Transfected NIH 3T3 Cells Research Articles

Akt activation by growth factors is a multiple-step process: the role of the PH domain.

The protein kinase encoded by the Akt proto-oncogene is activated by phospholipid binding, membrane translocation and phosphorylation. To address the relative roles of these mechanisms of Akt activation, we have employed a combination of genetic and pharmacological approaches. Transient transfection of NIH3T3 cells with wild-type Akt, pleckstrin homology (PH) domain mutants, generated on the basis of a PH domain structural model, and phosphorylation site Akt mutants provided evidence for a model of Akt activation consisting of three sequential steps: (1) a PH domain-dependent, growth factor-independent step, marked by constitutive phosphorylation of threonine 450 (T450) and perhaps serine 124 (S124), that renders the protein responsive to subsequent activation events; (2) a growth factor-induced, PI3-K-dependent membrane-translocation step; and (3) a PI3-K-dependent step, characterized by phosphorylation at T308 and S473, that occurs in the cell membrane and is required for activation. When forced to translocate to the membrane, wild-type Akt and PH domain Akt mutants that are defective in the first step become constitutively active, suggesting that the purpose of this step is to prepare the protein for membrane translocation. Both growth factor stimulation and forced membrane translocation, however, failed to activate a T308A mutant. This, combined with the finding that T308D/S473D double mutant is constitutively active, suggests that the purpose of the three-step process of Akt activation is the phosphorylation of the protein at T308 and S473. The proposed model provides a framework for a comprehensive understanding of the temporal and spatial requirements for Akt activation by growth factors.

Pp120, a Substrate of the Insulin Receptor Tyrosine Kinase, Is Associated with Phosphatase Activity

pp120, a plasma membrane glycoprotein, is phosphorylated on Tyr488by the insulin receptor tyrosine kinase. This requires basal phosphorylation on Ser503by cAMP-dependent serine kinase. Phe513, the other tyrosine residue in the intracellular domain, does not undergo insulin-stimulated phosphorylation. Phe488mutation abolished basal and insulin-stimulated phosphorylation, whereas, Phe513plus Phe488mutation markedly decreased the effect of insulin on pp120 phosphorylation without altering basal phosphorylation in intact cells. To investigate whether basal phosphorylation of pp120 is regulated by a phosphatase activity that requires Tyr513,in vitrophosphorylation assays using partially purified glycoproteins from stably transfected NIH 3T3 cells were performed in the absence of phosphatase inhibitors. Wild-type pp120 was promptly dephosphorylated, whereas, Y513F pp120 was not. Decreasing pp120 expression by antisense cDNA transfection proportionally decreased phosphatase activity in H4-II-E hepatoma cells measured by thep-nitrophenyl phosphate assay. This suggests that pp120 is associated with phosphatase activity that requires an intact Tyr513residue.

Effect of pp120 on Receptor-mediated Insulin Endocytosis Is Regulated by the Juxtamembrane Domain of the Insulin Receptor

pp120, a substrate of the insulin receptor tyrosine kinase, does not undergo ligand-stimulated phosphorylation by the insulin-like growth factor-1 (IGF-1) receptor. However, replacement of the C-terminal domain of the IGF-1 receptor beta-subunit with the corresponding segment of the insulin receptor restored pp120 phosphorylation by the chimeric receptor. Since pp120 stimulates receptor-mediated insulin endocytosis when it is phosphorylated, we examined whether pp120 regulates IGF-1 receptor endocytosis in transfected NIH 3T3 cells. pp120 failed to alter IGF-1 receptor endocytosis via either wild-type or chimeric IGF-1 receptors. Thus, the effect of pp120 on hormone endocytosis is specific to insulin, and the C-terminal domain of the beta-subunit of the insulin receptor does not regulate the effect of pp120 on insulin endocytosis. Mutation of Tyr960 in the juxtamembrane domain of the insulin receptor abolished the effect of pp120 to stimulate receptor endocytosis, without affecting pp120 phosphorylation by the insulin receptor. These findings suggest that pp120 interacts with two separate domains of the insulin receptor as follows: a C-terminal domain required for pp120 phosphorylation and a juxtamembrane domain required for internalization. We propose that the interaction of pp120 with the juxtamembrane domain is indirect and requires one or more substrates that bind to Tyr960 in the insulin receptor.

Structure and regulation of a polymorphic gene encoding folate receptor type gamma/gamma'.

The human folate receptor (hFR) type gamma and gamma' are constitutively secreted proteins that are expressed primarily in hematopoietic tissues and are potential serum markers for certain hematopoietic malignancies. hFR-gamma' is a variant of hFR-gamma with a two base deletion in its cDNA resulting in a truncated polypeptide. The gene encoding hFR-gamma' was isolated from a placental genomic library. The gene has five exons, four introns and a 5' flanking sequence which contains multiple putative regulatory elements. From RNase protection assay and RACE analysis, the major site of transcriptional initiation was identified at -56 nt. Systematically deleted fragments in the 5' region of the genomic DNA of FR-gamma' were ligated into the PGL3Basic plasmid and the reporter luciferase activity was assayed in cell lysates from transiently transfected NIH3T3 cells. From those results, putative positive and negative regulatory regions in the 5' flanking sequences were noted, and a TATA-less proximal promoter was located between -206 and -22 nt. Gel mobility shift and supershift analyses as well as mutagenesis experiments revealed that Sp1 and ets binding elements in the proximal promoter region confer transcriptional activity. From partial sequencing of genomic DNA, genomic Southern blots, RACE analysis and RNase protection assays, it appears that hFR-gamma shares the gene organization of hFR-gamma'. The results of the analysis of genomic DNA in spleen tissues from several individuals, were consistent with the interpretation that hFR-gamma and hFR-gamma' are encoded by a polymorphic gene.

Macrophage Inflammatory Protein-1β Induces Migration and Activation of Human Thymocytes

The CC chemokine macrophage inflammatory protein 1beta (MIP-1beta), has been shown to be a chemoattractant preferentially activating CD4(+) CD45RA+ T lymphocytes. Further analysis of chemokine action on lymphocytic cells has shown the potent migration-promoting capacity of MIP-1beta on human thymocytes. The responding cells were the CD4(+) and CD8(+) single-positive (SP), as well as the CD4(+) CD8(+) double-positive (DP) populations, with little if any migratory activity on the double-negative (DN) population. The activation of thymocytes by MIP-1beta appeared to be a direct, receptor-mediated event as evidenced by the rapid mobilization of intracellular calcium, increase in proteins phosphorylated on tyrosine, and activation of the mitogen-activated protein kinase (MAPK) pathway. Radioligand binding analyses showed specific and displaceable binding of MIP-1beta to thymocytes with a Kd of approximately 1 nmol/L, a profile that was comparable with MIP-1beta binding to CCR-5-transfected NIH 3T3 cells. In addition, CCR-5 mRNA was detected in total thymocyte populations indicating that activation of thymocytes by MIP-1beta may occur through binding to CCR-5. Further dissection of the subpopulations showed that only the DP and CD8(+) SP populations expressed CCR-5 and expression data on these two populations was confirmed using anti-CCR-5 monoclonal antibody. These data may be suggestive of a role for MIP-1beta in human thymocyte activation, and show a potential route for HIV infectivity in the developing immune system.

Transformation suppressor activity of C3G is independent of its CDC25-homology domain.

The guanine nucleotide releasing protein C3G was initially identified as a Crk SH3-binding protein and recently shown to exhibit exchange activity on Rap1 proteins. Overexpression in NIH3T3 cells of a full-length C3G cDNA isolated from human placenta markedly reduced the focus forming activity of cotransfected, malignantly activated, ras oncogenes (5-7-fold). C3G also had a reverting effect on sis-mediated transformation, decreasing the number of c-sis-induced foci by a factor of 5-10-fold. The observed inhibitory effect of C3G on focus-forming activity of Ras and Sis was always higher than that observed with Rap1A, a known target of C3G. The inhibition of focus formation observed in the presence of C3G was not due to toxic effects on cell viability, since transfected C3G cells exhibited the same survival and growth rates as untransfected NIH3T3 cells or cells transfected with plasmid vector alone. Surprisingly, as opposed to Rap1A, which has no effect on Raf-1 oncogene-mediated transformation, C3G also reduced dramatically (6-8-fold) the number of v-raf-induced foci in transfected NIH3T3 cells. The inhibitory effect on Raf-induced transformation suggests that C3G has other functional targets in addition to Rap1. A C3G mutant (C3G deltaCat) lacking the catalytic domain (CDC25-H) but retaining the rest of the N-terminal sequences, including the Crk-binding domain, exhibited similar ability than full length C3G to inhibit focus formation. In contrast, a C3G mutant (C3G Cat), containing the catalytic domain only but lacking the rest of the N-terminal sequences, did not have any inhibitory effect on transformation mediated by the oncogenes tested. The C3G-derived gene products overexpressed in our transfected cell lines localized to the cytoplasm and did not change the basal MAPK or JNK activity of those cell lines nor their ability to activate the kinases in response to agonists. Our results suggest that the N-terminal region of C3G, and not its catalytic domain, may be responsible for the inhibitory effects observed.

Comparison of the promoters of the mouse (APEX) and human (APE) apurinic endonuclease genes

We investigated the minimal promoter of APEX, which encodes mouse apurinic DNA repair endonuclease. A 1.85-kb fragment with APEX upstream sequences and ∼290 bp of the transcribed region linked to a chloramphenicol acetyltransferase (CAT) reporter gene was assayed by transient transfection in NIH-3T3 cells. The minimal APEX promoter was comprised of ∼190 bp of upstream and ∼170 bp of transcribed DNA (exon 1 and most of intron 1). This ∼360-bp region contains two CCAAT boxes and other consensus protein binding sites, but no TATA box. Deletion of the 5′-most CCAAT box decreased activity ∼5-fold. The second CCAAT box (situated in exon 1) may play an independent role in APEX expression. Transcription start sites have been identified downstream of the second CCAAT box, and DNase I footprinting demonstrated NIH-3T3 nuclear proteins binding this region, including an Sp1 site located between the CCAAT boxes. Electrophoretic mobility-shift assays indicated binding by purified Sp1. Mouse proteins did not bind three myc-like (USF) sites in the APEX promoter, in contrast to the APE promoter. The APEX and APE promoter had similar activity in Hela cells, but in mouse cells, the murine promoter had ∼5-fold higher activity than did the human promoter. Both the APEX and APE promoters exhibited bidirectional activity in their cognate cells.

The differential transforming activities of Tal1 oncoproteins.

TAL1 (Or SCL) oncogene produces both full-length pp42 and truncated pp22 phosphoproteins in T-cell acute lymphoblastic leukemia cells. To investigate the transforming properties of these two oncoproteins, permanent transfected NIH3T3 cell lines were generated that constitutively express either pp42 or pp22. Both cell lines exhibited similar morphology as parental NIH3T3 cells. However, they showed differential anchorage independent growth in soft-agar. The clonogenicity of pp22 expressing cells was ten folds higher than that of pp42 expressing cells. This result might implicate the differential transformation potential of the two Tal1 oncoproteins.

Differential effect of pp120 on insulin endocytosis by two variant insulin receptor isoforms.

The insulin receptor is expressed as two variably spliced isoforms that differ by the absence (isoform A) or presence (isoform B) of a 12-amino acid sequence encoded by exon 11 at the carboxy terminus of the alpha-subunit. Coexpression of the A isoform and pp120, a substrate of the insulin receptor tyrosine kinase, in NIH 3T3 fibroblasts increased receptor A-mediated insulin endocytosis and degradation by two- to threefold compared with cells expressing receptors alone. Because B is the predominant isoform in the liver and binds insulin with lower affinity than A, we have examined the effect of pp120 on receptor B-mediated endocytosis. In contrast to isoform A, the effect of pp120 on isoform B-mediated insulin internalization and degradation in stably transfected NIH 3T3 cells was minimal.

Granulocyte-macrophage colony stimulating factor receptor alpha and beta chain complexes can form both high and intermediate affinity functional receptors.

Scatchard analysis of primary human haemopoietic cells using iodinated GM-CSF suggests that there are low, intermediate and high affinity classes of the GM-CSF receptor. To investigate the molecular basis of this, we generated a clone of transfected NIH3T3 cells that constitutively expressed the human granulocyte-macrophage colony stimulating factor receptor (GM-CSF R) beta chain and inducibly expressed the human GM-CSF R alpha chain. In the cells fully induced to express the alpha chain the overall level of expression of the alpha and beta chains at the cell surface was comparable with that found in primary haemopoietic cells and cell lines. When cells were partially induced to express the alpha chain, the alpha:beta ratio determined by antibody binding was approximately 1:1 and Scatchard analysis revealed a single class of intermediate affinity receptors (Kd = 614+/-88 pM). In cells with fully induced alpha chain expression, the alpha:beta ratio was approximately 3:1 and there was a switch to a dual high and low affinity receptor with K(d)s of 67+/-32 pM and 1.7+/-0.56 nM respectively. The change from intermediate to high affinity was not associated with changes in alphabeta stoichiometry as detected by cross-linking with radiolabelled GM-CSF and gel electrophoresis. Both the high and intermediate affinity receptors were able to activate the STAT 5 and the MAP kinase pathways, although there was a difference in the ligand dose-response curves which was compatible with the different affinities of the receptors. It is proposed that the switch from an intermediate to high affinity receptor was due to the availability of free alpha chains presenting ligand to the alphabeta chain complexes at the surface of the cell membrane.

Nerve growth factor mRNA stability is controlled by a cis-acting instability determinant in the 3'-untranslated region.

Nerve growth factor (NGF) mRNA is rapidly degraded in many non-neuronal cell types with a half-life of between 30 and 60 min. Similar to other short-lived mRNAs the 3'-untranslated region (3'-UTR) of the NGF mRNA contains a short AU nucleotide-rich sequence. To implicate this region as a cis-acting determinant of NGF mRNA instability, expression vectors containing NGF cDNA with and without the 3'-UTR, and vectors containing only the 3'-UTR were constructed and used in cell transfection experiments. Transfection of HEK293 or NIH3T3 cells with these expression vectors followed by measurement of NGF mRNA half-life indicated that NGF mRNA without the AU-rich 3'-UTR was approximately 3-fold more stable than NGF mRNA containing the 3'-UTR. Similar results were seen in a polysome-based cell-free RNA decay assay using NGF mRNA with and without the 3'-UTR prepared from transfected cells. Addition of a short RNA containing the AU-rich 3'-UTR to the cell-free RNA decay system prolonged the half-life of the full-length NGF mRNA, suggesting competition between these two RNA species for polysome-associated factors which degrade the NGF mRNA. Moreover, transfection of HEK293 or astroglial cells with vectors designed to express only the AU-rich region of the 3'-UTR resulted in enhanced expression of NGF mRNA. The results indicate that the 3'-UTR of the NGF mRNA contains a cis-acting instability determinant which, perhaps by interacting with trans-acting RNA-binding proteins, controls the rate of NGF mRNA turnover.

Control of mouse U1 snRNA gene expression during in vitro differentiation of mouse embryonic stem cells.

Early in mouse development, two classes of U1 RNAs, mU1a and mU1b, are synthesized, but as development proceeds, transcription of the embryo-specific mU1b genes is selectively down-regulated to a barely detectable level. We show here that during in vitro differentiation of mouse embryonic stem (ES) cells, both exogenously introduced and endogenous U1b genes are subject to normal developmental regulation. Thus, ES cells represent a convenient isogenic system for studying the control of expression of developmentally regulated snRNA genes. Using this system, we have identified a region in the proximal 5'flanking region, located outside the PSE element, that is responsible for differential transcription of the mU1a and mU1b genes in both developing cells and transiently transfected NIH 3T3 cells.

Marked elevation of hypusine formation activity on eukaryotic initiation factor 5A in v-HA-RAS transformed mouse NIH3T3 cells.

Hypusine formation on the eukaryotic initiation factor 5A (eIF-5A) precursor is ubiquitously present in eukaryotic cells and archebacteria. In this reaction, deoxyhypusine synthase catalyzes the conversion of one unique lysine residue on eIF-5A to deoxyhypusine using spermidine as the substrate. Hydroxylation of the deoxyhypusine residue completes hypusine formation on eIF-5A. Hypusine formation activity can be measured by an in vitro labeling technique in polyamine-depleted cells. In addition, an in vitro cross-labeling assay can be employed to measure simultaneously the relative deoxyhypusine synthase activity and protein substrate amount. Using these approaches, together with Western blot analysis, we showed that hypusine formation activity is serum-responsive and significantly elevated in Ras oncogene transfected NIH3T3 cells as compared to NIH3T3 cells. The large difference, >30-fold, in hypusine formation activity between these two cells is mainly due to difference in the amount of newly synthesized eIF-5A precursor rather than deoxyhypusine synthase. The deoxyhypusine synthase activity is about three-fold higher in Ras-3T3 cells than in 3T3 cells, and remains constant throughout serum stimulation in both cells. Despite the significant difference in eIF-5A protein amounts, the eIF-5A mRNA levels in 3T3 cells and in Ras-3T3 cells are almost identical. Furthermore, unlike serum-dependent increase in eIF-5A precursor protein, the eIF-5A mRNA in both cells is constitutively expressed after serum stimulation, suggesting that eIF-5A gene is regulated at posttranscriptional/translational level during serum stimulation and cell transformation.

Insulin secretion and its modulation by antiarrhythmic and sulfonylurea drugs.

Cardiovascular drugs such as antiarrhythmic agents with Vaughan Williams class Ia action have been found to induce a sporadic hypoglycemia. Recent investigation has revealed that these drugs induce insulin secretion from pancreatic beta-cells by inhibiting ATP-sensitive K+ (KATP) channels in a manner similar to sulfonylurea drugs. The mechanism underlying block of KATP channels by antiarrhythmic drugs was different, however, from that of sulfonylureas: firstly, because binding of radioactive glibenclamide could not be inhibited by unlabelled antiarrhythmic agents, and vice versa; secondly, because the two compounds differ in the kinetics and sidedness of drug action-antiarrhythmic drugs act on the channel from the inner surface of the cell membrane, whereas glibenclamide binds through the intramembrane pathway; finally, it was shown that functional KATP channels in beta-cells are composed of two distinct molecules-a sulfonylurea receptor (SUR) and a channel pore-forming subunit, an inwardly-rectifying K channel with two transmembrane regions (Kir6.2). Antiarrhythmic drugs reversibly inhibit the K+ conductance displayed by the Kir6.1 (a putative KATP channel clone)-transfected NIH3T3 cells. Therefore they appear to interact directly with the pore-forming subunit, thereby inhibiting KATP channel currents and exerting an insulinotrophic effect.

Inducible high-level expression vector for mammalian cells, pEF-LAC carrying human elongation factor 1α promoter and lac operator

We have constructed an inducible high-level expression vector, pEF-LAC. pEF-LAC has a modified human polypeptide chain elongation factor 1α (EF-1α) promoter containing three lactose operator sequences. Using the cat reporter gene, we characterized the transcriptional activity of pEF-LAC. In the transient transfection of NIH3T3 and BaF3 cells, the transcriptional activity of pEF-LAC was higher than that of the original human elongation factor 1α promoter, simian virus 40 (SV40) promoter, and Rous sarcoma virus (RSV) long terminal repeat (LTR). Cotransfection of the lactose repressor expression plasmid effectively suppressed the promoter activity of pEF-LAC, and the activity was fully recovered by addition of isopropyl β- d-thiogalactopyranoside (IPTG). Even in the stable transfection of Rat-1 cells, the promoter activity of the integrated pEF-LAC was much higher than that of the RSV-LTR and regulated in an IPTG-dependent manner. These results suggest that pEF-LAC is a useful vector for the inducible high-level expression of the cloned gene in a variety of mammalian cells.

Conventional PKC-α, Novel PKC-ε and PKC-θ, but Not Atypical PKC-λ Are MARCKS Kinases in Intact NIH 3T3 Fibroblasts

Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in intact cells has been employed as an indicator for activation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological conditions, however, remained to be identified. In our present study using stably transfected NIH 3T3 cell clones we demonstrate that expression of constitutively active mutants of either conventional cPKC-alpha or novel nPKC-epsilon increased phosphorylation of endogenous MARCKS in the absence of phorbol 12,13-dibutyrate in intact mouse fibroblasts, implicating that each of these PKC isoforms itself is sufficient to induce enhanced MARCKS phosphorylation. Similarly, ectopic expression of a constitutively active mutant of PKC-theta significantly increased MARCKS phosphorylation compared to vector controls, identifying PKC-theta as a MARCKS kinase. The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduced MARCKS phosphorylation in intact cells at a similar dose-response as enzymatic activity of recombinant isoenzymes cPKC-alpha, nPKC-epsilon, and nPKC-theta in vitro. Consistently, phorbol 12,13-dibutyrate-dependent MARCKS phosphorylation was significantly reduced in cell lines expressing dominant negative mutants of either PKC-alpha K368R or (dominant negative) PKC-epsilon K436R. The fact, that the constitutively active PKC-lambda A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not involved in this process. We conclude that under physiological conditions, conventional cPKC-alpha and novel nPKC-epsilon, but not atypical aPKC-lambda are responsible for MARCKS phosphorylation in intact NIH 3T3 fibroblasts.

Inhibition of colony formation of NIH 3T3 cells by the expression of the small molecular weight heat shock protein HSP27: involvement of its phosphorylation and aggregation at the C-terminal region.

The ectopic expression of the small molecular weight heat shock protein HSP27 reportedly confers resistance to heat and other types of stress, but our recent findings indicated that it rendered human immortalized fibroblast cells (KMST-6) more sensitive to oxidative stress and caused irreversible growth arrest (Arata et al., 1995, J. Cell. Physiol., 163:458-465). To clarify the relationship between HSP27 and growth regulation, we investigated the effect of overexpression of HSP27 and its mutants on the growth potential of several cell lines. Mammalian expression vectors of the wild-type, hypophosphorylatable, or C-terminal deletion mutants of human HSP27 were constructed from the pRc/CMV plasmid that contained the neomycin-resistant gene. The plasmid was introduced into mouse fibroblasts (NIH 3T3), normal human fibroblasts (TIG-3), Chinese hamster ovary (CHO-K1), or mammary tumor cells (MCF-7), which were then selected in medium containing G418. The number of drug-resistant colonies was significantly decreased by transfection with the expression vector for wild-type HSP27 compared with vector alone, whereas the overexpression of HSP27 in CHO-K1 cells had essentially no effect. The expression vectors of an hypophosphorylatable mutant (pKSm, human HSP27 gene in which codons for Ser-15, -78, and -82 were converted to code for Gly by site-directed mutagenesis) as well as C-terminal deletion mutants in which 12-36 amino acid residues from the C-terminus were deleted had no significant effect on the colony-forming efficiency of NIH 3T3 cells. Cells isolated from G418-resistant colonies formed by transfection of NIH 3T3 cells with the HSP27 expression vector expressed no detectable levels of wild-type HSP27 and did not form stable clonal transformants expressing high levels of HSP27 from NIH 3T3 cells. In contrast, several clones expressing high levels of HSP27 were obtained from CHO-K1 cells transfected with the HSP27 expression vector. In KMST-6 clones expressing high levels of HSP27, the wild-type HSP27 formed aggregates with a mean molecular mass of about 200 kDa as determined by gel filtration, and the size of the oligomers changed with oxidative stress. On the other hand, the size of aggregates of HSP27 encoded by pKSm or C-terminal deletion mutants did not change. These observations indicated that the forced expression of wild-type HSP27 participates in inhibiting the growth of some cell types and that the inhibition may be associated with its phosphorylation and aggregation.

CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus.

Canine distemper virus (CDV), a lymphotropic and neurotropic negative-stranded RNA virus of the Morbillivirus genus, causes a life-threatening disease in several carnivores, including domestic dogs. To identify the cellular receptor(s) involved in the uptake of CDV by susceptible cells, we isolated a monoclonal antibody (MAb K41) which binds to the cell surface and inhibits the CDV infection of several cell lines from various species. Pretreatment of cells with MAb K41 reduces the number of infectious centers and the size of the syncytia. Using affinity chromatography with MAb K41, we purified from HeLa and Vero cell extracts a 26-kDa protein which contained the amino acid sequence TKDEPQRETLK of human CD9, a member of the tetraspan transmembrane or transmembrane 4 superfamily of cell surface proteins. Transfection of NIH 3T3 or MDBK cells with a CD9 expression plasmid rendered these cells permissive for viral infection and raised virus production by a factor of 10 to 100. The mechanism involved is still unclear, since we were unable to detect direct binding of CDV to CD9 by using immunoprecipitation and a virus overlay protein binding assay. These findings indicate that human CD9 and its homologs in other species are necessary factors for the uptake of CDV by target cells, the formation of syncytia, and the production of progeny virus.

Human cytomegalovirus mtrII oncoprotein binds to p53 and down-regulates p53-activated transcription.

The 79-amino-acid (79-aa) open reading frame (UL111a) gene within morphological transforming region II (mtrII) of human cytomegalovirus strain Towne has been shown to transform rodent cells in vitro (J. Thompson, J. Doniger, and L. J. Rosenthal, Arch. Virol. 136:161-172, 1994). Moreover, a translation termination linker (TTL) mutant of mtrII that coded for the first 49 aa of mtrII oncoprotein (designated TTL49) was sufficient for malignant transformation, whereas a TTL mutant that coded for the first 24 aa (designated TTL24) was not. The current study demonstrates the binding of mtrII oncoprotein to the tumor suppressor protein p53 both in vivo using transiently transfected cells and in vitro using labeled proteins. Furthermore, the C-terminally truncated mtrII protein TTL49, but not truncated protein TTL24, bound to p53. The mtrII binding domain mapped to the N-terminal region of p53, residues 1 to 106, with a critical region from aa 27 to 44, whereas the p53 binding domain of mtrII protein was the first 49 aa. Furthermore, mtrII inhibited p53-activated transcription, indicating its ability to alter p53-directed cellular regulatory mechanisms. mtrII oncoprotein was detected both in stably transfected NIH 3T3 cell lines and human cytomegalovirus-infected HEL 299 cells (as early as 12 h after infection) in the perinuclear region and in the nucleus. mtrII-transformed cell lines, at both early and late passage, exhibited high levels of p53 with a 15-fold-extended half-life. However, p53-activated transcription was suppressed in these cells in spite of the increased p53 levels. Finally, the results with wild-type mtrII and its TTL mutants with respect to p53 binding, p53-activated transcription, and transforming ability suggest that the mechanism of mtrII transformation is linked to both p53 binding and disruption of p53 cell regulation.

Bcl-2 protects cells from cytokine-induced nitric-oxide-dependent apoptosis.

Cytokine-mediated cell death in tumor cells can be achieved through endogenous nitric oxide (NO) from within tumor cells or exogenous NO from either activated macrophages or endothelial cells. The purpose of this study was to determine the role of Bcl-2 in NO-mediated apoptosis. The incubation of murine L929 and NIH3T3 cells with interleukin-1 alpha (IL-1 alpha) and interferon gamma (IFN gamma) induced high endogenous NO production only in the L929 cells that also underwent apoptosis. NIH3T3 cells were not resistant to NO-mediated apoptosis. In fact, the incubation of L929 and NIH3T3 cells with exogenous NO derived from NO donors, sodium nitroprusside, or S-nitroso-N-acetyl-DL-penicillamine (SNAP) induced death, characterized by typical apoptotic morphology and DNA fragmentation, in both cell types, but to a higher degree in NIH3T3 cells than in the L929 cells. We then measured the effect of Bcl-2 expression on exogenous NO-induced apoptosis. At both the mRNA and protein levels, L929 fibroblasts expressed higher levels of endogenous mouse Bcl-2 than did NIH3T3 cells. At the same time, L929 cells were much more resistant to exogenous NO-induced cell death than were NIH3T3 cells. The inverse correlation between mouse Bcl-2 expression and sensitivity to exogenous NO-mediated cell death was also found in the murine K-1735 melanoma C-23 and X-21 clonal populations. Transfection of both NIH3T3 cells and L929 cells with the human bcl-2 gene led to resistance to both exogenous and endogenous NO-mediated apoptosis. These data demonstrate that NO-mediated apoptosis can be suppressed by expression of Bcl-2, suggesting that abnormal expression of Bcl-2 may influence the efficacy of tumor immunotherapy.