Targeting carbonic anhydrase IX depletes breast cancer stem cells within the hypoxic niche.

The sub-population of tumor cells termed 'cancer stem cells' (CSCs) possess the capability to generate tumors, undergo epithelial-mesenchymal transition (EMT) and are implicated in metastasis, making treatments to specifically target CSCs an attractive therapeutic strategy. Tumor hypoxia plays a key role in regulating EMT and cancer stem cell function. Carbonic anhydrase IX (CAIX) is a hypoxia-inducible protein that regulates cellular pH to promote cancer cell survival and invasion in hypoxic microenvironments and is a biomarker of poor prognosis for breast cancer metastasis and survival. Here, we demonstrate that inhibition of CAIX expression or activity with novel small-molecule inhibitors in breast cancer cell lines, or in primary metastatic breast cancer cells, results in the inhibition of breast CSC expansion in hypoxia. We identify the mTORC1 axis as a critical pathway downstream of CAIX in the regulation of cancer stem cell function. CAIX is also required for expression of EMT markers and regulators, as well as drivers of 'stemness', such as Notch1 and Jagged1 in isolated CSCs. In addition, treatment of mice bearing orthotopic breast tumors with CAIX-specific small-molecule inhibitors results in significant depletion of CSCs within these tumors. Furthermore, combination treatment with paclitaxel results in enhanced tumor growth delay and eradication of lung metastases. These data demonstrate that CAIX is a critical mediator of the expansion of breast CSCs in hypoxic niches by sustaining the mesenchymal and 'stemness' phenotypes of these cells, making CAIX an important therapeutic target for selectively depleting breast CSCs.

Both lamin A and lamin C mutations cause lamina instability as well as loss of internal nuclear lamin organization.

We have applied the fluorescence loss of intensity after photobleaching (FLIP) technique to study the molecular dynamics and organization of nuclear lamin proteins in cell lines stably transfected with green fluorescent protein (GFP)-tagged A-type lamin cDNA. Normal lamin A and C proteins show abundant decoration of the inner layer of the nuclear membrane, the nuclear lamina, and a generally diffuse localization in the nuclear interior. Bleaching studies revealed that, while the GFP-tagged lamins in the lamina were virtually immobile, the intranuclear fraction of these molecules was partially mobile. Intranuclear lamin C was significantly more mobile than intranuclear lamina A. In search of a structural cause for the variety of inherited diseases caused by A-type lamin mutations, we have studied the molecular organization of GFP-tagged lamin A and lamin C mutants R453W and R386K, found in Emery-Dreifuss muscular dystrophy (EDMD), and lamin A and lamin C mutant R482W, found in patients with Dunnigan-type familial partial lipodystrophy (FPLD). In all mutants, a prominent increase in lamin mobility was observed, indicating loss of structural stability of lamin polymers, both at the perinuclear lamina and in the intranuclear lamin organization. While the lamin rod domain mutant showed overall increased mobility, the tail domain mutants showed mainly intranuclear destabilization, possibly as a result of loss of interaction with chromatin. Decreased stability of lamin mutant polymers was confirmed by flow cytometric analyses and immunoblotting of nuclear extracts. Our findings suggest a loss of function of A-type lamin mutant proteins in the organization of intranuclear chromatin and predict the loss of gene regulatory function in laminopathies.

Mapping of the water quality of Lake Erken, Sweden, from imaging spectrometry and Landsat Thematic Mapper.

Hyperspectral data have been collected by the Compact Airborne Spectrographic Imager (CASI) and multispectral data by the Landsat Thematic Mapper (TM) instrument for the purpose of mapping lake water quality. Field campaigns have been performed on Lake Erken in Sweden during the summer of 1997. Water samples have been collected and analysed in laboratory. Continuously measured variables from a boat have added a spatial dimension to the ground truth dataset. The data have been used to construct algorithms, based on remotely sensed data, for the retrieval of water quality parameters. The correlation between the continuous data and the collected CASI data has been investigated. Algorithms using both the point sampling results and the continuous data have been developed. Maps based on data from each instrument, showing the distribution of chlorophyll, are presented. Problems of having few water sampling stations, and the potential of using sub-water optics models are addressed as well. Tests were performed on MERIS bands and found useful for mapping chlorophyll and turbidity, and algorithms have been suggested for future use with MERIS.

Statistical analysis of hyperspectral data from two Swedish lakes.

CASI data has been collected from two lakes in Sweden. In this paper, some statistical properties of CASI spectral data have been discussed. Principal component analysis is used for assessing the dimensionality of the data and the principal components were used for making chlorophyll maps. The quality of the reconstruction of the spectra from the principal components was demonstrated. Examples of the accuracy of the radiative transfer code 6S in atmospheric correction applications have been given. Furthermore, the widths and positions of the spectral bands based on the studied dataset were proposed for chlorophyll mapping. Robustness aspects of regression models have been discussed. Algorithms derived from one lake have been used to map water quality parameters in another lake. Algorithms based on principal components, as well as algorithms based on image bands, have been used.

Properties of lamin A mutants found in Emery-Dreifuss muscular dystrophy, cardiomyopathy and Dunnigan-type partial lipodystrophy.

Autosomal dominant Emery-Dreifuss muscular dystrophy is caused by mutations in the LMNA gene, which encodes lamin A and lamin C. Mutations in this gene also give rise to limb girdle muscular dystrophy type 1B, dilated cardiomyopathy with atrioventricular conduction defect and Dunnigan-type partial lipodystrophy. The properties of the mutant lamins that cause muscular dystrophy, lipodystrophy and dilated cardiomyopathy are not known. We transfected C2C12 myoblasts with cDNA encoding wild-type lamin A and 15 mutant forms found in patients affected by these diseases. Immunofluorescence microscopy showed that four mutants, N195K, E358K, M371K and R386K, could have a dramatically aberrant localization, with decreased nuclear rim staining and formation of intranuclear foci. The distributions of endogenous lamin A/C, lamin B1 and lamin B2 were also altered in cells expressing these four mutants and three of them caused a loss of emerin from the nuclear envelope. In the yeast two-hybrid assay, the 15 lamin A mutants studied interacted with themselves and with wild-type lamin A and lamin B1. Pulse-chase experiments showed no decrease in the stability of several representative lamin A mutants compared with wild-type. These results indicate that some lamin A mutants causing disease can be aberrantly localized, partially disrupt the endogenous lamina and alter emerin localization, whereas others localize normally in transfected cells.

Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy protein.

Emerin is an integral protein of the inner nuclear membrane that is mutated or not expressed in patients with Emery-Dreifuss muscular dystrophy. Confocal immunofluorescence microscopy studies of the intracellular targeting of truncated forms of emerin, some of which are found in patients with Emery-Dreifuss muscular dystrophy, show that the nucleoplasmic, amino-terminal domain is necessary and sufficient for nuclear retention. When this domain is fused to a transmembrane segment of an integral membrane protein of the ER/plasma membrane, the chimeric protein is localized in the inner nuclear membrane. The transmembrane segment of emerin is not targeted to the inner nuclear membrane. Fluorescence photobleaching experiments of emerin fused to green fluorescent protein demonstrate that the diffusional mobility (D) of emerin is decreased in the inner nuclear membrane (D=0.10+/-0.01 microm2/second) compared to the ER membrane (D=0.32+/-0.01 microm2/second). This is in agreement with a model where integral proteins reach the inner nuclear membrane by lateral diffusion and are retained there by association with nucleoplasmic components. Some overexpressed emerin-green fluorescent protein also reaches the plasma membrane of transfected cells, where its diffusion is similar to that in the inner nuclear membrane, suggesting that emerin may also associate with non-nuclear structures.

Autoantibodies in human chronic graft-versus-host disease after hematopoietic cell transplantation.

Primary biliary cirrhosis (PBC) and graft-versus-host disease (GVHD) are thought to have common immunopathologic features and previous studies have reported that 5.2 to 81% of patients with chronic GVHD after allogeneic hematopoietic cell transplant have antimitochondrial antibodies (AMA). We studied a total of 89 patients with chronic GVHD and 60 controls for AMA reactivity by ELISA and immunoblotting using recombinant PDC-E2, BCOADC-E2, and OGDC-E2, immunoblotting of beef heart mitochondrial proteins, and reactivity to nuclei, smooth muscle (ASMA), ribonucleoprotein JO-1, extractable nuclear antigen, nuclear proteins SSA/ SSB, ribonucleic P proteinase III, cardiolipin (ACA), liver kidney microsomal, thyroid microsomal, myeloperoxidase, and the reactivity of rheumatoid factor. A subset of 60 chronic GVHD sera were tested for reactivity to gp210 and LBR. Finally, liver tissue from patients with chronic GVHD and PBC was studied by immunohistochemistry to determine whether there was comparable abnormal apical staining of biliary epithelial cells using PDC-E2-specific monoclonal antibodies. Surprisingly, there were no AMA found in the sera from the 89 patients with chronic GVHD. Review of published data on AMA in GVHD suggests that previous results were primarily false positives. In contrast, sera from the patients with GVHD did have a variety of other autoantibodies and, in particular, 20/89 (22.4%) positive ANA, 23/89 (25.8%) positive ASMA, and 9/89 (10.1%) positive ACA. The other autoantibodies assayed were not statistically different from controls. Finally, abnormal biliary epithelial luminal staining of bile ducts was found, as expected, in liver tissue of patients with PBC but was absent in chronic GVHD.

Process-scale purification from cell culture supernatants: monoclonal antibodies.

A method for processing monoclonal antibodies (mAb) from large volumes of cell culture supernatants using recently developed high performance and fast flow chromatography media is described. A high-antibody producing mouse hybridoma cell line was adapted to low serum containing medium (1% foetal calf serum) for the production of anti-tissue Plasminogen Activator (anti-tPA) monoclonal antibody (murine subclass IgG1). The process consisted of three main chromatographic steps: desalting, cation exchange on S Sepharose Fast Flow and gel filtration on Superose 6 prep grade. With this process for the purification of anti-tPA monoclonal antibody, the final product was greater than 95% pure with a total recovery of 75% i.e. 1.4 g was recovered in 0.345 l from 35 l of culture supernatant originally containing 1.9 g of mAb. The adaptation of this process for purification of other monoclonal antibodies is discussed.

Iodinated fibroblast beta-glucuronidase as a ligand for receptor-mediated endocytosis.

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 X 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 X 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.

Quantification of unscheduled DNA synthesis in mononuclear leukocytes of the horse.

This study compares the relationship between N-acetoxy-2-acetylaminofluorene (NA-AAF) and u.v. induced unscheduled DNA synthesis (UDS) and their respective relationships to age and blood pressure in horse mononuclear leukocytes with earlier, similar investigations on human leukocytes. U.v. induced UDS was found to proceed more rapidly than NA-AAF induced UDS. A pronounced lag period associated with the rapid demand for 3H-dThd into DNA after u.v. damage was observed. NA-AAF induced UDS correlated significantly with NA-AAF binding, age and the blood pressure of male horses. UDS values, induced by either method, were about half the level calculated for human leukocytes.

Reproducibility and the influence of age on interspecimen determinations of blood pressure in the horse.

1. The reproducibility of blood pressure determinations on 103 male horses gave an average coefficient of variation of 5.0%. 2. Different parameters affecting the methodology of blood pressure measurements were separately analysed; i.e. size of specimen, size of cuff in relationship to tail circumference and temperature of the environment. 3. A strong positive linear correlation between age and blood pressure in the horse was established for two breeds with widely varying genetic background--Swedish Warmbloods and Arabians.

The effect of an individual's blood pressure on the percentage of total 7,12-dimethylbenz[a] anthracene metabolites that bind covalently to DNA in cultured resting lymphocytes.

A method for the quantitative analysis of the percent metabolism that results in covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) to DNA in viable resting human lymphocytes is described. The inter- and intra-experimental reproducibility as judged by the coefficient of variation and examined in the same individual over a 3-month period was 31.4% and 13.9%, respectively. When the lymphocytes from 30 hypertensive individuals were exposed to 1 microM DMBA for 18 h, the percent of total DMBA metabolites that bind DNA covalently was correlated to the blood pressures of the patients at the time of sampling (r = 0.53, P less than 0.005). No influences on the data from the type or duration of hypertensive drug treatment could be statistically determined for this sample of hypertensive patients. It was concluded that high blood pressure is a strong determinant in predisposing lymphocytes to increased genetic risk from induced DNA damage and that this relationship is not statistically affected by hypertensive drug therapy.

Direct comparison, in humans resting lymphocytes, of the inter-individual variations in unscheduled DNA synthesis induced by N-acetoxy-2-acetylaminofluorene and ultraviolet irradiation.

Unscheduled DNA synthesis (UDS) in human leukocytes has become a popular tool for estimating an individual's DNA-repair capacity and sensitivity to mutagens. The physical induction of UDS by UV-irradiation, as opposed to the chemical induction of UDS by N-acetoxy-2-acetylaminofluorene (NA-AAF), has yielded contradictory reports in the literature regarding inter-individual variations relating to risk factors such as age. In an effort to resolve this anomaly, we have compared directly, in duplicate lymphocyte samples from 29 male individuals, the UDS induced by 10 microM NA-AAF and UV-irradiation of 10 J . m-2. The kinetics of UV-induced UDS was very "fast", being 50% complete within 2.5 h whereas the corresponding value for NA-AAF-induced UDS was 7 h. Consistent with our previous studies, individual variations in NA-AAF-induced UDS were positively correlated to age. However, a strong tendency toward both positive and negative correlations to an individual's age could be calculated depending on whether "fast"- or "slow"-UV-induced DNA repair was taken into consideration. These data, therefore, question the validity of previous reports establishing DNA-repair deficiencies when UV-induced UDS was used for quantitation and the "fast" UV repair was only partially estimated because of early delays in the 3H-dThd pulse. If appropriately pulsed with 3H-dThd, then the inter-individual fluctuations in NA-AAF- and UV-induced UDS correlated to each other in a highly significant linear manner (r = 0.51, p less than 0.01). Basic similarities between NA-AAF- and UV-induced UDS were also observed when both parameters correlated to the phytohemagglutinin responsiveness of the lymphocytes. A working hypothesis, based on environmentally influenced changes in chromatin structure regulating the relative accessibility of NA-AAF- and UV-induced lesions for repair, was formulated to explain the parallel inter-individual fluctuations of NA-AAF- and UV-induced UDS.