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The second domain of Pseudomonas exotoxin A (PEAII, residues 253-364) has been shown to facilitate translocation of extracellular and vesicular contents into the cytoplasm, and can transport heterologous molecules into target cells. Because full length PEAII may elicit a host immune response, we tried to identify the minimal PEAII translocation motif and use this fragment in combination with an antibody and constitutively active granzyme B (ImmunoGrB) to kill HER2-positive tumor cells. We constructed four ImmunoGrB fusion proteins containing different PEAII deletions and tested their abilities to kill HER2-positive cells. Our data showed that while a complete deletion of PEAII in ImmunoGrB resulted in an inability to kill cancer cells, ImmunoGrBs containing either PEAII (253-358aa) or PEAII (275-358aa) could efficiently kill HER2-positive SK-BR-3 cells. Most interestingly, the construct which contains only a furin cleavage site, named PEAII (275-280aa), could still induce SK-BR-3 apoptosis, although less efficiently. Moreover, delivery of the recombinant proteins by intramuscular plasmid injection led to an apparent tumor regression and prolonged animal survival in a nude mouse xenograft SK-BR-3 tumor model, indicating in vivo antitumor activity of the different PEAII containing ImmunoGrBs. Our results may help in understanding PEAII translocation and may lead to the development of useful tools or alternative therapy.

YKL-40 is a new prognostic biomarker in cancer. The biological function is only poorly understood. This study aimed at determining the subcellular localization of YKL-40, using immunogold labeling, in normal epithelial cells and in malignant tumor cells of the breast by immunoelectron microscopy. YKL-40 protein expression was redistributed in carcinoma versus normal glandular tissue of the breast. A reduced expression of YKL-40 in relation to intermediate filaments and desmosomes was found in tumor cells. Changes in YKL-40 expression suggest that the function of YKL-40 in cells of epithelial origin may be related to cell motility and cell-cell adhesion, features associated with invasion and migration potential of tumor cells.

Results of clinical trials over the past 15 years demonstrate that the taxanes are among the most effective new class of cytotoxic drugs to treat breast cancer and other solid tumors. Moreover, the efficacy of the taxanes added further credence to the relevance of the microtubule as a tumor target. In spite of the significant benefits observed in early and advanced breast cancer, a number of factors contribute to disease relapse and, perhaps more discouragingly, disease refractoriness. After exhausting cytotoxic chemotherapy, hormonal therapy, and other molecular-based-therapies, patients whose tumors exhibit taxane resistance had virtually no additional options. This paper, a product of the ongoing advances in the treatment of breast cancer, reviews two important areas: first, molecular concepts and relevance of the microtubule in breast cancer and second, clinical implications of ixabepilone, a novel, nontaxane tubulin-stabilizing agent in patients with taxane-resistant breast cancer.

Background: Fibroadenomas with stromal giant cell reaction have been described in the literature, but cytologic atypia including giant cell reaction due to chickenpox giving rise to suspicious cytology has not been reported., Case Report: A 25-year-old woman, recovering from chickenpox, presented with a 1.5 x 1.5-cm mass in the lower outer quadrant of her right breast. Fine needle aspiration smears showed sheets of benign ductal cells with overlapping myoepithelial cells and many bipolar bare nuclei. Cells showing nuclear enlargement, prominent nucleoli and multilobated or multinucleated giant cell formation occurred in separate sheets or dispersed among groups of benign ductal cells. Cytodiagnosis was suspicion for malignancy; excision biopsy was advised. Histopathologic examination showed fibroadenoma with evidence of epithelial hyperplasia, nuclear enlargement and multilobated giant cell formation. Atypical ductal cells, including the giant cells, were immunohistochemically positive for epithelial membrane antigen, estrogen receptor and progesterone receptor and negative for smooth muscle actin, indicating epithelial origin. Both cytologic and histologic specimens showed focal positive reaction with HSV-1 and HSV-2 antibodies. Ultrastructural examination of aspirated material revealed cytoplasmic viral particles with characteristic surface projections., Conclusion: Herpes zoster virus can produce morphologic alteration mimicking a malignancy. Pathologists should be aware of these changes to avoid a false positive diagnosis.

Polypyrrole-Fe3O4 nanospheres were synthesized via an emulsion polymerization method with hyaluronic acid as the surfactant. Hyaluronic acid offers the advantages of biocompatibility, cell adhesive property and the availability of functional groups for attachment of other molecules. The nanospheres were further functionalized with herceptin, and the efficacy of uptake of the functionalized nanospheres by human breast cancer cells was evaluated. It is envisioned that the combination of hyaluronic acid with its cell adhesive property and herceptin would result in high efficacy of internalization of the nanospheres by the cancer cells via a HER-2-mediated endocytosis. Our results showed that this is indeed the case and that the high concentration of herceptin-functionalized magnetic nanospheres in the cancer cells offers great potential in cancer cell targeting and treatment. In addition, the magnetic property of these nanospheres was also critically investigated and the magnetization was found to be affected by the particles' environment. The combination of these cell-targeting magnetic carriers with chemotherapeutic agents will be highly advantageous for the preferential killing of cancer cells in hyperthermia treatment.

Lamellipodia formation necessary for cell invasion is regulated by Rac1. We report here that lamellipodia formation and three-dimensional invasion were significantly promoted by HGF and serum, respectively, in invasive human breast cancer cells. Rac1 formed a complex with CLIP-170, IQGAP1, and kinesin in serum-starved cells, and stimulation of the cells with HGF and serum caused the partial release of IQGAP1 and kinesin from Rac1-CLIP-170 complex. The HGF-induced release of the proteins and promotion of lamellipodia formation were inhibited by an inhibitor of PI3K. Moreover, downregulation of CLIP-170 by siRNA released IQGAP1 and kinesin from Rac1 and promoted lamellipodia formation and invasion, independent of HGF and serum. The results suggest that promotion of lamellipodia formation and invasion by HGF or serum requires PI3K-dependent release of IQGAP1 and kinesin from Rac1-CLIP-170 complex and that CLIP-170 prevents cells from the extracellular stimulus-independent lamellipodia formation and invasion by tethering IQGAP1 and kinesin to Rac1.

A new method for the identification of intracellular structures of a living cell and obtaining the quantitative parameters characterizing these structures by means of coherent phase microscopy is proposed. The method is based on the analysis of the histogram of a cell phase image and its decomposition by phase height levels. In the spherical and cylindrical approximation of the cell, the method makes it possible to separate the contributions of phase-contrast intracellular structures to the integral refractive index of the whole cell. The calculation of refractive indices of intracellular structures is illustrated on a two-component model of a spherical cell. The possibility of determining the refractive indices of cellular organelles was shown by an example of cyanobacterium Anabaena variabilis ATCC 29413 cells and Cancer mammae breast cancer cells. Three most contrast cellular structures in the phase images of the cells were identified, and their refractive indices were determined. For Anabaena variabilis cells, these structures were the cell wall ( = 1.39), tylakoids ( = 1.47), and the nucleoid ( = 1.58); for breast cancer cells, these were the cytoplasm ( = 1.34), the nucleus ( = 1.36), and the nucleolus ( = 1.44). Using this method is possible to identify phase-contrast intracellular structures and calculate their refractive indices. This enables one to follow morphofunctional changes of not only the whole cell but also its individual organelles.

The metabolic and morphological characteristics of two human epithelial breast cell populations--MCF7 cells, a cancerous cell line, and 48R human mammary epithelial cells (48R HMECs), a noncancerous, finite lifespan cell strain--were compared at identical growth rates. Both cell types were induced to grow rapidly in nutrient-rich media containing 13C-labeled glucose, and the isotopic enrichment of cellular metabolites was quantified to calculate metabolic fluxes in key pathways. Despite their similar growth rates, the cells exhibited distinctly different metabolic and morphological profiles. MCF7 cells have an 80% smaller exposed surface area and contain 26% less protein per cell than the 48R cells. Surprisingly, rapidly proliferating 48R cells exhibited a 225% higher per-cell glucose consumption rate, a 250% higher per-cell lactate production rate, and a nearly identical per-cell glutamine consumption rate relative to the cancer cell line. However, when fluxes were considered on the basis of exposed area, the cancer cells were observed to have higher glucose, lactate, and glutamine fluxes, demonstrating superior transport capabilities per unit area of cell membrane. MCF7 cells also consumed amino acids at rates much higher than are generally required for protein synthesis, whereas 48R cells generally did not. Pentose phosphate pathway activity was higher in MCF7 cells, and the flux of glutamine to glutamate was less reversible. Energy efficiency was significantly higher in MCF7 cells, as a result of a combination of their smaller size and greater reliance on the TCA cycle than the 48R cells. These observations support evolutionary models of cancer cell metabolism and suggest targets for metabolic drugs in metastatic breast cancers.

Caveolin-1 is a principal component of caveolae, invaginations of the plasma membrane that are enriched in cholesterol and sphingolipids. The expression of caveolin-1 has been shown to be tightly correlated to the progression of breast cancer tumors. However, the consequences of altered caveolin-1 expression during tumor progression still remain unclear. Modification of caveolin-1 expression modulates store-operated Ca(2+) entry (SOCE) in various cell types. SOCE is a ubiquitous Ca(2+) entry pathway that previous studies have linked to apoptosis and tumor progression in prostate cancer cells. In this study, we tested the effect of altering caveolin-1 expression on SOCE in Hs578/T breast cancer cells. Through overexpression of caveolin-1 and small hairpin RNA (shRNA) knockdown, we generated four stable cell lines that have 3 different caveolin-1 protein levels. Cav-1 overexpression could increase SOCE activity, while knockdown of caveolin-1 significantly reduced SOCE activity. These functional consequences were correlated with changes in caveolae number in Hs578/T cells. Our results suggest alteration of SOCE by caveolin-1 expression changes could be one of the mechanisms contributing to the progression of breast cancer.

Two novel oestrogen receptor (ER) negative breast cancer cell lines, BCa-11 (familial) and BCa-15 (sporadic) were successfully established from primary tumours. Characterisation of these cell lines showed expression of epithelial specific antigen and cytokeratins confirming their epithelial lineage. Analysis of ultrastructure and anchorage independent growth confirmed the epithelial nature and transformed phenotype of these cells. Both cell lines showed loss of pRb, Dab2 and ERalpha and elevated levels of proliferation marker Ki67. In addition, BCa-11 cells showed loss of HOXA5, tumour suppressor genes p16(INK4A) and RARbeta as well as overexpression of CyclinD1. Elevation of DNMT1 and DNMT3B transcript levels, promoter hypermethylation of RASSF1A, RARbeta2, and HOXA5 further support their neoplastic origin. In conclusion, the two ERalpha negative breast cancer cell lines established herein have certain useful characteristics that may make them valuable for understanding the mechanism of oestrogen receptor negative breast tumours and testing new drugs.

Cancer cells migrating within a 3D microenvironment are able to adopt either a mesenchymal or amoeboid mode of migration. Amoeboid migration is characterised by membrane blebbing that is dependent on the Rho effectors, ROCK1/2. We identify LIMK2 as the preferred substrate for ROCK1 but find that LIMK2 did not induce membrane blebbing, suggesting that a LIMK2 pathway is not involved in amoeboid-mode migration. In support of this hypothesis, novel FRET data demonstrate a direct interaction between ROCK1 and LIMK2 in polarised but not blebbing cells. Our results point to a specific role for the ROCK1:LIMK2 pathway in mesenchymal-mode migration.

Radiotherapy has become an indispensable tool in the effective management of most of the cancers. There have been efforts earlier to study the differential radio-sensitivity patterns in patients undergoing radiation treatment to correlate with treatment induced complications such as tissue injury, cell death, and chromosomal aberration frequencies etc. The present study is an attempt to correlate the radiation-induced damage in the peripheral blood lymphocytes (PBLs) of breast cancer patients with the frequency of telomere mediated chromosomal damage. Blood samples from 55 patients with (Gr-II and Gr-III) CA-breast were obtained pre- and post-radiotherapy. The patients were treated with external beam radiotherapy of 50.4 Gy over a period of 6 weeks. Chromosome damage was measured by analysing micronucleus (MN) frequency in PBLs. The MN-frequency of the irradiated patients increased significantly compared to the patients being self-controls. The micronuclei were hybridized with telomere probes to study the extent of telomere damage. The fluorescence signals of the telomere regions in the first generation of the binucleated cells were significantly higher in the post-radiotherapy patients. There was also significant correlation observed in the patients with higher-grade tumours. Inter-individual variability was observed in the radiation-induced MN frequency in lymphocytes of patients after six weeks of radiotherapy. There was a significant correlation between functionally intact telomeres and the cellular response to ionising radiation. Our findings suggest that fluorescence in situ hybridisation on micronuclei could be effectively used as routine clinical application to determine the individual sensitivity to ionising radiation with respect to telomere damage.

FRAP (fluorescence recovery after photobleaching) and FCS (fluorescence correlation spectroscopy) are spectroscopic methods for monitoring the dynamic distribution of proteins inside the nucleus of living cells. As an example we report our studies on the intracellular mobility of the actin-binding protein CapG in live breast cancer cells. This Gelsolin-related protein is a putative oncogene. It appears to be overexpressed especially in metastasizing breast cancer. Furthermore, the CapG protein is known to be involved in the motility control of non-muscle benign cells. Its increased expression triggers an increase in cell motility of benign cells. Thus it can be expected that in cancer cells overexpressing the CapG protein, motility, invasiveness and metastasis might be particularly promoted. Since the nuclear CapG fraction seems to be pivotal to the increase in cell motility, we focused our studies on the CapG mobility in cell nuclei of live breast cancer cells. Using FCS and FRAP we showed that the eGFP-tagged CapG is monomeric and characterized its diffusional properties on the microsecond to minute timescale. This information about the mobility and compartmentalization of CapG might help to provide insight into its function within the cell nucleus and give clues about its altered cellular function in malignant dedifferentiation.

Decorin is a prototype member of the small leucine-rich proteoglycan family widely distributed in the extracellular matrices of many connective tissues, where it has been shown to play multiple important roles in the matrix assembly process, as well as in some cellular activities. A major interest for decorin function concerns its role in tumorigenesis, as growth-inhibitor of different neoplastic cells, and potential antimetastatic agent. The aim of our research was to investigate wide-ranged effects of transgenic decorin on breast cancer cells. To this purpose we utilized the well-characterized 8701-BC cell line, isolated from a ductal infiltrating carcinoma of the breast, and two derived decorin-transfected clones, respectively, synthesizing full decorin proteoglycan or its protein core. The responses to the ectopic decorin production were examined by studying morphological changes, cell proliferation rates, and proteome modulation. The results revealed new important antioncogenic potentialities, likely exerted by decorin through a variety of distinct biochemical pathways. Major effects included the downregulation of several potential breast cancer biomarkers, the reduction of membrane ruffling, and the increase of cell-cell adhesiveness. These results disclose original aspects related to the reversion of malignant traits of a prototype of breast cancer cells induced by decorin. They also raise additional interest for the postulated clinical application of decorin.

The genetic program that drives tumor metastasis and the mode and timing of its initiation are of great practical significance to clinical management. Modern technical advances open new opportunities for gaining useful relevant information. Gene expression profiles of histologically-verified viable tissue from lymph node metastases were compared with those of matched primary breast cancers from 10 different patients, among samples from over 400 cases, using high-throughput oligonucleotide arrays comprising probes for 22,000 genes. It was observed that metastases have very similar expression signatures to their parent tumors. However, detailed computational analysis revealed that a small number of genes were consistently differentially expressed between 100% of tumors and metastases, suggesting that these are mechanistically important. Lists of such candidate genes, of potential clinical interest, are provided. We interpret these results in the framework of a meta-analysis of previous investigations by others and ourselves and of existing clinical knowledge on the behavior of human tumors. The collective data show that metastases resemble their primary tumors but the signatures obtained in different studies are not sufficiently reproducible or informative to be prognostically useful, although they do give valuable insights into the pathogenesis and biology of human tumor metastasis. The findings indicate that the genetic program encoding metastasis is implemented progressively over time although, occasionally, this evolution can occur rapidly, early in the life of the neoplasm. The important clinical significance of this deduction is that, in most patients, early detection provides time for appropriate therapeutic intervention to be effective in obstructing metastasis.

Distributions of ErbB receptors on membranes of SKBR3 breast cancer cells were mapped by immunoelectron microscopy. The most abundant receptor, ErbB2, is phosphorylated, clustered and active. Kinase inhibitors ablate ErbB2 phosphorylation without dispersing clusters. Modest co-clustering of ErbB2 and EGFR, even after EGF treatment, suggests that both are predominantly involved in homointeractions. Heregulin leads to dramatic clusters of ErbB3 that contain some ErbB2 and EGFR and abundant PI 3-kinase. Other docking proteins, such as Shc and STAT5, respond differently to receptor activation. Levels of Shc at the membrane increase two- to five-fold with EGF, whereas pre-associated STAT5 becomes strongly phosphorylated. These data suggest that the distinct topography of receptors and their docking partners modulates signaling activities.

Dual-focus imaging optics for three-dimensional tracking of individual quantum dots has been developed to study the molecular mechanisms of motor proteins in cells. The new system has a high spatial and temporal precision, 2 nm in the x-y sample plane and 5 nm along the z-axis at a frame time of 2 ms. Three-dimensional positions of the vesicles labeled with quantum dots were detected in living cells. Vesicles were transported on the microtubules using 8-nm steps towards the nucleus. The steps had fluctuation of approximately 20 nm which were perpendicular to the axis of the microtubule but with the constant distance from the microtubule. The most of perpendicular movement was not synchronized with the 8-nm steps, indicating that dynein moved on microtubules without changing the protofilaments. When the vesicles changed their direction of movement toward the cell membrane, they moved perpendicular with the constant distance from the microtubule. The present method is powerful tool to investigate three dimensional movement of molecules in cells with nanometer and millisecond accuracy.

Background: We present an analysis of the utility of multispectral versus standard RGB imagery for routine H&E stained histopathology images, in particular for pixel-level classification of nuclei. Our multispectral imagery has 29 spectral bands, spaced 10 nm within the visual range of 420-700 nm. It has been hypothesized that the additional spectral bands contain further information useful for classification as compared to the 3 standard bands of RGB imagery. We present analyses of our data designed to test this hypothesis., Results: For classification using all available image bands, we find the best performance (equal tradeoff between detection rate and false alarm rate) is obtained from either the multispectral or our "ccd" RGB imagery, with an overall increase in performance of 0.79% compared to the next best performing image type. For classification using single image bands, the single best multispectral band (in the red portion of the spectrum) gave a performance increase of 0.57%, compared to performance of the single best RGB band (red). Additionally, red bands had the highest coefficients/preference in our classifiers. Principal components analysis of the multispectral imagery indicates only two significant image bands, which is not surprising given the presence of two stains., Conclusion: Our results indicate that multispectral imagery for routine H&E stained histopathology provides minimal additional spectral information for a pixel-level nuclear classification task than would standard RGB imagery.

Mucinous carcinoma of the breast (MCB) is histologically classified into 2 groups: (1) pure MCB and (2) mixed MCB. Pure MCB carries a better diagnosis than mixed MCB. This research relates to the cell surface topography and ultrastructure of the cells in the above cases and aims to find the differences between them, by means of two methods: scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For the SEM examination, it was necessary to initially culture the MCB tissues and then proceed with the usual SEM method. In contrast, for the TEM technique, MCB tissues were initially fixed followed by the classic TEM method. The authors found the topography of pure MCB cases to be without nodes. The cell membrane was smooth, with numerous pores and small ruffles that covered the entire cell. The ultrastructural appearance of the same cases was with a normal cell membrane containing abundant collagen fibers. They also had many small vesicles containing mucin as well as secretory droplets. In contrast the mixed MCB had a number of lymph nodes and their cell surface topography showed stronger changes such as microvilli, numerous blebs, ruffles and many long projections. Their ultrastructure showed very long microvilli with large cytoplasmic inclusions and extracellular mucin collections, electron-dense material vacuoles, and many important cytoplasmic organelles. An important fact is that mixed MCB also contains areas of infiltrating ductal carcinoma. These cells of the cytoplasmic organelles are clearly responsible for the synthesis, storage, and secretion of the characteristic mucin of this tumor type. Evidently, this abnormal mucin production and the abundance of secretory granules along with the long projections observed in the topographical structure might be responsible for transferring tumor cells to neighboring organs, thus being responsible for metastatic disease.

Three cell lines, that is, the human breast cancer cell line (MCF-7) and the human mammary epithelial cell line (S-1) and its malignant form (T4-2) were embedded in a reconstituted basement membrane (Matrigel) that had 20-nL pyramid-shaped silicon microstructures. The proliferative behavior of the MCF-7 cells was dependent on the surrounding conditions (2-D, collagen gel, or Matrigel), whereas the respiratory activity of a single cell (F(c)) was almost identical under different culture conditions. The F(c) value changed with cellular polarity. The F(c) value for the S-1 cells was observed to decrease slightly, whereas that of the T4-2 cells increased 2 days after cultivation in the microstructures within the Matrigel. However, when the T4-2 cells were cultured in the presence of tyrphostin AG 1478 (T4-2 tyr) to inhibit epidermal growth factor (EGF) signaling, the F(c) value decreased slightly and remained almost constant for an additional 1 week; this was similar to the behavior of the S-1 cells. Further, fluorescence images showed that the T4-2 tyr cells formed polar structures that were similar to those formed by the S-1 cells whereas the T4-2 cells did not form such structures. These results indicate that cellular polarity can be assessed by measuring cellular respiratory activity.

We report an easy and stable transfection technique using electrogene transfer with a nonviral Epstein-Barr (EB) virus-based vector. To achieve stable transfection of human breast cancer cells, we conducted electrogene transfer of an EB virus-based plasmid vector (reduced size of oriP) containing the enhanced green fluorescence protein (eGFP) gene. Because the EB virus-based vector exhibits high transfer efficiency and strong persistent transgene expression as a result of autonomous replication in human cells, and as Nucleofector electrogene transfer can achieve highly efficient gene transfection, this method is particularly suitable for generation of stably transfected cell lines.

Breast cancer cells are known to overexpress Glut5, a sugar transporter responsible for the transfer of fructose across the cell membrane. Since Glut5 transporter is not significantly expressed in normal breast cells, fructose uptake can potentially be used to differentiate between normal and cancerous cells. Fructose was labeled with two fluorophores at the C-1 position: 7-nitro-1,2,3-benzadiazole (NBD) and Cy5.5. The labeling site was chosen on the basis of the presence and substrate specificity of the key proteins involved in the first steps of fructose metabolism. Using fluorescence microscopy, the uptake of the probes was studied in three breast cancer cell lines: MCF 7, MDA-MB-435, and MDA-MB-231. Both fluorescent fructose derivatives showed a very good uptake in all tested cell lines. The level of uptake was comparable to that of the corresponding glucose analogs, 2-NBDG and Cy5.5-DG. Significant uptake of 1-NBDF derivative was not observed in cells lacking Glut5 transporter, while the uptake of the 1-Cy5.5-DF derivative was independent of the presence of a fructose-specific transporter. While 1-NBDF showed Glut5-specific accumulation, the coupling of a large fluorophore such as Cy5.5 likely introduces big structural and electronic changes, leading to a fructose derivative that does not accurately describe the uptake of fructose in cells.

The importance of microenvironment and context in regulation of tissue-specific genes is well established. DNA exposure to or the sequestration from nucleases detects differences in higher order chromatin structure in intact cells without disturbing cellular or tissue architecture. To investigate the relationship between chromatin organization and tumor phenotype, we used an established three-dimensional assay in which normal and malignant human breast cells can be easily distinguished by the morphology of the structures they make (acinus-like versus tumor-like, respectively). We show that these phenotypes can be distinguished also by sensitivity to AluI digestion in which the malignant cells resist digestion relative to nonmalignant cells. Treatment of T4-2 breast cancer cells in three-dimensional culture with cAMP analogs or a phosphatidylinositol 3-kinase inhibitor not only reverted their phenotype from nonpolar to polar acinar-like structures but also enhanced chromatin sensitivity to AluI. By using different cAMP analogs, we show that cAMP-induced phenotypic reversion, polarization, and shift in DNA organization act through a cAMP-dependent protein-kinase A-coupled signaling pathway. Importantly, inhibitory antibody to fibronectin produced the same effect. These experiments underscore the concept that modifying the tumor microenvironment can alter the organization of tumor cells and demonstrate that architecture and global chromatin organization are coupled and highly plastic.

Background: We compared the Nottingham histological grade (H-grade) and the Japanese nuclear grade (N-grade) to select the better prognostic factor for breast cancers., Methods: The series included 1786 patients with breast cancers with the exception of non-invasive and stage 4 cancers. They were classified according to the H- and N-grade. We analyzed their survival curves and also performed multivariate Cox regression analyses., Results: According to the H-grade classification, 476 cases were grade 1, 647 cases were grade 2 and 663 cases were grade 3. According to the N-grade, 381 cases were grade 1, 215 cases were grade 2, and 1129 cases were grade 3. In the survival curves of those cases with lymph node metastases (N+) and recurrent cases, there were statistically significant differences in different categories of the H-grades, but not in the N-grades. The survival curves of all the cases and those cases without lymph node metastases (N-) always exhibited statistically significant differences. According to the 2003 St Gallen consensus, the N- group was classified as a minimal risk and an average risk groups. Both H- and N-grade exhibited statistically significant differences between the minimal risk and the average risk groups in the disease-free survival. The multivariate analyses proved that the H-grade was a statistically significant prognostic factor in all the cases and N+ group, but the N-grade was not significant in any of the studies., Conclusions: The H-grade is clearly proved to be a more significant prognostic factor for wider stage cases than the N-grade.

Background: Gliotoxin belongs to a group of compounds produced by fungi, all of them having a bridged polysulfide piperazine ring in their chemical structure. This internal polysulfide bridge enables them to carry out various biofunctions, but so far, the toxicity of these compounds limited them to be used as medicines in clinic. However, the toxicities of these compounds are quite different and determined by their different part of chemical structures. Therefore, it is still possible to find a suitable low toxic compound for drug use. As for anticancer drug developing, the first need is to confirm the anticancer activity in vivo., Materials and Methods: The morphological changes of human breast cancer MCF-7 cells affected by gliotoxin in culture, and the structural damages of human cancer xenograft tissue in SCID mice after intra-tumor injection of gliotoxin were observed after histological stain and transmission electromicroscopic treatment. The DNA changes of the human colon cancer xenograft were observed in 1.2% agarose gel electrophoresis., Results: Gliotoxin 1 or 5 microM in medium for 24 hours induced typical apoptotic structural changes to MCF-7 cells, the cell surface membrane showed blebbing clearly. Injection of 1 mg gliotoxin into the tumor tissue directly did not induce noticeable side-effects to the host mice but induced complete damage of the cell structure, the cell surface membran broken down and the components of the nuclei segmented. The whole cancer tissue shrinked and finally formed a dark color scab which came off from the skin few days later. The cured mice showed no tumor recurrence in the six months following observation. The apoptotic DNA damage was also found in human colon cancer xenograft C1-2 tissues after gliotoxin was injected inside the tumor tissue., Conclusion: The anticancer activity of gliotoxin is confirmed in vivo.

Purpose: The distribution and significance of caveolin 1 (CAV1) expression in different breast cell types and role in breast carcinogenesis remain poorly understood. Both tumor-suppressive and oncogenic roles have been proposed for this protein. The aims of this study were to characterize the distribution of CAV1 in normal breast, benign breast lesions, breast cancer precursors, and metaplastic breast carcinomas; to assess the prognostic significance of CAV1 expression in invasive breast carcinomas; and to define whether CAV1 gene amplification is the underlying genetic mechanism driving CAV1 overexpression in breast carcinomas., Experimental Design: CAV1 distribution in frozen and paraffin-embedded whole tissue sections of normal breast was evaluated using immunohistochemistry, immunofluorescence, and immunoelectron microscopy. CAV1 expression was immunohistochemically analyzed in benign lesions, breast cancer precursors, and metaplastic breast carcinomas and in a cohort of 245 invasive breast carcinomas from patients treated with surgery followed by anthracycline-based chemotherapy. In 25 cases, CAV1 gene amplification was assessed by chromogenic in situ hybridization., Results: In normal breast, CAV1 was expressed in myoepithelial cells, endothelial cells, and a subset of fibroblasts. Luminal epithelial cells showed negligible staining. CAV1 was expressed in 90% of 39 metaplastic breast carcinomas and in 9.4% of 245 invasive breast cancers. In the later cohort, CAV1 expression was significantly associated with 'basal-like' immunophenotype and with shorter disease-free and overall survival on univariate analysis. CAV1 gene amplification was found in 13% of cases with strong CAV1 expression., Conclusions: The concurrent CAV1 amplification and overexpression call into question its tumor-suppressive effects in basal-like breast carcinomas.

Aims: To evaluate the nuclear morphometric features of breast columnar cell lesions (CCLs) observed on mammotome core biopsies, to determine if there are significant measurable differences between those with atypia and those without. Correlation with follow-up open excision specimens was made., Methods: Mammotome core biopsies performed on patients that contained CCLs were derived from the departmental case files. Histological material was reviewed and foci of CCLs demarcated for nuclear morphometric assessment, which was accomplished using an imaging system. Nuclear parameters studied were nuclear area and perimeter, circularity factor and feret's diameter. Statistical analysis used the GraphPad Prism software, with p<0.05 indicating significance., Results: On examination of core biopsies of 40 patients with CCLs, 8 lesions were benign, 4 showed atypical lobular hyperplasia, 8 showed CCLs with nuclear atypia, 19 disclosed atypical ductal hyperplasia (ADH) and 1 showed ductal carcinoma in situ (DCIS). The nuclear area, perimeter and feret's diameter of CCLs with atypia were significantly greater than those without (p = 0.04, 0.03 and 0.019, respectively), whereas no difference was observed in the circularity factor. Follow-up open excision biopsy specimens in 24 patients showed upgrading to DCIS in 40% of cases diagnosed initially with ADH on core biopsy compared with 20% of CCLs with atypia., Conclusions: Nuclear morphometry in CCLs confirms nuclear size as the key parameter in the assessment of nuclear atypia. Whether it can be potentially used as an adjunctive tool depends on the establishment of appropriate cut-offs.

Transendothelial migration of cancer cells from the vasculature into tissue stroma is a final step in the metastatic cascade, prior to formation of secondary tumors. Due to its role in 2-dimensional migration of cells on extracellular matrix proteins, we hypothesized that focal adhesion kinase (FAK) promotes transendothelial migration of cancer cells. AU-565 cells are weakly invasive metastatic breast adenocarcinoma cells that migrate through bovine lung microvessel endothelial cell monolayers. Electric cell-substrate impedance sensing detects a significant decrease in monolayer resistance upon addition of AU-565 cells. Immunofluorescence microscopy and filter-based migration assays demonstrate that this drop in resistance correlates with transendothelial migration. Transfection of AU-565 cells with FAK siRNA results in significantly diminished transendothelial migration of AU-565 cells within 15h. Expression of the dominant negative FAK inhibitor FAK-related non-kinase (FRNK) also results in delayed AU-565 transendothelial migration, whereas over-expression of wildtype FAK does not impact transendothelial migration substantially. These results demonstrate that FAK affects the rate of a key step in the metastatic cascade.

Photodynamic therapy (PDT) is a novel and promising cancer treatment which employs a combination of a photosensitizing chemical and visible light to induce apoptosis in cancer cells. Singlet oxygen has been recognized as the main origin of oxidative stress in PDT. However, the precise mechanism of PDT-induced apoptosis is not well characterized, especially the dualistic role of nitric oxide (NO). To dissect the apoptosis pathways triggered by PDT, the intracellular free radicals in MCF-7 cells were investigated by examining a novel photosensitizer 2-butylamino-2-demethoxyhypocrellin B (2-BA-2-DMHB)-mediated PDT. It was found that exposure of the cells to 2-BA-2-DMHB and irradiation resulted in a significant increase of intracellular ROS in minutes, and then followed by cytoplasmic free calcium enhancement, mitochondrial nitric oxide synthase (mtNOS) activation, cytochrome c release, and apoptotic death. Scavengers of singlet oxygen or NO could attenuate PDT-induced cell viability loss, nucleus morphology changes, cytochrome c release, mitochondria swelling, and apo-apoptosis gene p53 and p21 mRNA levels. The results suggested that both ROS and NO played important roles in the apoptosis-induced by PDT., (Copyright 2006 Elsevier Inc.)

We successfully established a breast scirrhous carcinoma cell line (designated as NABCA) derived from metastatic tumors of the lymph node. The cells grew as multi-layered cultures without contact inhibition. The population doubling time was approximately 66 h. G-band karyotype of NABCA revealed 66% diploid, XX. Surprisingly, the cells had a number of secretory granules and straight microvilli as a brash border. In heterotransplantation, the cells produced a tumor resembling the original tumor. The NABCA is sensitive to Adriamycin (doxorubicin; KYOWA HAKKO KOGYO, Tokyo, Japan) and Taxol (paclitaxel; Bristol-Myers KK, Tokyo, Japan). This cell line is useful for studying the mechanism of lymphatic metastasis and susceptibility of anticancer drugs in human breast scirrhous cancer.

Diffuse near-infrared tomography of tissue reveals scattering changes that originate from the submicroscopic features of the tissue; yet the existing tools to use this information to predict which features contribute to the scattering spectrum are limited by the lack of direct data quantifying the particle sizes. Breast tissue was examined with electron microscopy, and analysis showed that the distributions of particle sizes appear in double exponential functions for most cellular tissues. The average particle size histograms of high-grade cancer, low-grade cancer, fibroglandular tissue, and adipose tissue were examined. The particle histograms were progressively decreasing in magnitude for these tissue types, and the average size of the particles increased, for these four tissues, respectively. Typical particle sizes in the range of 10 to 500 nm for these tissue types, with biexponential fitting, gave two particle distributions: one near 20 to 25 nm for the smaller size and one at 110 to 230 nm for the larger distributions. Mie scatter theory was used to take these particle distributions and calculate scattering spectra. The ability to image reduced scattering coefficient spectra of bulk breast tissues exists, and so this data provides insight into how bulk imaging may be mapped over to predict factors related to the tissue ultrastructure.

PRIMA-1 has emerged as a small molecule that restores the wild type function to mutant p53. To identify molecular targets that are involved in PRIMA-1-induced apoptosis, we used a proteomics approach with two-dimensional gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry for protein identification. By comparing the proteome of the PRIMA-1-treated MDA-231 breast carcinoma cells with that of MCF-7 cells, we have identified seven proteins that upregulated only in MDA-231 cells as a result of PRIMA-1-induced apoptosis. The identified proteins are involved in anaerobic glycolysis and in mitochondrial intrinsic apoptosis. Treatment of MDA-231 cells with PRIMA-1 resulted in the release of mitochondrial cytochrome c as well as the activation of caspase-3, which are essential for the execution of apoptosis. We present evidence to suggest that PRIMA-1-induced apoptosis in breast cancer cells with mutated p53 function involved the expression of proteins required for the activation of mitochondrial intrinsic pathway that is glycolysis-relevant.

Gene amplification and protein overexpression of MDM2, which is often found in certain types of cancers, indicate that MDM2 plays an important role in tumorigenesis. Interestingly, several clinical reports have demonstrated that amplification of the MDM2 gene correlates with the metastatic stage. Using an antibody array assay, we identified E-cadherin as an MDM2-binding protein and confirmed that E-cadherin is a substrate for the MDM2 E3 ubiquitin ligase. We demonstrate that MDM2 interacts in vivo with E-cadherin, resulting in its ubiquitination and degradation. This regulation appears to be clinically relevant, as we found a significant correlation between high MDM2 and low E-cadherin protein levels in resected tumor specimens recovered from breast cancer patients with lymph node metastases. Ectopic expression of MDM2 in breast cancer cells was found to disrupt cell-cell contacts and enhance cell motility and invasive potential. We found that E-cadherin and MDM2 colocalized on the plasma membrane and in the early endosome, where ubiquitin moieties were attached to E-cadherin. Blocking endocytosis with dominant-negative mutants of dynamin abolished the association of MDM2 with E-cadherin, prevented E-cadherin degradation, and attenuated cell motility as observed by fluorescence microscopy. Thus, we provide evidence to support a novel role for MDM2 in regulating cell adhesions by a mechanism that involves degrading and down-regulating the expression of E-cadherin via an endosome pathway. This novel MDM2-regulated pathway is likely to play a biologically relevant role in cancer metastasis.

Crude extracts of the marine sponge Geodia corticostylifera from Brazilian Coast have previously shown antibacterial, antifungal, cytotoxic, haemolytic and neurotoxic activities. The present work describes the isolation of the cyclic peptides geodiamolides A, B, H and I (1-4) from G. corticostylifera and their anti-proliferative effects against sea urchin eggs and human breast cancer cell lineages. Its structure-activity relationship is discussed as well. In an initial series of experiments these peptides inhibited the first cleavage of sea urchin eggs (Lytechinus variegatus). Duplication of nuclei without complete egg cell division indicated the mechanism of action might be related to microfilament disruption. Further studies showed that the geodiamolides have anti-proliferative activity against human breast cancer cell lines (T47D and MCF7). Using fluorescence techniques and confocal microscopy, we found evidence that the geodiamolides A, B, H and I act by disorganizing actin filaments of T47D and MCF7 cancer cells, in a way similar to other depsipeptides (such as jaspamide 5 and dolastatins), keeping the normal microtubule organization. Normal cells lines (primary culture human fibroblasts and BRL3A rat liver epithelial cells) were not affected by the treatment as tumor cells were, thus indicating the biomedical potential of these compounds.

Background: Distant spread from breast cancer is commonly found in bones, lungs, liver and central nervous system. Metastatic involvement of peritoneum and retroperitoneum is unusual and unexpected., Case Presentation: We report the case of a 67 year-old-woman who presented with gastrointestinal symptoms which revealed to be the clinical manifestations of peritoneal and retroperitoneal metastatic spread of an invasive lobular breast cancer diagnosed 15 years before., Conclusion: To the best of our knowledge, the case presented is the third one reported in literature showing a wide peritoneal and extraperitoneal diffusion of an invasive lobular breast cancer. The long and complex diagnostic work up which led us to the diagnosis is illustrated, with particular emphasis on the multidisciplinary approach, which is mandatory to obtain such a result in these cases. Awareness of such a condition by clinicians is mandatory in order to make an early diagnosis and start a prompt and correct therapeutic approach.

Carrier-mediated delivery of drugs into the cytosol is often limited by either release from the carrier or release from an internalizing endolysosome. Here, loading, delivery, and cytosolic uptake of drug mixtures from degradable polymersomes are shown to exploit both the thick membrane of these block copolymer vesicles and their aqueous lumen as well as pH-triggered release within endolysosomes. Our initial in vivo studies demonstrate growth arrest and shrinkage of rapidly growing tumors after a single intravenous injection of polymersomes composed of poly(ethylene glycol)-polyester. Vesicles are shown to break down into membrane-lytic micelles within hours at 37 degrees C and low pH, although storage at 4 degrees C allows retention of drug for over a month. It is then shown that cell entry of the polymersomes into endolysosomes is followed by copolymer-induced endolysosomal rupture with release of cytotoxic drugs. Above a critical poration concentration (CCPC) that is easily achieved within endolysosomes and that scales with copolymer proportions and molecular weight, the copolymer micelles are seen to disrupt lipid membranes and thereby enhance drug activity. Neutral polymersomes and related macrosurfactant assemblies can thus create novel pathways within cells for controlled release and delivery.

This paper describes the use of fluorescent silica nanospheres as luminescent signal amplifiers in biological assays based on digital counting of individual particles instead of measuring averaged fluorescence intensity. We recently described a simple method to prepare highly fluorescent mono-dispersed silica nanospheres that avoids microemulsion formulations and the use of surfactants. Modification of the Stöber method was used successfully to prepare fluorescent silica spheres with the inorganic dye dichlorotris(1,10-phenanathroline)ruthenium (II) hydrate encapsulated during the condensation of tetraethylorthosilicate in ethanol and dye aqueous mixtures. Modifications in the ammonia and water content in the reaction mixture resulted in mono-dispersed silica spheres of 65, 440 and 800 nm in diameter. The dye-encapsulating particles emit intense red luminescence when excited at 460 nm. We observed an increased photostability and longer fluorescence lifetime in our particles that we attributed to increased protection of the encapsulated dye molecules from molecular oxygen. The newly prepared fluorescent silica particles were easily modified using trialkoxysilane reagents for covalent conjugation of anti-HER2/neu. We demonstrated the utility of the fluorescent nanospheres to detect the cancer marker HER2/neu in a glass slide based assay. The assay was shown to be simple but highly sensitive with a limit of detection approaching 1 ng/mL and a linear range between 1 ng/mL and 10 microg/mL of HER2/neu.

Growth of FRM cells was inhibited by the addition of pyridoxine in a dose-dependent manner. Use of 5 mM pyridoxine caused an almost complete arrest of cell growth. Pyridoxal was as effective as pyridoxine, but pyridoxamine showed weak inhibitory action. Electron-microscopic examination of control cells revealed large nuclei and cellular membranes with villi, but, in pyridoxine-treated cells, condensed or degraded nuclei were observed. Many vacuoles and cholesterol crystals were widely distributed inside the cellular membrane of pyridoxine-treated cells. One of the vacuoles was identified as a lipid droplet. The DNA ladder was observed in the pyridoxine-treated cells. It is suggested that pyridoxine treatment of FRM cells causes cytolysis of cells by apoptosis.

The chemotherapeutic drug paclitaxel induces microtubular stabilization and mitotic arrest associated with increased survivin expression. Survivin is a member of the inhibitor of apoptosis (iap) family which is highly expressed in during G2/M phase where it regulates spindle formation during mitosis. It is also constitutively overexpressed in most cancer cells where it may play a role in chemotherapeutic resistance. MCF-7 breast cancer cells stably overexpressing the sense strand of survivin (MCF-7(survivin-S) cells) were more resistant to paclitaxel than cells depleted of survivin (MCF-7(survivin-AS) despite G2/M arrest in both cell lines. However, survivin overexpression did not protect cells relative to control MCF-7(pcDNA3) cells. Paclitaxel-induced cytotoxicity can be enhanced by retinoic acid and here we show that RA strongly reduces survivin expression in MCF-7 cells and prevents paclitaxel-mediated induction of survivin expression. Mitochondrial release of cytochrome c after paclitaxel alone or in combination with RA was weak, however robust Smac release was observed. While RA/paclitaxel-treated MCF-7 (pcDNA3) cultures contained condensed apoptotic nuclei, MCF-7(survivin-S) nuclei were morphologically distinct with hypercondensed disorganized chromatin and released mitochondrial AIF-1. RA also reduced paclitaxel-associated levels of cyclin B1 expression consistent with mitotic exit. MCF-7(survivin-S) cells displayed a 30% increase in >2N/<4N ploidy while there was no change in this compartment in vector control cells following RA/paclitaxel. We propose that RA sensitizes MCF-7 cells to paclitaxel at least in part through survivin downregulation and the promotion of aberrant mitotic progression resulting in apoptosis. In addition we provide biochemical and morphological data which suggest that RA-treated MCF-7(survivin-S) cells can also undergo catastrophic mitosis when exposed to paclitaxel.

Background & Objective: Electrochemotherapy mediated by electric pulse has become a multidisciplinary biomedical engineering technique in modern medical science. Its main mechanisms are enhancing the diffusion of chemotherapeutic drugs, antibodies, or genes into the inner part of tumor cells mediated by membrane-electropermeabilization caused by electric pulse. Our previous studies confirmed that steep pulsed electric field (SPEF) could irreversibly cause membrane electropermeabilization, and lead to death of tumor cells. This study was to explore the acute killing effects of SPEF on lymphatic capillaries in VX2 implanted breast cancer in rabbits., Methods: Tumor model of VX2 implanted breast cancer was successfully established in rabbits. Isosulfan blue staining, 5'-AMP-ALPase enzymohistochemical double staining, and electron microscopy was used to observe the morphologic changes of local lymphatic capillaries around cancer tissues exposed to SPEF., Results: After exposed to SPEF, no lymphatic vessels were found with isosulfan blue staining, only blurred structures were observed; enzymohistochemistry showed no positively stained lymphatic vessels, only fragmental structures around cancer tissues were observed; integrity and continuity of lymphatic endothelium were destroyed under transmission electron microscope., Conclusion: SPEF has the potential to destroy lymphatic capillaries around VX2 implanted breast cancer, and can decrease the possibility of post-treatment lymphatic metastasis.

The development of effective cancer therapies has been hampered, in part, by the inability to noninvasively follow tumor progression from the initial cancerous lesion through to metastasis. We have previously shown that superparamagnetic iron oxide particles can be used as magnetic resonance imaging contrast agents to label embryonic, mesenchymal and hematopoietic stem cells in vivo. Improving the capacity to non-invasively image cancer progression is an appealing method that could be useful for assessing the efficacy of anticancer therapies. We have established that human prostate (LNCaP, DU145, PC3), rodent prostate (TRAMPC1, YPEN-1), human breast (MDA-MB-231) and mouse mammary (Myc/VEGF) cancer cell lines were readily labeled by fluorescent superparamagnetic sub-micron particles of iron oxide (MPIOs). The MPIOs were essentially inert with respect to cell proliferation and tumor formation. Fluorescence stereomicroscopy and three dimensional magnetic resonance imaging (MRI) determined that subcutaneous, intramuscular or orthotopically implanted labeled cancer cells could be imaged, in vivo, despite in some cases being undetectable by manual palpation. The MPIO-labeled cancer cells could also be imaged, in vivo, at least 6 weeks after implantation. The fluorescent MPIOs further allowed for the ex vivo identification of tumors cells from histological sections. This study demonstrates the feasibility of using fluorescent MPIOs in prostate and breast cancer cell lines as both a negative contrast agent for in vivo MRI as well as a fluorescent tumor marker for optical imaging in vivo and ex vivo.

Objective: To compare the cytologic features benign and malignant papillary breast lesions., Study Design: We reviewed the clinical and cytologic features in 29 cases of intraductal papilloma and 26 cases of atypical papilloma or papillary carcinoma that had been diagnosed by histologic examination. The diameter of the mass was examined as a clinical feature. The cytologic features evaluated were as follows: bloody background, row of tall columnar cells, naked bipolar nuclei, hemosiderin-laden macrophages, myoepithelial cells, single scattered atypical cells, cellularity, nuclear atypia, nuclear grade, apocrine metaplasia, eosinophilic cytoplasmic granules, papillary clusters, small papillae, cell balls and large sheets., Results: Of the features evaluated, the diameter of the mass, naked bipolar nuclei and cell balls differed significantly between benign and atypical or malignant papillary neoplasms. The average diameter of a benign papillary neoplasm was 1.8 cm, and that of an atypical or malignant papillary neoplasm was 2.2 cm (p = 0.042). Naked bipolar nuclei were found in 27 cases of benign papillary neoplasm (93.1%) versus 19 cases of atypical or malignant papillary neoplasm (73.1%) (p = 0.050). Cell balls were found in 14 (48.3%) and 21 (80.8%) cases, respectively (p = 0.012). All 6 cases in which cell balls were present and naked bipolar nuclei were absent proved to be atypical or malignant papillary neoplasms. Of 17 cases in which cell balls were absent and naked bipolar nuclei present, 13 (76.5%) were benign papillary neoplasms., Conclusion: Most cytologic features overlapped in benign and atypical or malignant papillary neoplasms. Although they were not pathognomonic, naked bipolar nuclei and cell balls were cytologic features that differed significantly between benign and atypical or malignant papillary neoplasms. When papillary neoplasms of the breast are suspected in a cytologic smear, the combination of clinical examination, mammography and cytologic features should be considered to make the correct diagnosis.

Microtubules (MT) are dynamic polymers that rapidly transition between states of growth, shortening, and pause. These dynamic events are critical for many microtubule functions such as intracellular trafficking and signaling. In addition, cancer chemotherapy drugs that target microtubules, such as the taxanes and the vinca alkaloids, are known to suppress microtubule dynamics at low doses, leading to mitotic arrest and cell death. Quantification of microtubule dynamics can be used as a read-out of anticancer-drug activity and can be a surrogate marker of drug sensitivity/resistance. The emerging nanotechnology such as quantum dots has provided properties such as less photo bleaching, higher probe imaging intensity, better specificity and sensitivity, which finally makes visualizing subcellular events over long enough time a possibility. But it also results in big increase in data acquisition. The traditional way of annotating MT manually is becoming a daunting task. Thus, the goal is to research and develop an efficient, reliable, and rapid MT tracking. In this paper, we describe active contour-based tracking methods to automatically track MT. We redefine the internal energy terms specifically for open snake, and examine different external energy terms for locating the end tips of a microtubule. This algorithm has been validated using simulated images, images of untreated MCF-7 breast cancer cells, and image of cells treated with the microtubule-targeting chemotherapeutic agent, Taxol.

The normal mammary gland can be roughly divided into the large duct close to the nipple and the terminal duct located within the lobulus. Both the large duct and the terminal duct are composed of epithelial cells and myoepithelial cells. The epithelial cells can be divided into light and dark cells using electron density. Heterochromatin is the predominant type of chromatin found in normal mammary glands. The cytoplasm of myoepithelial cells contains a number of fine filaments that possess dense patches. The myoepithelial cells of the large duct have a large process with a crablike appearance that protrudes from the cytoplasm. The myoepithelial cells of the terminal duct, by contrast, assume a relatively flat form and are approximately parallel to the epithelial-stromal junction. If the nuclei of the epithelial cells of normal mammary glands and benign breast lesions are compared with those of malignant breast lesions, the latter are primarily oval or circular in shape whereas the former often show marked notches. The predominant chromatin is heterochromatin in noncancer cells and euchromatin in cancer cells. The intracytoplasmic lumen (ICL) can be roughly divided into two types. The ICL is frequently seen in breast cancers, especially scirrhous carcinoma and lobular carcinoma. Invasive ductal carcinoma can be divided into three types: papillotubular carcinoma, solid-tubular carcinoma, and scirrhous carcinoma. Scirrhous carcinoma can be divided into two subtypes: scirrhous carcinoma in the broader sense of the term (characterized by scirrhous invasion of the stroma by papillotubular carcinoma or solid-tubular carcinoma), and scirrhous carcinoma in the narrower sense of the term (characterized by linear or cluster-like invasion of the stroma without forming ducts). Ultrastructural characteristics of scirrhous carcinoma in the narrow sense are bright cytoplasm (seen in most cells) and euchromatin (seen in all cells of this type of carcinoma). In cases of papillotubular carcinoma, solid-tubular carcinoma, and scirrhous carcinoma in the broad sense, euchromatin is predominant but sporadic cells with heterochromatin are also seen. Adenoid cystic carcinoma and carcinoid tumor of the breast are histological types of breast carcinoma that show characteristic features under an electron microscope. Ultrastructurally, the former shows a pseudocyst and true lumen whereas the latter presents numerous neuroendocrine granules within the cytoplasm. Breast carcinoma shows several ultrastructural characteristics that are useful in differential diagnosis. Therefore, it is advisable to take electron microscopic findings into account when evaluating or diagnosing breast lesions.

Exosomes are nanovesicles that are released into the extracellular environment during the fusion of multivesicular bodies with the plasma membrane. Exosomes released from dendritic cells, dexosomes, have several biological functions, for example as immunostimulants. Some tumor cells also secrete exosomes (Tu-exosomes). Although experimental data obtained with the use of dexosomes suggest a biological function of Tu-exosomes, this still remains poorly understood. To examine the function of Tu-exosomes, we established a method for collecting highly purified Tu-exosomes, using paramagnetic beads coated with antibodies against tumor-specific proteins such as HER2/neu. With these antibody-coated beads (Ab-beads), it was possible to collect HER2-expressing Tu-exosomes of high purity. Tu-exosomes were also collected from malignant ascites, which contain exosomes secreted from various types of cells such as tumor cells, lymphoid cells and mesothelial cells. The isolation of Tu-exosomes was confirmed by FACS analysis. With regard to their biological functions, Tu-exosomes cultured with a human breast cancer cell line bound to the cell surface and increased tumor cell proliferation. These data indicate that Tu-exosomes may have physiological functions.

New therapeutic strategies are required to overcome the limitations of conventional breast cancer treatment. Suicide gene therapy offers a potential approach to this type of tumour, since systems based on the use of prodrugs may present some drawbacks related to toxicity, drug release and bioavailability. The gef gene has cell-killing functions in Escherichia coli and does not depend on the use of a prodrug for its action, making it an attractive target for suicide gene therapy. We created a gef-overexpressing human breast cancer cell line (MCF-7TG) by transfecting the gef gene under the control of a pMAMneo promotor. Dexamethasone-induction of gef gene expression in MCF-7TG cells produced a significant decrease in Ki-67 expression, which is a known proliferation marker. In addition, annexin-V-FITC and propidium iodide assays showed the presence of apoptotic cell death, which was confirmed by scanning electron microscopy. The most significant finding was the presence of "craters" in the cell membrane, as previously described in other apoptotic breast cancer cells. These results demonstrate the ability of the gef gene to down regulate Ki-67 expression and induce apoptosis in a breast cancer cell line, suggesting its potential application as a new gene therapy strategy for this type of tumor.