Clinical Data as an Adjunct to Ultrasound Reduces the False-Negative Malignancy Rate in BI-RADS 3 Breast Lesions.

PURPOSE: Ultrasound (US) is a well-established diagnostic procedure for breast examination. We investigated the malignancy rate in solid breast lesions according to their BI-RADS classification with a particular focus on false-negative BI-RADS 3 lesions. We examined whether patient history and clinical findings could provide additional information that would help determine further diagnostic steps in breast lesions. MATERIALS AND METHODS: We conducted a retrospective study by exploring US BI-RADS in 1469 breast lesions of 1201 patients who underwent minimally invasive breast biopsy (MIBB) from January 2002 to December 2011. RESULTS: The overall sensitivity and specificity of BI-RADS classification was 97.4% and 66.4%, respectively, with a positive (PPV) and negative predictive value (NPV) of 65% and 98%, respectively. In 506 BI-RADS 3 lesions, histology revealed 15 malignancies (2.4% malignancy rate), which corresponds to a false-negative rate (FNR) of 2.6%. Clinical evaluation and patient requests critically influenced the further diagnostic procedure, thereby prevailing over the recommendation given by the BI-RADS 3 classification. CONCLUSION: Clinical criteria including age, family and personal history, clinical examination, mammography and patient choice ensure adequate diagnostic procedures such as short-term follow-up or MIBB in patients with lesions classified as US-BI-RADS 3.

Conformation-specific antibodies reveal distinct actin structures in the nucleus and the cytoplasm.

For many years the existence of actin in the nucleus has been doubted because of the lack of phalloidin staining as well as the failure to document nuclear actin filaments by electron microscopy. More recent findings reveal actin to be a component of chromatin remodeling complexes and of the machinery involved in RNA synthesis and transport. With distinct functions for nuclear actin emerging, the quest for its conformation and oligomeric/polymeric structure in the nucleus has resumed importance. We used chemically cross-linked 'lower dimer' (LD) to generate mouse monoclonal antibodies specific for different actin conformations. One of the resulting antibodies, termed 1C7, recognizes an epitope that is buried in the F-actin filament, but is surface-exposed in G-actin as well as in the LD. In immunofluorescence studies with different cell lines, 1C7 selectively reacts with non-filamentous actin in the cytoplasm. In addition, it detects a discrete form of actin in the nucleus, which is different from the nuclear actin revealed by the previously described 2G2 [Gonsior, S.M., Platz, S., Buchmeier, S., Scheer, U., Jockusch, B.M., Hinssen, H., 1999. J. Cell Sci. 112, 797]. Upon latrunculin-induced disassembly of the filamentous cytoskeleton in Rat2 fibroblasts, we observed a perinuclear accumulation of the 1C7-reactive actin conformation. In addition, latrunculin treatment led to the assembly of phalloidin-staining actin structures in chromatin-free regions of the nucleus in these cells. Our results indicate that distinct actin conformations and/or structures are present in the nucleus and the cytoplasm of different cell types and that their distribution varies in response to external signals.

Polymerization, three-dimensional structure and mechanical properties of Ddictyostelium versus rabbit muscle actin filaments.

To assess more systematically functional differences among non-muscle and muscle actins and the effect of specific mutations on their function, we compared actin from Dictyostelium discoideum (D-actin) with actin from rabbit skeletal muscle (R-actin) with respect to the formation of filaments, their three-dimensional structure and mechanical properties. With Mg(2+) occupying the single high-affinity divalent cation-binding site, the course of polymerization is very similar for the two types of actin. In contrast, when Ca(2+ )is bound, D-actin exhibits a significantly longer lag phase at the onset of polymerization than R-actin. Crossover spacing and helical screw angle of negatively stained filaments are similar for D and R-F-actin filaments, irrespective of the tightly bound divalent cation. However, three-dimensional helical reconstructions reveal that the intersubunit contacts along the two long-pitch helical strands of D-(Ca)F-actin filaments are more tenuous compared to those in R-(Ca)F-actin filaments. D-(Mg)F-actin filaments on the other hand exhibit more massive contacts between the two long-pitch helical strands than R-(Mg)F-actin filaments. Moreover, in contrast to the structure of R-F-actin filaments which is not significantly modulated by the divalent cation, the intersubunit contacts both along and between the two long-pitch helical strands are weaker in D-(Ca)F-actin compared to D-(Mg)F-actin filaments. Consistent with these structural differences, D-(Ca)F-actin filaments were significantly more flexible than D-(Mg)F-actin. Taken together, this work documents that despite being highly conserved, muscle and non-muscle actins exhibit subtle differences in terms of their polymerization behavior, and the three-dimensional structure and mechanical properties of their F-actin filaments which, in turn, may account for their functional diversity.

Structure, assembly, and dynamics of actin filaments in situ and in vitro.

Actin, though highly conserved, exhibits a myriad of diverse functions, most of which ultimately depend on its intrinsic ability to rapidly assemble and disassemble filamentous structures. Many organisms synthesize multiple actin isoforms even within the same cell. Tissue-specific expression patterns and tight developmental regulation as well as a high conservation across species emphasize the functional importance of isoforms. The detailed knowledge of the structure, assembly, and dynamic behavior of actin provides important pieces in solving the puzzle of how the different isoforms can be so versatile despite their extremely high sequence identity.

Differential epitope tagging of actin in transformed Drosophila produces distinct effects on myofibril assembly and function of the indirect flight muscle.

We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments.

Relative microelastic mapping of living cells by atomic force microscopy.

The spatial and temporal changes of the mechanical properties of living cells reflect complex underlying physiological processes. Following these changes should provide valuable insight into the biological importance of cellular mechanics and their regulation. The tip of an atomic force microscope (AFM) can be used to indent soft samples, and the force versus indentation measurement provides information about the local viscoelasticity. By collecting force-distance curves on a time scale where viscous contributions are small, the forces measured are dominated by the elastic properties of the sample. We have developed an experimental approach, using atomic force microscopy, called force integration to equal limits (FIEL) mapping, to produce robust, internally quantitative maps of relative elasticity. FIEL mapping has the advantage of essentially being independent of the tip-sample contact point and the cantilever spring constant. FIEL maps of living Madine-Darby canine kidney (MDCK) cells show that elasticity is uncoupled from topography and reveal a number of unexpected features. These results present a mode of high-resolution visualization in which the contrast is based on the mechanical properties of the sample.

Structural changes in native membrane proteins monitored at subnanometer resolution with the atomic force microscope: a review.

Three membrane proteins, OmpF porin from Escherichia coli, bacteriorhodopsin from Halobacterium salinarium, and the hexagonally packed intermediate (HPI) layer from Deinoccocus radiodurans, were investigated with the atomic force microscope in buffer solution. A resolution of up to 0.8 nm allowed structural differences of individual proteins to be detected. OmpF porin exhibits different static conformations on the outer surface, which possibly represent the two conductive states of the ion channels. Reversible structural changes in the cytoplasmic surface of purple membrane have been induced by changing the force applied to the scanning stylus: doughnut-shaped bacteriorhodopsin trimers transformed into a structure with three pronounced protrusions when the force was reduced from 300 to 100 pN. Furthermore, individual pores of the inner surface of the HPI layer were observed to switch from an "open" to a "closed" state. Together, the structural changes in proteins monitored under physiological conditions suggest that direct observation of function-related conformational changes of biomolecules with the atomic force microscope is feasible.

High resolution imaging of native biological sample surfaces using scanning probe microscopy.

The possibility of acquiring high resolution topographs using scanning probe microscopes under physiological conditions allows the observation of biomolecules at work. Progress has recently been made in imaging protein-DNA complexes, individual oligomers and protein arrays. Scanning probe microscopes are now tools that complement X-ray crystallography and electron microscopy.

Immuno-atomic force microscopy of purple membrane.

The atomic force microscope is a useful tool for imaging native biological structures at high resolution. In analogy to conventional immunolabeling techniques, we have used antibodies directed against the C-terminus of bacteriorhodopsin to distinguish the cytoplasmic and extracellular surface of purple membrane while imaging in buffer solution. At forces > or = 0.8 nN the antibodies were removed by the scanning stylus and the molecular topography of the cytoplasmic purple membrane surface was revealed. When the stylus was retracted, the scanned membrane area was relabeled with antibodies within 10 min. The extracellular surface of purple membrane was imaged at 0.7 nm resolution, exhibiting a major and a minor protrusion per bacteriorhodopsin monomer. As confirmed by immuno-dot blot analysis and sodium dodecyl sulfate-gel electrophoresis, labeling of the purple membrane was not observed if the C-terminus of bacteriorhodopsin was cleaved off by papain.

Slow cellular dynamics in MDCK and R5 cells monitored by time-lapse atomic force microscopy.

We have examined dynamic events that occur on a time scale of minutes in an epithelial monolayer of Madine-Darby Canine Kidney (MDCK) cells and in ras-transformed MDCK cells by atomic force microscopy (AFM). Cells were imaged under physiological conditions, and time-lapse movies representing approximately 60 s real time per frame were assembled. In normal MDCK cells, two types of protrusions in the apical plasma membrane exhibit dynamic behavior. First, smooth bulges formed transiently over the time scale of minutes to tens of minutes. Second, spike-like protrusions appear initially as bulges, extend well above the apical surface and, finally, seem to detach. R5, an oncogenic transformant derived from MDCK cells, grows very flat on glass. During AFM imaging, these cells sometimes round up and detach from the substrate. In light microscopic observations of parallel preparations, cells rarely detach, suggesting that this is an active response of these cells to irritation by the AFM tip. R5 cells often extend processes that are supported by actin stress fibers. During imaging with the AFM, these processes withdraw at a rate of 1-5 microns/min, similar to that observed by light microscopy. During the withdrawal, movement of the stress fibers can be clearly seen. In the flat periphery of these cells, the transport of intracellular particles along cytoskeletal elements was seen. In addition, we have observed two types of wave-like movements through the cell, which appear to be an organized rearrangement of cytoplasm. One type of wave moves radially out from center of the cell while the other moves circularly along the cell periphery.

Surface morphology and mechanical properties of MDCK monolayers by atomic force microscopy.

We describe the morphology and mechanical stability of the apical surface of MDCK monolayers by atomic force microscopy (AFM). Living cells could be imaged in physiological solution for several hours without noticeable deterioration. Cell boundaries appear as ridges that clearly demarcate neighboring cells. In some cases the nucleus of individual cells could be seen, though apparently only in very thin areas of the monolayer. Two types of protrusions on the surface could be visualized. Smooth bulges that varied in width from a few hundred nanometers to several micrometers, which appear to represent relatively rigid subapical structures. Another type of protrusion extended well above the membrane and was swept back and forth during the imaging. However, the microvilli that are typically present on the apical surface could not be resolved. For comparison, a transformed MDCK cell line expressing the K-ras oncogene was also examined. When cultured on solid substrata at low density, the R5 cells spread out and are less than 100 nm thick over large areas with both extensive processes and rounded edges. Many intracellular structures such as the nucleus, cytoskeletal elements and vesicles could be visualized. None of the intracellular structures seen in the AFM images could be seen by scanning electron microscopy. Both R5 cells and MDCK monolayers required imaging forces of > 2 nN for good image contrast. Force measurements on the MDCK monolayers show that they are very soft, with an effective spring constant of approximately 0.002 N/m for the apical plasma membrane, over the first micrometer of deformation, resulting in a height deformation of approximately 500 nm per nanoNewton of applied force. The mechanical properties of the cells could be manipulated by addition of glutaraldehyde. These changes were monitored in real time by collecting force curves during the fixation reaction. The curves show a stiffening of the apical plasma membrane that was completed in approximately 1 minute. On the basis of these measurements and the imaging forces required, we conclude that deformation of the plasma membrane is an important component of the contrast mechanism, in effect 'staining' structures based on their relative rigidity.

Integrin expression and localization in normal MDCK cells and transformed MDCK cells lacking apical polarity.

Epithelial cells polarize in response to contacts with the extracellular matrix and with neighboring cells. Interactions of cells with the extracellular matrix are mediated mainly by the integrin family of receptors. To begin to understand the role of integrins in polarization, we have investigated the expression and localization of three integrin families in the polarized Madin-Darby canine kidney (MDCK) epithelial cell line and in transformed MDCK cells lacking apical polarity. We find that MDCK cells express several beta 1 integrins, including alpha 2 beta 1, alpha 3 beta 1, and an unidentified integrin designated alpha x beta 1. The beta 1 integrins are the major receptors for collagens I and IV and laminin in MDCK cells, since a blocking anti-beta 1 antibody almost totally abolishes adhesion to these proteins. They also express a vitronectin receptor tentatively identified as alpha v beta 3, and the epithelial-specific integrin alpha 6 beta 4. The latter is not a laminin receptor in MDCK cells because a function blocking anti-alpha 6 antibody has no effect on cell adhesion to laminin. All three integrin families are expressed exclusively on both the basal and lateral surfaces, as determined by immunofluorescence microscopy and surface biotinylation. Transformed MDCK cells express beta 1 integrins as well as alpha v beta 3 and alpha 6 beta 4, but show alterations in the beta 1 family. Expression of alpha x is lacking, and the relative amount of the beta 1 subunit is diminished, resulting in the accumulation of Endo-H-sensitive alpha 3. In addition, surface biotinylation and immunofluorescence indicate that significant amounts of both alpha 2 beta 1 and alpha 3 beta 1 appear on not only the basolateral but also the apical plasma membrane. These results indicate that integrins are the major receptors for the extracellular matrix in MDCK cells, and that they may affect epithelial cell polarization by mediating not only cell-substratum but also cell-cell contacts.

Direct in situ structural analysis of recombinant outer membrane porins expressed in an OmpA-deficient mutant Escherichia coli strain.

An OmpA-deficient mutant of an OmpF/OmpC-free Escherichia coli B strain was selected using phage K3. The mutant strain was characterized by SDS-gel electrophoresis, immunoblotting, and electron microscopy. All major outer membrane proteins, including OmpA, were absent. This strain was then transformed with the plasmid pMY222 encoding the K12 OmpF porin or with pBlue-script-derived plasmids, encoding the porins OmpC, PhoE, and maltoporin, respectively. Following SDS extraction of outer membrane sacculi from strains expressing individual porins, crystalline porin arrays that allowed in situ structural analysis to be performed were observed. Furthermore, the absence of endogenous major outer membrane proteins facilitated the purification of native porin-lipopolysaccharide complexes, the functionally active channels, from the sacculi of transformed strains.

Cell polarity and epithelial oncogenesis.

Pathologists have long recognized that tumour formation in epithelia leads to disruption of normal epithelial cell polarity. Despite this, few studies have taken advantage of new information on the biogenesis of cell polarity to analyse the process of epithelial oncogenesis. Recent studies of epithelial cell lines now indicate that the pattern of breakdown of polarity during oncogenesis may reflect the way in which normal epithelial cells achieve polarity. These results suggest not only a novel way to study the development of polarity in vitro, but also new ideas for the early detection of cancer.

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras.

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.

Induction of the endogenous whey acidic protein (Wap) gene and a Wap-myc hybrid gene in primary murine mammary organoids.

In rodents, the whey acidic protein (Wap) is the major whey protein expressed in mammary glands in response to lactogenic hormones. The regulation of the Wap gene differs from that of other milk protein genes, with one consequence being that little or no Wap expression is detectable in cell culture. Here we describe the efficient in vitro induction of the Wap gene in mammary organoids isolated from midpregnant mice. Mammary organoids were isolated as intact epithelial subcomponents which retained the glandular microarchitecture. If organoids were cultured in contact with a monolayer of 3T3-L1 adipocytes, significant levels of Wap mRNA were induced upon hormonal stimulation, with the highest level of Wap mRNA being induced by a combination of hydrocortisone, prolactin, and insulin. Dissociation of the three-dimensional organization abrogated Wap inducibility. Organoids cultured on plastic or hydrated type I collagen did not transcribe Wap mRNA even after hormonal stimulation. Addition of hormones was required to maintain low levels of Wap mRNA in organoids cultured on reconstituted basement membrane, however, Wap mRNA was not induced. Organoid-adipocyte interactions as well as cell-cell interactions inherent in the structure of organoids promote hormone-dependent Wap mRNA expression. In order to study the Wap promoter region in vitro, we cocultured organoids from transgenic mice harboring a chimeric Wap-myc gene with 3T3-L1 adipocytes. Lactogenic hormones induced the Wap-myc transgene in vitro. The kinetics of induction were similar for both the transgene and the endogenous Wap gene indicating that the 2.5-kb regulatory Wap region present in the hybrid gene contains the sequence elements required for hormone-induced gene expression in vitro.

Chromogenic labeling of milk oligosaccharides: purification by affinity chromatography and structure determination.

Oligosaccharides from human milk were derivatized with 4'-N,N-dimethylamino-4-amino-azobenzene (DAAB) by reductive amination and purified by affinity chromatography on immobilized antibodies followed by resolution of the retained antigenic molecules by adsorption chromatography on HPLC. The visibility to the naked eye and the favorable handling properties of the DAAB-oligosaccharides (desalting, quantification) offered distinctive advantages over underivatized oligosaccharides. Analysis by MS and NMR identified the two major antigens as the Lewis a active pentasaccharide and the Lewis b active hexasaccharide, respectively. Further derivation of DAAB-oligosaccharides by palmitoylamidoacetaldehyde generated glycolipid-like compounds suitable for immunological detection by in situ overlay techniques after separation by thin-layer chromatography.

Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line.

In order to study the hormonal regulation of gene expression in mammary epithelial cells, we isolated a prolactin-responsive cell clone, HC11, from the COMMA-1D mouse mammary epithelial cell line. Clone HC11 was selected as a unique example of a cloned mouse mammary epithelial cell which has no requirement for complex, exogenously added, extracellular matrix or co-cultivation with other cell types for the prolactin-dependent in vitro induction of the endogenous beta-casein gene by lactogenic hormones. Induction of beta-casein mRNA is rapid and was detected 3 h after hormone stimulation. A prolactin-dependent increase in the rate of transcription of the beta-casein gene was shown in an in vitro nuclear transcription assay. beta-Casein protein was detected in an immunoblot assay after 24 h, and further accumulated during 5 days of hormone treatment. To identify low-abundance proteins induced directly after prolactin stimulation, mRNA was accumulated during 5 h of stimulation of HC11 cells with prolactin in the presence of cycloheximide. Following cycloheximide removal, the mRNA was translated into protein during a 60-min [35S]methionine pulse and the proteins were resolved by DEAE ion exchange HPLC and SDS-PAGE. A strong induction of a 120-kd cytosolic protein was detected which was maximally expressed within 6 h of hormone stimulation.

Targeted c-myc gene expression in mammary glands of transgenic mice induces mammary tumours with constitutive milk protein gene transcription.

We investigated the consequences of augmented c-myc gene expression in the mammary gland of transgenic mice. For this purpose we directed the expression of a mouse c-myc transgene to the differentiating mammary epithelial cells by subjecting the protein coding region to the 5' regulatory sequences of the murine whey acidic protein gene (Wap). Analogous to the expression pattern of the endogenous Wap gene, the Wap-myc transgene is abundantly expressed in the mammary gland during lactation. The tissue-specific and hormone-dependent expression of the Wap-myc transgene results in an 80% incidence of mammary adenocarcinomas. As early as two months after the onset of Wap-myc expression, tumours occur in the mammary glands of the transgenic animals. The tumours express not only the Wap-myc transgene, but also the endogenous Wap and beta casein genes. The expression of the milk protein genes becomes independent of the lactogenic hormonal stimuli and persists even in transplanted nude mouse tumours.