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Biomedical subjects

Mark I Greene

Publications and source records attributed to Mark I Greene.

At least 19 recordsLinked to original sources
PubMed2007

Targeted antireceptor therapy with monoclonal antibodies leads to the formation of inactivated tetrameric forms of ErbB receptors.

mAbs capable of disabling heterodimeric kinase complexes of the epidermal growth factor receptor (EGFR) and human EGFR type 2/neu have therapeutic relevance to various human cancers. In this study, we demonstrate that in addition to the dimer, EGFR and human EGFR type 2 can associate as homo- and heterotetramers. EGF-induced phosphorylation of the tetramers was significantly lower than that of the dimers, indicating that the tetrameric receptor complexes have impaired signaling activity. Targeting v-erb-b2 erythroblastic leukemia viral oncogene homolog (erbB) receptors with mAbs promoted erbB tetrameric assembly, suggesting that a component of the antitumor activity may be mediated by the ability of Abs to shift the equilibrium from active dimeric to impaired tetrameric receptor complex states. This study suggests a novel therapeutic approach to disable signaling of erbB and potentially other receptors in tumors by biologic agents capable of inducing receptor tetramerization.

Animals↗
PubMed2006

Test for detection of disease-associated prion aggregate in the blood of infected but asymptomatic animals.

We have developed a sensitive in vitro assay for detecting disease-associated prion aggregates by combining an aggregation-specific enzyme-linked immunosorbent assay (AS-ELISA) with the fluorescent amplification catalyzed by T7 RNA polymerase technique (FACTT). The new assay, named aggregation-specific FACTT (AS-FACTT), is much more sensitive than AS-ELISA and could detect prion aggregates in the brain of mice as early as 7 days after an intraperitoneal inoculation of PrP(Sc). However, AS-FACTT was still unable to detect prion aggregates in blood of infected mice. To further improve the detection limit of AS-FACTT, we added an additional prion amplification step (Am) and developed a third-generation assay, termed Am-A-FACTT. Am-A-FACTT has 100% sensitivity and specificity in detecting disease-associated prion aggregates in blood of infected mice at late but still asymptomatic stages of disease. At a very early stage, Am-A-FACTT had a sensitivity of 50% and a specificity of 100%. Most importantly, Am-A-FACTT also detects prion aggregates in blood of mule deer infected with the agent causing a naturally occurring prion disease, chronic wasting disease. Application of this assay to cattle, sheep, and humans could safeguard food supplies and prevent human contagion.

Animals↗
PubMed2006

DIPA, which can localize to the centrosome, associates with p78/MCRS1/MSP58 and acts as a repressor of gene transcription.

DIPA (delta-interacting protein A) was initially identified as a protein that associates with the hepatitis delta antigen. In this study, we found that DIPA can associate with p78/MCRS/MSP58, a Forkhead-associated domain containing protein implicated in malignant transformation as well as in regulation of gene transcription and translation. We analyzed the interaction between DIPA and p78 by co-immunoprecipitation and identified the structural regions involved in the interaction. Consistent with the physical interaction, we found that DIPA is predominant co-localized with p78 to the nucleus. In addition, a fraction of DIPA can be detected on the centrosome. Furthermore, we demonstrate that DIPA can act as a repressor of gene transcription, an activity that appears to be enhanced by p78. Taken together, our results revealed a novel protein complex that plays a role in regulation of gene expression and cell proliferation. We propose that dysfunction of DIPA may contribute to malignant transformation by affecting the functions of p78.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Characterization of Su48, a centrosome protein essential for cell division.

The centrosome functions as the major microtubule-organizing center and plays a vital role in guiding chromosome segregation during mitosis. Centrosome abnormalities are frequently seen in a variety of cancers, suggesting that dysfunction of this organelle may contribute to malignant transformation. In our efforts to identify the protein components of the centrosome and to understand the structure features involved in the assembly and functions of this organelle, we cloned and characterized a centrosome-associated protein called Su48. We found that a coiled coil-containing subdomain of Su48 was both sufficient and required for its centrosome localization. In addition, this structure also modulates Su48 dimerization. Moreover, ectopic expression of Su48 causes abnormal mitosis, and a mutant form of Su48 disrupts the localization of gamma-tubulin to the centrosome. Finally, by microinjection of an anti-Su48 antibody, we found that disruption of normal Su48 functions leads to mitotic failure, possibly due to centrosome defects or incomplete cytokinesis. Thus, Su48 represents a previously unrecognized centrosome protein that is essential for cell division. We speculate that Su48 abnormalities may cause aberrant chromosome segregation and may contribute to aneuploidy and malignant transformation.

Animals↗
PubMed2006

A sensitive and high-throughput assay to detect low-abundance proteins in serum.

The ability to detect antigens immunologically is limited by the affinity of the antibodies and the amount of antigens. We have now succeeded in creating a modular, facile amplification system, termed fluorescent amplification catalyzed by T7 polymerase technique (FACTT). Such a system can detect protein targets specifically at subfemtomolar levels ( approximately 0.08 fM). We describe here the detection of Her2 (also known as Neu) from rodent and human sera. FACTT is adaptable to high-throughput screening and automation and provides a practical method to enhance current ELISAs in medical practice.

Animals↗
PubMed2006

The gefitinib-sensitizing mutant epidermal growth factor receptor enables transformation of a mouse fibroblast cell line.

A specific inhibitor of the Epidermal Growth Factor Receptor (EGFR), Gefitinib, displays significant antitumor effects against non-small cell lung cancers (NSCLC) that express EGFR with mutations in their tyrosine kinase domain. Although previous reports have already demonstrated that oncogenic transformation can be induced by some mutant EGFR forms, the precise differences between mutant and wild-type EGFR in terms of mechanisms of transformation have not been fully elucidated. We show here that a murine fibroblast cell line, NR6 becomes transformed by an expression level of the mutant EGFR form lacking E746-A750 that is far less than that needed with transfected wild-type EGFR. However, the mutant EGFR was unable to transform NR6 in a ligand-independent manner, as was seen with the wild-type EGFR. The consequent biological features after transformation, including DNA synthesis or cell cycle progression and biochemical characteristics such as MAPK activation mediated by the mutant EGFR are comparable and equivalent to those mediated by wild-type EGFR. These data suggest that the mutant EGFR possesses greater ligand-dependent transformation when compared with wild-type EGFR, although the exact mechanisms to account for this characteristic remain to be defined.

Animals↗
PubMed2006

The extracellular domain of p185(c-neu) induces density-dependent inhibition of cell growth in malignant mesothelioma cells and reduces growth of mesothelioma in vivo.

EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance.

Animals↗
PubMed2006

Characterization of the structures involved in localization of the SUN proteins to the nuclear envelope and the centrosome.

The nuclear envelope forms a selective barrier that separates the cytoplasm from the nucleus. During mitosis the nuclear envelope breaks down so that the microtubule network can form contacts with the kinetochore and guide chromosome segregation. Previous studies have suggested a model in which the centrosome and the microtubule network may play a role in nuclear envelope breakdown through as yet unidentified interactions with proteins localized to the nuclear envelope. In the current study we characterized a nuclear envelope protein SUN2 and identified a substructure involved in its localization to the nuclear envelope. We found that a structurally related protein, SUN1, may be localized to the nuclear envelope through a different mechanism. Furthermore, the SUN2 protein can form different assemblies, including homodimers and heterodimers with SUN1. Finally, we provide evidence indicating that SUN1 and SUN2 may form a physical interaction between the nuclear envelope and the centrosome.

Animals↗
PubMed2006

FOXP3 ensembles in T-cell regulation.

Our recent studies have identified dynamic protein ensembles containing forkhead box protein 3 (FOXP3) that provide insight into the molecular complexity of suppressor T-cell activities, and it is our goal to determine how these ensembles regulate FOXP3's transcriptional activity in vivo. In this review, we summarize our current understanding of how FOXP3 expression is induced and how FOXP3 functions in vivo as a transcriptional regulator by assembling a multisubunit complex involved in histone modification as well as chromatin remodeling.

Animals↗
PubMed2005

Sialylation regulates peripheral tolerance in CD4+ T cells.

Decreased binding by the 6C10 auto-antibody serves as a unique marker for CD4+ T cell unresponsiveness after the induction of T cell tolerance in Vbeta8.1 TCR transgenic mice. We further define the nature of the epitope recognized by the 6C10 antibody to be a subset of Thy-1 bearing incompletely sialylated N-linked glycans, and furthermore, we demonstrate that tolerant CD4+ T cells have an increased degree of cell-surface sialylation. To test the significance of the altered glycosylation state identified by the 6C10 auto-antibody in the tolerant CD4+ T cell population, surface sialic acid was cleaved enzymatically. Treatment of purified peripheral CD4+ T cells with Vibrio cholerae sialidase (VCS) leads to increased 6C10 binding, significantly enhances proliferation in the tolerant CD4+ population and corrects defects in phosphotyrosine signaling observed in the tolerant CD4+ T cell. Furthermore, in vivo administration of VCS enhances proliferation in both tolerant and naive CD4+ T cell subsets. These studies suggest that sialylation of glycoproteins on the surface of the CD4+ T cell contributes to the regulation of T cell responsiveness in the tolerant state.

Animals↗
PubMed2005

UXT is a novel centrosomal protein essential for cell viability.

Ubiquitously expressed transcript (UXT) is a prefoldinlike protein that has been suggested to be involved in human tumorigenesis. Here, we have found that UXT is overexpressed in a number of human tumor tissues but not in the matching normal tissues. We demonstrate that UXT is located in human centrosomes and is associated with gamma-tubulin. In addition, overexpression of UXT disrupts centrosome structure. Furthermore, abrogation of UXT protein expression by small interfering RNA knockdown leads to cell death. Together, our findings suggest that UXT is a component of centrosome and is essential for cell viability. We propose that UXT may facilitate transformation by corrupting regulated centrosome functions.

Amino Acid Sequence↗
PubMed2005

Disabling TNF receptor signaling by induced conformational perturbation of tryptophan-107.

We have disabled TNF receptor (TNFR) function by inducing allosteric modulation of tryptophan-107 (W107) in the receptor. The allosteric effect operates by means of an allosteric cavity found a short distance from a previously identified loop involved in ligand binding. Occupying this cavity by small molecules leads to perturbation of distal W107 and disables functions of the TNFR, a molecule not known to undergo conformational change upon binding TNF-alpha. TNF-alpha-induced NF-kappaB and p38 kinase activities and clinical symptoms of collagen-induced arthritis in mice were all diminished. Thus, disabling receptor function by induced conformational changes of active binding surfaces represents an innovative paradigm in structure-based drug design.

Allosteric Site↗
PubMed2005

EGFR enhances Survivin expression through the phosphoinositide 3 (PI-3) kinase signaling pathway.

The ErbB family of receptor tyrosine kinases includes the epidermal growth factor receptor (EGFR), p185/neu/c-erbB2, ErbB3, and ErbB4. Many of these receptors are overexpressed or amplified in various forms of cancers. Previous studies have indicated that activation of erbB molecules contributes to malignant transformation both by promoting cell proliferation through the mitogen-activated protein kinase (MAP kinase) signaling pathway and by preventing apoptosis through the Phosphoinositide 3 kinase (PI-3 kinase) pathway. Disabling erbB receptors converts malignant cells that were resistant to cell death caused by irradiation to cells that are sensitive to apoptosis. Here, we report that an activated form of EGFR can elevate the levels of Survivin, a member of the Inhibitor of Apoptosis Protein (IAP) family implicated in mitotic checkpoint control. Conversely, inactivation of the ErbB receptors reduces the expression levels of Survivin. Furthermore, we found that upregulation of Survivin by EGFR is dependent on the PI-3 kinase pathway but not on the MAP kinase pathway. Indeed, inhibition of PI-3 kinase can diminish Survivin at both the mRNA and the protein levels. Combined with previous findings that Survivin plays a role in control of chromosome segregation and that it is overexpressed in various cancers, our results suggest that EGFR may cause transformation by directly affecting mitosis and increasing chromosome instability.

Apoptosis↗
PubMed2005

Differential localization of ErbB receptor ensembles influences their signaling in hippocampal neurons.

Our studies indicate that ErbB complexes participate in both survival and synaptic plasticity signals of hippocampal neurons but in a manner that depends on the subcellular localization of the receptor ensembles. Using dissociated hippocampal cultures, we found that neurons, rather than glial cells, are the primary targets of ErbB receptor ligands such as epidermal growth factor and heregulin. Further investigation demonstrated that ErbB receptors distribute differentially in hippocampal neurons with the epidermal growth factor receptor confined to neural cell bodies and the p185(c-neu) and ErbB4 receptors distributed to both neural soma and neurites. Activation of ErbB receptor and downstream signaling molecules were observed in neurites only after heregulin stimulation. The receptor complex which mediated neurite located signals was the p185(c-neu)/ErbB4 heterodimer. Colocalization of p185(c-neu), but not epidermal growth factor receptor, with postsynaptic density protein 95 suggests that the heregulin signaling contributes to synapse specific activities. However, the epidermal growth factor receptor complex mediates physiological survival signals, as neuronal survival was enhanced by epidermal growth factor, rather than heregulin. Collectively, these studies indicate that different ErbB ensembles localize to different locations on the neuron to mediate distinct signals and functions.

Animals↗
PubMed2004

Ca(2+) and K(+) (BK) channels in chick hair cells are clustered and colocalized with apical-basal and tonotopic gradients.

Electrical resonance is a mechanism used by birds and many vertebrates to discriminate between frequencies of sound, and occurs when the intrinsic oscillation in the membrane potential of a specific hair cell corresponds to a specific stimulus sound frequency. This intrinsic oscillation results from an interplay between an inward Ca(2+) current and the resultant activation of a hyperpolarizing Ca(2+)-activated K(+) current. These channels are predicted to lie in close proximity owing to the fast oscillation in membrane potential. The interplay of these channels is widespread in the nervous system, where they perform numerous roles including the control of synaptic release, burst frequency and circadian rhythm generation. Here, we used confocal microscopy to show that these two ion channels are clustered and colocalized in the chick hair cell membrane. The majority of Ca(2+) channels were colocalized while the proportion of colocalized BK channels was markedly less. In addition, we report both an apical-basal gradient of these clusters in individual hair cells, as well as a gradient in the number of clusters between hair cells along the tonotopic axis. These results give physical confirmation of previous predictions. Since the proportion of colocalized channels was a constant function of Ca(2+) channels, and not of BK channels, these results suggest that their colocalization is determined by the former. The molecular mechanisms underpinning their clustering and colocalization are likely to be common to other neuronal cells.

Animals↗
PubMed2004

Fas-disabling small exocyclic peptide mimetics limit apoptosis by an unexpected mechanism.

Fas ligand- (FasL) mediated apoptosis is an important element of tissue-specific organ damage. We have developed biologically active small exocyclic peptide mimetics that disable apoptotic functions of Fas. The most effective mimetic binds to both its receptor and FasL with comparable affinity. In vitro, the most effective antagonist blocked FasL-induced cytotoxicity completely and specifically. In vivo, the antagonistic mimetic also prevented Concanavilin A (Con A) induced hepatitis, a CD4(+) T cell-mediated animal model of liver injury. Although current approaches prevent Fas receptor signaling by excluding FasL binding to Fas, the small molecule mimetics reported here disable Fas by promoting a defective Fas-FasL receptor complex. This event desensitizes FasL-mediated apoptosis by inhibiting extracellular signal regulated kinase activity and up-regulating NF-kappaB.

Amino Acid Sequence↗