1/2 in PECs from the ad libitum group (ERK1, 199 +/- 41%; ERK2, 211 +/- 49%; p < .03) but not in those from diet-restricted mice (ERK1, 98 +/- 10%; ERK2, 91 +/- 9%). Mean fluorescence intensity (MFI) of pERK in PECs obtained by LSC was significantly elevated after fMLP in the ad libitum group (from 19.4 +/- 1.5 MFI to 22.4 +/- 1.2 MFI; p < .05) but did not change in the diet-restricted group (from 19.4 +/- 1.8 MFI to 19.1 +/- 1.5 MFI). CONCLUSIONS: ERK activation is essential for survival in this murine sepsis model. Impaired ERK activation of PECs may, at least in part, impair host defense during malnutrition.

Animals↗
PubMed2003

Both calcium and ROS as common signals mediate Na(2)SeO(3)-induced apoptosis in SW480 human colonic carcinoma cells.

Recent studies have shown that reactive oxygen species (ROS) play a crucial role in Se-induced cell apoptosis. A number of studies have demonstrated that perturbed cellular calcium homeostasis has been implicated in apoptosis. The main objective of this study was to evaluate the role of Ca(2+) in Na(2)SeO(3)-induced apoptosis and the relationship between Ca(2+) and ROS in human colonic carcinoma cells SW480. When SW480 cells were exposed to 25-100 microM Na(2)SeO(3), both cell apoptosis and growth inhibition were observed by flow cytometric analysis and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Na(2)SeO(3) was able to induce increase of [Ca(2+)](i) and ROS production and disrupt mitochondrial membrane potential (Delta Psi m) in SW480 cells monitored by using a confocal laser scanning microscope. Ca(2+) channel inhibitor CoCl(2) and an intracellular Ca(2+) chelator o-phtalaldehyde, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA) completely inhibited [Ca(2+)](i) increase, but catalase had no effect on Na(2)SeO(3)-induced increase of [Ca(2+)](i). BAPTA-AM, CoCl(2), and mitochondrial Ca(2+) uptake inhibitor ruthenium red blocked Delta Psi m dissipation. The increase of ROS was also suppressed by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine and catalase, respectively. The mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) completely inhibited Na(2)SeO(3)-induced ROS increase. This showed that ROS increase is due to mitochondrial Ca(2+) overload. The Na(2)SeO(3)-induced apoptosis of SW480 cells was also inhibited by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine, and catalase, respectively. The results mentioned above imply that both calcium and Ca(2+)-dependent ROS as a signal molecule mediate apoptosis induced by Na(2)SeO(3) in SW480 cells.

Acetylcysteine↗
PubMed1998

Modulation of the frequency of spontaneous sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks) by myoplasmic [Mg2+] in frog skeletal muscle.

The modulation by internal free [Mg2+] of spontaneous calcium release events (Ca2+ "sparks") from the sarcoplasmic reticulum (SR) was studied in depolarized notched frog skeletal muscle fibers using a laser scanning confocal microscope in line-scan mode (x vs. t). Over the range of [Mg2+] from 0.13 to 1.86 mM, decreasing the [Mg2+] induced an increase in the frequency of calcium release events in proportion to [Mg2+]-1.6. The change of event frequency was not due to changes in [Mg-ATP] or [ATP]. Analysis of individual SR calcium release event properties showed that the variation in event frequency induced by the change of [Mg2+] was not accompanied by any changes in the spatiotemporal spread (i.e., spatial half width or temporal half duration) of Ca2+ sparks. The increase in event frequency also had no effect on the distribution of event amplitudes. Finally, the rise time of calcium sparks was independent of the [Mg2+], indicating that the open time of the SR channel or channels underlying spontaneous calcium release events was not altered by [Mg2+] over the range tested. These results suggest that in resting skeletal fibers, [Mg2+] modulates the SR calcium release channel opening frequency by modifying the average closed time of the channel without altering the open time. A kinetic reaction scheme consistent with our results and those of bilayer and SR vesicle experiments indicates that physiological levels of resting Mg2+ may inhibit channel opening by occupying the site for calcium activation of the SR calcium release channel.

Adenine Nucleotides↗
PubMed2001

Alterations of platelet aggregation kinetics with ultraviolet laser emission: the "stunned platelet" phenomenon.

Platelets, a major constituent of thrombus, play a crucial role in the pathogenesis of acute ischemic coronary syndromes. The effect of ultraviolet laser emission on platelets within thrombi is unknown. The effects of increasing levels of laser energy on platelets in whole blood were investigated. Blood samples were obtained by aseptic venipuncture and anticoagulated with 3.8% sodium citrate. Samples were exposed to increased levels (0, 30, 45, 60 mJ/mm2; 25 Hz) of ultraviolet excimer laser fluence (308 nm wave-length) and then tested for ADP and collagen induced platelet aggregation, platelet concentration, and for platelet contractile force (PCF) development. Scanning electron microscopy was used to detect laser induced morphologic changes of platelets and by flow cytometric analysis to detect changes in expression of platelet surface antigens p-selectin (CD 62) and glycoprotein IIb/IIIa (CD 43). Exposure to excimer laser energy produced dose dependent suppression of platelet aggregation and force development ("stunned platelets"). ADP aggregation decreased from 8.0+/-1.1 Ohms (mean+/-SEM) to 3.7+/-0.8 Ohms (p<0.001) to 2.7+/-0.6 Ohms (p <0.001) and to 1.8+/-0.5 Ohms (p <0.001) as the laser energy increased from 0 to 30 to 45 to 60 mJ/mm2, respectively. Collagen induced aggregation decreased from 21.4+/-1.4 Ohms to 15.7+/-1.2 Ohms (p <0.001) to 11.7+/-1.1 Ohms (p <0.001) and to 9.9+/-1.0 Ohms (p <0.001), in response to the same incremental range of laser energy. Platelet contractile forces declined from 34,500+/-3700 to 27.800+/-2700 dynes as laser energy increased from 0 to 60 mJ/mm2 (p <0.03). Platelet concentration did not change with increasing laser energy. The expression of platelet surface antigen p-selectin (CD 62) remained stable through increasing levels of laser energy exposures while the percentage of CD 43 positive platelets significantly increased with exposure to laser energy, yet the level of expression did not exceed 0.5% of cells. Thus, aggregation kinetics are altered in platelets exposed to ultraviolet laser energy as manifested by decreased platelet aggregation and reduction in platelet force development capability. The response is dose dependent and most pronounced at higher energy levels such as 60 mJ/mm2.

Adenosine Diphosphate↗
PubMed2002

Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy.

Toxoplasma gondii is an intracellular parasite that proliferates within most nucleated cells, an important human pathogen, and a model for the study of human and veterinary parasitic infections. We used a stable yellow fluorescent protein-alpha-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-alpha-tubulin parasites by laser-scanning confocal microscopy (LSCM) failed to resolve the 22 subpellicular microtubules characteristic of the parasite cytoskeleton. To understand this result, we analyzed sources of noise in the LSCM and identified illumination fluctuations on time scales from microseconds to hours that introduce significant amounts of noise. We confirmed that weakly fluorescent structures could not be imaged in LSCM by using fluorescent bead standards. By contrast, wide-field microscopy (WFM) did visualize weak fluorescent standards and the individual microtubules of the parasite cytoskeleton. We therefore measured the fluorescence per unit length of microtubule by using WFM and used this information to estimate the tubulin content of the conoid (a structure important for T. gondii infection) and in the mitotic spindle pole. The conoid contains sufficient tubulin for approximately 10 microtubule segments of 0.5-microm length, indicating that tubulin forms the structural core of the organelle. We also show that the T. gondii mitotic spindle contains approximately 1 microtubule per chromosome. This analysis expands the understanding of structures used for invasion and intracellular proliferation by an important human pathogen and shows the advantage of WFM combined with image deconvolution over LSCM for quantitative studies of weakly fluorescent structures in moderately thin living cells.

Algorithms↗
PubMed1995

Plasmin abrogates alpha v beta 5-mediated adhesion of a human keratinocyte cell line (HaCaT) to vitronectin.

At cellular surfaces, urokinase-type plasminogen activator (uPA) is bound to a specific receptor (uPA-R). When bound to this receptor, uPA activates plasminogen, which is derived from plasma or the interstitial fluids. Thus, plasmin is provided for proteolysis of pericellular proteinaceous substrates. Here we demonstrate by immunocytology and laser scan microscopy that in the human keratinocyte cell line HaCaT uPA-R and uPA are localized together with the integrin alpha v beta 5 in focal contacts. Via the integrin alpha v beta 5, HaCaT cells adhere to vitronectin in a RGD-dependent manner. Plasmin interfered with the alpha v beta 5-mediated keratinocyte adhesion to vitronectin, most likely via cleavage of vitronectin and destruction of its cell binding function. Our findings demonstrate that plasmin, when generated by the uPA-dependent cell surface-associated pathway of plasminogen activation, can abrogate the cell-binding function of vitronectin and can thus disturb the adhesive interaction with this matrix molecule. In focal contacts molecules are assembled that are crucial for adhesion to vitronectin (i.e., the integrin alpha v beta 5), as well as for the generation of plasmin (i.e., uPA-R and uPA), which can negatively influence the binding interaction. We suggest that the plasmin-mediated abrogation of the interaction between the integrin alpha v beta 5 and vitronectin is a pathway of negative regulation; the codistribution of uPA-R/uPA and alpha v beta 5 in focal contacts may restrict this process to areas of cell/matrix contact.

Amino Acid Sequence↗
PubMed2006

Changes in fluid secretion rate alter net transepithelial transport of MRP2 and P-glycoprotein substrates in Malpighian tubules of Drosophila melanogaster.

The effects of stimulants of fluid secretion on net transepithelial transport of the MRP2 substrate Texas Red and the p-glycoprotein substrate daunorubicin were examined in Malpighian tubules of Drosophila melanogaster. Fluid secretion rates were determined using the Ramsay assay and secreted fluid concentrations of Texas Red and daunorubicin were determined using a microfluorometric technique. Nanoliter droplets of secreted fluid were collected in optically flat glass capillaries and dye concentration was determined from fluorescence intensity measured by confocal laser scanning microscopy. Net transepithelial flux of each compound was then calculated as the product of its concentration in the secreted fluid and the fluid secretion rate. Net transepithelial flux of Texas Red increased when fluid secretion was stimulated by tyramine, cyclic AMP or hypoosmotic saline. Net flux decreased when fluid secretion rate of cAMP-stimulated tubules was reduced by elevating saline osmolality with sucrose. Net transepithelial flux of daunorubicin increased when fluid secretion was stimulated by cAMP. Significant increases in dye flux were seen only when the dyes were present at concentrations close to or greater than the concentration required for half maximal transport. Regression analyses showed that 57- 88% of the change in dye flux was attributable to the change in fluid secretion rate when tubules were stimulated with cAMP, cGMP, or tyramine. The results do not suggest that the effects of tyramine and cAMP are mediated through changes in transepithelial potential, nor do they indicate the direct effects of the stimulants on MRP2-like or p-glycoprotein-like transporters (e.g., via protein kinases). Instead, the results suggest that increases in fluid secretion rate minimize diffusive backflux of these dyes and, thus, facilitate higher rates of net transepithelial transport indirectly.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2004

Lovastatin inhibits tumor growth and lung metastasis in mouse mammary carcinoma model: a p53-independent mitochondrial-mediated apoptotic mechanism.

The effects of lovastatin, a potent inhibitor of hydroxymethylglutaryl-coenzyme A reductase, were studied in a mouse model of metastatic mammary cancer carrying a p53 mutation. Mice bearing mammary tumors, induced by inoculation of syngeneic BALB/c mice with BJMC3879 cells, were treated with lovastatin at 0, 25 and 50 mg/kg three times a week. Tumor volumes were significantly reduced in a dose-dependent manner throughout the 6 week study and were associated with both a decrease in DNA synthesis and an increase in apoptosis. The high dose of lovastatin also inhibited lung metastasis. In a corollary in vitro study, flow cytometric analyses of lovastatin-treated mammary cancer cells additionally showed cell cycle arrest at G1 phase and decreases in S and G2/M phases. Laser scanning cytometric analyses further demonstrated that cancer cells in S and G2/M were particularly susceptible to the effects of lovastatin. Transmission electron microscopic evaluation of TUNEL-confirmed apoptotic bodies in lovastatin-treated mammary carcinoma cells revealed many free 3'-OH ends of DNA in condensed chromatin within fragmented nuclei that occasionally assumed a characteristic half-moon shape. Consistent with initiation of apoptosis, cellular caspase-8, caspase-9 and caspase-3 activities were elevated in lovastatin-treated cells. The mitochondrial membrane potential was also decreased, with subsequent release of cytochrome c. However, lovastatin-induced cell death was significantly reduced by the broad spectrum caspase inhibitor z-VAD-fmk, as well as the caspase-9 inhibitor z-LEHD-fmk and the caspase-3 inhibitor z-DEVD-fmk, but not by the specific caspase-8 inhibitor z-IETD-fmk. Since immunoelectron microscopy showed translocation of Bax to the mitochondria in lovastatin-treated cells, lovastatin-induced apoptosis may, therefore, be ultimately dependent on Bax induction of cytochrome c release. These results suggest that lovastatin may be useful as an adjuvant therapy in breast cancers containing p53 mutations due to its ability to both suppress DNA synthesis and induce p53-independent mitochondria-mediated apoptosis.

Animals↗
PubMed2005

Insulin and angiotensin II induce the translocation of scavenger receptor class B, type I from intracellular sites to the plasma membrane of adipocytes.

Scavenger receptor class B, type I (SR-BI) mediates the selective uptake of lipids from high density lipoproteins and is expressed in several types of tissues. However, to date little is known about its role in adipocytes. In this study, we investigated the cellular distribution of SR-BI in 3T3-L1 adipocytes and its regulation by hormones known to increase lipid storage such as angiotensin II (Ang II) and insulin. SR-BI was mainly distributed in the cytoplasm as determined by laser-scanning confocal analysis of the immunofluorescence labeling of SR-BI or the study of an enhanced green fluorescent protein-tagged SR-BI fusion protein. Exposure of cells to either insulin or Ang II (1-2 h) induced the mobilization of SR-BI from intracellular pools to the plasma membrane. This was further confirmed by Western blotting on purified plasma membrane and by fluorescence-activated cell sorter analysis of the SR-BI receptor. Similar results were also observed in primary adipocytes. We also demonstrated that, in the presence of either insulin or Ang II, SR-BI translocation to the cell membrane is functional, because insulin and Ang II induced a significant increase in the high density lipoprotein-delivered 22-(N-7-nitrobenz-2-oxa-1,3-diazo-4-yl)-amino-23,24-bisnor-5-cholen-3-ol uptake and in total cholesterol content. These data demonstrate that SR-BI can be acutely mobilized from intracellular stores to the cell surface by insulin or Ang II, two hormones that exert lipogenic effects in adipocytes. This suggests that SR-BI might participate in the storage of lipids in the adipose tissue.

3T3-L1 Cells↗
PubMedDate not supplied

Developmental changes of calcium transients and contractility during the cultivation of rat neonatal cardiomyocytes.

Neonatal cardiomyocytes of the rat were investigated (a) by Confocal Laser Scanning Microscopy (CLSM) using the Ca(2+)-sensitive dye fluo-3/AM to measure calcium transients, and (b) by a Laser Doppler Microscope (LSC-1) to obtain data of the cell culture's contractility. Our experiments resulted in: (1) About 20% of the freshly prepared cardiomyocytes exhibited spontaneous but not rhythmically appearing calcium transients. None of these cells was found to be active mechanically. The remainder of 80% showed neither calcium transients nor cell movements. (2) At the latest after four days of cultivation, the cells showed spontaneous calcium transients of constant frequency and concomitant contractions. (3) During the cultivation, spontaneous Ca2+ transients became steeper and shorter. The time course of the calcium transient is abbreviated by a factor of at least two in cells after four days when compared with cardiac cells after one day of cultivation. (4) Addition of 100 nM ryanodine caused an increase of the cytosolic calcium concentration and a decrease of the amplitude of the Ca2+ transients. This effect became more significant with increasing time of cultivation and ran parallel to a decrease of the cell's contractility. (5) Addition of 1 microM thapsigargin yielded a similar increase of the cytosolic calcium concentration and a decrease of the Ca2+ peak accompanied by a smaller lowering of the contractility (in comparison with the mentioned influence of ryanodine). The effects of thapsigargin were practically independent of the time of cultivation.

Animals↗
PubMed1994

Expression of integrin and organization of F-actin in epithelial cells depends on the underlying surface.

PURPOSE: To evaluate the role of ionic interactions in the cell surface expression of integrins and the organization of F-actin. Understanding these interactions will allow the development of surfaces for prosthetic purposes that will promote the normal expression of adhesion proteins. METHODS: Hema (hydroxyethylmethacrylate) hydrogels were used to mimic the charges present on extracellular matrix proteins. The surfaces were modified by the addition of amines (N,N-dimethylaminoethylmethacrylate; NDAM) or carboxyl moieties (methacrylic acid). The effects of ionic interactions on cellular spreading and on the expression of proteins were examined by modification of the stoichiometrically defined amounts of positive and negative charges on the Hemas. Changes in intracellular pH and the distribution and localization of protein were monitored using fluorescent markers, spectrofluorometry, and confocal laser scanning microscopy, respectively. The immunohistochemical studies were confirmed by flow cytometric analysis. RESULTS: The data indicate that although cells adhered to all the surfaces, the number of cells possessing adhesion receptors is significantly greater on surfaces with amine functionalities. Cell seeding and plating efficiency after 2 hours were identical on all surfaces. The intracellular pH of epithelial cells grown on surfaces containing NDAM, a tertiary amine, was higher than that of cells grown on Hemas containing only methacrylic acid. Lamellipodial extensions and an extensive actin network were present on surfaces containing 5% NDAM. The alpha 6 subunit was localized along the lateral cell membranes. The alpha 2 and 3 subunits were present along cell membranes and at lamellipodial extensions. Cells cultured on surfaces containing only methacrylic acid did not spread. Actin filaments were not detected, and alpha 6 was negligible on these surfaces. CONCLUSIONS: This is a novel approach to understanding cell-substrate interactions, and one that allows quantitative evaluation of the response of cells to defined surfaces. The organization of F-actin is altered by the substrates containing only carboxyl moieties. The distribution of integrin subunits is also altered by the substrate. These results indicate that epithelial cell spreading and protein expression may be regulated by ionic interactions.

Actins↗