The electrical resistance breakdown assay determines the role of proteinases in tumor cell invasion

Ludwig, Thomas; Ossig, Rainer; Graessel, Susanne; Wilhelmi, Marianne; Oberleithner, Hans; Schneider, Stefan W.

doi:10.1152/ajprenal.00327.2001

Cited by 38 publications

(63 citation statements)

References 40 publications

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“…In the first set of experiments we investigated the invasive properties of all three cell lines with an electrophysiological cell-based invasion assay previously established in our laboratory 24,25 . One of the key features of this assay is the high sensitivity to evaluate very early steps of invasion 26 . Although the invasive behaviour of the three cell lines was comparable in siCon cells, we found that knockdown of HPSE attenuates the invasive properties in BLM and MV3 cells as indicated by a delayed decrease of electrical resistance (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity

et al. 2015

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Heparanase-1 (HPSE) plays a pivotal role in structural remodeling of the ECM and glycocalyx thus conferring protumorigenic, proangiogenic and prometastatic properties to many cancer entities. In addition to its extracellular function, recent studies suggest an intracellular activity of HPSE with a largely unknown significance during tumor progression. Therefore, we investigated the relevance of HPSE duality in malignant melanoma in vitro as well as in mouse melanoma models basing on the intradermal injection of transfected melanoma cells. In line with its extracellular action, HPSE-deficiency led to a reduced shedding of the glycocalyx accompanied by a reduced availability of VEGF affecting tumor growth and vascularization. In contrast, we measured an elevated expression of the protumorigenic factors pentraxin-3, tissue factor, TNF-and most prominently MMP-9 upon HPSE knockdown. In vivo, HPSE-deficiency was related to increased lymph node metastasis. While inhibition of its extracellular function heparin was unable to block the gene regulatory impact of HPSE we proposed an intracellular mechanism. Immunostainings revealed a counter-staining of HPSE and NF-B in the nucleus suggesting a close relationship between both proteins. This finding was further supported by the discovery of a direct charge-driven molecular interaction between HPSE and DNA by using atomic force microscopy and a co-precipitation approach. Our findings are novel and point towards a dual identity of HPSE in malignant melanoma with a protumorigenic extracellular activity and a tumor suppressive nuclear action. Identification of molecular strategies to shuttle extracellular HPSE into the nuclei of cancer cells envisions new therapeutic options. AbstractHeparanase-1 (HPSE) plays a pivotal role in structural remodeling of the ECM and glycocalyx thus conferring protumorigenic, proangiogenic and prometastatic properties to many cancer entities. In addition to its extracellular function, recent studies suggest an intracellular activity of HPSE with a largely unknown significance during tumor progression.Therefore, we investigated the relevance of HPSE duality in malignant melanoma in vitro as well as in mouse melanoma models basing on the intradermal injection of transfected melanoma cells. In line with its extracellular action, HPSE-deficiency led to a reduced shedding of the glycocalyx accompanied by a reduced availability of VEGF affecting tumor growth and vascularization. In contrast, we measured an elevated expression of the protumorigenic factors pentraxin-3, tissue factor, TNF-α and most prominently MMP-9 upon HPSE knockdown. In vivo, HPSE-deficiency was related to increased lymph node metastasis.While inhibition of its extracellular function heparin was unable to block the gene regulatory impact of HPSE we proposed an intracellular mechanism. Immunostainings revealed a counter-staining of HPSE and NF-κB in the nucleus suggesting a close relationship between both proteins. This finding was further supported by the discovery of a dir...

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Section: Discussionmentioning

confidence: 99%

“…We previously demonstrated that MMP-2 and MMP-9 secreted from melanoma cells are strongly involved in the breakdown of TEER 25,26 . Therefore, we analyzed the secreted amounts of both proteinases in supernatants of melanoma cells by gelatin gel zymography.…”

Section: Knockdown Of Hpse Enhances the Expression Of Mmp But Reducesmentioning

confidence: 99%

Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity

et al. 2015

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“…The quantification of proteolytic activity in vitro and even more in situ is hampered by two major obstacles: excessive posttranslational regulation and spatial restriction (Fig. 12b), both of which are major topics of ongoing research themselves [85,88,89,136].…”

Section: Cellular Microecology and Proteolytic Activitymentioning

confidence: 99%

“…As a result, protease action must obviously occur in the immediate cellular surrounding to contribute to the regulation of the pericellular microenvironment [89]. Not surprisingly, mechanisms that confine and concentrate protease activity in the pericellular space have been found to be essential in tumor cell invasion [85,88]. Among others, these mechanisms comprise the expression of membrane-anchored proteases and receptors for soluble ones by cancer cells [85,86].…”

Section: Cellular Microecology and Proteolytic Activitymentioning

confidence: 99%

Probing cellular microenvironments and tissue remodeling by atomic force microscopy

Ludwig

Kirmse

Poole

et al. 2007

Pflugers Arch - Eur J Physiol

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The function of cells is strongly determined by the properties of their extracellular microenvironment. Biophysical parameters like environmental stiffness and fiber orientation in the surrounding matrix are important determinants of cell adhesion and migration. Processes like tissue maintenance, wound repair, cancer cell invasion, and morphogenesis depend critically on the ability of cells to actively sense and remodel their surroundings. Pericellular proteolytic activity and adaptation of migration tactics to the environment are strategies to achieve this aim. Little is known about the distinct regulatory mechanisms that are involved in these processes. The system's critical biophysical and biochemical determinants are well accessible by atomic force microscopy (AFM), a unique tool for functional, nanoscale probing and morphometric, highresolution imaging of processes in live cells. This review highlights common principles of tissue remodeling and focuses on application examples of different AFM techniques, for example elasticity mapping, the combination of AFM and fluorescence microscopy, the morphometric imaging of proteolytic activity, and force spectroscopy applications of single molecules or individual cells. To achieve a more complete understanding of the processes underlying the interaction of cells with their environments, the combination of AFM force spectroscopy experiments will be essential.

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“…An epithelial MDCK-C7 cell monolayer serves as a test barrier for malignant cells and develops a high transepithelial electrical resistance (TEER), which can be measured continuously using STX-2 electrode (WPI, Sarasota, FL). Added invasive cells will disturb monolayer integrity followed by decrease and breakdown of TEER (23,31). C7 cells were grown in membrane filter cups (growth area, 4.2 cm 2 ; pore diameter, 0.4 m; thickness, 20 m; Falcon, Heidelberg, Germany).…”

Section: Melanoma Cells and Madin-darby Canine Kidney Cells (Mdck)-c7mentioning

confidence: 99%

Microtubule-Dependent Matrix Metalloproteinase-2/Matrix Metalloproteinase-9 Exocytosis

et al. 2004

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Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave and degrade a wide spectrum of extracellular matrix components. By enhancing turnover of extracellular matrix, MMP activity is also known to play a key role in tumor cell invasion. Because extracellular protease activity requires efficient release of these proteases to the cellular surface, we investigated storage, transport, and exocytosis of MMP-2 and MMP-9 in human melanoma cells using immunofluorescence, electrical, and biochemical techniques. Immunolabeling of melanoma cells with antibodies specific for MMP-2 and MMP-9 led to the identification of two distinct populations of small cytoplasmatic vesicles containing MMP-2 or MMP-9, respectively. In combination with ␣-tubulin-specific antibodies, both vesicle populations were found to be aligned along the microtubular network. Moreover, the molecular motor protein kinesin is shown to be localized on most of these vesicles, providing evidence that the identified vesicles are actively propelled along microtubules toward the plasma membrane. The functional relevance of these findings is demonstrated using low dosage (5.9 nmol/L) of paclitaxel to affect the microtubular function of melanoma cells. Although cell proliferation is not altered, paclitaxel treatment impairs secretion of MMP-2/MMP-9 and significantly reduces invasive activity in our new cell invasion assay. In conclusion, we demonstrate in melanoma cells that microtubule-dependent traffic of MMP-containing vesicles and exocytosis are critical steps for invasive behavior and therefore are potential targets for specific antitumor drugs.

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The electrical resistance breakdown assay determines the role of proteinases in tumor cell invasion

Cited by 38 publications

References 40 publications

Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity

Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity

Probing cellular microenvironments and tissue remodeling by atomic force microscopy

Microtubule-Dependent Matrix Metalloproteinase-2/Matrix Metalloproteinase-9 Exocytosis

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