Michael A. Partridge

Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase

Imaging studies implicate microtubule targeting of focal adhesions in focal adhesion disassembly, although the molecular mechanism is unknown. Here, we develop a model system of focal adhesion disassembly based on the finding that microtubule regrowth after nocodazole washout induces disassembly of focal adhesions, and that this disassembly occurs independently of Rho and Rac, but depends on focal adhesion kinase (FAK) and dynamin. During disassembly, dynamin interacts with FAK and colocalizes with focal adhesions. Inhibition of dynamin prevents migration of cells with a focal adhesion phenotype. Our results show that focal adhesion disassembly involves microtubules, dynamin and FAK, and is not simply the reversal of focal adhesion formation.

Resveratrol sensitizes melanomas to TRAIL through modulation of antiapoptotic gene expression.

Although many human melanomas express the death receptors TRAIL-R2/DR5 or TRAIL-R1/DR4 on cell surface, they often exhibit resistance to exogenous TRAIL. One of the main contributors to TRAIL-resistance of melanoma cells is upregulation of transcription factors STAT3 and NF-kappaB that control the expression of antiapoptotic genes, including cFLIP and Bcl-xL. On the other hand, the JNK-cJun pathway is involved in the negative regulation of cFLIP (a caspase-8 inhibitor) expression. Our observations indicated that resveratrol, a polyphenolic phytoalexin, decreased STAT3 and NF-kappaB activation, while activating JNK-cJun that finally suppressed expression of cFLIP and Bcl-xL proteins and increased sensitivity to exogenous TRAIL in DR5-positive melanomas. Interestingly, resveratrol did not increase surface expression of DR5 in human melanomas, while gamma-irradiation or sodium arsenite treatment substantially upregulated DR5 expression. Hence, an initial increase in DR5 surface expression (either by gamma-irradiation or arsenite), and subsequent downregulation of antiapoptotic cFLIP and Bcl-xL (by resveratrol), appear to constitute an efficient approach to reactivate apoptotic death pathways in TRAIL-resistant human melanomas. In spite of partial suppression of mitochondrial function and the mitochondrial death pathway, melanoma cells still retain the potential to undergo the DR5-mediated, caspase-8-dependent death pathway that could be accelerated by either an increase in DR5 surface expression or suppression of cFLIP. Taken together, these results suggest that resveratrol, in combination with TRAIL, may have a significant efficacy in the treatment of human melanomas.

Arsenic induced mitochondrial DNA damage and altered mitochondrial oxidative function: implications for genotoxic mechanisms in mammalian cells.

Arsenic is a well-established human carcinogen that is chronically consumed in drinking water by millions of people worldwide. Recent evidence has suggested that arsenic is a genotoxic carcinogen. Furthermore, we have shown that mitochondria mediate the mutagenic effects of arsenic in mammalian cells, as arsenic did not induce nuclear mutations in mitochondrial DNA (mtDNA)-depleted cells. Using the human-hamster hybrid A(L) cells, we show here that arsenic alters mitochondrial function by decreasing cytochrome c oxidase function and oxygen consumption but increasing citrate synthase function. These alterations correlated with depletion in mtDNA copy number and increase in large heteroplasmic mtDNA deletions. In addition, mtDNA isolated periodically from cultures treated continuously with arsenic did not consistently display the same deletion pattern, indicating that the mitochondrial genome was subjected to repeated and continuous damage. These data support the theory that the mitochondria, and particularly mtDNA, are important targets of the mutagenic effects of arsenic in mammalian cells.

Src SH2 arginine 175 is required for cell motility: specific focal adhesion kinase targeting and focal adhesion assembly function.

ABSTRACT Src kinase is a crucial mediator of adhesion-related signaling and motility. Src binds to focal adhesion kinase (FAK) through its SH2 domain and subsequently activates it for phosphorylation of downstream substrates. In addition to this binding function, data suggested that the SH2 domain might also perform an important role in targeting Src to focal adhesions (FAs) to enable further substrate phosphorylations. To examine this, we engineered an R175L mutation in cSrc to prevent the interaction with FAK pY397. This constitutively open Src kinase mediated up-regulated substrate phosphorylation in SYF cells but was unable to promote malignant transformation. Significantly, SrcR175L cells also had a profound motility defect and an impaired FA generation capacity. Importantly, we were able to recapitulate wild-type motile behavior and FA formation by directing the kinase to FAs, clearly implicating the SH2 domain in recruitment to FAK and indicating that this targeting capacity, and not simply Src-FAK scaffolding, was critical for normal Src function.

Inhibition of Ataxia Telangiectasia Mutated Kinase Activity Enhances TRAIL-Mediated Apoptosis in Human Melanoma Cells

The aim of the present study was to elucidate the effects of ataxia telangiectasia mutated (ATM) kinase on the regulation of the extrinsic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2/DR5-mediated death pathway in human melanoma cells. We revealed that total ATM protein levels were high in some human melanoma lines compared with normal cells. The basal levels of active form ATM phospho-Ser(1981) were also detectable in many melanoma lines and could be further up-regulated by gamma-irradiation. Pretreatment of several melanoma lines just before gamma-irradiation with the inhibitor of ATM kinase KU-55933 suppressed p53 and nuclear factor-kappaB (NF-kappaB) activation but notably increased radiation-induced DR5 surface expression, down-regulated cFLIP (caspase-8 inhibitor) levels, and substantially enhanced exogenous TRAIL-induced apoptosis. Furthermore, gamma-irradiation in the presence of KU-55933 rendered TRAIL-resistant HHMSX melanoma cells susceptible to TRAIL-mediated apoptosis. In addition, suppression of ATM expression by the specific short hairpin RNA also resulted in down-regulation of cFLIP levels, up-regulation of surface DR5 expression, and TRAIL-mediated apoptosis in melanoma cells. Besides p53 and NF-kappaB, crucial regulators of DR5 expression, transcription factor STAT3 is known to negatively regulate DR5 expression. Suppression of Ser(727) and Tyr(705) phosphorylation of STAT3 by KU-55933 reduced STAT3 transacting activity accompanied by elevation in DR5 expression. Dominant-negative STAT3beta also efficiently up-regulated the DR5 surface expression and down-regulated cFLIP levels in melanoma cells in culture and in vivo. Taken together, our data show the existence of an ATM-dependent STAT3-mediated antiapoptotic pathway, which on suppression sensitizes human melanoma cells to TRAIL-mediated apoptosis.

TGFBI expression reduces in vitro and in vivo metastatic potential of lung and breast tumor cells

Controversy has arisen as to the role of transforming growth factor-β-induced protein (TGFBI) in the regulation of tumor metastasis. Using lung and breast cancer cell lines (H522 and MCF-7, respectively), we established that TGFBI induced cell adhesion to extracellular matrix proteins by activating adhesion-associated signaling and subsequent structure reformation, ultimately leading to cells less motile; whereas TGFBI reduced abilities of colony formation in soft agar, penetration through matrix gel, and activation of matrix metalloproteinases 2 and 9. Furthermore, injection of TGFBI-expressing cells into immuno-deficient mice resulted in a significant reduction in tumor metastasis in vivo. Taken together, these data suggest that TGFBI moderates the metastatic potential of cancer cells.

Mitochondria-Derived Reactive Intermediate Species Mediate Asbestos-Induced Genotoxicity and Oxidative Stress-Responsive Signaling Pathways

Background: The incidence of asbestos-induced human cancers is increasing worldwide, and considerable evidence suggests that reactive oxygen species (ROS) are important mediators of these diseases. Our previous studies suggested that mitochondria might be involved in the initiation of oxidative stress in asbestos-exposed mammalian cells. Objective: We investigated whether mitochondria are a potential cytoplasmic target of asbestos using a mitochondrial DNA–depleted (ρ0) human small airway epithelial (SAE) cell model: ρ0 SAE cells lack the capacity to produce mitochondrial ROS. Methods: We examined nuclear DNA damage, micronuclei (MN), intracellular ROS production, and the expression of inflammation-related nuclear genes in both parental and ρ0 SAE cells in response to asbestos treatment. Results: Asbestos induced a dose-dependent increase in nuclear DNA oxidative damage and MN in SAE cells. Furthermore, there was a significant increase in intracellular oxidant production and activation of genes involved in nuclear factor κB and proinflammatory signaling pathways in SAE cells. In contrast, the effects of asbestos were minimal in ρ0 SAE cells. Conclusions: Mitochondria are a major cytoplasmic target of asbestos. Asbestos may initiate mitochondria-associated ROS, which mediate asbestos-induced nuclear mutagenic events and inflammatory signaling pathways in exposed cells. These data provide new insights into the molecular mechanisms of asbestos-induced genotoxicity.

The complete nucleotide sequence of Chinese hamster (Cricetulus griseus) mitochondrial DNA

Mammalian mitochondria contain their own ∼16.5 kb circular genome. Mitochondrial DNA (mtDNA) encodes for a subset of the proteins involved in the electron transport chain and depletion or mutation of the sequence is implicated in a number of human disease processes. The recent finding is that mitochondrial damage mediates genotoxicity after exposure to chemical carcinogens has focused attention on the role of mtDNA mutations in the development of cancer. Although the entire genome has been sequenced for a number of mammals, only a small fraction of the mtDNA sequence is available for hamsters. We have obtained here the entire 16,284 bp sequence of the Chinese hamster mitochondrial genome, which will enable detailed analysis of mtDNA mutations caused by exposure to mutagens in hamster-derived cell lines.

Transforming growth factor-β-induced protein (TGFBI) suppresses mesothelioma progression through the Akt/mTOR pathway

As an uncommon cancer, mesothelioma is very hard to treat with a low average survival rate owing to its usual late detection and being highly invasive. The link between asbestos exposure and the development of mesothelioma in humans is unequivocal. TGFBI, a secreted protein that is induced by transforming growth factor-β in various human cell types, has been shown to be associated with tumorigenesis in various types of tumors. It has been demonstrated that TGFBI expression is markedly suppressed in asbestos-induced tumorigenic cells, while an ectopic expression of TGFBI significantly suppresses tumorigenicity and progression in human bronchial epithelial cells. In order to delineate a potential role of TGFBI in mediating the molecular events that occur in mesothelioma tumorigenesis, we generated stable TGFBI knockdown mutants from the mesothelium cell line Met-5A by using an shRNA approach, and secondly created ectopic TGFBI overexpression mutants from the mesothelioma cell line H28 in which TGFBI is absent. We observed that in the absence of TGFBI, the knockdown mesothelial and mesothelioma cell lines exhibited an elevated proliferation rate, enhanced plating efficiency, increased anchorage-independent growth, as well as an increased cellular protein synthesis rate as compared with their respective controls. Furthermore, cell cycle regulatory proteins c-myc/cyclin D1/phosphor-Rb were upregulated; a more active PI3K/Akt/mTOR signaling pathway was also detected in TGFBI-depleted cell lines. These findings suggest that TGFBI may repress mesothelioma tumorigenesis and progression via the PI3K/Akt signaling pathway.

High-throughput antibody-based assays to identify and quantify radiation-responsive protein biomarkers

Purpose: After a radiological ‘dirty bomb’ incident in a major metropolitan center, substantial numbers of people may be exposed to radiation. However, only a fraction of those individuals will need urgent medical attention. Consequently, a rapid screening test is needed to identify those people who require immediate treatment. Material and methods: Ten normal human cell lines were screened by enzyme-linked immunosorbent assay (ELISA) for the expression of a dozen secreted cytokines that have been reported to have changes in protein or mRNA levels at 1, 2, and 3 days after 0–10 Gy irradiation using 137Cs gamma rays at 0.82 Gy min−1. After this systematic in vitro screen, we measured changes in the level of a subset of these candidate proteins in plasma from irradiated C57BL/6 mice (n = 3 per group), comparing shams with a single radiation dose (5 Gy X-rays) at 3.7 Gy min−1 at 6 h after irradiation. Results: We identified four cytokine molecules that had altered levels after radiation exposure, one of which, Interleukin (IL) 6, was consistently elevated after irradiation in vitro and in vivo. Conclusions: Our findings underscore the potential for IL6 as a marker for an immunoassay-based, rapid, high-throughput biodosimeter.