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Biomedical subjects

J S Gutkind

Publications and source records attributed to J S Gutkind.

At least 19 recordsLinked to original sources
PubMed2006

Gene expression changes in a patient presenting nonleukaemic nasal granulocytic sarcoma to acute myelogenous leukaemia using 40 K cDNA microarray.

This is a case report of granulocytic sarcoma occurring as a nasal lesion prior to the onset of acute myelogenous leukaemia (AML). To understand this case in more detail, we used 40,000 human cDNA microarray to identify the gene expression patterns of nonleukaemic stage bone marrow (BM), AML stage BM and AML stage peripheral blood cells and subsequently define the molecular basis of this disease progression. Of significance, we have tracked the expression profile of BM samples during the course of nonleukaemic to leukaemic progression, and identified a number of genes that may account for the growth potential of leukaemia cells and indicate poor prognosis of this case.

Aged, 80 and over↗
PubMed2005

Proteome-wide analysis of head and neck squamous cell carcinomas using laser-capture microdissection and tandem mass spectrometry.

Remarkable progress has been made to identify genes expressed in squamous cell carcinomas of the head and neck (HNSCC). However, limited information is available on their corresponding protein products, whose expression, post-translational modifications, and activity are ultimately responsible for the malignant behavior of this tumor type. We have combined laser-capture microdissection (LCM) with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins expressed in histologically normal squamous epithelium and matching SCC. The protein fraction from approximately 10,000-15,000 normal and tumor cells was solubilized, digested with trypsin, and the resulting peptides were analyzed by LC-MS/MS. Database searching of the resulting sequence information identified 30-55 proteins per sample. Keratins were the most abundant proteins in both normal and tumor tissues. Among the proteins differentially expressed, keratin 13 was much lower in tumors, whereas heat-shock (Hsp) family members were highly expressed in neoplastic cells. Wnt-6 and Wnt-14 were identified in both normal and tumor tissues, respectively, and placental growth factor (PIGF) was detected only in tumors. Immunohistochemical analysis of HNSCC tissues revealed lack of keratin 13 in tumor tissues, and strong staining in normal epithelia, and high expression of Hsp90 in tumors. Our study, by combining LCM and proteomic technologies, underscores the advantages of this approach to investigate complex changes at the protein level in HNSCC, thus complementing existing and emerging genomic technologies. These efforts may likely result in the identification of new biomarkers for HNSCC that can be used to diagnose disease, predict susceptibility, and monitor progression in individual patients.

Aged↗
PubMed2003

Gene discovery in oral squamous cell carcinoma through the Head and Neck Cancer Genome Anatomy Project: confirmation by microarray analysis.

The near completion of the human genome project and the recent development of novel, highly sensitive high-throughput techniques have now afforded the unique opportunity to perform a comprehensive molecular characterization of normal, precancerous, and malignant cells, including those derived from squamous carcinomas of the head and neck (HNSCC). As part of these efforts, representative cDNA libraries from patient sets, comprising of normal and malignant squamous epithelium, were generated and contributed to the Head and Neck Cancer Genome Anatomy Project (HN-CGAP). Initial analysis of the sequence information indicated the existence of many novel genes in these libraries [Oral Oncol 36 (2000) 474]. In this study, we surveyed the available sequence information using bioinformatic tools and identified a number of known genes that were differentially expressed in normal and malignant epithelium. Furthermore, this effort resulted in the identification of 168 novel genes. Comparison of these clones to the human genome identified clusters in loci that were not previously recognized as being altered in HNSCC. To begin addressing which of these novel genes are frequently expressed in HNSCC, their DNA was used to construct an oral-cancer-specific microarray, which was used to hybridize alpha-(33)P dCTP labeled cDNA derived from five HNSCC patient sets. Initial assessment demonstrated 10 clones to be highly expressed (>2-fold) in the normal squamous epithelium, while 14 were highly represented in the malignant counterpart, in three of the five patient sets, thus suggesting that a subset of these newly discovered transcripts might be highly expressed in this tumor type. These efforts, together with other multi-institutional genomic and proteomic initiatives are expected to contribute to the complete understanding of the molecular pathogenesis of HNSCCs, thus helping to identify new markers for the early detection of preneoplastic lesions and novel targets for pharmacological intervention in this disease.

Aged↗
PubMed2001

MAPK and Akt act cooperatively but independently on hypoxia inducible factor-1alpha in rasV12 upregulation of VEGF.

Oncogenic ras upregulates the expression of VEGF through the activation of the transcriptional enhancer hypoxia inducible factor-1alpha (HIF-1alpha) by a still poorly understood mechanism. Here, we demonstrate that both the Raf/MEK/MAPK and the PI3 kinase/Akt signaling pathways potently and additively stimulate the expression from a hypoxia response element (HRE) within the 5'flanking region of the VEGF promoter. Interestingly, while MAPK appears to specifically upregulate the transactivation activity of HIF-1alpha through direct phosphorylation of its regulatory/inhibitory domain, GSK-3, a downstream target of Akt, directly phosphorylates the HIF-1alpha oxygen-dependent degradation domain. These results suggest a novel mechanism whereby two divergent signaling pathways emerging from Ras may cooperatively but independently regulate the activity of a HIF-1alpha, thereby promoting the expression of a potent angiogenic mediator.

3T3 Cells↗
PubMed2001

Structure of the RGS-like domain from PDZ-RhoGEF: linking heterotrimeric g protein-coupled signaling to Rho GTPases.

BACKGROUND: The multidomain PDZ-RhoGEF is one of many known guanine nucleotide exchange factors that upregulate Rho GTPases. PDZ-RhoGEF and related family members play a critical role in a molecular signaling pathway from heterotrimeric G protein-coupled receptors to Rho proteins. A approximately 200 residue RGS-like (RGSL) domain in PDZ-RhoGEF and its homologs is responsible for the direct association with Galpha12/13 proteins. To better understand structure-function relationships, we initiated crystallographic studies of the RGSL domain from human PDZ-RhoGEF. RESULTS: A recombinant construct of the RGSL domain was expressed in Escherichia coli and purified, but it did not crystallize. Alternative constructs were designed based on a novel strategy of targeting lysine and glutamic acid residues for mutagenesis to alanine. A triple-point mutant functionally identical to the wild-type protein was crystallized, and its structure was determined by the MAD method using Se-methionine (Se-Met) incorporation. A molecular model of the RGSL domain was refined at 2.2 A resolution, revealing an all-helical tertiary fold with the mutations located at intermolecular lattice contacts. CONCLUSIONS: The first nine helices adopt a fold similar to that observed for RGS proteins, although the sequence identity with other such known structures is below 20%. The last three helices are an integral extension of the RGS fold, packing tightly against helices 3 and 4 with multiple hydrophobic interactions. Comparison with RGS proteins suggests features that are likely relevant for interaction with G proteins. Finally, we conclude that the strategy used to produce crystals was beneficial and might be applicable to other proteins resistant to crystallization.

Amino Acid Sequence↗
PubMed2001

The mood stabilizer valproic acid activates mitogen-activated protein kinases and promotes neurite growth.

The mood-stabilizing agents lithium and valproic acid (VPA) increase DNA binding activity and transactivation activity of AP-1 transcription factors, as well as the expression of genes regulated by AP-1, in cultured cells and brain regions involved in mood regulation. In the present study, we found that VPA activated extracellular signal-regulated kinase (ERK), a kinase known to regulate AP-1 function and utilized by neurotrophins to mediate their diverse effects, including neuronal differentiation, neuronal survival, long term neuroplasticity, and potentially learning and memory. VPA-induced activation of ERK was blocked by the mitogen-activated protein kinase/ERK kinase inhibitor PD098059 and dominant-negative Ras and Raf mutants but not by dominant-negative stress-activated protein kinase/ERK kinase and mitogen-activated protein kinase kinase 6 mutants. VPA also increased the expression of genes regulated by the ERK pathway, including growth cone-associated protein 43 and Bcl-2, promoted neurite growth and cell survival, and enhanced norepinephrine uptake and release. These data demonstrate that VPA is an ERK pathway activator and produces neurotrophic effects.

Base Sequence↗
PubMed2001

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor promotes endothelial cell survival through the activation of Akt/protein kinase B.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor (KSHV-GPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma, playing a central role in the promotion of vascular endothelial growth factor (VEGF)-driven angiogenesis and spindle cell proliferation. We previously have shown that KSHV-GPCR has oncogenic potential when overexpressed in fibroblasts and is responsible for the expression and secretion of VEGF through the regulation of different intracellular signaling pathways (A. Sodhi et al., Cancer Res., 60: 4873-4880, 2000; C. Bais et al., Nature, 391: 86-89, 1998). Here, we describe that this constitutively active G protein-coupled receptor is able to promote cell survival in primary human umbilical vein endothelial cells and that this effect is independent of its ability to secrete VEGF because it is not prevented by the expression of antisense constructs for VEGF or the addition of VEGF-blocking antibodies. Instead we found that ectopic expression of KSHV-GPCR potently induces the kinase activity of Akt/protein kinase B in a dose-dependent manner and triggers its translocation to the plasma membrane. This signaling pathway requires the function of phosphatidylinositol 3'-kinase and is dependent on betagamma subunits released from both pertussis toxin-sensitive and -insensitive G proteins. Furthermore, we found that KSHV-GPCR is able to protect human umbilical vein endothelial cells from the apoptosis induced by serum deprivation and that both wortmannin and the expression of a kinase-deficient Akt K179M mutant are able to block this effect. Finally, we observed that the Akt K179M protein also inhibits the activation of nuclear factor-KB induced by KSHV-GPCR, suggesting that this transcription factor may represent one of the putative downstream targets for Akt in the survival-signaling pathway. These results provide further knowledge in the elucidation of the signal transduction pathways activated by KSHV-GPCR and support its key role in promoting the survival of viral-infected cells. Moreover, the present findings also emphasize the importance of this G protein-coupled receptor in the development of KSHV-related neoplasias.

Animals↗
PubMed2001

Regulation of gene expression by the small GTPase Rho through the ERK6 (p38 gamma) MAP kinase pathway.

Small GTP-binding proteins of the Rho-family, Rho, Rac, and Cdc42, have been traditionally linked to the regulation of the cellular actin-based cytoskeleton. Rac and Cdc42 can also control the activity of JNK, thus acting in a molecular pathway transmitting extracellular signals to the nucleus. Interestingly, Rho can also regulate gene expression, albeit by a not fully understood mechanism. Here, we found that activated RhoA can stimulate c-jun expression and the activity of the c-jun promoter. As the complexity of the signaling pathways controlling the expression of c-jun has begun to be unraveled, this finding provided a unique opportunity to elucidate the biochemical routes whereby RhoA regulates nuclear events. We found that RhoA can initiate a linear kinase cascade leading to the activation of ERK6 (p38 gamma), a recently identified member of the p38 family of MAPKs. Furthermore, we present evidence that RhoA, PKN, MKK3/MKK6, and ERK6 (p38 gamma) are components of a novel signal transduction pathway involved in the regulation of gene expression and cellular transformation.

3T3 Cells↗
PubMed2001

Granulocyte colony-stimulating factor induces ERK5 activation, which is differentially regulated by protein-tyrosine kinases and protein kinase C. Regulation of cell proliferation and survival.

Granulocyte colony-stimulating factor (G-CSF) plays a major role in the regulation of granulopoiesis. Treatment of cells with G-CSF has been shown to activate multiple signal transduction pathways. We show here that Erk5, a novel member of the MAPK family, and its specific upstream activator MEK5 were activated in response to incubation of cells with G-CSF. Different from other members of the MAPK family including Erk1/2, JNK, and p38, maximal activation of Erk5 by G-CSF required the C-terminal region of the G-CSF receptor. Genistein, a specific inhibitor of protein-tyrosine kinases, blocked G-CSF-induced Erk5 activation. In contrast, inhibition of protein kinase C activity increased G-CSF-mediated activation of Erk5 and MEK5, whereas stimulation of protein kinase C activity inhibited activation of the two kinases by G-CSF. The proliferation of BAF3 cells in response to G-CSF was inhibited by expression of a dominant-negative MEK5 but potentiated by expression of a constitutively active MEK5. Expression of the constitutively active MEK5 also increased the survival of BAF3 cells cultured in the absence of or in low concentrations of G-CSF. Together, these data implicate Erk5 as an important signaling component in the biological actions of G-CSF.

Animals↗
PubMed2001

Proteomic profiling of the cancer microenvironment by antibody arrays.

Critical changes in protein expression that enable tumors to initiate and progress originate in the local tissue microenvironment, and there are increasing indications that these microenvironmental alterations in protein expression play critical roles in shaping and directing this process. As a model to better understand how patterns of protein expression shape the tissue microenvironment, we analyzed protein expression in tissue derived from squamous cell carcinoma of the oral cavity through an antibody microarray approach for high-throughput proteomic analysis. Utilizing laser capture microdissection to procure total protein from specific microscopic cellular populations, we demonstrate that quantitative, and potentially qualitative, differences in expression patterns of multiple proteins within epithelial cells reproducibly correlate with oral cavity tumor progression. Furthermore, differential expression of multiple proteins was also found in stromal cells surrounding and adjacent to regions of diseased epithelium that directly correlated with tumor progression of the epithelium. Most of the proteins identified in both cell types are involved in signal transduction pathways, thus we hypothesize that extensive molecular communication involving complex cellular signaling between epithelium and stroma play a key role in driving oral cavity cancer progression.

Antibodies, Neoplasm↗
PubMed2001

G-protein-coupled receptors and signaling networks: emerging paradigms.

G-protein-coupled receptors (GPCRs) constitute the largest family of cell-surface molecules involved in signal transmission. These receptors play key physiological roles and their dysfunction results in several diseases. Recently, it has been shown that many of the cellular responses mediated by GPCRs do not involve the sole stimulation of conventional second-messenger-generating systems, but instead result from the functional integration of an intricate network of intracellular signaling pathways. Effectors for GPCRs that are independent of G proteins have now also been identified, thus changing the conventional view of the GPCR-heterotrimeric-G-protein-associated effector. The emerging information is expected to help elucidate the most basic mechanism by which these receptors exert their numerous physiological roles, in addition to determining why the perturbation of their function results in many pathological conditions.

Animals↗
PubMed2001

Regulation of c-myc expression by PDGF through Rho GTPases.

Src family protein-tyrosine kinases have a central role in several biological functions, including cell adhesion and spreading, chemotaxis, cell cycle progression, differentiation and apoptosis. Surprisingly, these kinases also participate in mitogenic signalling by receptors that themselves exhibit an intrinsic protein-tyrosine kinase activity, including those for platelet-derived growth factor (PDGF), epidermal growth factor and colony-stimulating factor-1. Indeed, Src kinases are strictly required for the nuclear expression of the c-myc proto-oncogene and thus for DNA synthesis in response to PDGF. However, the nature of the signalling pathways by which Src kinases participate in the induction of c-myc expression by tyrosine kinase receptors is still unknown. Here we show that PDGF enhances c-myc expression and stimulates the c-myc promoter in a Src-dependent manner, and that neither Ras nor the mitogen-activated protein kinase pathway mediate these effects. In contrast, we present evidence that PDGF stimulates Vav2 through Src, thereby initiating the activation of a Rac-dependent pathway that controls the expression of the c-myc proto-oncogene.

3T3 Cells↗
PubMed2001

New approaches to the understanding of the molecular basis of oral cancer.

Cancers of the oral cavity, salivary glands, larynx, and pharynx, collectively referred to as squamous cell carcinomas of the head and neck (HNSCC), are the sixth most common cancer among men in the developed world. The prognosis of HNSCC patients is still poor, which reflects the fact that although the risk factors for HNSCC are well-recognized, very little is known about the molecular mechanisms responsible for this malignancy. This review describes some of the current efforts and technological advances that have focused on the creation of a complete information infrastructure for genes expressed during squamous cell carcinogenesis. These include: the recently described HNSCC-specific chromosomal alterations (cCAP); the Head and Neck Cancer Genome Anatomy Project (HN-CGAP), whose goal is the systematic identification and cataloguing of known and novel genes expressed during tumor development; and the use of laser-capture microdissection (LCM), which is pivotal for the comprehensive molecular characterization of normal, pre-cancerous, and malignant cells by means of DNA-array technology. The latter provides the means for the analysis of expression patterns of thousands of genes simultaneously. The use of LCM for proteomics and DNA analysis is also included in this review. These revolutionary approaches are likely to have an unprecedented impact on cancer biology, and provide exciting opportunities to unravel the still-unknown mechanisms involved in squamous cell carcinogenesis. They are also expected to provide a molecular blueprint for HNSCC, thus helping to identify suitable markers for the early detection of preneoplastic lesions, as well as novel targets for pharmacological intervention in this disease.

Carcinoma, Squamous Cell↗
PubMed2000

Signaling from E-cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell-cell contact formation.

E-cadherins are well characterized cell surface molecules expressed in epithelial cells, which play a major role in cell adhesion through the establishment of calcium-dependent homophilic interactions at sites of cell-cell contacts. They are also integral components of morphogenetic programs controlling the maintenance of the structural and functional integrity of epithelia. Accumulated evidence indicates that the E-cadherin-mediated cell adhesion system is highly regulated from inside the cells by a number of intracellular signaling pathways. Recently available information suggests that E-cadherins may also play a role in the transduction of signals from the outside of the cell to the cytoplasm. However, the nature of the biochemical routes regulated by E-cadherins is still largely unknown. In this study, we set out to explore the possibility that E-cadherins may regulate the activity of MAPK, a key signaling pathway involved in cell fate decisions, upon the formation of cell-cell contacts among neighboring cells. By using an immortalized non-tumorigenic keratinocyte cell line, HaCat, as a model system, we provide evidence that the assembly of calcium-dependent adherens junctions leads to a rapid and remarkable increase in the state of activation of MAPK and that this event is mediated by E-cadherins. Furthermore, we found that E-cadherins stimulate the MAPK pathway through the ligand-independent activation of epidermal growth factor receptors and the consequent activation of a biochemical route leading to the stimulation of MAPKs. These findings suggest that E-cadherins can initiate outside-in signal transducing pathways through the engagement of tyrosine kinase receptors for epidermal growth factor, thus providing a novel molecular mechanism whereby these cell adhesion molecules may ultimately control the fate of normal and transformed epithelial cells.

Cadherins↗
PubMed2000

Induction of apoptosis in head-and-neck squamous carcinoma cells by gamma-irradiation and bleomycin is p53-independent.

We have examined the ability of gamma-irradiation and bleomycin to induce apoptosis in a model system consisting of cell lines derived from naturally occurring human head-and-neck squamous-cell carcinomas with contrasting p53 status and expression levels of pro- and anti-apoptotic molecules. Following exposure to gamma-irradiation (20 Gy) or bleomycin (3.5 microM) for 0 to 96 hr, cells expressing either transcriptionally inactive mutant p53 (HN6) or a truncated p53 molecule (HN19) underwent apoptosis, as assessed by fluorescence-activated cell sorting and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, in contrast to cells that express wild-type p53 (HN30), suggesting that apoptosis induced by these agents occurs by p53-independent mechanisms. Apoptosis in HN6 and HN19 cells was preceded by a G(2)/M cell-cycle block, as analyzed by DNA content and BrdU staining. In contrast, HN30 cells remained blocked in both G(1) and G(2)/M and failed to re-enter the cell cycle. Levels of Bcl-2 were elevated in 3 of 10 cell lines, and only marginal differences were observed for Bcl-x(L). Pro-apoptotic proteins bax and Bcl-x(S) were detectable in normal keratinocytes and 4 tumor cell lines. Bax-delta (16 kDa) was highly represented in normal keratinocytes, and levels of bak were variable between cell lines. Elevated expression of Bcl-2 failed to protect HN19 cells from either gamma-irradiation or bleomycin-induced apoptosis. Our data support the existence of p53- and Bcl-2-independent pathways regulating apoptosis in keratinocytes and suggest that efficacy of either radiotherapy or bleomycin treatment for oral squamous-cell neoplasms may not, therefore, be influenced solely by endogenous p53 status.

Antibiotics, Antineoplastic↗
PubMed2000

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G(12) family to Rho.

A putative guanine nucleotide exchange factor (GEF), termed leukemia-associated RhoGEF (LARG), was recently identified upon fusion to the coding sequence of the MLL gene in acute myeloid leukemia. Although the function of LARG is still unknown, it exhibits a number of structural domains suggestive of a role in signal transduction, including a PDZ domain, a LH/RGS domain, and a Dbl homology/pleckstrin homology domain. Here, we show that LARG can activate Rho in vivo. Furthermore, we present evidence that LARG is an integral component of a novel biochemical route whereby G protein-coupled receptors (GPCRs) and heterotrimeric G proteins of the G alpha(12) family stimulate Rho-dependent signaling pathways.

Cloning, Molecular↗
PubMed2000

Loss of PTEN expression leading to high Akt activation in human multiple myelomas.

Mouse plasma cell tumor (PCT) and human multiple myeloma (MM) are terminal B-cell malignancies sharing many similarities. Our recent work demonstrated that activation of the insulin-like growth factor receptor (IGF-IR)/insulin receptor substrate (IRS)/phosphatidylinositol 3' kinase (PI 3'K) pathway was evident in the tumor lines derived from both species. Although PI 3'K activity was higher in mouse tumor lines than that in human tumors, activation of Akt serine/threonine kinase was markedly lower in mouse lines. This discrepancy prompted us to test the status of PTEN tumor suppressor gene, as it has been shown to be a negative regulator of PI 3'K activity. Although all the mouse lines expressed intact PTEN, 2 of the 4 human lines (Delta47 and OPM2) possessing the highest Akt activity lost PTEN expression. Sequencing analysis demonstrated that the PTEN gene contains a deletion spacing from exon 3 to exon 5 or 6 in the Delta47 line and from exon 3 to 7 in the OPM2 line. Restoration of PTEN expression suppressed IGF-I-induced Akt activity, suggesting that loss of PTEN is responsible for uncontrolled Akt activity in these 2 lines. Despite the expression of PTEN with the concomitant low Akt activity in all mouse PCT lines, their p70S6K activities were generally higher than those in 3 human MM lines, arguing for specific negative regulation of Akt, but not p70S6K by PTEN. These results suggest that p70S6K and Akt may be differentially used by the plasma cell tumors derived from mice and humans, respectively.

Animals↗