Characterization of the 5' untranslated region of the human c-fgr gene and identification of the major myelomonocytic c-fgr promoter.

In this study, we have characterized the 5' region of the human c-fgr proto-oncogene and identified the major myelomonocytic c-fgr promoter. Seven distinct 5' untranslated exons were identified and localized to a region extending 13 kb upstream from the first coding exon. Two major promoters were identified, one utilized exclusively in Epstein-Barr virus (EBV)-infected B-lymphocyte cell lines, and the other functional only in myelomonocytic cells. Differential promoter utilization and alternative splicing of the 5' untranslated exons give rise to at least six distinct c-fgr mRNA species that differ only in their 5' untranslated regions. Two major mRNAs were identified, c-fgr A and c-fgr 4; these two mRNAs were detected exclusively in EBV-infected B-lymphocyte cell lines and myelomonocytic cells respectively. We have previously demonstrated that c-fgr is transcriptionally activated in U937 cells treated with either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or cycloheximide (CHX). We now show that a DNA fragment extending from -772 to +97 (with respect to the transcription initiation site upstream from exon M4) is responsive to TPA but not CHX treatment in U937 cells. These results suggest that TPA and CHX induce c-fgr mRNA accumulation by different mechanisms.

Characterization of Brx, a novel Dbl family member that modulates estrogen receptor action.

Regulation of gene activation by the estrogen receptor (ER) is complex and involves co-regulatory proteins, oncoproteins (such as Fos and Jun), and phosphorylation signaling pathways. Here we report the cloning and initial characterization of a novel protein, Brx, that contains a region of identity to the oncogenic Rho-guanine nucleotide exchange (Rho-GEF) protein Lbc, and a unique region capable of binding to nuclear hormone receptors, including the ER. Western and immunohistochemistry studies showed Brx to be expressed in estrogen-responsive reproductive tissues, including breast ductal epithelium. Brx bound specifically to the ER via an interaction that required distinct regions of ER and Brx. Furthermore, overexpression of Brx in transfection experiments using an estrogen-responsive reporter revealed that Brx augmented gene activation by the ER in an element-specific and ligand-dependent manner. Moreover, activation of ER by Brx could be specifically inhibited by a dominant-negative mutant of Cdc42Hs, but not by dominant negative mutants of RhoA or Rac1. Taken together, these data suggest that Brx represents a novel modular protein that may integrate cytoplasmic signaling pathways involving Rho family GTPases and nuclear hormone receptors.

Scientific progress in understanding oral and pharyngeal cancers.

Oral and pharyngeal cancers result from a complex interaction between genetic susceptibility and behavioral factors. Improved understanding of the underlying genetic events has led to insights about how oral and pharyngeal cancers develop and suggests promising new treatments. Tobacco and alcohol consumption are associated with most oral and pharyngeal cancers. Dental professionals' efforts to modify their patients' tobacco and alcohol use and to detect oral lesions at an early stage, together with scientific advances, will help reduce the impact of these cancers.

Reducing the burden of oral and pharyngeal cancers.

In the United States, oral and pharyngeal cancers continue to result in significant morbidity and mortality. Dental professionals play a pivotal role in all facets of controlling the burden of oral and pharyngeal cancer-from efforts to prevent its occurrence, to ensuring that oral cancers are detected at the earliest possible stage, to treating these cancers, and to ensuring maximum quality of life and function for oral and pharyngeal cancer survivors. Individually and by making linkages within the community and beyond, dentists can help patients modify their risk of these cancers and can take steps to screen for them, thereby potentially improving survival and function of those who develop oral cancer. Creative partnerships between community dentists and academic and other research centers will help move knowledge of the biological processes involved in carcinogenesis and innovations in treatment into clinical practice. Partnerships between dental and medical professionals may also help efforts to reduce the morbidity related to oral and pharyngeal cancers. Local, state and national multidisciplinary initiatives are emerging that focus more broadly on risk factor control or oral and pharyngeal cancer issues. These many forms of cooperative approaches offer excellent opportunities to make a significant impact on reducing the incidence of and in treating these debilitating and disfiguring malignancies.

Dual transduction signaling by a Xenopus muscarinic receptor: adenylyl cyclase inhibition and MAP kinase activation.

Using transient transfection of COS-7 and human embryonic kidney 293 cells, we studied the functional properties of a previously cloned muscarinic Xenopus receptor [Herrera et al. (1994): FEBS Lett 352:175-179] and its coupling to adenylyl cyclase (AC) and mitogen-activated protein kinase (MAPK) pathways. Expression of the Xenopus muscarinic receptor results in the inhibition of AC activity and activation of the MAPK pathway through a mechanism that involves a Pertussis-toxin-sensitive G-protein and the G beta gamma subunits. The signal transduction properties of this receptor are similar to the mammalian m2 and m4 muscarinic receptors. These results strongly support the idea that inhibition of AC and MAPK activation, signaled out from the muscarinic oocyte receptor, are involved in the oocyte maturation process.

Characterization of AT2 receptor expression in NIH 3T3 fibroblasts.

1. A high expression of angiotensin II receptors and of angiotensin-converting enzyme (ACE) activity was detected in confluent NIH 3T3 fibroblasts. 2. Characterization with selective ligands, dithiothreitol, and GTP gamma S, indicated that only the AT2 subtype was expressed. 3. AT2 receptors and ACE expression were strictly dependent on the cell density and growth phase of the cells, with AT2 receptors being expressed earlier than ACE. In contrast, high expression of AT2 receptors irrespective of their growth state was observed in NIH 3T3 cells lacking contact inhibition upon neoplastic transformation with ras. 4. Our results imply a possible relation of AT2 receptors to cell growth and cell-cell contact.

Insulin-like growth factor-I inhibits the stress-activated protein kinase/c-Jun N-terminal kinase.

The pathways involved in the cellular responses to the insulin-like growth factors (IGFs) are numerous and vary according to cell type. Following activation of the IGF-I receptor, the mitogen-activated protein kinase and phosphatidylinositide 3'-kinase (PI3'K) pathways are activated and result in cellular proliferation and inhibition of apoptosis. In this study, we analyzed the IGF-I effect on the stress-activated protein kinase/c-Jun N-terminal kinase (JNK) activity using human embryonic kidney 293 cells, 293 cells transiently expressing hemagglutinin-JNK, and 293 cells stably expressing a hemagglutinin-JNK transgene. In all cell types, endogenous or transfected JNK activity was strongly stimulated by anisomycin or tumor necrosis factor-alpha, and 10 nM IGF-I pretreatment suppressed the induced JNK activity. To determine whether the effect of IGF-I on JNK activity involves the mitogen-activated protein kinase or PI3'K pathway, we used the specific MEK1 inhibitor PD098059 and the PI3'K inhibitor LY 294002. PD098059 did not alter the IGF-I suppressive effect on stressor-induced JNK activity, but LY 294002 suppressed the IGF-I effect. Moreover, in transiently transfected parental 293 cells expressing dominant-negative Akt, anisomycin-increased JNK activity was not suppressed by pretreatment with IGF-I. Our results demonstrate that the action of IGF-I on JNK in these cells is via PI3'K and Akt.

Carboxyl-terminal domain of p27Kip1 activates CDC2.

A variant form of p27 was unexpectedly detected in a synchronized culture of NIH3T3 cells treated with serum. The expression levels of this form of p27 which lacked its amino (NH2)-terminal region reached maximum during G2/M phase. Since the appearance of the NH2-terminal truncated form of p27 coincided with increased expression of Cdc2, we hypothesized that p27 may play a role in regulating Cdc2 catalytic activity. To test this hypothesis, wild type p27, as well as the amino-terminal (Np27) and carboxyl-terminal (Cp27), were individually expressed, purified, and examined for their ability to regulate CDC2 kinase activity in vitro. Our data showed that both p27 and Np27 inhibited CDC2 kinase activity. However, in marked contrast, Cp27 enhanced the CDC2 kinase activity. In vitro kinase assays showed that Cp27 and p27 were phosphorylated by CDC2, whereas Np27 was not. In addition, we demonstrated that deletion of the putative CDC2 phosphorylation site in the carboxyl-terminal domain of Cp27 diminished activation of CDC2 kinase activity otherwise stimulated by Cp27. A similar deletion did not have any effect on the inhibitory function of p27. Together these results suggest that the carboxyl-terminal domain of p27 may activate CDC2 kinase activity in vivo during G2/M and that this effect may be regulated by serine/threonine phosphorylation.

Effect of activating and inactivating mutations of Gs- and Gi2-alpha protein subunits on growth and differentiation of 3T3-L1 preadipocytes.

Previous investigations have demonstrated that both Gs- and the Gi-family of GTP-binding proteins are implicated in differentiation of the 3T3-L1 preadipocyte. In order to further analyze the role of Gs alpha vs. Gi2 alpha, which are both involved in adenylate cyclase modulation, we transfected undifferentiated 3T3-L1 cells with two sets of G-protein cDNA: the pZEM vector with either wild type, the activating (i.e., GTP-ase inhibiting) R201C-Gs alpha or the inactivating G226A(H21a)-Gs alpha point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the Gi2 alpha wild type or the missense mutations R179E-Gi2 alpha, Q205L-Gi2 alpha, and G204A(H21a)-Gi2 alpha. The activating [R201C]Gs alpha-mutant did not significantly affect the differentiation process, i.e., increase in the steady-state levels of G-protein subunits, gross appearance, or insulin-elicited deoxy-glucose uptake into 3T3-L1 adipocytes, despite a marked initial increase in hormone-elicited adenylate cyclase activity. The [H21a]Gs alpha-mutant, on the other hand, enhanced the degree of differentiation slightly, as evidenced by an augmented production of lipid vesicles and insulin-stimulated deoxy-glucose uptake. However, an expected increase in mRNA for hormone-sensitive lipase was not seen. Secondly, it appeared that both activating [R179E]Gi2 alpha or [Q205L]Gi2 alpha mutants reduced cell doubling time in non-confluent 3T3-L1 cell cultures, while [H21a]Gi2 alpha slowed proliferation rate. Furthermore, it seemed that cell proliferation, as evidenced by thymidine incorporation, ceased at a much earlier stage prior to cell confluency when cultures were transfected with the [R179E]Gi2 alpha or [Q205L]Gi2 alpha mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]Gi2 alpha and [Q205L]Gi2 alpha mutants consistently enhanced adenylate cyclase activation and cAMP accumulation stimulated by isoproterenol and corticotropin over controls. Deoxy-glucose uptake was also super-activated by the [R179E]Gi2 alpha and [Q205L]Gi2 alpha mutants. Finally, steady-state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]Gi2 alpha and [Q205L]Gi2 alpha over differentiated controls. The inactivating [H21a]Gi2 alpha-mutant obliterated all signs of preadipocyte differentiation. It is concluded that Gi2 plays a positive and much more important role than Gs in 3T3-L1 preadipocyte differentiation. Cyclic AMP appears to play no role in this process.

Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate.

Ceramide is an important regulatory participant of programmed cell death (apoptosis) induced by tumour-necrosis factor (TNF)-alpha and Fas ligand, members of the TNF superfamily. Conversely, sphingosine and sphingosine-1-phosphate, which are metabolites of ceramide, induce mitogenesis and have been implicated as second messengers in cellular proliferation induced by platelet-derived growth factor and serum. Here we report that sphingosine-1-phosphate prevents the appearance of the key features of apoptosis, namely intranucleosomal DNA fragmentation and morphological changes, which result from increased concentrations of ceramide. Furthermore, inhibition of ceramide-mediated apoptosis by activation of protein kinase C results from stimulation of sphingosine kinase and the concomitant increase in intracellular sphingosine-1-phosphate. Finally sphingosine-1-phosphate not only stimulates the extracellular signal-regulated kinase (ERK) pathway, it counteracts the ceramide-induced activation of stress-activated protein kinase (SAPK/JNK). Thus, the balance between the intracellular levels of ceramide and sphingosine-1-phosphate and their regulatory effects on different family members of mitogen-activated protein kinases determines the fate of the cell.

Interactions of protein kinase CK2 subunits.

Several approaches have been used to study the interactions of the subunits of protein kinase CK2. The inactive mutant of CK2alpha that has Asp 156 mutated to Ala (CK2alphaA156) is able to bind the CK2beta subunit and to compete effectively in this binding with wild-type subunits alpha and alpha'. The interaction between CK2alphaA156 and CK2beta was also demonstrated by transfection of epitope-tagged cDNA constructs into COS-7 cells. Immunoprecipitation of epitope-tagged CK2alphaA156 coprecipitated the beta subunit and vice-versa. The assay of the CK2 activity of the extracts obtained from cells transiently transfected with these different subunits yielded some surprising results: The CK2 specific phosphorylating activity of these cells transfected with the inactive CK2alphaA156 was considerably higher than the control cells transfected with vectors alone. Assays of the immunoprecipitated CK2alphaA156 expressed in these cells, however, demonstrated that the mutant was indeed inactive. It can be concluded that transfection of the inactive CK2alphaA156 affects the endogenous activity of CK2. Transfection experiments with CK2alpha and beta subunits and CK2alphaA156 were also used to confirm the interaction of CK2 with the general CDK inhibitor p21WAF1/CIP1 co-transfected into these cells. Finally a search in the SwissProt databank for proteins with properties similar to those derived from the amino acid composition of CK2beta indicated that CK2beta is related to protein phosphatase 2A and to other phosphatases as well as to a subunit of some ion-transport ATPases.

Regulation of c-Myc and Max in megakaryocytic and monocytic-macrophagic differentiation of K562 cells induced by protein kinase C modifiers: c-Myc is down-regulated but does not inhibit differentiation.

We have studied the regulation and role of c-Myc and Max in the differentiation pathways induced in K562 cells by 12-O-tetradecanoyl phorbol-13 acetate (TPA) and staurosporine, an activator and inhibitor, respectively, of protein kinase C (PKC). We found that staurosporine induced megakaryocytic differentiation, as revealed by the cellular ultrastructure, platelet formation, and DNA endoreduplication. In contrast, TPA induced a differentiated phenotype that more closely resembled that of the monocyte-macrophage lineage. c-myc expression was down-regulated in K562 differentiated by both TPA and staurosporine, whereas max expression did not change in either case. Although PKC enzymatic activity was low in cells terminally differentiated with TPA and staurosporine, inhibition of PKC activity by itself did not induce c-myc down-regulation. We conclude that the c-myc gene is switched off as a consequence of the differentiation process triggered by these drugs in a manner independent from PKC. Ectopic overexpression of c-Myc in K562 cells did not affect the monocytic-macrophagic and megakaryocytic differentiation, indicating that c-Myc suppression is not required for these processes in K562. Similarly, both differentiation pathways were not affected by Max overexpression or by concomitant overexpression of c-Myc and Max. This result is in contrast with the inhibition of erythroid differentiation of K562 exerted by c-Myc, suggesting divergent roles for c-Myc/Max, depending on the differentiation pathway.

EPS8 and E3B1 transduce signals from Ras to Rac.

The small guanine nucleotide (GTP)-binding protein Rac regulates mitogen-induced cytoskeletal changes and c-Jun amino-terminal kinase (JNK), and its activity is required for Ras-mediated cell transformation. Epistatic analysis placed Rac as a key downstream target in Ras signalling; however, the biochemical mechanism regulating the cross-talk among these small GTP-binding proteins remains to be elucidated. Eps8 (relative molecular mass 97,000) is a substrate of receptors with tyrosine kinase activity which binds, through its SH3 domain, to a protein designated E3b1/Abi-1. Here we show that Eps8 and E3b1/Abi-1 participate in the transduction of signals from Ras to Rac, by regulating Rac-specific guanine nucleotide exchange factor (GEF) activities. We also show that Eps8, E3b1 and Sos-1 form a tri-complex in vivo that exhibits Rac-specific GEF activity in vitro. We propose a model in which Eps8 mediates the transfer of signals between Ras and Rac, by forming a complex with E3b1 and Sos-1.