A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis.

Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

A versatile system to introduce clusters of genomic double-strand breaks in large cell populations.

In vitro assays for clustered DNA lesions will facilitate the analysis of the mechanisms underlying complex genome rearrangements such as chromothripsis, including the recruitment of repair factors to sites of DNA double-strand breaks (DSBs). We present a novel method generating localized DNA DSBs using UV irradiation with photomasks. The size of the damage foci and the spacing between lesions are fully adjustable, making the assay suitable for different cell types and targeted areas. We validated this setup with genomically stable epithelial cells, normal fibroblasts, pluripotent stem cells, and patient-derived primary cultures. Our method does not require a specialized device such as a laser, making it accessible to a broad range of users. Sensitization by 5-bromo-2-deoxyuridine incorporation is not required, which enables analyzing the DNA damage response in post-mitotic cells. Irradiated cells can be cultivated further, followed by time-lapse imaging or used for downstream biochemical analyses, thanks to the high throughput of the system. Importantly, we showed genome rearrangements in the irradiated cells, providing a proof of principle for the induction of structural variants by localized DNA lesions.

Altered nuclear envelope structure and proteasome function of micronuclei.

Micronuclei are extra-nuclear bodies containing whole chromosomes that were not incorporated into the nucleus after cell division or damaged chromosome fragments. Even though the link between micronuclei and DNA damage is described for a long time, little is known about the functional organization of micronuclei and their contribution to tumorigenesis. We showed fusions between micronuclear membranes and lysosomes by electron microscopy and linked lysosome function to DNA damage levels in micronuclei. In addition, micronuclei drastically differ from primary nuclei in nuclear envelope composition, with a significant increase in the relative amount of nuclear envelope proteins LBR and emerin and a decrease in nuclear pore proteins. Strikingly, micronuclei lack active proteasomes, as the processing subunits and other factors of the ubiquitin proteasome system. Moreover, micronuclear chromatin shows a higher degree of compaction as compared to primary nuclei. The specific aberrations identified in micronuclei and the potential functional consequences of these defects may contribute to the role of micronuclei in catastrophic genomic rearrangements.

Lamin A and lamin C form homodimers and coexist in higher complex forms both in the nucleoplasmic fraction and in the lamina of cultured human cells.

We have investigated and quantified the nuclear A-type lamin pool from human HeLa S3 suspension cells with respect to their distribution to detergent soluble and insoluble fractions. We devised a sequential extraction protocol and found that maximally 10% of A-type lamins are recovered in the soluble fraction. Notably, lamin C is enriched in low detergent fractions and only with 0.5% Nonidet P-40 lamin A and C are recovered in ratios nearly equivalent to those found in whole cell extracts and in the lamina fraction. Authentic nucleoplasmic proteins such as LAP2a, pRB and p53 are co-extracted to a large part together with the A-type lamins in these fractions. By sucrose density centrifugation we revealed that the majority of lamins co-sedimented with human IgG indicating they form rather small complexes in the range of dimers and slightly larger complexes. Some lamin A - but not lamin C - is obtained in addition in a much faster sedimenting fraction. Authentic nuclear proteins such as PCNA, p53 and LAP2a were found both in the light and the heavy sucrose fractions together with lamin A. Last but not least, immunoprecipitation experiments from both soluble fractions and from RIPA lysates of whole cells revealed that lamin A and lamin C do not form heterodimers but segregate practically completely. Correspondingly, immunofluorescence microscopy of formaldehyde-fixed cells clearly demonstrated that lamin A and C are localized at least in part to distinct patches within the lamina. Hence, the structural segregation of lamin A and C is indeed retained in the nuclear envelope to some extent too.

Vimentin coil 1A-A molecular switch involved in the initiation of filament elongation.

Interestingly, our previously published structure of the coil 1A fragment of the human intermediate filament protein vimentin turned out to be a monomeric alpha-helical coil instead of the expected dimeric coiled coil. However, the 39-amino-acid-long helix had an intrinsic curvature compatible with a coiled coil. We have now designed four mutants of vimentin coil 1A, modifying key a and d positions in the heptad repeat pattern, with the aim of investigating the molecular criteria that are needed to stabilize a dimeric coiled-coil structure. We have analysed the biophysical properties of the mutants by circular dichroism spectroscopy, analytical ultracentrifugation and X-ray crystallography. All four mutants exhibited an increased stability over the wild type as indicated by a rise in the melting temperature (T(m)). At a concentration of 0.1 mg/ml, the T(m) of the peptide with the single point mutation Y117L increased dramatically by 46 degrees C compared with the wild-type peptide. In general, the introduction of a single stabilizing point mutation at an a or a d position did induce the formation of a stable dimer as demonstrated by sedimentation equilibrium experiments. The dimeric oligomerisation state of the Y117L peptide was furthermore confirmed by X-ray crystallography, which yielded a structure with a genuine coiled-coil geometry. Most notably, when this mutation was introduced into full-length vimentin, filament assembly was completely arrested at the unit-length filament (ULF) level, both in vitro and in cDNA-transfected cultured cells. Therefore, the low propensity of the wild-type coil 1A to form a stable two-stranded coiled coil is most likely a prerequisite for the end-to-end annealing of ULFs into filaments. Accordingly, the coil 1A domains might "switch" from a dimeric alpha-helical coiled coil into a more open structure, thus mediating, within the ULFs, the conformational rearrangements of the tetrameric subunits that are needed for the intermediate filament elongation reaction.

Differential expression pattern of protein ARVCF in nephron segments of human and mouse kidney.

The protein ARVCF is a member of the p120 subfamily of armadillo proteins whose members have been described to occur in junction-bound and non-junction-bound forms. Studies on ARVCF were constrained because the endogenous protein was difficult to detect with the available reagents. We have generated novel monoclonal and polyclonal antibodies usable for biochemical and localization studies. By systematic immunohistochemical analysis of various tissues protein ARVCF is prominently detected in mouse, bovine and human kidney. Using antibodies against specific markers of nephron segments protein ARVCF is localized in proximal tubules according to double label immunofluorescence. Besides its occurrence in proximal tubules of adult kidney and in renal cell carcinoma derived from proximal tubules ARVCF is also detected in maturing nephrons in early mouse developmental stages such as, for example, 15 days of gestation (E15). Immunoblotting of total extracts of cultured cells of renal origin showed that ARVCF is detected in all human and murine cultured cells analyzed. Upon immunolocalization ARVCF is mostly detected in the cytoplasm occurring in a fine granular form. This prominent cytoplasmic localization of ARVCF in cultured cells and its occurrence in proximal tubules implies an involvement of ARVCF in specific functional processes of proximal tubules of kidney.

Drebrin, an actin-binding, cell-type characteristic protein: induction and localization in epithelial skin tumors and cultured keratinocytes.

Isoform E2 of drebrin, an actin-binding protein originally identified in neuronal cells, has recently been identified in diverse non-neuronal cells, mostly in association with cell processes and intercellular junctions. Here, we report on the presence of drebrin in normal human skin, epithelial skin cancers, and cultured keratinocytes. Keratinocytes of normal epidermis contain almost no drebrin but the protein is readily seen in hair follicles. By immunohistochemistry and immunoblot, basal cell carcinomas (BCC) are rich in drebrin, and confocal laser scanning and immunoelectron microscopy show accumulation at adhering junctions, in co-localization with actin and partially with plaque proteins. In squamous cell carcinomas, keratoacanthomas, and in epidermal precancers, drebrin is heterogeneously distributed, appearing as mosaics. Primary keratinocyte cultures contain significant amounts of drebrin enriched at adhering junctions. When epithelium-derived cells devoid of drebrin are transfected with drebrin-enhanced green fluorescent protein, constructs accumulate in the cell periphery, and immunoprecipitation shows complexes with actin. During epidermal growth factor induced formation of cell processes, drebrin retains this junction association, as observed by live cell microscopy. Our results suggest novel functions of drebrin such as an involvement in cell-cell adhesion and tumorigenesis and a potential value in diagnosis of BCC.

Urothelial umbrella cells of human ureter are heterogeneous with respect to their uroplakin composition: different degrees of urothelial maturity in ureter and bladder?

Urothelial umbrella cells are characterized by apical, rigid membrane plaques, which contain four major uroplakin proteins (UP Ia, Ib, II and III) forming UPIa/UPII and UPIb/UPIII pairs. These integral membrane proteins are thought to play an important role in maintaining the physical integrity and the permeability barrier function of the urothelium. We asked whether the four uroplakins always coexpress in the entire human lower urinary tract. We stained immunohistochemically (ABC-peroxidase method) paraffin sections of normal human ureter (n = 18) and urinary bladder (n = 10) using rabbit antibodies against UPIa, UPIb, UPII and UPIII; a recently raised mouse monoclonal antibody (MAb), AU1, and two new MAbs, AU2 and AU3, all against UPIII; and mouse MAbs against umbrella cell-associated cytokeratins CK18 and CK20. Immunoblotting showed that AU1, AU2 and AU3 antibodies all recognized the N-terminal extracellular domain of bovine UPIII. By immunohistochemistry, we found that in 15/18 cases of human ureter, but in only 2/10 cases of bladder, groups of normal-looking, CK18-positive umbrella cells lacked both UPIII and UPIb immunostaining. The UPIb/UPIII-negative cells showed either normal or reduced amounts of UPIa and UPII staining. These data were confirmed by double immunofluorescence microscopy. The distribution of the UPIb/UPIII-negative umbrella cells was not correlated with localized urothelial proliferation (Ki-67 staining) or with the distribution pattern of CK20. Similar heterogeneities were observed in bovine but not in mouse ureter. We provide the first evidence that urothelial umbrella cells are heterogeneous as some normal-looking umbrella cells can possess only one, instead of two, uroplakin pairs. This heterogeneity seems more prominent in the urothelium of human ureter than that of bladder. This finding may indicate that ureter urothelium is intrinsically different from bladder urothelium. Alternatively, a single lineage of urothelium may exhibit different phenotypes resulting from extrinsic modulations due to distinct mesenchymal influence and different degrees of pressure and stretch in bladder versus ureter. Additional studies are needed to distinguish these two possibilities and to elucidate the physiological and pathological significance of the observed urothelial and uroplakin heterogeneity.

NO66, a highly conserved dual location protein in the nucleolus and in a special type of synchronously replicating chromatin.

It has recently become clear that the nucleolus, the most prominent nuclear subcompartment, harbors diverse functions beyond its classic role in ribosome biogenesis. To gain insight into nucleolar functions, we have purified amplified nucleoli from Xenopus laevis oocytes using a novel approach involving fluorescence-activated cell sorting techniques. The resulting protein fraction was analyzed by mass spectrometry and used for the generation of monoclonal antibodies directed against nucleolar components. Here, we report the identification and molecular characterization of a novel, ubiquitous protein, which in most cell types appears to be a constitutive nucleolar component. Immunolocalization studies have revealed that this protein, termed NO66, is highly conserved during evolution and shows in most cells analyzed a dual localization pattern, i.e., a strong enrichment in the granular part of nucleoli and in distinct nucleoplasmic entities. Colocalizations with proteins Ki-67, HP1alpha, and PCNA, respectively, have further shown that the staining pattern of NO66 overlaps with certain clusters of late replicating chromatin. Biochemical experiments have revealed that protein NO66 cofractionates with large preribosomal particles but is absent from cytoplasmic ribosomes. We propose that in addition to its role in ribosome biogenesis protein NO66 has functions in the replication or remodeling of certain heterochromatic regions.

Detection of the human organic anion transporters SLC21A6 (OATP2) and SLC21A8 (OATP8) in liver and hepatocellular carcinoma.

Transport proteins mediating the selective uptake of organic anions into human hepatocytes include the organic anion transporters SLC21A6 (also termed OATP2, OATP-C, or LST-1) and SLC21A8 (OATP8). Both transporters are localized to the basolateral membrane of human hepatocytes. Because of the importance of these transporters for hepatobiliary elimination, including the removal of bilirubin and its conjugates from the blood circulation, we have generated monoclonal antibodies for studies on the expression and localization of these transport proteins. We describe two antibodies, designated monoclonal antibody MDQ (mMDQ) and monoclonal antibody ESL (mESL), directed against the amino terminus and the carboxyl terminus of human SLC21A6, respectively. Both antibodies have been characterized by immunoblot analysis, immunoprecipitation, and immunofluorescence microscopy. While mESL reacted specifically with SLC21A6, mMDQ detects both SLC21A6 and SLC21A8. Neither of the two antibodies reacted with other human, or with dog, rat, or mouse liver SLC21A family members. Antibody mMDQ may be used for the simultaneous detection of SLC21A6 and SLC21A8 in immunoblotting because of its immunoreactivity with both molecules and because of the different molecular masses of both glycosylated proteins in human hepatocytes. This is exemplified in hepatocellular carcinomas where SLC21A6 and SLC21A8 were differentially synthesized and showed an irregular staining pattern. Both transport proteins have not been detected in human hepatoma HepG2 cells. In routine paraffin sections, 10 of 12 hepatocellular carcinomas were focally positive with antibody mMDQ. In contrast, cholangiocarcinomas and liver metastases of colorectal and pancreatic adenocarcinoma were negative without exception. This suggests the usefulness of SLC21A6/SLC21A8 within a panel of tumor markers for hepatocellular carcinomas. Moreover, both antibodies should be useful in studies on the expression and localization of two important uptake transporters of human hepatocytes under physiologic and pathophysiologic conditions.

The evolutionarily conserved single-copy gene for murine Tpr encodes one prevalent isoform in somatic cells and lacks paralogs in higher eukaryotes.

Vertebrate Tpr and its probable homologs in insects and yeast are heptad repeat-dominated nuclear proteins of M(r) 195,000 to M(r) 267,000 the functions of which are still largely unknown. Whereas two homologs exist in Saccharomyces cerevisiae, it has remained uncertain whether metazoans possess different paralogs or isoforms of Tpr that might explain controversial reports on the subcellular localization of this protein. To address these possibilities, we first determined the sequence and structure of the murine tpr gene, revealing a TATA box-less gene of approximately 57 kb and 52 exons. Southern hybridization of genomic DNA and radiation hybrid mapping showed that murine tpr exists as a single-copy gene on chromosome 1; RNA blotting analyses and EST (expressed sequence tag) database mining revealed that its expression results in only one major mRNA in embryonic and most adult tissues. Accordingly, novel antibodies against the N- and C-terminus of Tpr identified the full-length protein as the major translation product in different somatic cell types; reinvestigation of Tpr localization by confocal microscopy corroborated a predominant localization at the nuclear pore complexes in these cells. Antibody specificity and reliability of Tpr localization was demonstrated by post-transcriptional tpr gene silencing using siRNAs that eliminated the Tpr signal at the nuclear periphery but did not affect intranuclear staining of Tpr-unrelated proteins. Finally, we defined several sequence and structural features that characterize Tpr polypeptides in different species and used these as a guideline to search whole-genome sequence databases for putative paralogs of Tpr in higher eukaryotes. This approach resulted in identification of the Tpr orthologs in Arabidopsis thaliana and Caenorhabditis elegans, but also in the realization that no further paralogs of Tpr exist in several metazoan model organisms or in humans. In summary, these results reveal Tpr to be a unique protein localized at the nuclear periphery of the somatic cell in mammals.