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.     

The 1.8 A crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species

Proteins from organisms living in extreme conditions are of particular interest because of their potential for being templates for redesign of enzymes both in biotechnological and other industries. The crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species has been solved to 1.8 A. The structure has been compared with the structures of proteinase K from Tritirachium album Limber and Vibrio sp. PA44 in order to reveal structural explanations for differences in biophysical properties. The Serratia peptidase shares around 40 and 64% identity with the Tritirachium and Vibrio peptidases, respectively. The fold of the three enzymes is essentially identical, with minor exceptions in surface loops. One calcium binding site is found in the Serratia peptidase, in contrast to the Tritirachium and Vibrio peptidases which have two and three, respectively. A disulfide bridge close to the S2 site in the Serratia and Vibrio peptidases, an extensive hydrogen bond network in a tight loop close to the substrate binding site in the Serratia peptidase and different amino acid sequences in the S4 sites are expected to cause different substrate specificity in the three enzymes. The more negative surface potential of the Serratia peptidase, along with a disulfide bridge close to the S2 binding site of a substrate, is also expected to contribute to the overall lower binding affinity observed for the Serratia peptidase. Clear electron density for a tripeptide, probably a proteolysis product, was found in the S' sites of the substrate binding cleft.     

Self-fitting shape memory polymer foam inducing bone regeneration: A rabbit femoral defect study

Although tissue engineering has been attracted greatly for healing of critical-sized bone defects, great efforts for improvement are still being made in scaffold design. In particular, bone regeneration would be enhanced if a scaffold precisely matches the contour of bone defects, especially if it could be implanted into the human body conveniently and safely. In this study, polyurethane/hydroxyapatite-based shape memory polymer (SMP) foam was fabricated as a scaffold substrate to facilitate bone regeneration. The minimally invasive delivery and the self-fitting behavior of the SMP foam were systematically evaluated to demonstrate its feasibility in the treatment of bone defects in vivo. Results showed that the SMP foam could be conveniently implanted into bone defects with a compact shape. Subsequently, it self-matched the boundary of bone defects upon shape-recovery activation in vivo. Micro-computed tomography determined that bone ingrowth initiated at the periphery of the SMP foam with a constant decrease towards the inside. Successful vascularization and bone remodeling were also demonstrated by histological analysis. Thus, our results indicate that the SMP foam demonstrated great potential for bone regeneration.     

The leukemia-specific fusion gene ETV6/RUNX1 perturbs distinct key biological functions primarily by gene repression

Background

ETV6/RUNX1 (E/R) (also known as TEL/AML1) is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL) and also most likely the crucial factor for disease initiation; its role in leukemia propagation and maintenance, however, remains largely elusive. To address this issue we performed a shRNA-mediated knock-down (KD) of the E/R fusion gene and investigated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions in two E/R-positive leukemic cell lines.

Findings

Microarray analyses identified 777 genes whose expression was substantially altered. Although approximately equal proportions were either up- (KD-UP) or down-regulated (KD-DOWN), the effects on biological processes and pathways differed considerably. The E/R KD-UP set was significantly enriched for genes included in the “cell activation”, “immune response”, “apoptosis”, “signal transduction” and “development and differentiation” categories, whereas in the E/R KD-DOWN set only the “PI3K/AKT/mTOR signaling” and “hematopoietic stem cells” categories became evident. Comparable expression signatures obtained from primary E/R-positive ALL samples underline the relevance of these pathways and molecular functions. We also validated six differentially expressed genes representing the categories “stem cell properties”, “B-cell differentiation”, “immune response”, “cell adhesion” and “DNA damage” with RT-qPCR.

Conclusion

Our analyses provide the first preliminary evidence that the continuous expression of the E/R fusion gene interferes with key regulatory functions that shape the biology of this leukemia subtype. E/R may thus indeed constitute the essential driving force for the propagation and maintenance of the leukemic process irrespective of potential consequences of associated secondary changes. Finally, these findings may also provide a valuable source of potentially attractive therapeutic targets.