Showing papers on "Cyclin-dependent kinase 8 published in 2000"

Role of Transforming Growth Factor-β Signaling in Cancer

Abstract: Signaling from transforming growth factor-beta (TGF-beta) through its unique transmembrane receptor serine-threonine kinases plays a complex role in carcinogenesis, having both tumor suppressor and oncogenic activities. Tumor cells often escape from the antiproliferative effects of TGF-beta by mutational inactivation or dysregulated expression of components in its signaling pathway. Decreased receptor function and altered ratios of the TGF-beta type I and type II receptors found in many tumor cells compromise the tumor suppressor activities of TGF-beta and enable its oncogenic functions. Recent identification of a family of intracellular mediators, the Smads, has provided new paradigms for understanding mechanisms of subversion of TGF-beta signaling by tumor cells. In addition, several proteins recently have been identified that can modulate the Smad-signaling pathway and may also be targets for mutation in cancer. Other pathways such as various mitogen-activated protein kinase cascades also contribute substantially to TGF-beta signaling. Understanding the interplay between these signaling cascades as well as the complex patterns of cross-talk with other signaling pathways is an important area of investigation that will ultimately contribute to understanding of the bifunctional tumor suppressor/oncogene role of TGF-beta in carcinogenesis.

A Novel Positive Feedback Loop Mediated by the Docking Protein Gab1 and Phosphatidylinositol 3-Kinase in Epidermal Growth Factor Receptor Signaling

Abstract: The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4, 5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR.

PTEN, a unique tumor suppressor gene.

Abstract: For many years, it has been thought that the chromosome region 10q22-24 includes one or more genes that appear to play a role in several human malignancies. PTEN is a new tumor suppressor gene encoding a dual-specificity phosphatase that was cloned simultaneously by three groups (Li & Sun 1997, Li et al. 1997, Steck et al. 1997), two of which used a positional cloning approach to identify genes in chromosome 10 (Li et al. 1997, Steck et al. 1997). While several protein kinases have been implicated as oncogenes, and phosphatases have long been known frequently to antagonize their function, there has been no direct demonstration of the role of phosphatases in tumor development (Myers & Tonks 1997). PTEN characterization as a bona fide tumor suppressor gene has confirmed that a deficient phosphatase activity can lead to cancer, as detailed by studies that are described below.

Neoplastic transformation by Notch requires nuclear localization.

Abstract: Notch proteins are plasma membrane-spanning receptors that mediate important cell fate decisions such as differentiation, proliferation, and apoptosis. The mechanism of Notch signaling remains poorly understood. However, it is clear that the Notch signaling pathway mediates its effects through intercellular contact between neighboring cells. The prevailing model for Notch signaling suggests that ligand, presented on a neighboring cell, triggers proteolytic processing of Notch. Following proteolysis, it is thought that the intracellular portion of Notch (N(ic)) translocates to the nucleus, where it is involved in regulating gene expression. There is considerable debate concerning where in the cell Notch functions and what proteins serve as effectors of the Notch signal. Several Notch genes have clearly been shown to be proto-oncogenes in mammalian cells. Activation of Notch proto-oncogenes has been associated with tumorigenesis in several human and other mammalian cancers. Transforming alleles of Notch direct the expression of truncated proteins that primarily consist of N(ic) and are not tethered to the plasma membrane. However, the mechanism by which Notch oncoproteins (generically termed here as N(ic)) induce neoplastic transformation is not known. Previously we demonstrated that N1(ic) and N2(ic) could transform E1A immortalized baby rat kidney cells (RKE) in vitro. We now report direct evidence that N1(ic) must accumulate in the nucleus to induce transformation of RKE cells. In addition, we define the minimal domain of N1(ic) required to induce transformation and present evidence that transformation of RKE cells by N1(ic) is likely to be through a CBF1-independent pathway.

A regulatory shortcut between the Snf1 protein kinase and RNA polymerase II holoenzyme.

Abstract: RNA polymerase II holoenzymes respond to activators and repressors that are regulated by signaling pathways. Here we present evidence for a “shortcut” mechanism in which the Snf1 protein kinase of the glucose signaling pathway directly regulates transcription by the yeast holoenzyme. In response to glucose limitation, the Snf1 kinase stimulates transcription by holoenzyme that has been artificially recruited to a reporter by a LexA fusion to a holoenzyme component. We show that Snf1 interacts physically with the Srb/mediator proteins of the holoenzyme in both two-hybrid and coimmunoprecipitation assays. We also show that a catalytically hyperactive Snf1, when bound to a promoter as a LexA fusion protein, activates transcription in a glucose-regulated manner; moreover, this activation depends on the integrity of the Srb/mediator complex. These results suggest that direct regulatory interactions between signal transduction pathways and RNA polymerase II holoenzyme provide a mechanism for transcriptional control in response to important signals.

The paradox of E2F1: oncogene and tumor suppressor gene.

Abstract: Cancer cells often contain mutations that lead to the loss of retinoblastoma tumor suppressor (Rb) function and the activation of E2F-dependent transcription As a result, proliferation is deregulated, and sensitivity to apoptotic stimuli is increased In cell culture studies, the transcription factor E2F1 has been shown to be equally adept at inducing proliferation and apoptosis Several groups using mouse models have been examining how these E2F1-regulated processes impact the development of cancer The conclusion from these studies is that E2F1 can function as both oncogene and tumor suppressor gene and that both positive and negative effects on tumorigenesis can be observed whether E2F1 is absent or overexpressed These findings are discussed in the context of a model in which pathways controlling cell-cycle progression and apoptosis are intimately linked

Multiple signals regulate GAL transcription in yeast.

Abstract: Gal4p activates transcription of the Saccharomyces GAL genes in response to galactose and is phosphorylated during interaction with the RNA polymerase II (Pol II) holoenzyme. One phosphorylation at S699 is necessary for full GAL induction and is mediated by Srb10p/CDK8 of the RNA Pol II holoenzyme mediator subcomplex. Gal4p S699 phosphorylation is necessary for sensitive response to inducer, and its requirement for GAL induction can be abrogated by high concentrations of galactose in strains expressing wild-type GAL2 and GAL3. Gal4p S699 phosphorylation occurs independently of Gal3p and is responsible for the long-term adaptation response observed in gal3 yeast. SRB10 and GAL3 are shown to represent parallel mechanisms for GAL gene induction. These results demonstrate that Gal4p activity is controlled by two independent signals: one that acts through Gal3p-galactose and a second that is mediated by the holoenzyme-associated cyclin-dependent kinase Srb10p. Since Srb10p is regulated independently of galactose, our results suggest a function for CDK8 in coordinating responses to specific inducers with the environment through the phosphorylation of gene-specific activators.

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

Abstract: Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness.

Modulation of EGFR gene transcription by secondary structures, a polymorphic repetitive sequence and mutations--a link between genetics and epigenetics.

Abstract: The epidermal growth factor receptor (EGFR) plays a crucial role in growth, differentiation and motility of normal as well as tumor cells. The transduction of extracellular signals to the cytoplasm via the receptor not only depends on ligand binding, but is also determined by the receptor density on the cell surface. Therefore, in terms of cancer diagnosis and therapeutic approaches targeting EGFR it is decisive to know how the expression level of EGFR is controlled. We found that transcription activity declines with increasing numbers of CA dinucleotides of a highly polymorphic CA repeat in the first intron epidermal growth factor receptor gene. In vivo data from cultured cell lines support these findings, although other regulation mechanisms can compensate this effect. In addition, we showed that RNA elongation terminates at a site closely downstream of the simple sequence repeat (SSR) and that there are two separate major transcription start sites. Model calculations for the helical DNA conformation revealed a high bendability in the EGFR polymorphic region, especially if the CA stretch is extended. These data suggest that the CA-SSR can act like a joint bringing the promoter in proximity to a putative repressor protein bound downstream of the CASSR. The data suggest that this polymorphism is a marker for cancer linking genetic and epigenetic risk. Furthermore in breast cancer, heterozygous tumours with short CA-SSR showed an elevated EGFR-expression in contrast to tumours with longer CA-SSR. Tumours with loss of heterozygosity in intron 1 of egfr revealed an increased EGFR expression if the longer allele was lost. Moreover, deceased egfr gene dosages were significantly correlated to poor prognosis in breast cancer.

RGS domain in the amino-terminus of G protein-coupled receptor kinase 2 inhibits Gq-mediated signaling.

Abstract: We have previously shown that not only G protein-coupled receptor kinase (GRK) 2, but also a catalytically inactive Lys220Trp GRK2 decreases endothelin (ET)-1-induced inositol 1,4,5-trisphosphate (IP3) formation, and demonstrated the presence of phosphorylation-independent desensitization mechanism. To clarify the role of GRK2 other than that as a kinase, we characterized an RGS (regulator of G protein signaling)-like domain in the amino-terminus of GRK2. Both GRK2(1-181) and GRK2(54-174) suppressed Ca2+ responses induced by angiotensin II (Ang II) and ET-1, and bound directly with Galphaq but not Galphas nor Galphai3 in the presence of GDP and AlF4-. These results demonstrate that GRK2 regulates Gq-mediated signaling negatively by direct interaction between its RGS domain and the transitional state of Galphaq, as well as through phosphorylation of activated receptors by its kinase domain.

Inhibitory effects of EGFR antisense oligodeox ynucleotide in human colorectal cancer cell line.

Abstract: Epidermal-growth-factor receptor[1] (EGFR) is a polypeptide with 1186 amino acids, which binds to EGF family growth factors. Two major natural ligands in the family interact with EGFR: one is EGF, the other is transforming growth factor-α (TGF-α)[2]. When EGF or TGF-α, binds to EGFR, tyrosine kinase activity is induced which in turn triggers a series of events regulating the cell growth[3-8]. The importance of EGFR in growth regulating pathways was confirmed by the fact that enhanced expression of this receptor was found in brain glioblastomas, breast, lung, ovarian, colorectal, and renal carcinomas[9,10]. Elevated EGFR levels correlated with poor prognosis in human tumors[11-17], for this reason, it seemed to be that EGFR would be a logical target for cancer therapy. Previous reports had shown that monoclonal antibodies to EGFR were effective in the treatment of many human carcinoma cells[18-20]. Other drug therapies which targeted the EGFR had also been successful. Kunkel had shown a drug that inhibited EGFR tyrosine kinase activity could inhibit the growth of A431 cells in nude mice[10]. Yoneda also reported that selective inhibitors of EGFR tyrosine kinase activity, such as tyrphostins, could inhibit the growth of squamous carcinoma in nude mice[21]. Antisense oligodeoxynucleotides inhibit gene expression on a highly selective and target sequence in a specific manner[22-25]. Specific oligonucleotides hybridize to complementary mRNA and decrease protein expression[26-29]. Antisense oligonucleotides against proto-oncogenes of growth factors had already been shown to be successful in cell lines[18,30]. For example, an antisense oligonucleotide to the erB2 gene product had been shown to inhibit protein production in a breast cancer cell line[31]. Akino reported inhibition of in vivo growth and metastases in malignant pituitary tumors with an antisense compound to the PTHrp (parathyroid hormone-related peptide)[32]. An oligonucleotide to the c-myc gene inhibited the growth of thyroid carcinoma cell lines[33]. Phosphorothioate antisense oligodeoxynucleotides targeted against human c-raf-1 kinase producing potent antiproliferative effects on cell culture and in vivo antitumor effects against a variety of tumor types[34]. The co-expression of EGFR along with TGF-α in human colon cancer cell lines, also in colon carcinoma tissue, had led to the suggestion that the autocrine stimulation of EGFR by its ligands could be a mechanism for tumor cells to escape from normal growth controls[35]. Previous studies in our laboratory confirmed over-expression of EGFR in HR8348 cells[36]. In this investigation, we hypothesized that growth and proliferation of HR8348 could be inhibited by EGFR ASODN. In this report 15-mer EGFR ASODN was synthesized and the effects of EGFR ASODN on cell proliferation and tumorigenic rate of HR8348 cells were observed.

Tumor Suppressor Genes

Abstract: Tumor suppressor genes (TSGs) are genes whose protein products function in the control of cellular proliferation. In contrast to oncogenes, which are activated in cancer, TSGs are functionally inactivated in cancer. In general, TSGs are characterized by loss-offunction mutations in human tumors. These loss-of-fuunction mutations can be missense mutations that alter critical amino acid residues, mutations that cause premature protein truncation resulting in the loss of functional domains or unstable products, or deletion of the entire gene. Loss-of-function mutations are often accompanied by loss of heterozygosity (LOH) at the TSG locus; LOH can be detected by comparing the pattern of a polymorphic DNA marker in tumor versus normal DNA (see Chapter 1).

Gene expression pattern Notch gene expression during pancreatic organogenesis

Abstract: Notch receptors are involved in regulating the balance between cell differentiation and stem cell proliferation during the development of numerous tissues (Artavanis-Tsakonas, S., Matsuno, K., Fortini, M.E., 1995. Notch signaling. Science 268, 225‐232). Here the expression of all four vertebrate Notch genes, their ligands, and some down-stream targets is analyzed during mouse pancreatic organogenesis. Notch 1 is the first Notch gene expressed in the pancreatic epithelium, and coexpression with HES 1 suggests that the Notch 1 pathway is activated. Notch 2 expression follows later when pancreatic buds branch and is restricted to embryonic ducts, believed to be the source for endocrine and exocrine stem cells. Notch 3 and Notch 4 are expressed in pancreatic mesenchyme and later in endothelial cells. Together these descriptive data comprise a framework for understanding the cellular basis for Notch function during pancreatic development. q 2000 Elsevier Science Ireland Ltd. All rights reserved.

Immunohistochemical analysis of expression of a 65 kDa oncofetal protein (p65), epidermal growth factor receptor (EGFR), oncogene c-erb B2 and tumor suppressor gene p53 protein products in breast cancer patients.

Abstract: Paraffin-embedded tissue slides from 88 infiltrating ductal breast carcinoma were examined by immunohistochemistry technique with the use of monoclonal antibody against human p65 antigen and polyclonal antibody against p65-like protein present in fetal bovine serum. Immunohistochemical analysis of expression of growth factor receptors (EGFR), protein product of oncogene c-erb B2 as well as protein product of mutated anti-oncogene p53 was also done. It was established that there is no correlation between p65 and c-erbB2, EGFR or p53 expression. In low differentiated tumors (grade III) high p53 index and high EGFR and c-erbB2 expression was connected with low p65 expression. The lack of c-erbB2 and EGFR and low p53 expression was combined usually with high p65 oncoprotein levels.

Regulation of ErbB Receptor Tyrosine Kinase Activities in Breast Cancer by the Kek Proteins

Abstract: : We have found that the Drosophila transmembrane molecule kekkon 1 (kek1) acts in a negative feed back loop to modulate the activity of the Epidermal Growth Factor Receptor (EGFR) tyrosine kinase. kek1 is expressed in response to activation of the Gurken/EGFR signaling pathway during oogenesis. While loss of kek1 activity is associated with a phenotype reminiscent of increased Grk/EGFR signaling, ectopic overexpression of kek1 mimics the complete loss of EGFR activity. We found that the extracellular domain of Kek1 physically associates with the EGFR providing the basis for this inhibitory mechanism. Interestingly, we found that Kek1 is also a potent inhibitor of the EGFR in mammalian cells. First, Kek1 binds the EGFR and related proteins ErbB2, ErbB3 and ErbB4. Kek1 interferes with EGF mediated receptor tyrosine phosphorylation and activation of the downstream signaling molecules PI3-kinase and Erk/MAP kinases. Kek1 can also inhibit transformation in mouse mammary tumor cells with deregulated expression of receptors and ligands of the ErbB family.

Antibodies against the tumor suppressor gene ING1

Abstract: The invention provides novel tumor suppressor genes, methods for making and using these and related tumor suppressor genes and proteins and peptides, and nucleic acids encoding these and related tumor suppressor proteins and peptides.