Corinna Herz

Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.

A novel family of focal adhesion proteins, the kindlins, is involved in attachment of the actin cytoskeleton to the plasma membrane and in integrin-mediated cellular processes. Deficiency of kindlin-1, as a result of loss-of-function mutations in the KIND1 gene, causes Kindler syndrome, an autosomal recessive genodermatosis characterized by skin blistering, progressive skin atrophy, photosensitivity and, occasionally, carcinogenesis. Here we characterized authentic and recombinantly expressed kindlin-1 and show that it is localized in basal epidermal keratinocytes in a polar fashion, close to the cell surface facing the basement membrane, in the areas between the hemidesmosomes. We identified two forms of kindlin-1 in keratinocytes, with apparent molecular masses of 78 and 74 kDa, corresponding to phosphorylated and desphosphorylated forms of the protein. In kindlin-1-deficient skin, basal keratinocytes show multiple abnormalities: cell polarity is lost, proliferation is strongly reduced, and several cells undergo apoptosis. In vitro, deficiency of kindlin-1 in keratinocytes leads to strongly reduced cell proliferation, decreased adhesion, undirected motility, and intense protrusion activity of the plasma membrane. Taken together, these results show that kindlin-1 plays a role in keratinocyte adhesion, polarization, proliferation, and migration. It is involved in organization and anchorage of the actin cytoskeleton to integrin-associated signaling platforms.

Kindlin-1 Is required for RhoGTPase-mediated lamellipodia formation in keratinocytes.

Kindlin-1 is an epithelial-specific member of the novel kindlin protein family, which are regulators of integrin functions. Mutations in the gene that encodes Kindlin-1, FERMT1 (KIND1), cause the Kindler syndrome (KS), a human disorder characterized by mucocutaneous fragility, progressive skin atrophy, ulcerative colitis, photosensitivity, and propensity to skin cancer. Our previous studies indicated that loss of kindlin-1 resulted in abnormalities associated with integrin functions, such as adhesion, proliferation, polarization, and motility of epidermal cells. Here, we disclosed novel FERMT1 mutations in KS and used them, in combination with small-interfering RNA, protein, and imaging studies, to uncover new functions for kindlin-1 in keratinocytes and to discern the molecular pathology of KS. We show that kindlin-1 forms molecular complexes with beta1 integrin, alpha-actinin, migfilin, and focal adhesion kinase and regulates cell shape and migration by controlling lamellipodia formation. Kindlin-1 governs these processes by signaling via Rho family GTPases, and it is required to maintain the pool of GTP-bound, active Rac1, RhoA and Cdc42, and the phosphorylation of their downstream effectors p21-activated kinase 1, LIM kinase, and cofilin. Loss of these kindlin-1 functions forms the biological basis for the epithelial cell fragility and atrophy in the pathology of KS.

Pharmacokinetics and pharmacodynamics of isothiocyanates

Isothiocyanates from Brassica vegetables are of great interest for use in the cure of bacterial infections, as is their potential application in the prevention and treatment of cancer. Although much information is available on their mode of action within the cell, when it comes to the question of whether the necessary pharmacologic concentration has been reached at the target organ, detailed knowledge is still lacking. However, a basic prerequisite for clinical application to humans is knowledge of isothiocyanate pharmacokinetic and dynamic behavior in the human body (e.g., to define intake intervals or to ascertain constant levels of the active compound). In this context, we, therefore, reviewed the available literature on in vitro studies, as well as animal and human intervention trials conducted with isothiocyanate and isothiocyanate-containing food preparations.

Occurrence of multipolar mitoses and association with Aurora-A/-B kinases and p53 mutations in aneuploid esophageal carcinoma cells

BackgroundAurora kinases and loss of p53 function are implicated in the carcinogenesis of aneuploid esophageal cancers. Their association with occurrence of multipolar mitoses in the two main histotypes of aneuploid esophageal squamous cell carcinoma (ESCC) and Barretts adenocarcinoma (BAC) remains unclear. Here, we investigated the occurrence of multipolar mitoses, Aurora-A/-B gene copy numbers and expression/activation as well as p53 alterations in aneuploid ESCC and BAC cancer cell lines.ResultsA control esophageal epithelial cell line (EPC-hTERT) had normal Aurora-A and -B gene copy numbers and expression, was p53 wild type and displayed bipolar mitoses. In contrast, both ESCC (OE21, Kyse-410) and BAC (OE33, OE19) cell lines were aneuploid and displayed elevated gene copy numbers of Aurora-A (chromosome 20 polysomy: OE21, OE33, OE19; gene amplification: Kyse-410) and Aurora-B (chromosome 17 polysomy: OE21, Kyse-410). Aurora-B gene copy numbers were not elevated in OE19 and OE33 cells despite chromosome 17 polysomy. Aurora-A expression and activity (Aurora-A/phosphoT288) was not directly linked to gene copy numbers and was highest in Kyse-410 and OE33 cells. Aurora-B expression and activity (Aurora-B/phosphoT232) was higher in OE21 and Kyse-410 than in OE33 and OE19 cells. The mitotic index was highest in OE21, followed by OE33 > OE19 > Kyse-410 and EPC-hTERT cells. Multipolar mitoses occurred with high frequency in OE33 (13.8 ± 4.2%), followed by OE21 (7.7 ± 5.0%) and Kyse-410 (6.3 ± 2.0%) cells. Single multipolar mitoses occurred in OE19 (1.0 ± 1.0%) cells. Distinct p53 mutations and p53 protein expression patterns were found in all esophageal cancer cell lines, but complete functional p53 inactivation occurred in OE21 and OE33 only.ConclusionsHigh Aurora-A expression alone is not associated with overt multipolar mitoses in aneuploid ESCC and BAC cancer cells, as specifically shown here for OE21 and OE33 cells, respectively. Additional p53 loss of function mutations are necessary for this to occur, at least for invasive esophageal cancer cells. Further assessment of Aurora kinases and p53 interactions in cells or tissue specimens derived from non-invasive dysplasia (ESCC) or intestinal metaplasia (BAC) are necessary to disclose a potential causative role of Aurora kinases and p53 for development of aneuploid, invasive esophageal cancers.

The MAPK Pathway Signals Telomerase Modulation in Response to Isothiocyanate-Induced DNA Damage of Human Liver Cancer Cells

4-methylthiobutyl isothiocyanate (MTBITC), an aliphatic, sulphuric compound from Brassica vegetables, possesses in vitro and in vivo antitumor activity. Recently we demonstrated the potent growth inhibitory potential of the DNA damaging agent MTBITC in human liver cancer cells. Here we now show that MTBITC down regulates telomerase which sensitizes cells to apoptosis induction. This is mediated by MAPK activation but independent from production of reactive oxygen species (ROS). Within one hour, MTBITC induced DNA damage in cancer cells correlating to a transient increase in hTERT mRNA expression which then turned into telomerase suppression, evident at mRNA as well as enzyme activity level. To clarify the role of MAPK for telomerase regulation, liver cancer cells were pre-treated with MAPK-specific inhibitors prior to MTBITC exposure. This clearly showed that transient elevation of hTERT mRNA expression was predominantly mediated by the MAPK family member JNK. In contrast, activated ERK1/2 and P38, but not JNK, signalled to telomerase abrogation and consequent apoptosis induction. DNA damage by MTBITC was also strongly abolished by MAPK inhibition. Oxidative stress, as analysed by DCF fluorescence assay, electron spin resonance spectroscopy and formation of 4-hydroxynonenal was found as not relevant for this process. Furthermore, N-acetylcysteine pre-treatment did not impact MTBITC-induced telomerase suppression or depolarization of the mitochondrial membrane potential as marker for apoptosis. Our data therefore imply that upon DNA damage by MTBITC, MAPK are essential for telomerase regulation and consequent growth impairment in liver tumor cells and this detail probably plays an important role in understanding the potential chemotherapeutic efficacy of ITC.

hTERT: another brick in the wall of cancer cells.

In human cancer, expression of telomerase is positively correlated with tumour aggressiveness and metastatic potential. There is accumulating evidence that hTERT (the catalytic subunit of telomerase) favours an immortal phenotype by blocking programmed cell death (apoptosis) independently of its protective function at the telomere ends. This review summarized existing evidence for the anti-apoptotic role of hTERT in the context of tumour-cell resistance against DNA damage and aims to put hTERT in the context of cell-signal-transduction pathways leading either to survival or cell death. We found evidence that telomerase is cross-linked with many different signalling pathways that regulate cell proliferation, DNA damage repair, and also cell death. Thereby, hTERT survival function seems to occur at early stages of DNA damage recognition. We found some discrepancies in the published data though. Based on our findings, we suggest further exploration is needed of the interplay of the DNA damage response signalling network, including MAPK and p53 family activation, on telomerase regulation. This interaction is probably an important factor for fine tuning of the sensitivity of the cell to genotoxic stress. Using anti-neoplastic agents, further dose relationships on timing and extent of DNA damage, cellular repair and death should be established and correlated with hTERT expression/telomerase activation. Closing the data gaps identified here could profoundly improve our understanding of the relevance of telomerase for protecting the cell against anti-cancer agents and would contribute to developing new strategies for cancer therapy.

The Brassica epithionitrile 1‐cyano‐2,3‐epithiopropane triggers cell death in human liver cancer cells in vitro

SCOPE Glucosinolates are secondary metabolites present in Brassica vegetables. Alkenyl glucosinolates are enzymatically degraded forming nitriles or isothiocyanates, but in the presence of epithiospecifier protein, epithionitriles are released. However, studies on the occurrence of epithionitriles in Brassica food and knowledge about their biological effects are scarce. METHODS AND RESULTS Epithionitrile formation from glucosinolates of seven Brassica vegetables was analyzed using GC-MS and HPLC-DAD. Bioactivity of synthetic and plant-derived 1-cyano-2,3-epithiopropane (CETP) - the predominant epithionitrile in Brassica vegetables - in three human hepatocellular carcinoma (HCC) cell lines and primary murine hepatocytes was also evaluated. The majority of the Brassica vegetables were producers of nitriles or epithionitriles as hydrolysis products and not of isothiocyanates. For example, Brussels sprouts and savoy cabbage contained up to 0.8 μmol CETP/g vegetable. Using formazan dye assays, concentrations of 380-1500 nM CETP were observed to inhibit the mitochondrial dehydrogenase activity of human HCC cells without impairment of cell growth. At 100-fold higher CETP concentrations, cell death was observed. Presence of plant matrix increased CETP-based toxicity. CONCLUSION These in vitro data provide no indication that epithionitriles will severely affect human health by Brassica consumption. In contrast to isothiocyanates, no evidence of selective toxicity against HCC cells was found.

Harmonic Filters for 3D Multichannel Data: Rotation Invariant Detection of Mitoses in Colorectal Cancer

In this paper, we present a novel approach for a trainable rotation invariant detection of complex structures in 3D microscopic multichannel data using a nonlinear filter approach. The basic idea of our approach is to compute local features in a window around each 3D position and map these features by means of a nonlinear mapping onto new local harmonic descriptors of the local window. These local harmonic descriptors are then combined in a linear way to form the output of the filter. The optimal combination of the computed local harmonic descriptors is determined in previous training step, and allows the filter to be adapted to an arbitrary structure depending on the problem at hand. Our approach is not limited to scalar-valued images and can also be used for vector-valued (multichannel) images such as gradient vector flow fields. We present realizations of a scalar-valued and a vector-valued multichannel filter. Our proposed algorithm was quantitatively evaluated on colorectal cancer cell lines (cells grown under controlled conditions), on which we successfully detected complex 3D mitotic structures. For a qualitative evaluation we tested our algorithms on human 3D tissue samples of colorectal cancer. We compare our results with a steerable filter approach as well as a morphology-based approach.

Elevated endogenous expression of the dominant negative basic helix-loop-helix protein ID1 correlates with significant centrosome abnormalities in human tumor cells

BackgroundID proteins are dominant negative inhibitors of basic helix-loop-helix transcription factors that have multiple functions during development and cellular differentiation. Ectopic (over-)expression of ID1 extends the lifespan of primary human epithelial cells. High expression levels of ID1 have been detected in multiple human malignancies, and in some have been correlated with unfavorable clinical prognosis. ID1 protein is localized at the centrosomes and forced (over-)expression of ID1 results in errors during centrosome duplication.ResultsHere we analyzed the steady state expression levels of the four ID-proteins in 18 tumor cell lines and assessed the number of centrosome abnormalities. While expression of ID1, ID2, and ID3 was detected, we failed to detect protein expression of ID4. Expression of ID1 correlated with increased supernumerary centrosomes in most cell lines analyzed.ConclusionsThis is the first report that shows that not only ectopic expression in tissue culture but endogenous levels of ID1 modulate centrosome numbers. Thus, our findings support the hypothesis that ID1 interferes with centrosome homeostasis, most likely contributing to genomic instability and associated tumor aggressiveness.

Occurrence of Aurora A positive multipolar mitoses in distinct molecular classes of colorectal carcinomas and effect of Aurora A inhibition.

Aurora A “over‐”expression may induce supernumerary centrosomes, respective multipolar mitoses, and aneuploidy. Here, we examined Aurora A positive multipolar mitoses in aneuploid, microsatellite‐stable (MSS, “CIN‐type”) versus near‐diploid, microsatellite‐instable (MSI, “MIN‐type”) colorectal carcinomas (CRC) and CRC cell lines as well as the effect of Aurora A inhibition in CRC cell lines. In situ, three‐dimensional immunofluorescence (3D‐IF) revealed Aurora A positive multipolar mitoses in both CIN‐ (n = 8) and MIN‐ (n = 10) type primary CRCs with similar frequencies (CIN: 27 ± 14%; MIN: 34 ± 14%, P = 0.224). In vitro, Aurora A positive multipolar mitoses were detected in asynchronized or thymidine synchronized CIN‐type (HT29, CaCo‐2), but not MIN‐type (HCT116, DLD‐1) CRC cells. Nocodazole treatment arrested mitotic cells with multiple centrosomal Aurora A signals in CIN‐ and MIN‐type CRC cells, albeit to a lower extent in CaCo‐2 cells. This was associated with concomitant activation of Aurora A (T288 phosphorylation) and Polo‐like kinase 1 (PLK‐1, T210 phosphorylation). Aurora A inhibition by siRNA resulted in increased apoptosis (>50%) in all cell lines, but did not abolish PLK‐1 expression. Double 3D‐IF revealed that Aurora A siRNA treated, still viable CIN‐type (HT29, CaCo‐2) CRC cells were Aurora A negative and mostly in prophase/(pro)metaphase with maintained phosphorylated PLK‐1 T210 expression. Aurora A positive multipolar mitoses occur in both aneuploid, CIN‐ and near‐diploid MIN‐type CRCs. This appears to be largely independent of Aurora A expression alone. Although Aurora A inhibition causes apoptosis in both CIN‐ and MIN‐type CRC cells, remaining PLK‐1 activation by other factors may affect therapeutic Aurora inhibition.