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Biomedical subjects

Alan Wells

Publications and source records attributed to Alan Wells.

At least 37 records · Page 2Linked to original sources
PubMed2005

Erythropoietin-mediated activation of JAK-STAT signaling contributes to cellular invasion in head and neck squamous cell carcinoma.

Originally characterized as a growth factor for erythrocytes, erythropoietin (EPO) is used to treat anemia and fatigue in cancer patients receiving radiation therapy and chemotherapy. EPO and the EPO receptor (EPOR) are expressed in nonhematopoietic cells and cancers. However, the role of EPO and EPOR within nonhematopoietic cancer cells remains incompletely understood. Although a recent clinical trial demonstrated worse tumor control and survival in head and neck cancer patients treated with EPO, the role of EPO and EPOR in head and neck squamous cell carcinoma (HNSCC) has not been examined. In the present study, we demonstrate the previously unrecognized EPO-mediated invasion by HNSCC cells through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. Furthermore, we confirmed the expression of EPO and EPOR in a panel of human HNSCC cell lines and tissue specimens. Pharmacological doses of EPO also had a limited proliferation effect in these cell lines. These results define a novel role for EPO in mediating tumor cell invasion. Increased levels of EPO and EPOR in lymph node metastases as compared to primary tumors from HNSCC patients further support the role of EPO/EPOR in HNSCC disease progression and metastasis.

Aged↗
PubMed2005

Modeling of signal-response cascades using decision tree analysis.

MOTIVATION: Signal transduction cascades governing cell functional responses to stimulatory cues play crucial roles in cell regulatory systems and represent promising therapeutic targets for complex human diseases. however, mathematical analysis of how cell responses are governed by signaling activities is challenging due to their multivariate and non-linear nature. diverse computational methods are potentially available, but most are ineffective for protein-level data that is limited in extent and replication. RESULTS: We apply a decision tree approach to analyze the relationship of cell functional response to signaling activity across a spectrum of stimulatory cues. as a specific example, we studied five intracellular signals influencing fibroblast migration under eight conditions: four substratum fibronectin levels and presence versus absence of epidermal growth factor. we propose techniques for preprocessing and extending the experimental measurement set via interpolative modeling in order to gain statistical reliability. for this specific case study, our approach has 70% overall classification accuracy and the decision tree model reveals insights concerning the combined roles of the various signaling activities in governing cell migration speed. we conclude that decision tree methodology may facilitate elucidation of signal-response cascade relationships and generate experimentally testable predictions, which can be used as directions for future experiments.

Algorithms↗
PubMed2005

Calpain proteases in cell adhesion and motility.

Cell adhesion and its role during cell spreading and motility are central to normal development and homeostasis, including its effects on immune response and wound repair and tissue regeneration. Disruption of cell adhesion impacts not only the healing process but promotes tumor invasion and metastasis. A family of intracellular, limited proteases, the calpains, has recently been shown to be a key molecular control point in attachment of cells to the surrounding matrix. Herein, the two main and ubiquitously expressed calpain isoforms will be introduced as to their modes of regulation and the current status of research will be discussed as to how these calpains might function in the biophysical process of adhesion and biological cellular responses of spreading and motility.

Animals↗
PubMed2005

Interferon-inducible protein 9 (CXCL11)-induced cell motility in keratinocytes requires calcium flux-dependent activation of mu-calpain.

Keratinocyte migration is critical to reepithelialization during wound repair. The motility response is promoted by growth factors, cytokines, and cytokines produced in the wound bed, including those that activate the epidermal growth factor (EGF) receptor. The Alu-Leu-Arg-negative CXC chemokine interferon-inducible protein 9 (IP-9; also known as CXCL11, I-TAC, beta-R1, and H-174) is produced by keratinocytes in response to injury. As keratinocytes also express the receptor, CXCR3, this prompted us to examine the role and molecular mechanism by which IP-9 regulates keratinocyte motility. Unexpectedly, as CXCR3 liganding blocks growth factor-induced motility in fibroblasts, IP-9 alone promoted motility in undifferentiated keratinocytes (37 +/- 6% of the level of the highly motogenic EGF) as determined in a two-dimensional in vitro wound healing assay. IP-9 even enhanced EGF-induced motility in undifferentiated keratinocytes (116 +/- 5%; P < 0.05 compared to EGF alone), suggesting two separate mechanisms of action. IP-9-increased motility and -decreased adhesiveness required the intracellular protease calpain. The increases in both motility and calpain activity by IP-9 were blocked by pharmacological and molecular inhibition of phospholipase C-beta3 and chelation of calcium, which prevented an intracellular calcium flux. Molecular downregulation or RNA interference-mediated depletion of mu-calpain (calpain 1) but not M-calpain (calpain 2) blocked IP-9-induced calpain activation and motility. In accord with elimination of IP-9-induced de-adhesion, RNA interference-mediated depletion of calpain 1 but not calpain 2 prevented cleavage of the focal adhesion component focal adhesion kinase and disassembly of vinculin aggregates. In comparison, EGF-induced motility of the same undifferentiated keratinocytes requires the previously described extracellular signal-regulated kinase to the M-calpain pathway. These data demonstrate that while both EGF- and IP-9-induced motility in keratinocytes requires calpain activity, the isoform of calpain triggered depends on the nature of the receptor for the particular ligand. Interestingly, physiological nonapoptotic calcium fluxes were capable of activating mu-calpain, implying that the calcium requirement of mu-calpain for activation is attained during cell signaling. This is also the first demonstration of differential activation of the two ubiquitous calpain isoforms in the same cell by different signals.

Calcium↗
PubMed2005

Glomerular effects of AVT on the in situ perfused trunk preparation of the dogfish.

The effects of arginine vasotocin on the pattern of glomerular perfusion in an elasmobranch fish were examined using an in situ perfused renal trunk preparation. A significant antidiuresis was coupled with a marked reduction in the proportion of filtering glomeruli (86%-25%) and an increase in the proportion of perfused but not filtering (10%-53%) and non-perfused glomeruli (4%-22%). However, the reduction in filtering glomeruli was greater than predicted by measurements of urine flow rate and glomerular filtration rate alone, suggesting that alterations in single nephron glomerular filtration rate may occur.

Animals↗
PubMed2004

Parsing ERK activation reveals quantitatively equivalent contributions from epidermal growth factor receptor and HER2 in human mammary epithelial cells.

HER2, a member of the epidermal growth factor receptor (EGFR) tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we applied a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2 and their downstream activation of ERK to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we could separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrated that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activate ERK were quantitatively equivalent. We found that HER2-mediated effects on EGFR dimerization and trafficking were sufficient to explain the observed HER2-mediated amplification of epidermal growth factor-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared with the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking yielding increased EGFR sparing cause the sustained HER2-mediated enhancement of ERK signaling.

Animals↗
PubMed2004

Attraction or repulsion: a matter of individual taste?

Cell migration along the correct paths is critical to normal organogenesis and tissue repair, and goes awry in tumor dissemination. Understanding the molecular cues that control such migration would provide opportunities to control these events. Recent findings in Drosophila highlight the role of the epidermal growth factor receptor (EGFR) in chemotaxis and pathfinding during development. These studies, and others performed in vertebrates, support a central role for EGFR-mediated chemotaxis. However, the signal or signals that then enable cells to further migrate independently of this chemotactic effect are not known. Three possible scenarios are discussed: that EGFR signaling sensitizes cells to new cues, that EGFR signaling is altered so that the cell responds predominantly to other motility signals that are now "unblinded," and that EGFR signaling still provides the motile signal but in a chemokinetic manner with matrix components providing the directionality. There are precedents for all three scenarios, but which one proves true will define the window of opportunity for regulating cell migration.

Animals↗
PubMed2004

DU145 human prostate carcinoma invasiveness is modulated by urokinase receptor (uPAR) downstream of epidermal growth factor receptor (EGFR) signaling.

Tumor cell motility and invasion have been linked to upregulated signaling from both the epidermal growth factor receptor (EGFR) and that for urokinase-type plasminogen activator (uPAR). However, we do not know whether these events are interdependent or unrelated, despite the obvious diagnostic and therapeutic implications. Gene microarray analyses have suggested that EGFR signaling via phospholipase C-gamma (PLCgamma) induces uPAR transcription. We utilized two sublines of the DU145 human prostate carcinoma cell line that are genetically engineered to differentially activate the EGFR/PLCgamma cascade and are variously invasive in vitro and in vivo. uPAR protein levels in these cells were found to be dependent on PLC signaling, pharmacologic inhibition of PLC signaling reduced uPAR expression. To determine whether uPAR was a required element in EGFR-mediated invasion, we stably expressed uPAR cDNA in either sense or antisense orientation in the two DU145 sublines. Interestingly, uPA production was modulated in parallel, although to a lesser degree, with uPAR in these sublines. Antisense to uPAR significantly restricted invasion of the highly invasive DU145 WT cells through Matrigel and reduced aggressiveness of tumors in nude mice. Up-regulation of uPAR significantly increased the invasiveness of the moderately invasive DU145 parental (DU145 P) cells through Matrigel, but this increased invasiveness was not seen in mice. uPA activity appears to contribute to invasiveness at least through Matrigel, as antibody to uPA or amiloride limited the transmigration. These results support a model of tumor invasion promoted by autocrine EGFR signaling involving reinforcing altered gene expression, of uPAR at least, that further induces cell motility. Herein, a number of key molecules whose expression levels are interrelated, including both EGFR and uPAR, are required but none are sufficient in the absence of other keys molecules in promoting tumor progression.

Amiloride↗
PubMed2004

Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C delta-dependent pathway.

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCdelta is the most strongly phosphorylated isoform, and the preferential PKCdelta inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCdelta is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCdelta activation, whereas lack of PLCgamma signaling resulted in delayed PKCdelta activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCdelta is required, a dominant-negative PKCdelta construct or RNAi-mediated PKCdelta depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCdelta activation. All of the interventions that blocked PKCdelta activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCdelta activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCdelta to promote the acute phase of EGF-induced MLC activation.

Acetophenones↗
PubMed2004

Webcasting pathology department conferences in a geographically distributed medical center.

As academic pathology departments have become increasingly based in large, regional medical systems spread across hundreds of square miles, new methods are needed to tie these increasingly distributed departments together as integrated units. An important part of that integration is the ability to share academic and teaching conferences across long distances. In this article we present an effective, low-cost webcasting system that has evolved at the University of Pittsburgh Medical Center Department of Pathology over the past several years based on inexpensive, widely available software. To date, the system has broadcast and archived more than 400 conferences and currently serves approximately 80 to 100 requests each week. Important factors in the success of the program include the creation of a faculty steering committee to control resources and manage growth, the availability of informatics faculty and support for technical staff, and the decision to operate the service as part of the core information technology infrastructure of the department. Webcasting will likely become an even more important academic and operational tool in the future as more of the department's conferences, seminars, and even working meetings are communicated through the webcasting infrastructure.

Academic Medical Centers↗
PubMed2004

Expression of human, mouse, and rat m-calpains in Escherichia coli and in murine fibroblasts.

The two best known calpains, micro- and m-calpain, are Ca(2+)-dependent cysteine proteases found in all mammalian tissues. They are probably involved in many Ca(2+)-linked signal pathways, although the details are not yet clear. The enzymes are heterodimers of a specific large subunit (micro-80k or m-80k) and a common small subunit (28k). Recombinant calpains have been obtained by co-expression of large and small subunits in Escherichia coli and in Sf9 cells, with variable success. Expression with the 28k subunit is very low, but is much higher with a C-terminal 21k fragment of this subunit. Rat m-calpain (m-80k/21k) is well expressed in E. coli but mouse m-calpain (m-80k/21k) is poorly expressed, even though the amino acid sequences of rat-m-80k and mouse-m-80k are 92% identical. It had also been reported that human m-calpain could be expressed in Sf9 cells but not in E. coli. To investigate these differences, hybrid rat/mouse and rat/human m-calpains were cloned and expressed in E. coli. It was shown that Ile-6 and Pro-127, which are specific to the mouse m-80k sequence, caused poor expression. High expression of human m-calpain in E. coli could be achieved by providing the correct Shine-Dalgarno ribosome binding site. The results provide a simple method to obtain approximately 10mg amounts of human m-calpain and a slightly modified mouse m-calpain. Expression of m-80k-EGFP fusions was also studied, both in E. coli and in mammalian cells, varying both the small subunit and the promoters. m-80k-EGFP alone was not active, but with 21k or 28k subunits was active in both cell types. The EGFP domain was partially cleaved during expression, releasing an active m-80k/21k calpain.

Amino Acid Sequence↗
PubMed2004

Expression of myopodin induces suppression of tumor growth and metastasis.

Myopodin was previously reported as a gene that was frequently deleted in prostate cancer. This gene shares significant homology with a cell shape-regulating gene, synaptopodin. Myopodin was shown to bind actin and to induce actin bundling when cells were stimulated. To clarify the functional role of myopodin in prostate cancer, several assays were performed to evaluate the tumor suppression activity of myopodin. Our results indicate that myopodin inhibits tumor growth and invasion both in vitro and in vivo. The activity of tumor suppression of myopodin is located at the C-terminus region. To further evaluate the role of myopodin in suppressing the invasiveness of prostate cancer, an expression analysis of myopodin protein was performed in prostate tissues. The results indicate that down-regulation of myopodin expression occurs mostly in invasive stages of prostate cancer, implying a potential invasion suppression role for myopodin in prostate cancer. In addition, hemizygous deletion and down-regulation of myopodin expression occur in three aggressive prostate cancer cell lines. All these results support the hypothesis that myopodin functions as a tumor suppressor gene to limit the growth and to inhibit the metastasis of cancer cells.

Agar↗
PubMed2003

Aging-related attenuation of EGF receptor signaling is mediated in part by increased protein tyrosine phosphatase activity.

As fibroblasts near senescence, their responsiveness to external signals diminishes. This well-documented phenomenon likely underlies physiological deterioration and limited tissue regeneration in aging individuals. Understanding the underlying molecular mechanisms would provide opportunities to ameliorate these situations. A key stimulus for human dermal fibroblasts are ligands for the epidermal growth factor receptor (EGFR). We have shown earlier that EGFR expression decreases by about half in near senescent fibroblasts (Shiraha et al., 2000, J. Biol. Chem. 275 (25), 19343-19351). However, as the cell responses are nearly absent near senescence, other aging-related signal attenuation changes must also occur. Herein, we show that EGFR signaling as determined by receptor autophosphorylation is diminished over 80%, with a corresponding decrease in the phosphorylation of the immediate postreceptor adaptor Shc. Interestingly, we found that this was due at least in part to increased dephosphorylation of EGFR. The global cell phosphotyrosine phosphatase activity increased some threefold in near senescent cells. An initial survey of EGFR-associated protein tyrosine phosphatases (PTPases) showed that SHP-1 (PTPIC, HCP, SHPTP-1) and PTPIB levels are increased in parallel in these cells. Concomitantly, we also discovered an increase in expression of receptor protein tyrosine phosphatase alpha (RPTPalpha). Last, inhibition of protein tyrosine phosphatases by sodium orthovanadate in near senescent cells resulted in increased EGFR phosphorylation. These data support a model in which, near senescence, dermal fibroblasts become resistant to EGFR-mediated stimuli by a combination of receptor downregulation and increased signal attenuation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Epidermal growth factor receptor-stimulated activation of phospholipase Cgamma-1 promotes invasion of head and neck squamous cell carcinoma.

Lymph node metastasis and local invasion of head and neck squamous cell carcinoma (HNSCC) is associated with a poor prognosis. However, little is known about the factors governing tumor cell invasion in HNSCC. Phospholipase Cgamma-1 (PLCgamma-1) contributes to tumor cell invasion in experimental systems when activated by the epidermal growth factor receptor (EGFR). We hypothesized that EGFR overexpression in HNSCC mediates invasion via PLCgamma-1. On EGFR ligand stimulation, phosphorylation of PLCgamma-1 increased in all of the HNSCC cell lines tested (4 of 4). In the presence of EGFR-specific tyrosine kinase inhibitor (PD153035) or an anti-EGFR antibody (C225), PLCgamma-1 activation was abrogated indicating that PLCgamma-1 was downstream of EGFR. Blocking cellular PLC with an inhibitor (U73122) reduced inositol phosphate turnover in all of the HNSCC cell lines examined, and treatment with the PLC inhibitor or antisense oligonucleotides targeting PLCgamma-1 significantly reduced in vitro invasiveness of HNSCC cell lines through Matrigel. To determine the clinical relevance of these findings, we compared levels of PLCgamma-1 in tumor and paired normal tissue from 33 patients with HNSCC. PLCgamma-1 levels were significantly higher (P < 0.0001) in the tumors compared with the normal mucosa of HNSCC patients. Levels of activated PLCgamma-1 were analyzed in 20 patients. Tumors expressed higher levels of phosphorylated PLCgamma-1 compared with normal adjacent mucosa (P = 0.05). Thus, PLCgamma-1 may mediate invasion and metastasis downstream of EGFR in HNSCC.

Aged↗
PubMed2003

Calpain-2 as a target for limiting prostate cancer invasion.

Mortality and morbidity of prostate cancer result from extracapsular invasion and metastasis. This tumor progression depends on active cell motility. Previous studies have shown that calpain-regulated rear detachment enabling forward locomotion is required for cell migration initiated by growth factor and adhesion receptors. Therefore, we asked whether calpain would be a target for limiting tumor progression, using as our model the PA DU-145 human prostate carcinoma cell line and a highly invasive subline, wild-type DU-145, derived from it. In vitro, the calpain-specific inhibitor CI-I (ALLN) and the preferential-but-less-specific inhibitor leupeptin decreased transmigration of both cell lines across a Matrigel barrier. These calpain inhibitors limited epidermal growth factor-induced motility but did not alter the growth rate of the tumor cells, as expected. Antisense down-regulation of the growth factor-activated calpain-2 (m-calpain) isoform also reduced transmigration and cell motility. These in vitro findings were then buttressed by in vivo studies, in which i.p. DU-145 tumor xenografts were treated with leupeptin. Tumor invasion into the diaphragm was reduced by leupeptin treatment for both the PA and wild-type DU-145 cells (from 1.7 to 0.78 for the parental line and 2.3 to 1.2 for the invasive derivative, respectively). Tumor cells of both types engineered to express calpain-2 antisense constructs also demonstrated a similar 50% reduced invasiveness in vivo. Finally, we found by gene expression survey of 53 human prostate tumors and 23 normal prostates that calpain was not up-regulated in relationship to invasiveness or metastatic activity, consistent with expectation from the biological role of this effector. Taken together, these results strongly suggest that epigenetic activation of calpain plays an important role in the invasion of human prostate cancer and that it can be targeted to reduce tumor progression.

Animals↗
PubMed2003

Directional motility induced by epidermal growth factor requires Cdc42.

Cell motility is actuated by a host of intracellular signaling cascades that result in movement of the cell in one direction, even without an external gradient. Phospholipase C-gamma (PLCgamma) has been shown to be important for growth factor-induced lamellipodial protrusion at the front of the cell while Cdc42 has been implicated in both filopodium formation at the leading edge and control of polarity of migrating cells. We asked whether these asymmetries in effector molecules may be linked. When we overexpressed either constitutively active, dominant negative, or GFP-tagged Cdc42, wild-type NR6 fibroblasts lost directionality, as expected. On epidermal growth factor (EGF) exposure these cells produced multiple, transient protrusions in every direction; these extensions failed to result in productive motility. GFP-tagged Cdc42 appeared transiently at edges of newly formed protrusions in EGF-stimulated cells while they moved haphazardly. While PLCgamma is distributed throughout the cell, the ratio of active, tyrosyl-phosphorylated PLCgamma was increased at the leading edge, where phosphatidylinositol (4,5)-bisphosphate (PIP(2)) hydrolysis is concentrated. This co-localization of activities may be due to Cdc42 directing PLCgamma to the cell front, as PLCgamma associated with Cdc42 in an EGF-dependent manner. We conclude that Cdc42 controls cell polarity, likely in part, through its binding to active PLCgamma.

Animals↗
PubMed2003

Glutamate substitutions at a PKA consensus site are consistent with inactivation of calpain by phosphorylation.

Regulation of calpain by phosphorylation has often been suggested, but has proved difficult to detect. Calpains extracted from mammalian tissue are reported to contain 2-4 mol phosphate/mol of enzyme distributed over multiple sites, but phosphate groups are not detectable in the X-ray structures of recombinant calpain. Some serine and threonine residues in the large subunit of rat m-calpain were converted to aspartic or glutamic acid residues, at sites suggested by previous studies, to assess the probable effects of phosphate groups on the enzyme. Expression of the mutant calpains in Escherichia coli, and their heat stabilities, did not differ from those of the wild-type enzyme. m-Calpains with the mutations Ser50Asp, Ser50Glu, Ser67Glu, and Thr70Glu had the same specific activity and Ca(2+) requirement as the wild-type enzyme. In contrast, Ser369Asp-, Ser369Glu-, and Thr370Glu-m-calpain were inactive. This result is consistent with the recent report that phosphorylation at position 369 or 370 in vivo reduced m-calpain activation.

Amino Acid Substitution↗