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Biomedical subjects

M McMahon

Publications and source records attributed to M McMahon.

At least 19 recordsLinked to original sources
PubMed2006

Oncogenic BRAF regulates beta-Trcp expression and NF-kappaB activity in human melanoma cells.

Mutational activation of BRAF is a frequent event in human malignant melanomas suggesting that BRAF-dependent signaling is conducive to melanoma cell growth and survival. Previously published work reported that melanoma cells exhibit constitutive anti-apoptotic nuclear factor kappaB (NF-kappaB) transcription factor activation triggered by proteolysis of its inhibitor IkappaB. IkappaB degradation is dependent upon its phosphorylation by the IkappaB kinase (IKK) complex and subsequent ubiquitination facilitated by beta-Trcp E3 ubiquitin ligase. Here, we report that melanocytes expressing a conditionally oncogenic form of BRAF(V600E) exhibit enhanced beta-Trcp expression, increased IKK activity and a concomitant increase in the rate of IkappaBalpha degradation. Conversely, inhibition of BRAF signaling using either a broad-spectrum Raf inhibitor (BAY 43-9006) or by selective knock-down of BRAF(V600E) expression by RNA interference in human melanoma cells leads to decreased IKK activity and beta-Trcp expression, stabilization of IkappaB, inhibition of NF-kappaB transcriptional activity and sensitization of these cells to apoptosis. Taken together, these data support a model in which mutational activation of BRAF in human melanomas contributes to constitutive induction of NF-kappaB activity and to increased survival of melanoma cells.

Cell Line, Tumor↗
PubMed2006

Identification of a novel germline MET mutation in dogs.

The MET proto-oncogene encodes a transmembrane tyrosine kinase receptor that mediates multiple functions such as migration, cycling and survival by binding to hepatocyte growth factor (HGF). Dysregulation of MET through inappropriate expression or mutation has been shown to play an important role in human cancers. Furthermore, inherited mutations in MET are known to contribute to the development of gastric and renal cancer in humans. Lastly, mouse models of MET mutations lead to the development of a wide variety of cancers including lymphomas, sarcomas and some forms of carcinoma. In the process of cloning canine MET, a novel germline point mutation was found in the juxtamembrane domain (G966S) in two of the templates used for cloning, both of which were derived from Rottweiler dogs, a breed believed to be at high risk for the development of several cancers. Screening of germline DNA from a variety of breeds revealed that this mutation was present in approximately 70% of Rottweiler dogs and <5% of all other breeds examined, suggesting a breed-specific heritable mutation. Stable transfection of the G966S mutant form of MET into NIH3T3 cells resulted in enhanced baseline scattering and migration of the cells, which was further increased in the presence of HGF. This study supports the notion that particular dog breeds may carry germline mutations that contribute to high rates of cancer in a manner similar to heritable, cancer-associated mutations in humans.

Animals↗
PubMed2003

Perceptual indicators of environmental health, future land use, and stewardship.

There are important linkages between the health of humans and the environment, restoration of degraded lands, and long-term stewardship of public lands, yet most environmental indicators deal only with assessing the physical and biological aspects of ecosystems. In this article, we examine the ratings of perceptions of several environmental problems for their utility as indicators of environmental quality, and examine perceptions of future land use by people interviewed in Santa Fe, New Mexico, near the Department of Energy's (DOE) Los Alamos National Laboratory (LANL). Overall, people with lower incomes rated environmental problems as more severe than others, were more willing to spend federal funds to solve them, and were consistent in their ratings of severity of environmental problems and their willingness to spend federal funds. Cleaning up LANL and other Department of Energy sites, received the highest rating for expenditure of federal funds. The highest rated future uses for DOE sites were for recreation and for National Environmental Research Parks. People with less education generally gave higher ratings to most future land uses for DOE than did those with more education. However, those with higher education gave higher ratings to nuclear reprocessing, and nuclear material storage. Where there were differences, the people interviewed at Santa Fe rated all environmental problems (except pesticides) as more severe than did those previously interviewed in Albuquerque (located farther from the LANL site), and they were more willing to spend federal funds on these problems. Ratings for all future land uses did not differ between the Santa Fe and Albuquerque respondents. These perception-based indicators show general agreement among people living close and farther away from LANL with respect to cleaning up LANL and the future land uses for the site. These indicators should be considered by regulators, site personnel, and policy makers in future management and land use decisions.

Adult↗
PubMed2003

Requirement for the PI3K/Akt pathway in MEK1-mediated growth and prevention of apoptosis: identification of an Achilles heel in leukemia.

The Raf/MEK/ERK kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using DeltaMEK1:ER, a conditionally active form of MEK1 which responds to either beta-estradiol or the estrogen receptor antagonist 4 hydroxy-tamoxifen (4HT), we previously documented the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of human (TF-1) and murine (FDC-P1 and FL5.12) hematopoietic cells lines. Here we demonstrate the ability of DeltaMEK1:ER to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/p70 ribosomal S6 kinase (p70(S6K)) pathway and the importance of this pathway in MEK1-mediated prevention of apoptosis. MEK1-responsive cells can be maintained long term in the presence of beta-estradiol, 4HT or IL-3. Removal of hormone led to the rapid cessation of cell proliferation and the induction of apoptosis in a manner similar to cytokine deprivation of the parental cells. Stimulation of DeltaMEK1:ER by 4HT resulted in ERK, PI3K, Akt and p70(S6K) activation. Treatment with PI3K, Akt and p70(S6K) inhibitors prevented MEK-responsive growth. Furthermore, the apoptotic effects of PI3K/Akt/p70(S6K) inhibitors could be enhanced by cotreatment with MEK inhibitors. Use of a PI3K inhibitor and a constitutively active form of Akt, [DeltaAkt(Myr(+))], indicated that activation of PI3K was necessary for MEK1-responsive growth and survival as activation of Akt alone was unable to compensate for the loss of PI3K activity. Cells transduced by MEK or MEK+Akt displayed different sensitivities to signal transduction inhibitors, which targeted these pathways. These results indicate a requirement for the activation of the PI3K pathway during MEK-mediated transformation of certain hematopoietic cells. These experiments provide important clues as to why the identification of mutant signaling pathways may be the Achilles heel of leukemic cell growth. Leukemia treatment targeting multiple signal transduction pathways may be more efficacious than therapy aimed at inhibiting a single pathway.

Animals↗
PubMed2003

Differential effects of kinase cascade inhibitors on neoplastic and cytokine-mediated cell proliferation.

The Raf/MEK/ERK and PI3K/Akt pathways regulate proliferation and prevent apoptosis, and their altered expression is commonly observed in human cancer due to the high mutation frequency of upstream regulators. In this study, the effects of Raf, MEK, and PI3K inhibitors on conditionally transformed hematopoietic cells were examined to determine if they would display cytotoxic differences between cytokine- and oncogene-mediated proliferation, and whether inhibition of both pathways was a more effective means to induce apoptosis. In the hematopoietic model system employed, proliferation was conditional and occurred when either interleukin-3 (IL-3) or the estrogen receptor antagonist 4-hydroxytamoxifen (4HT), which activates the conditional oncoprotein (DeltaRaf:ER), were provided. Thus, upon the addition of the signal transduction inhibitors and either IL-3 or 4HT, the effects of these drugs were examined in the same cell under 'cytokine-' and 'oncoprotein' -mediated growth conditions avoiding genetic and differentiation stage heterogeneity. At drug concentrations around the reported IC(50) for the Raf inhibitor L-779,450, it suppressed DNA synthesis and induced apoptosis in hematopoietic FDC-P1 cells transformed to grow in response to either Raf-1 or A-Raf (FD/DeltaRaf-1:ER and FD/DeltaA-Raf:ER), but it displayed less effects on DNA synthesis and apoptosis when the cells were cultured in IL-3. This Raf inhibitor was less effective on B-Raf- or MEK1-responsive cells, demonstrating the specificity of this drug. MEK inhibitors also suppressed DNA synthesis and induced apoptosis in Raf-responsive cells and the effects were more significant on Raf-responsive compared to cytokine-mediated growth. The PI3K inhibitor LY294002 suppressed Raf-mediated growth, indicating that part of the long-term proliferative effects mediated by Raf are PI3K dependent. Simultaneous inhibition of both Raf/MEK/ERK and PI3K/Akt pathways proved a more efficient means to suppress DNA synthesis and induce apoptosis at lower drug concentrations.

Animals↗
PubMed2002

Exposure of South Carolinians to commercial meats and fish within their meat and fish diet.

There has been considerable interest in the public's exposure to a variety of contaminants through the consumption of wild fish and game, yet there is little information on consumption of commercial meats and fish, or the relationship between commercial and self-caught fish. We conducted a dietary survey in 1999 to estimate exposure levels of 464 individuals from people attending the Palmetto Sportsmen's Classic. Mean consumption was similar for beef, chicken/turkey, and wild-caught fish, and much lower for pork and store-bought fish, and still lower for restaurant fish. There were no ethnic differences in the consumption of most commercial fish and meats, although the differences for chicken approached significance. There were significant ethnic differences in consumption of wild-caught fish. Women ate significantly less of all meat types, except store-bought fish. People over 45 ate less beef than younger people, and people younger than 32 ate significantly more chicken than others. There were no significant differences in consumption patterns as a function of income, except for chicken and wild-caught fish; people with higher incomes ate more chicken than others, and people with lower incomes ate more wild-caught fish than others. When all wild-caught and commercial fish and meats are considered, there are significant differences only for ethnicity and gender. Blacks consume significantly more fish than Whites, and men consume significantly more than women.

Adult↗
PubMed2001

Molecular basis for the contribution of the antioxidant responsive element to cancer chemoprevention.

This article provides an overview of the mechanisms by which cancer chemopreventive blocking agents increase the expression of detoxication and antioxidant genes. These agents all appear capable of transcriptionally activating a gene battery that includes NAD(P)H:quinone oxidoreductase, aldo-keto reductases, glutathione S-transferases, gamma-glutamylcysteine synthetase, glutathione synthetase and heme oxygenase. Gene induction occurs through the antioxidant responsive element (ARE), a process that is dependent on the Nuclear Factor-Erythroid 2p45-related factors, Nrf1 and Nrf2. Under basal conditions, these basic region leucine zipper (bZIP) transcription factors are located in the cytoplasm of the cell bound to Keap1, and upon challenge with inducing agents, they are released from Keap1 and translocate to the nucleus. Within the nucleus, Nrf1 and Nrf2 are recruited to the ARE as heterodimers with either small Maf proteins, FosB, c-Jun, JunD, activating transcription factor 2 (ATF2) or ATF4. The role of protein kinases in transducing chemical stress signals to the bZIP factors that affect gene induction through the ARE is discussed.

Animals↗
PubMed2001

Formation of intracranial tumors by genetically modified human astrocytes defines four pathways critical in the development of human anaplastic astrocytoma.

The formation of human malignant gliomas is thought to involve the accumulation of multiple genetic alterations. To define the function of specific alterations in glioma formation, we serially introduced genetic alterations functionally equivalent to those noted in human malignant gliomas into normal human astrocytes (NHAs). We then monitored the ability of each of these alterations to contribute to the growth of otherwise genetically stable NHAs into intracranial malignant gliomas. Using this model, we show that expression of human telomerase catalytic component (hTERT), but not E7-mediated inactivation of pRb or E6/E7-mediated inactivation of p53/pRb, was sufficient to initiate the tumorigenic process by circumventing cellular senescence in astrocytes. hTERT expression, even in combination with inactivation of p53/pRb, did not transform astrocytes. These alterations together, however, cooperated with ras pathway activation (initiated by expression of mutant H-Ras), but not with phosphatidylinositol 3-kinase pathway activation (initiated by expression of myristoylated Akt) or epidermal growth factor receptor activation, to allow for the formation of intracranial tumors strongly resembling p53/pRb pathway-deficient, telomerase-positive, ras-activated human grade III anaplastic astrocytomas. These results identify four pathways as key in the development of human anaplastic astrocytomas.

Animals↗
PubMed2001

A conditional form of Bruton's tyrosine kinase is sufficient to activate multiple downstream signaling pathways via PLC Gamma 2 in B cells.

BACKGROUND: Bruton's tyrosine kinase (Btk) is essential for B cell development and function. Mutations of Btk elicit X-linked agammaglobulinemia in humans and X-linked immunodeficiency in the mouse. Btk has been proposed to participate in B cell antigen receptor-induced signaling events leading to activation of phospholipase C-gamma2 (PLCgamma2) and calcium mobilization. However it is unclear whether Btk activation is alone sufficient for these signaling events, and whether Btk can activate additional pathways that do not involve PLCgamma2. To address such issues we have generated Btk:ER, a conditionally active form of the kinase, and expressed it in the PLCgamma2-deficient DT40 B cell line. RESULTS: Activation of Btk:ER was sufficient to induce multiple B cell signaling pathways in PLCgamma2-sufficient DT40 cells. These included tyrosine phosphorylation of PLCgamma2, mobilization of intracellular calcium, activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways, and apoptosis. In DT40 B cells deficient for PLCgamma2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis. CONCLUSIONS: These data suggest that Btk:ER regulates downstream signaling pathways primarily via PLCgamma2 in B cells. While it is not known whether activated Btk:ER precisely mimics activated Btk, this conditional system will likely facilitate the dissection of the role of Btk and its family members in a variety of biological processes in many different cell types.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2001

Analysis of the transcriptional program induced by Raf in epithelial cells.

Activation of the Raf/MAP kinase pathway is a critical event in tumorigenesis induced by RAS and other oncogenes, a major role of this signaling system being the regulation of cellular transcription factors. To address the contribution of MAP kinase mediated transcriptional changes to the transformed phenotype, we used an inducible form of Raf to analyze early changes in the transcription of some 6000 genes following activation of the kinase in a normal human breast epithelial cell line. Of the more than 120 significant changes in mRNA level detected, genes promoting cell proliferation, invasiveness, and angiogenesis featured prominently. Some of the most strongly induced genes encoded growth factors of the EGF family: Autocrine activation of the EGF receptor was shown to be responsible for the ability of Raf activation to protect these cells from apoptosis induced by detachment of cells from extracellular matrix (anoikis), which is a critical component of the transformed phenotype.

Apoptosis↗
PubMed2001

The Cap'n'Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes.

Northern blotting has shown that mouse small intestine contains relatively large amounts of the nuclear factor-E2 p45-related factor (Nrf) 2 transcription factor but relatively little Nrf1. Regulation of intestinal antioxidant and detoxication enzymes by Nrf2 has been assessed using a mouse line bearing a targeted disruption of the gene encoding this factor. Both Nrf2-/- and Nrf2+/+ mice were fed a control diet or one supplemented with either synthetic cancer chemopreventive agents [butylated hydroxyanisole (BHA), ethoxyquin (EQ), or oltipraz] or phytochemicals [indole-3-carbinol, cafestol and kahweol palmitate, sulforaphane, coumarin (CMRN), or alpha-angelicalactone]. The constitutive level of NAD(P)H:quinone oxidoreductase (NQO) and glutathione S-transferase (GST) enzyme activities in cytosols from small intestine was typically found to be between 30% and 70% lower in samples prepared from Nrf2 mutant mice fed a control diet than in equivalent samples from Nrf2+/+ mice. Most of the chemopreventive agents included in this study induced NQO and GST enzyme activities in the small intestine of Nrf2+/+ mice. Increases of between 2.7- and 6.2-fold were observed in wild-type animals fed diets supplemented with BHA or EQ; increases of about 2-fold were observed with a mixture of cafestol and kahweol palmitate, CMRN, or alpha-angelicalactone; and increases of 1.5-fold were measured with sulforaphane. Immunoblotting confirmed that in the small intestine, the constitutive level of NQO1 is lower in the Nrf2-/- mouse, and it also showed that induction of the oxidoreductase was substantially diminished in the mutant mouse. Immunoblotting class-alpha and class-mu GST showed that constitutive expression of most transferase subunits is also reduced in the small intestine of Nrf2 mutant mice. Significantly, induction of class-alpha and class-mu GST by EQ, BHA, or CMRN is apparent in the gene knockout animal. No consistent change in the constitutive levels of the catalytic heavy subunit of gamma-glutamylcysteinyl synthetase (GCS(h)) was observed in the small intestine of Nrf2-/- mice. However, although the expression of GCS(h) was found to be increased dramatically in the small intestine of Nrf2+/+ mice by dietary BHA or EQ, this induction was essentially abolished in the knockout mice. It is apparent that Nrf2 influences both constitutive and inducible expression of intestinal antioxidant and detoxication proteins in a gene-specific fashion. Immunohistochemistry revealed that induction of NQO1, class-alpha GST, and GCS(h) occurs primarily in epithelial cells of the small intestine. This suggests that the variation in inducibility of NQO1, Gsta1/2, and GCS(h) in the mutant mouse is not attributable to the expression of the enzymes in distinct cell types but rather to differences in the dependency of these genes on Nrf2 for induction.

Animals↗
PubMed2001

Dual growth arrest pathways in astrocytes and astrocytic tumors in response to Raf-1 activation.

Normal human fibroblasts have been shown to undergo a p16(Ink4a)-associated senescence-like growth arrest in response to sustained activation of the Ras/Raf/MEK/ERK pathway. We noted a similar p16(Ink4a)-associated, senescence-like arrest in normal human astrocytes in response to expression of a conditional form of Raf-1. While HPV16 E7-mediated functional inactivation of the p16(Ink4a)/pRb pathway in astrocytes blocked the p16(Ink4a)-associated growth arrest in response to activation of Raf-1, it also revealed a second p21(Cip1)-associated, senescence-associated, beta-galactosidase-independent growth arrest pathway. Importantly, the p21(Cip1)-associated pathway was present not only in normal astrocytes but also in p53-, p14(ARF)-, and p16(Ink4a)/pRb-deficient high grade glioma cells that lacked the p16(Ink4a)-dependent arrest mechanism. These results suggest that normal human cells have redundant arrest pathways, which can be activated by Raf-1, and that even tumors that have dismantled p16(Ink4a)-dependent growth arrest pathways are potentially regulated by a second p21(Cip1)-dependent growth arrest pathway.

Astrocytes↗
PubMed2001

Role of the oncogenic Raf-1 in orchestration of discrete nuclear factor-kappaB-activating pathways.

Raf-1, a key kinase in the Ras signaling pathway, plays critical roles in cell differentiation, proliferation, and tumorigenesis. However, knowledge of the Raf-1 in inflammation is limited. Using an inducible oncogenic Raf-1, we show that the Raf-1 orchestrates the discrete NF-kappaB activating pathways. While the Raf-1 activation induces a modest IkappaB degradation by enhancing the basal IkappaB kinase activity, it contradictorily suppresses the proinflammatory cytokine inducible IkappaB kinase complex, leading to an inhibition of TNF-alpha- and IL-1beta-induced NF-kappaB activation. Despite considerable degrees of overlap, LPS signaling is not affected by Raf-1. By either conditionally reducing Raf-1 activity or completely disrupting the Raf-1 signaling by PD98059, a specific inhibitor of MEK1, the otherwise inhibited cytokine responses can be restored. Moreover, when the activity of Raf-1 is up-regulated during the cell cycle progression from the G(0) phase to the late G(1) phase, the enhanced Raf-1 activity suffices to shift the TNF-alpha response from the sensitive to the insensitive state. Together, these studies elucidate a mechanism by which signaling outputs are shaped by the intracellular Raf-1, thus explaining the "cellular context"-dependent cytokine response.

Animals↗
PubMed2001

Reduction of aflatoxin B1 dialdehyde by rat and human aldo-keto reductases.

Oxidation of the mycotoxin aflatoxin (AF) B1 yields the 8,9-epoxide, which nonenzymatically hydrolyzes rapidly to a dihydrodiol that in turn undergoes slow, base-catalyzed ring opening to a dialdehyde [Johnson, W. W., Harris, T. M., and Guengerich F. P. (1996) J. Am. Chem. Soc. 118, 8213-8220]. AFB1 dialdehyde does not bind to DNA but can react with protein lysine groups. One enzyme induced by cancer chemopreventive agents is AFB1 aldehyde reductase (AFAR), which catalyzes the NADPH-dependent reduction of the dialdehyde to a dialcohol. AFB1 dialdehyde is known to convert nonenzymatically to AFB1 dihydrodiol at neutral pH, and we reinvestigated the enzymatic reaction by preparing AFB1 dialdehyde at pH 10 and then used this to initiate reactions (at neutral pH) with rat and human AFAR isozymes. Two monoalcohols were identified as products, and their identities were established by NaB2H4 reduction, chemical cleavage, and mass spectrometry. The monoalcohol corresponding to reduction at C-8 formed first in reactions catalyzed by either the rat or the human AFAR. This C-8 monoalcohol was further reduced to AFB1 dialcohol by AFAR. The other monoalcohol (C-6a) was formed but not reduced to the dialcohol rapidly. Steady-state kinetic parameters were estimated for the reduction of AFB1 dialdehyde by rat and human AFAR to the monoalcohols. The apparent k(cat) and K(m) values were not adequate to rationalize the observed DeltaA(340) spectral changes in a kinetic model. Simulation fitting was done and yielded parameters indicative of greater enzyme efficiency. A survey of 12 human liver cytosol samples showed a variation of 2.3-fold in AFAR activity. Rats treated with AFB1 excreted the dialcohol and a monoalcohol in urine. The results of these studies are consistent with a role of (rat and human) AFAR in protection against AFB1 toxicity.

Aflatoxin B1↗
PubMed2001

Effects of inducible MEK1 activation on the cytokine dependency of lymphoid cells.

The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using deltaMEK1:ER, a conditionally active form of MEK1, we demonstrate the ability of this dual specificity protein kinase to abrogate the cytokine dependency of the murine lymphoid hematopoietic cell line FL5.12. Cytokine-independent cells were obtained from FL5.12 cells at a frequency of 1 x 10(-7), indicating that a low frequency of cells expressing deltaMEK1:ER were factor-independent. In general, cells that were converted to a cytokine-independent phenotype displayed a higher level of MAP kinase activity in response to deltaMEK1:ER activation than those that remained cytokine-dependent. deltaMEK1:ER-responsive cells could be maintained long-term in the presence of beta-estradiol, as well as the estrogen-receptor antagonist 4-hydroxy-tamoxifen. Removal of hormone led to the rapid cessation of cell growth in a manner similar to that observed when cytokine is withdrawn from the parental cells. GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that activated deltaMEK1:ER may induce a pathway leading to autocrine proliferation. Cytokine-dependent deltaMEK1:ER cells were found to increase the expression of GM-CSF receptor alpha (GM-CSFRalpha) in response to beta-estradiol. In contrast, MEK1-responsive cells were found to express constitutively lower levels of GM-CSFRalpha and beta common (betac) chains indicating that constitutive GM-CSF expression resulted in a decrease in GM-CSFR expression. Treatment of parental cells with supernatant from MEK1-responsive FL5.12 cells was sufficient to promote [3H]-thymidine incorporation. GM-CSF was found to enhance the viability of FL5.12 cells. The cell lines described here will be useful for elaborating the ability of the MAP kinase pathway to regulate cell proliferation in hematopoietic cells.

Cell Cycle↗