Quantitative methods to characterize morphological properties of cell lines.

Descriptive terms are often used to characterize cells in culture, but the use of nonquantitative and poorly defined terms can lead to ambiguities when comparing data from different laboratories. Although recently there has been a good deal of interest in unambiguous identification of cell lines via their genetic markers, it is also critical to have definitive, quantitative metrics to describe cell phenotypic characteristics. Quantitative metrics of cell phenotype will aid the comparison of data from experiments performed at different times and in different laboratories where influences such as the age of the population and differences in culture conditions or protocols can potentially affect cellular metabolic state and gene expression in the absence of changes in the genetic profile. Here, we present examples of robust methodologies for quantitatively assessing characteristics of cell morphology and cell-cell interactions, and of growth rates of cells within the population. We performed these analyses with endothelial cell lines derived from dolphin, bovine and human, and with a mouse fibroblast cell line. These metrics quantify some characteristics of these cells lines that clearly distinguish them from one another, and provide quantitative information on phenotypic changes in one of the cell lines over large number of passages.

Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission.

The final stage of cytokinesis is abscission, the cutting of the narrow membrane bridge connecting two daughter cells. The endosomal sorting complex required for transport (ESCRT) machinery is required for cytokinesis, and ESCRT-III has membrane scission activity in vitro, but the role of ESCRTs in abscission has been undefined. Here, we use structured illumination microscopy and time-lapse imaging to dissect the behavior of ESCRTs during abscission. Our data reveal that the ESCRT-I subunit tumor-susceptibility gene 101 (TSG101) and the ESCRT-III subunit charged multivesicular body protein 4b (CHMP4B) are sequentially recruited to the center of the intercellular bridge, forming a series of cortical rings. Late in cytokinesis, however, CHMP4B is acutely recruited to the narrow constriction site where abscission occurs. The ESCRT disassembly factor vacuolar protein sorting 4 (VPS4) follows CHMP4B to this site, and cell separation occurs immediately. That arrival of ESCRT-III and VPS4 correlates both spatially and temporally with the abscission event suggests a direct role for these proteins in cytokinetic membrane abscission.

Characterization of collagen fibrils films formed on polydimethylsiloxane surfaces for microfluidic applications.

Type I collagen fibrillar thin films have been prepared on hydrophobic recovered poly(dimethylsiloxane) (PDMS) surfaces and inside of irreversibly sealed PDMS microfluidic devices. Fibrillar films prepared on PDMS surfaces have been characterized with optical microscopy and atomic force microscopy and compared with films prepared using more traditional bulk methods on thiol-coated gold substrates. Collagen fibril films formed after 18 h of incubation on PDMS surfaces were observed to have similar underlying film thicknesses (15 nm), fibril size (67 nm), fibril coverage (45%), and physiologically supermolecular structure when compared to films on gold substrates. Collagen fibrils formed within devices were also determined to be usable across physiologically relevant cell perfusion rates. To validate the utility of these collagen fibril thin films for cell culture applications, vascular smooth muscle cells are shown to attach to collagen fibrils and exhibit cell spread areas equivalent to those seen on collagen fibrils created via bulk cell culture methods on thiol-coated gold substrates. These results extend the use and benefits of collagen fibril thin films into microfluidic-based cellular studies.

Nanomechanical properties of thin films of type I collagen fibrils.

The mechanical cues that adherent cells derive from the extracellular matrix (ECM) can effect dramatic changes in cell migration, proliferation, differentiation, and apoptosis. Model ECMs composed of collagen fibrils formed from purified collagen are an important experimental system to study cell responses to mechanical properties of the ECM. Using a self-assembled model system of a film composed of 100-200 nm diameter collagen fibrils overlaying a bed of smaller fibrils, we have previously demonstrated changes in cellular response to systematically controlled changes in mechanical properties of the collagen. In this study, we describe an experimental and modeling approach to calculate the elastic modulus of individual collagen fibrils, and thereby the effective stiffness of the entire collagen thin film matrix, from atomic force microscopy force spectroscopy data. These results demonstrate an approach to the analysis of fundamental properties of thin, heterogeneous, and organic films and add further insights into the mechanical and topographical properties of collagen fibrils that are relevant to cell responses to the ECM.

The relative roles of collagen adhesive receptor DDR2 activation and matrix stiffness on the downregulation of focal adhesion kinase in vascular smooth muscle cells.

Cells within tissues derive mechanical anchorage and specific molecular signals from the insoluble extracellular matrix (ECM) that surrounds them. Understanding the role of different cues that extracellular matrices provide cells is critical for controlling and predicting cell response to scaffolding materials. Using an engineered extracellular matrix of Type I collagen we examined how the stiffness, supramolecular structure, and glycosylation of collagen matrices influence the protein levels of cellular FAK and the activation of myosin II. Our results show that (1) cellular FAK is downregulated on collagen fibrils, but not on a non-fibrillar monolayer of collagen, (2) the downregulation of FAK is independent of the stiffness of the collagen fibrils, and (3) FAK levels are correlated with levels of tyrosine phosphorylation of the collagen adhesion receptor DDR2. Further, siRNA depletion of DDR2 blocks FAK downregulation. Our results suggest that the collagen receptor DDR2 is involved in the regulation of FAK levels in vSMC adhered to Type I collagen matrices, and that regulation of FAK levels in these cells appears to be independent of matrix stiffness.

The treatment of collagen fibrils by tissue transglutaminase to promote vascular smooth muscle cell contractile signaling.

The enzyme tissue transglutaminase 2 (TG2) appears to play an important role in several physiological processes such as wound healing, the progression of cancer and of vascular disease. Additionally, TG2 has been proposed as a means of stabilizing collagen extracellular matrix (ECM) scaffolds for tissue engineering applications. In this report, we examined the effect of TG2 treatment on the mechanical properties of the ECM, and associated cell responses. Using a model ECM of fibrillar collagen, we quantitatively examined vascular smooth muscle cell (vSMC) response to untreated, or TG2 treated collagen. We show that cells respond to TG2 treated collagen with increased spreading, an increase in contractile response as indicated by elevated F-actin polymerization and myosin light chain phosphorylation, and increased proliferation, without apparent changes in integrin specificity or matrix topography. Comparative atomic force microscopy loading studies indicate that TG2 treated fibrils are 3 times more resistant to shearing force from an AFM tip than untreated fibrils. The data suggest that TG2 treatment of collagen increases matrix mechanical stiffness, which apparently alters the contractile and proliferative response of vSMC.

Cell response to matrix mechanics: focus on collagen.

Many model systems and measurement tools have been engineered for observing and quantifying the effect of mechanics on cellular response. These have contributed greatly to our current knowledge of the molecular events by which mechanical cues affect cell biology. Cell responses to the mechanical properties of type 1 collagen gels are discussed, followed by a description of a model system of very thin, mechanically tunable collagen films that evoke similar responses from cells as do gel systems, but have additional advantages. Cell responses to thin films of collagen suggest that at least some of the mechanical cues that cells can respond to in their environment occur at the sub-micron scale. Mechanical properties of thin films of collagen can be tuned without altering integrin engagement, and in some cases without altering topology, making them useful in addressing questions regarding the roles of specific integrins in transducing or mitigating responses to mechanical cues. The temporal response of cells to differences in ECM may provide insight into mechanisms of signal transduction.

Effect of C60 on solid supported lipid bilayers.

We show that water-soluble fullerenes accumulate on the surface of zwitterionic and cationic supported bilayers to different extents. We propose on the basis of bilayer thicknesses, phase-transition temperatures, and fullerene movement that the water-soluble fullerenes do not penetrate into the hydrocarbon tails of supported bilayers. These findings are important to toxicity issues concerning fullerene materials and the development of decorated lipid bilayers for future drug delivery or sensor application.

Poly-L-lysine-induced morphology changes in mixed anionic/zwitterionic and neat zwitterionic-supported phospholipid bilayers.

Poly-L-lysine-induced morphological changes in liquid phase supported bilayers consisting of mixed anionic/zwitterionic and neat zwitterionic headgroup phospholipids were studied with atomic force microscopy and epifluorescence microscopy. Results obtained from these studies indicate that poly-L-lysine can induce domains, defects, and aggregate structures on both mixed bilayers and strictly zwitterionic bilayers. The structures formed on liquid phase supported bilayers were observed to be immobile from a timescale of 50 ms to several minutes. We propose that poly-L-lysine of sufficient length interacts with the mica substrate and phospholipids to create the stationary structures noted.

Electrostatic stitching in gel-phase supported phospholipid bilayers.

We show that mixing zwitterionic lipids with up to 20% mole % cationic lipids produces gel-phase supported lipid bilayers that are morphologically free of defects detectable using noncontact mode atomic force microscopy (AFM). This contrasts with the observation of massive defects when anionic lipid was added, and also when no charged lipid was added. Infrared measurements of headgroup orientation in the presence of cationic lipid show that the mean headgroup orientation changes only minimally when temperature is lowered from the fluid phase to the gel phase. This is consistent with a tentative explanation, based on simple electrostatic arguments, in which cationic lipids "stitch" the bilayers together. On the functional side, this study demonstrates a simple method by which to minimize defects in gel-supported phospholipid bilayers.

Direct visualization of asymmetric behavior in supported lipid bilayers at the gel-fluid phase transition.

We utilize in situ, temperature-dependent atomic force microscopy to examine the gel-fluid phase transition behavior in supported phospholipid bilayers constructed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. The primary gel-fluid phase transition at T(m) occurs through development of anisotropic cracks in the gel phase, which develop into the fluid phase. At approximately 5 degrees C above T(m), atomic force microscopy studies reveal the presence of a secondary phase transition in all three bilayers studied. The secondary phase transition occurs as a consequence of decoupling between the two leaflets of the bilayer due to enhanced stabilization of the lower leaflet with either the support or the water entrained between the support and the bilayer. Addition of the transmembrane protein gramicidin A or construction of a highly defected gel phase results in elimination of this decoupling and removal of the secondary phase transition.

Organic nanoparticles whose size and rigidity are finely tuned by cross-linking the end groups of dendrimers.

Dendrimers with molecular weights ranging from ca. 2700 to 11 000 and from 16 to 64 homoallyl ether end groups were cross-linked using the Grubbs ring-closing metathesis reaction. A combination of SEC, MALDI-TOF-MS, and AFM were used to characterize the cross-linked nanoparticles. The data suggest a significant decrease in volume with cross-linking and a concomitant increase in rigidity, both of which can be controlled independently with a fair degree of precision.

In situ FT-IR measurements of competitive vapor adsorption into porous thin films containing silica nanoparticles.

Vapor adsorption into porous ultrathin films on a gold surface is investigated with in situ surface plasmon resonance (SPR) and polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS). The thin films are prepared by the electrostatic self-assembly of oppositely charged poly(L-lysine) (PL) and silica nanoparticles on a chemically modified gold surface. Characterization with ex situ SPR and PM-IRRAS demonstrates the buildup of multiple PL/SiO2 bilayers as well as an excellent correlation between the quantitative results from these two techniques. In situ vapor adsorption experiments with these thin films show evidence of porosity, reproducibility, and rapid reversibility. Exposure to acetone vapor (P/P0 = 0.032) causes the film to adsorb 9% acetone by volume, which corresponds to coverage of approximately one-half of the silica nanoparticle surface area. In situ PM-IRRAS provides much information about the molecular interactions occurring in the film upon adsorption or desorption of vapors. Dosing with a mixture of vapors leads to a competition for adsorption into the film, and PM-IRRAS results show that acetone slightly outcompetes nitromethane. These experiments with nanoparticle thin films demonstrate the advantages of using in situ PM-IRRAS for studying reversible adsorption in the presence of vapor mixtures.

Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair in human tumour cells.

Cellular survival following ionising radiation-mediated damage is primarily a function of the ability to successfully detect and repair DNA double-strand breaks (DSBs). Previous studies have demonstrated that radiosensitivity, determined as a reduction in colony forming ability in vitro, may be related to the incorrect repair (misrepair) of DSBs. The novel rapid dual fluorescence (RDF) assay is a plasmid-based reporter system that rapidly assesses the correct rejoining of a restriction-enzyme produced DSBs within transfected cells. We have utilised this novel assay to determine the fidelity of DSB repair in the prostate tumour cell line LNCaP, the bladder tumour cell line MGH-U1 and a radiosensitive subclone S40b. The two bladder cell lines have been shown in previous studies to differ in their ability to correctly repair plasmids containing a single DSB. Using the RDF assay we found that a substantial portion of LNCaP cells [80.4 +/- 5.3(standard error)%] failed to reconstitute reporter gene expression; however, there was little difference in this measure of DSB repair fidelity between the two bladder cell lines (48.3 +/- 3.5% for MGH-U1; 39.9 +/- 8.2% for S40b). The RDF assay has potential to be developed to study the relationship between DSB repair fidelity and radiosensitivity as well as the mechanisms associated with this type of repair defect.

Aneuploid colon cancer cells have a robust spindle checkpoint.

Colon cancer cells frequently display minisatellite instability (MIN) or chromosome instability (CIN). While MIN is caused by mismatch repair defects, the lesions responsible for CIN are unknown. The observation that CIN cells fail to undergo mitotic arrest following spindle damage suggested that mutations in spindle checkpoint genes may account for CIN. However, here we show that CIN cells do undergo mitotic arrest in response to spindle damage. Although the maximum mitotic index achieved by CIN lines is diminished relative to MIN lines, CIN cells clearly have a robust spindle checkpoint. Consistently, mutations in spindle checkpoint genes are rare in human tumours. In contrast, the adenomatous polyposis coli (APC) gene is frequently mutated in CIN cells. Significantly, we show here that expression of an APC mutant in MIN cells reduces the mitotic index following spindle damage to a level observed in CIN cells, suggesting that APC dysfunction may contribute to CIN.

Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells.

The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20-50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to gamma-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of approximately 2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the alpha parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.

Retinoic acid induced growth arrest of human breast carcinoma cells requires protein kinase C alpha expression and activity.

Retinoic acid inhibits proliferation of hormone-dependent, but not hormone-independent breast cancer cells. Retinoic acid-induced changes in cellular proliferation and differentiation are associated with disturbances in growth factor signaling and frequently with changes in protein kinase C expression. PKC delta, epsilon, and zeta are expressed in both hormone-dependent (T-47D) and hormone-independent (MDA-MB-231) cell lines. Retinoic acid arrested T-47D proliferation, induced PKC alpha expression and concomitantly repressed PKC zeta expression. The changes in PKC alpha and PKC zeta reflect retinoic acid-induced changes in mRNA. In contrast, retinoic acid had no effect on growth, or PKC expression in MDA-MB-231 cells. Growth arrest and the induction of PKC alpha, but not the reduction in PKC zeta, resulted from selective activation of RAR alpha. In total, these results support an important role for PKC alpha in mediating the anti-proliferative action of retinoids on human breast carcinoma cells.

Effect of caloric restriction on colonic proliferation in obese persons: implications for colon cancer prevention.

Dietary intervention to prevent colon cancer is a major health issue. At present it is not clear which dietary factors modify colon cancer risk. Caloric restriction reduces the incidence of many spontaneous and carcinogen-induced tumors in rodents, but its role in human carcinogenesis is unknown. The relationships of body mass index (BMI), body composition, and resting metabolic rate (RMR) to colon cancer risk are also undefined. In this study involving obese persons, we measured the effect of reducing caloric intake on rectal cell proliferation, a biomarker in colon carcinogenesis, and studied the relation of BMI, body composition, and RMR to rectal cell proliferation. Colonic cell proliferation was measured in rectal biopsies from persons weighing more than 130% of ideal body weight. Follow-up biopsies were performed in patients who enrolled in and completed a 16-week behavior modification weight-reduction program in which caloric intake was reduced. Baseline measurements included body composition by total body electrical conductance, RMR, and BMI. Rectal biopsies were processed for autoradiography following incubation with [3H]thymidine. Epithelial proliferation measurements were evaluable in 35 persons at baseline and in 8 persons before and after caloric restriction. Before caloric restriction, mean (+/- SD) BMI was 38 +/- 4 kg/m2 and percentage of body fat 41 +/- 2%. Subjects reduced their caloric intake by a mean of 34 +/- 4% and their weight by 8.6 +/- 1%. Caloric restriction resulted in a 39% reduction in whole-crypt labeling index (P < 0.001) and a 57% reduction in upper crypt labeling index (P < 0.05) without reduction in crypt depth. Labeling index was unrelated to BMI, RMR, or body composition. We conclude that caloric restriction reduced rectal cell proliferation measurements--intermediate biomarkers related to colon carcinogenesis. BMI, RMR, and body composition were unrelated to colonic proliferation. Caloric restriction may have a role in colon cancer prevention.