In a study on gene deregulation in ovarian carcinoma we found a mRNA coding for a 350 kDa protein, Drop1, to be downregulated 20‐ to 180‐fold in the majority of ovarian and mammary carcinomas. The mRNA is encoded by a set of exons in the 5′ region of the SYNE1 gene. Immunohistochemical staining for Drop1 protein by a specific monoclonal antibody corresponds to the pattern seen for the mRNA. cDNA arrays of matched pairs of tumor and normal tissue and in situ hybridizations confirmed the drastic loss of Drop1 mRNA as a common feature in uterus, cervix, kidney, lung, thyroid and pancreas carcinomas, already at early tumor stages and in all metastases. Two‐hybrid studies suggest a role of this deficiency in the malignant progression of epithelial tumors. © 2008 Wiley‐Liss, Inc.

Microglia phagocytic activity for apoptotic glioma cells is hardly analysed inspite of its relevance to tissue damage prevention. We provide evidence for a phosphatidylserine-independent clearance of mouse glioma cells at an advanced stage of death, suggesting microglia recognition of late apoptotic markers. Dying cells were immediately cleared or stayed for hours in that stage before engulfment occurred. This phagocytic activity was restricted to a microglia subset representing 30 to 70% of the population according to the used strain. Expression of receptors involved in late apoptotic markers recognition therefore seems confined to a subpopulation of microglia and to be strain-dependent.

Axon growth inhibitory CNS matrix proteins, such as tenascin-R (TN-R), have been supposed to contribute to the poor regenerative capacity of adult mammalian CNS. With regard to TN-R function in low vertebrates capable of CNS regeneration, questions of particular interest concern the (co)evolution of ligand-receptor pairs and cellular response mechanisms associated with axon growth inhibition and oligodendrocyte differentiation. We address here these questions in a series of comparative in vivo and in vitro analyses using TN-R proteins purified from different vertebrates (from fish to human). Our studies provide strong evidence that unlike TN-R of higher vertebrates, fish TN-R proteins are not repellent for fish and less repellent for mammalian neurons and do not interfere with F3/contactin- and fibronectin-mediated mammalian cell adhesion and axon growth. However, axonal repulsion is induced in fish neurons by mammalian TN-R proteins, suggesting that the intracellular inhibitory machinery induced by TN-R-F3 interactions is already present during early vertebrate evolution. In contrast to TN-R-F3, TN-R-sulfatide interactions, mediating oligodendrocyte adhesion and differentiation, are highly conserved during vertebrate evolution. Our findings thus indicate the necessity of being cautious about extrapolations of the function of ligand-receptor pairs beyond a species border and, therefore, about the phylogenetic conservation of a molecular function at the cellular/tissue level.

Cystatins regulate tumour-associated cysteine proteases, however, their role in tumour progression is not clear yet. To assess their relevance in the progression of non-small cell lung cancer (NSCLC) the protein level, cysteine protease activity (CPI) and localization of type I (stefins A and B) and type II (C, E/M and F) cystatins were defined in tumours and control lung counterparts from 165 patients. The medians of CPI activity, stefins A and B were significantly greater in tumour than in lung tissue (2.1-fold, 1.7-fold, 1.2-fold, respectively, all p<0.001). The median levels of cystatin C and cystatin E/M were lower in tumour tissue (0.9-fold, p=0.06; 0.6-fold, p<0.01). In all the samples the levels of cystatin F were below the detection limit. Immunohistochemical analysis revealed the presence of all cystatins in tumour cells and infiltrated inflammatory cells such as macrophages and neutrophils. In univariate survival analysis patients with high levels of stefin A, stefin B and CPI activity exhibited a better survival probability (p=0.05, p=0.05, p<0.01, respectively). In contrast, cystatins C and E/M provided no prognostic information. In multivariate analysis the most powerful predictor of survival was the pTNM stage (p<0.0001; RR 3.5), followed by stefin A, stefin B and CPI activity (all p=0.03; RR 1.5). Our results suggest that only stefins A and B, i.e. type I cystatins, are up-regulated in lung tumours and thus able to counteract harmful tumour-associated proteolytic activity. As biological markers they may add independent prognostic information for better assessment of low- and high-risk patients with NSCLC.

Abstract Background Splicing variants of human cathepsinB primary transcripts (CB(-2,3)) result in an expression product product which lacks the signal peptide and parts of the propeptide. This naturally truncated Δ51CB is thus unable to follow the regular CB processing and sorting pathway. It is addressed to the mitochondria through an activated N-terminal mitochondrial targeting signal instead. Although Δ51CB is supposed to be devoid of the typical CB enzymatic activity, it might play a role in malignancies and trigger cell death/apoptosis independent from the function of the regular enzyme. Cytoplasmic presence of the mature CB might occur as a result of lysosomal damage. Results We investigated such "aberrant" proteins by artificial CB-GFP chimeras covering various sequence parts in respect to their enzymatic activity, their localization in different cell types, and the effects on the cell viability. Unlike the entire full length CB form, the artificial single chain form was not processed and did not reveal typical enzymatic CB activity during transient overexpression in large cell lung carcinoma cells. Δ51CB was found predominantly in mitochondria. In contrast, the shorter artificial CB constructs localized in the cytoplasm, inside the cell nucleus, and in the midbodies of dividing cells. Bleaching experiments revealed both mobile and immobile fractions of these constructs in the nucleus. Nuclear accumulation of artificially truncated CB variants led to disintegration of nuclei, followed by cell death. Conclusion We propose that cell death associated with CB is not necessarily triggered by its regular enzymatic activity but alternatively by a yet unknown activity profile of truncated CB. Cytoplasmic CB might be able to enter the cell nucleus. According to a mutational analysis, the part of CB that mediates its nuclear import is a signal patch within its heavy chain domain. The results suggest that besides the N-terminal signal peptide also other CB domains contain patterns which are responsible for a differentiated targeting of the molecule, e.g. to the mitochondria, to the nucleus, or to vesicles. We propose a hierarchy of targeting signals depending on their strength and availability. This implies other possible transport mechanisms besides the usual trafficking via the mannose-6-℗ pathway.

2713 In a study employing differential display analysis to identify differentially expressed genes in ovarian carcinomas, we found an 11 kb mRNA coding for a 350 kDa Protein suppressed in over 70% of ovarian and mammary carcinomas. Its expression is downregulated 20-1000 fold in these tumors. cDNA arrays of matched pairs of tumor and normal tissue showed that the mRNA of Drop 1 is downregulated in more than 80% of epithelial tumors, such as ovary (>80%), breast (>50%), kidney (>90%), lung (>70%) and pancreas (85%). Incubation of cell lines expressing Drop 1 at a low lever with methyltransferase inhibitors resulted in an increase in Drop 1expression of up to 18-fold. This indicates that Drop 1 can be downregulated through methylation of the promoter. Mouse monoclonal antibodies raised to a C-terminal fragment of Drop1recognized a 350 kDa protein on Western Blots as well as a nucleus-associated protein on cytospins. Drop 1 is associated with the polar region of the mitotic spindle during mitosis. An N-terminal fragment of Drop 1 fused to GFP and expressed in COS-7 cells also shows association with the nucleus. This location as well as the structural homology to dystrophin make Drop1 an interesting candidate for processes involved in the integration of the nuclear structure during the cell cycle. RNAi knockdown experiments and yeast two hybrid screening are in progress to elucidate Drop1 function.

Ubiquitously expressed calpains are Ca(2+)-dependent, intracellular cysteine proteases comprising a large catalytic subunit (domains DI-DIV) and a noncovalently bound small regulatory subunit (domains DV and DVI). It is unclear whether Ca(2+)-induced calpain activation is followed by subunit dissociation or not. Here, we have applied advanced fluorescence microscopy techniques to study calpain subunit interactions in living cells using recombinant calpain subunits or domains fused to enhanced cyan and enhanced yellow fluorescent reporter proteins. All of the overexpressed variants of the catalytic subunit (DI-IV, DI-III, and DI-IIb) were active and Ca(2+)-dependent. The intact large subunit, but not its truncated variants, associates with the small subunit under resting and ionomycin-activated conditions. All of the variants were localized in cytoplasm and nuclei, except DI-IIb, which accumulates in the nucleus and in nucleoli as shown by microscopy and cell fractionation. Localization studies with mutated and chimeric variants indicate that nuclear targeting of the DI-IIb variant is conferred by the two N-terminal helices of DI. Only those variants that contain DIII migrated to membranes upon the addition of ionomycin, suggesting that DIII is essential for membrane targeting. We propose that intracellular localization and in particular membrane targeting of activated calpain, but not dissociation of its intact subunits, contribute to regulate its proteolytic activity in vivo.

Prion diseases are characterized by the accumulation of an abnormal, proteinase K-resistant isoform of the prion protein, PrP(Sc), which is generated by a post-translational conversion of the protease-sensitive normal cell-surface glycoprotein PrP(c) involving major conformational changes. The conversion is thought to occur at the plasma membrane or along the endocytic pathway towards the lysosome. PrP(Sc) aggregates have been found to accumulate in secondary lysosomes. In our study, the activities of two major lysosomal cysteine proteases, cathepsins B and L, were found to be significantly increased in scrapie-infected Neuro2a cells compared with uninfected cells using biochemical and cytochemical methods. We hypothesize that lysosomal proteases may be involved in a 'second autocatalytic loop' of PrP(Sc) formation, acting in concert with the well-known autocatalytic enhancement of PrP conversion in the presence of PrP(Sc).

Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.

Several GFP variants have been developedfor multicolor labeling in vivo. Here we report that simultaneous co-transfection of fluorescent protein chimeras can give false-positive results caused by the conversion of spectral properties. Under standard transfection conditions, approximately 8% of cells produce false-positive results, but, depending on the conditions, up to 26% of the cells permanently express altered fusion proteins. This compromises the interpretation of the results. The conversion is independent of transfection methods or cell types. Our results show that the effect is based on homologous recombination/repair/replication process events that occur between the nucleotide sequences of the fluorescent proteins. Consecutive transfection or low sequence similarities avoided recombination. The appearance of conversion facilitates exchanges of spectral properties infusion proteins, the creation of libraries, or the assembly of DNA fusion constructs in vivo. The detailed quantification of the conversion rate allows the investigation of recombination/repair/replication processes in general.

Cellular orders in normal tissue and in tumors can be described on the basis of physical terms. Assuming that the peptidase cathepsin B is involved as an essential factor in tumor progression we analyzed the corresponding distribution pattern and determined the entropy of this cell fraction in tumors by syntactic structure analysis using nearest neighbor relationships. Of the 120 surveyed sections from primary lung tumors 62 (51.7 %) were identified with cathepsin B expressing cells. Cathepsin B-positive tumor cells have significantly shorter mean cell-cell distances (p<0.01) and form significantly higher number of tumor cell clusters (p<0.01) when compared with cathepsin B-negative tumor cells. The diameters of the cathepsin B positive tumor cell clusters are increased in moderately and intensively stained tumor cell areas compared to the cathepsin B negative ones (p<0.1 and p<0.05, respectively). The mean cell-cell distance between positive tumor cells was significantly shorter in squamous cell carcinomas (SCC) compared to adenocarcinomas (AC) (p<0.01). We found an increased number of tumor cell clusters in intensively stained areas of SCC compared with AC (p<0.05). Interestingly, in dedifferentiated tumors cells which strongly expressed cathepsin B, have significantly (p<0.01) shorter mean cell-cell distances compared with those well differentiated tumors. In conclusion, cathepsin B positive tumor cells have shorter mean cell-cell distances and form higher number of tumor cell clusters. Our findings indicate that cathepsin B positive tumor cells of primary lung tumors seem to detach as tumor cell clusters instead of single tumor cells. This process appears to be more pronounced in squamous cell carcinomas than in adenocarcinomas.

The chapter presents a discussion on cytotomography and some practical instructions on its use. The chapter mentions to use the term “cytotomography” for the through-focus technique rendering a three-dimensional (3-D) impression from cells and their interior by light microscopical image acquisition. In electron microscopy, the tomography is performed on living objects. The sectioning is a noninvasive and nondestructive process leaving the integrity of the object undamaged. 3-D impressions of microscopical objects have so far been obtained by scanning electron microscopy and transmission electron microscopy of embedded and dissected objects or by taking images from tilted objects. By using cytotomography, a more general comprehension will be possible as applications in morphology and physiology of cells clearly reveal. The first choice in targeting organelles is specific antibodies coupled to fluorochromes. The chapter presents an example on the migration of a human tumor cell (SB 3) into a reconstituted collagen matrix to demonstrate the possible contribution of the lysosomal cysteine protease cathepsin B to the process. Multidimensional microscopy shows its full potential only in combination with state of the art data visualization approaches.