Establishment of three human breast epithelial cell lines derived from carriers of the 999del5 <Emphasis Type="Italic">BRCA2</Emphasis> icelandic founder mutation

Summary Germ line mutations in BRCA1 and BRCA2 account for a large proportion of inherited breast and ovarian cancer. Both genes are involved in DNA repair by homologous recombination and are thought to play a vital role in maintaining genomic stability. A major drawback for long-term functional studies of BRCA in general and BRCA2 in particular has been a lack of representative human breast epithelial cell lines. In the present study, we have established three cell lines from two patients harboring the 999del5 germ line founder mutation in the BRCA2 gene. Primary cultures were established from cellular outgrowth of explanted tissue and subsequently transfected with a retroviral construct containing the HPV-16 E6 and E7 oncogenes. Paired cancer-derived and normal-derived cell lines were established from one patient referred to as BRCA2-999del5-2T and BRCA2-999del5-2N, respectively. In addition, one cell line was derived from cancer-associated normal tissue from another patient referred to as BRCA2-999del5-1N. All three cell lines showed characteristics of breast epithelial cells as evidenced by expression of breast epithelial specific cytokeratins. Cytogenetic analysis showed marked chromosomal instability with tetraploidy and frequent telomeric association. In conclusion, we have established three breast cpithelial cell lines from two patients carrying the BRCA2 Icelandic 999del5 founder mutation. These cell lines from the basis for further studies on carcinogenesis and malignant progression of breast cancer on a defined genetic background. Agla J. Rubner Fridriksdottir and Thorarinn Gudjonsson contributed equally to this study.     

Production of an active recombinant thrombomodulin derivative in transgenic tobacco plants and suspension cells

Thrombomodulin is a membrane-bound protein that plays an active role in the blood coagulation system by binding thrombin and initiating the protein C anticoagulant pathway. Solulin™ is a recombinant soluble derivative of human thrombomodulin. It is used for the treatment of thrombotic disorders. To evaluate the production of this pharmaceutical protein in plants, expression vectors were generated using four different N-terminal signal peptides. Immunoblot analysis of transiently transformed tobacco leaves showed that intact Solulin™ could be detected using three of these signal peptides. Furthermore transgenic tobacco plants and BY2 cells producing Solulin™ were generated. Immunoblot experiments showed that Solulin™ accumulated to maximum levels of 115 and 27 μg g⁻¹ plant material in tobacco plants and BY2 cells, respectively. Activity tests performed on the culture supernatant of transformed BY2 cells showed that the secreted Solulin™ was functional. In contrast, thrombomodulin activity was not detected in total soluble protein extracts from BY2 cells, probably due to inhibitory effects of substances in the cell extract. N-terminal sequencing was carried out on partially purified Solulin™ from the BY2 culture supernatant. The sequence was identical to that of Solulin™ produced in Chinese hamster ovary cells, confirming correct processing of the N-terminal signal peptide. We have demonstrated that plants and plant cell cultures can be used as alternative systems for the production of an active recombinant thrombomodulin derivative.     

Early and stable upregulation of collagen type II, collagen type I and YKL40 expression levels in cartilage during early experimental osteoarthritis occurs independent of joint location and histological grading

While morphologic and biochemical aspects of degenerative joint disease (osteoarthritis [OA]) have been elucidated by numerous studies, the molecular mechanisms underlying the progressive loss of articular cartilage during OA development remain largely unknown. The main focus of the present study was to gain more insight into molecular changes during the very early stages of mechanically induced cartilage degeneration and to relate molecular alterations to histological changes at distinct localizations of the joint. Studies on human articular cartilage are hampered by the difficulty of obtaining normal tissue and early-stage OA tissue, and they allow no progressive follow-up. An experimental OA model in dogs with a slow natural history of OA (Pond-Nuki model) was therefore chosen. Anterior cruciate ligament transection (ACLT) was performed on 24 skeletally mature dogs to induce joint instability resulting in OA. Samples were taken from different joint areas after 6, 12, 24 and 48 weeks, and gene expression levels of common cartilage molecules were quantified in relation to the histological grading (modified Mankin score) of adjacent tissue. Histological changes reflected early progressive degenerative OA. Soon after ACLT, chondrocytes responded to the altered mechanical conditions by significant and stable elevation of collagen type II, collagen type I and YKL40 expression, which persisted throughout the study. In contrast to the mild to moderate histological alterations, these molecular changes were not progressive and were independent of the joint localization (tibia, femur, lateral, medial) and the extent of matrix degeneration. MMP13 remained unaltered until 24 weeks, and aggrecan and tenascinC remained unaltered until 48 weeks after ACLT. These findings indicate that elevated collagen type II, collagen type I and YKL40 mRNA expression levels are early and sensitive measures of ACLT-induced joint instability independent of a certain grade of morphological cartilage degeneration. A second phase of molecular changes in OA may begin around 48 weeks after ACLT with altered expression of further genes, such as MMP13, aggrecan and tenascin. Molecular changes observed in the present study suggest that dog cartilage responds to degenerative conditions by regulating the same genes in a similar direction as that observed for chondrocytes in late human OA.     

Cell-cell communication in Gram-negative bacteria

Over the last decade or so, a wealth of research has established that bacteria communicate with one another using small molecules. These signals enable the individuals in a population to coordinate their behaviour. In the case of pathogens, this behaviour may include decisions such as when to attack a host organism or form a biofilm. Consequently, such signalling systems are excellent targets for the development of new antibacterial therapies. In this review, we assess how Gram-negative bacteria use small molecules for cell-cell communication, and discuss the main approaches that have been developed to interfere with it.     

Functional measurement of local proteolytic activity in living cells of invasive and non-invasive tumors

Proteolytic cleavage of extracellular matrix (ECM) and disruption of tissue architecture are fundamental features of tumor cell invasion. The proteolytic activity is focused in close proximity to the tumor cells. Here, we describe the possibility to quantify local proteolytic activity in the microenvironment of larger cell populations by the electrical resistance breakdown assay. The assay utilizes the transepithelial electrical resistance (TEER) of an epithelial monolayer as a sensitive indicator of monolayer integrity and permeability. Local destruction of ECM by single tumor cells was demonstrated by a second assay, based on a fluorescent matrix coating on cover slides. Local digestion of the matrix results in a reduction of fluorescence. Primary cells derived from high and low grade brain tumors as well as established cell lines of malignant gliomas and non-neural tumors of different origin (melanoma, cervical carcinoma, and breast carcinoma) were compared. Differences in proteolytic activity between tumor entities were demonstrated in both assays. Primary cells of high grade gliomas and cell lines showed TEER breakdown and local matrix destruction, while low grade brain tumors lacked matrix disintegration and disruption of cell monolayers. Taken together, both assays are capable of demonstrating local proteolytic activity and thus are versatile tools for distinguishing high and low invasive tumor cells with a potential application as diagnostic and prognostic markers in clinical investigations. The advantage of the matrix digestion assay is the requirement of only very low tumor cell numbers, whereas measurement of TEER enables precise quantification of local proteolytic processes in large and even heterogeneous tumor cultures.     

Possible role of factor XIII subunit A in Fcgamma and complement receptor-mediated phagocytosis

Besides its traditional role in hemostasis, factor XIII subunit A (FXIII-A) is supposed to function as a cellular transglutaminase and to be involved in certain intracellular processes, including cytoskeletal remodeling. To investigate its intracellular role, the aim of the present study was to follow changes in FXIII-A production in combination with the receptor-mediated phagocytic activities of monocytes/macrophages and to examine the phagocytic functions of monocytes in patients with FXIII-A deficiency. Human blood monocytes were isolated from the buffy coats of healthy volunteers and cultured for 4 days. The FcgammaR-mediated phagocytosis of sensitized erythrocytes (EA) and the complement receptor (CR)-mediated phagocytosis of complement-coated yeast particles were studied during monocyte/macrophage differentiation. Changes in the gene expression of FXIII-A were detected by real-time quantitative RT-PCR. FXIII-A protein production was investigated with fluorescent image analysis at single cell level and Western immunoblot analysis. Both the FcgammaR and CR-mediated phagocytosis increased during culturing, which peaked on day 3. The phagocytic activity of the cells could be markedly inhibited with monodansylcadaverine, an inhibitor of the transglutaminase-induced crosslinking of proteins. The phagocytosis of EA, complement-coated and uncoated yeast particles was found to be strongly diminished in monocytes of FXIII-A deficient patients. The phagocytic functions of cultured cells showed a change in parallel with the alterations in FXIII-A mRNA expression, as well as with that in FXIII-A in protein synthesis detected by image and Western immunoblot analyses in concert. Our results suggest that FXIII-A plays a role in the Fcgamma and complement receptor-mediated phagocytic activities of monocytes/macrophages.     

Interaction of the disintegrin and cysteine-rich domains of ADAM12 with integrin alpha7beta1

We describe a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Integrin alpha7beta1 extracted from human embryonic kidney 293 cells transfected with alpha7 cDNA was retained on an affinity column containing immobilized DC domain of ADAM12. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells, and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for Mn2+ than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase (FAK) at Tyr397. We also show that adhesion of L6 myoblasts to DC is mediated in part by the endogenous integrin alpha7beta1 expressed in these cells. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulate that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. We also conclude that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses.     

Physiological concept for a blood based CFTR test.

We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) could be involved in the volume regulation of human red blood cells (RBC). Experiments were based on two gadolinium (Gd(3+)) sensitive mechanisms, i.e. inhibition of ATP release (thetaATP(i)) and membrane destabilization. RBC of either cystic fibrosis (CF) patients or healthy donors (non-CF) were exposed to KCl buffer containing Gd(3+). A significantly larger quantity of non-CF RBC (2.55 %) hemolyzed as compared to CF RBC (0.89 %). It was found that both of the Gd(3+) mechanisms simultaneously are needed to achieve hemolysis, since either overriding thetaATP(i) by exogenous ATP addition prevented Gd(3+) induced hemolysis, or mimicking thetaATP(i) by apyrase in absence of Gd(3+) could not trigger hemolysis. Additionally, ion driven volume uptake was found to be a prerequisite for Gd3+ induced hemolysis as chloride and potassium channel blockers reduced the Gd(3+) response. The results show that in non-CF RBC Gd(3+) exerts its dual effect leading to hemolysis. On the contrary, in CF RBC, lacking CFTR dependent ATP release, the sole Gd(3+) effect of membrane destabilization is not sufficient to induce hemolysis similar to non-CF. This concept could form the basis of a novel method suitable for testing CFTR function in a blood sample.     

Induction of the 72-kilodalton heat shock protein and protection from ultraviolet B-induced cell death in human keratinocytes by repetitive exposure to heat shock or 15-deoxy-delta(12,14)-prostaglandin J2

It has been demonstrated that hyperthermia protects keratinocytes from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if keratinocytes become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal keratinocytes (NHEK) and the carcinoma-derived cell line A431 were exposed to either 42 degrees C or to 15dPGJ2 for 4 hours at 24-hour intervals for 4 consecutive days. The intracellular level of the 72-kDa heat shock protein (Hsp72) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ2 treatment, and survival was determined 24 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ2 are potent inducers of Hsp72 expression and lead to increased resistance to UVB-induced cell death in human keratinocytes; (2) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp72 and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ2 is enhanced by repeated exposure without a further increase of Hsp72; (4) repeated heat shock and 15dPGJ2 up to a concentration of 1 microg/mL have no influence on cell growth over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing factors, stress tolerance can be maintained without additional toxicity in human keratinocytes. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.