High-level expression of c-H-ras1 fails to fully transform rat-1 cells.

Rat-1 cells were transfected with plasmids encoding normal (Gly-12), nonactivated (Pro-12), and activated (Val-12 and Ile-12) p21H-ras in the presence of an amplifiable dihydrofolate reductase marker. The introduced DNA was amplified by selection in methotrexate to establish the relationship between p21H-ras expression and various hallmarks of cellular transformation. The maximum level of p21H-ras (Gly-12) consistent with cell viability was approximately 0.13% of total cell protein (approximately 60,000 molecules per cell); this is 44-fold greater than the level of the endogenous protein. The maximum tolerated level of a second nontransforming form of p21H-ras (pro-12) was about half of this. Amplification in Rat-1 cells of H-ras genes encoding the highly oncogenic Val-12 and Ile-12 forms of p21H-ras could not be achieved by methotrexate selection, providing strong evidence that synthesis of activated p21H-ras above a certain threshold (about 0.02% of total protein) in Rat-1 cells is incompatible with cell viability. Individual cell lines were isolated and their morphology, anchorage-independent growth, tumorigenicity, and response to and production of growth factors were studied. We report that cell lines expressing near-maximum tolerated levels of either the normal or pro-12 form of p21H-ras were not as transformed as cells expressing much more modest levels of the highly oncogenic (Val-12) form, suggesting that the complete elaboration of the transformed phenotype by ras depends, at least in part, on mutations that distinguish the cellular and viral proteins. We found that cells expressing elevated levels of the normal p21(H-ras) could be fully transformed by the activated (Val-12) form and that such cells continued to overexpress p21(H-ras) (Gly-12), arguing against a role for normal ras genes in suppression of the oncogenic potential of their mutationally activated counterparts.     

Recombinant herpes simplex virus type 1 (HSV-1) codelivering interleukin-12p35 as a molecular adjuvant enhances the protective immune response against ocular HSV-1 challenge

An important aspect of ocular herpes simplex virus type 1 (HSV-1) vaccine development is identification of an appropriate adjuvant capable of significantly reducing both virus replication in the eye and explant reactivation in trigeminal ganglia. We showed recently that a recombinant HSV-1 vaccine expressing interleukin-4 (IL-4) is more efficacious against ocular HSV-1 challenge than recombinant viruses expressing IL-2 or gamma interferon (IFN-gamma) (Y. Osorio and H. Ghiasi, J. Virol. 77:5774-5783, 2003). We have now constructed and compared recombinant HSV-1 viruses expressing IL-12p35 or IL-12p40 molecule with IL-4-expressing HSV-1 recombinant virus. BALB/c mice were immunized intraperitoneally with IL-12p35-, IL-12p40-, IL-12p35+IL-12p40-, or IL-4-expressing recombinant HSV-1 viruses. Controls included mice immunized with parental virus and mice immunized with the avirulent strain KOS. The efficacy of each vaccine in protecting against ocular challenge with HSV-1 was assessed in terms of survival, eye disease, virus replication in the eye, and explant reactivation. Neutralizing antibody titers, T-cell responses, and expression of 32 cytokines and chemokines were also evaluated. Mice immunized with recombinant HSV-1 expressing IL-12p35 exhibited the lowest virus replication in the eye, the most rapid virus clearance, and the lowest level of explant reactivation. The higher efficacy against ocular virus replication and explant reactivation correlated with higher neutralizing antibody titers, cytotoxic-T-lymphocyte activities, and IFN-gamma expression in recombinant HSV-1 expressing IL-12p35 compared to other vaccines. Mice immunized with both IL-12p35 and IL-12p40 had lower neutralizing antibody responses than mice immunized with IL-12p35 alone. Our results confirm that recombinant virus vaccines expressing cytokine genes can enhance the overall protection against infection, with the IL-12p35 vaccine being the most efficacious of those tested. Collectively, the results support the potential use of IL-12p35 as a vaccine adjuvant, without the toxicity-associated concerns of IL-12.     

p12(DOC-1) is a novel cyclin-dependent kinase 2-associated protein

Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12(DOC-1) is a growth suppressor isolated from normal keratinocytes. We report that p12(DOC-1) associates with CDK2. More specifically, p12(DOC-1) associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12(DOC-1) resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12(DOC-1) transfectants ( upward arrow G(1) and downward arrow S). The p12(DOC-1)-mediated decrease of CDK2 was prevented if the p12(DOC-1) transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin beta-lactone, suggesting that p12(DOC-1) may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12(DOC-1)-mediated, CDK2-associated cell cycle phenotypes. These data support p12(DOC-1) as a specific CDK2-associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2.     

Mutation of Cys105 inhibits dimerization of p12CDK2-AP1 and its growth suppressor effect

p12(CDK2-AP1) (p12) is a CDK2-associated protein that negatively regulates its kinase activity. Growth arrest of normal diploid cells by contact inhibition resulted in an induction of p27(kip1) and reduction of CDK2 levels. Interestingly, we observed concomitantly in growth-arrested cells, there was a reduction of nuclear p12 and the appearance of a nuclear 25-kDa molecule (p25) recognized by anti-p12 polyclonal antibody. Biochemical analysis showed that bacterial His-tagged p12 could be converted into a dimeric p25 in a reducing agent-dependent manner, and mutating the only cysteine residue of p12 (Cys(105) --> Ala(105)) abolished the dimerization. Transient transfection of wild type p12 into U2OS cells showed a reducing agent-sensitive dimerization that was also abolished by the C105A mutation. Furthermore, reduction of p12 expression by a short interfering RNA resulted in a parallel reduction of p25. These data supports the possibility that p25 is a homodimeric form of p12 through the cysteine residue. More interestingly, transient transfection of p12 (C105A) into the normal diploid lung fibroblast CCD18LU cells resulted in a reduction of the growth-inhibitory effect of p12 and abolished the inhibitory effect of p12 on CDK2 kinase activity. In addition, we found that the C105A mutation did not alter nuclear localization of p12, but it prevented association with CDK2. Taken together, our data suggest that p12 forms a nuclear homodimers in contact inhibited normal diploid cells and dimerization of p12 is a necessary process for the growth inhibition effect by p12.     

Posttranslational modification and subcellular localization of the p12 capsid protein of herpes simplex virus type 1.

We have previously shown that the 12-kDa capsid protein (p12) of herpes simplex virus type 1 (HSV-1) is a gamma 2 (true late) gene product encoded by the UL35 open reading frame (D. S. McNabb and R. J. Courtney, J. Virol. 66:2653-2663, 1992). To extend the characterization of p12, we have investigated the posttranslational modifications and intracellular localization of the 12-kDa polypeptide. These studies have demonstrated that p12 is modified by phosphorylation at serine and threonine residues. In addition, analysis of p12 by acid-urea gel electrophoresis has indicated that the protein can be resolved into three components, designated p12a, p12b, and p12c. Using isotopic-labeling and alkaline phosphatase digestion experiments, we have determined that p12a and p12b are phosphorylated forms of the protein, and p12c is likely to represent the unphosphorylated polypeptide. The kinetics of phosphorylation was examined by pulse-chase radiolabeling, and these studies indicated that p12c can be completely converted into p12a and p12b following a 4-h chase. All three species of p12 were found to be associated with purified HSV-1 virions; however, p12b and p12c represented the most abundant forms of the protein within viral particles. We have also examined the intracellular localization of p12 by cell fractionation and indirect immunofluorescence techniques. These results indicated that p12 is predominantly localized in the nucleus of HSV-1-infected cells and appears to be restricted to specific regions within the nucleus.     

p12(DOC-1), a growth suppressor, associates with DNA polymerase alpha/primase.

p12(DOC-1) is a growth suppressor identified and isolated from normal keratinocytes. Ectopic expression of p12(DOC-1) in squamous carcinoma cells led to the reversion of in vitro transformation phenotypes including anchorage independence, doubling time, and morphology. Here we report that p12(DOC-1) associates with DNA polymerase alpha/primase (pol-alpha:primase) in vitro and in cells. The pol-alpha:primase binding domain in p12(DOC-1) is mapped to the amino-terminal six amino acid (MSYKPN). The biological effect of p12(DOC-1) on pol-alpha:primase was examined using in vitro DNA replication assays. Using the SV40 DNA replication assay, p12(DOC-1) suppresses DNA replication, leveling at approximately 50%. Similar results were obtained using the M13 single-stranded DNA synthesis assay. Analysis of the DNA replication products revealed that p12(DOC-1) affects the initiation step, not the elongation phase. The p12(DOC-1) suppression of DNA replication is likely to be mediated either by a direct inhibitory effect on pol-alpha:primase or by its effect on cyclin-dependent kinase 2 (CDK2), a recently identified p12(DOC-1)-associated protein known to stimulate DNA replication by phosphorylating pol-alpha:primase. p12(DOC-1) suppresses CDK2-mediated phosphorylation of pol-alpha:primase. These data support a role of p12(DOC-1) as a regulator of DNA replication by direct inhibition of pol-alpha:primase or by negatively regulating the CDK2-mediated phosphorylation of pol-alpha:primase.     

Assembly factors of F1FO-ATP synthase across genomes

Work with respiration-deficient strains of Saccharomyces cerevisiae has provided evidence that assembly of the mitochondrial ATP synthase is dependent on proteins that serve substrate-specific, chaperone-type functions: Atp10p, Atp11p, Atp12p, Atp22p, and Fmc1p. Atp11p and Atp12p mediate the formation of the F1 moiety via interaction with subunits F1-beta and F1-alpha, respectively. The role of Fmc1p is less clear. Atp10p and Atp22p are essential for the formation of the F(O) part, during which Atp10p assists in the incorporation of the F(O)-a subunit. Here we present a comprehensive analysis of ATP synthase assembly factors from all available genomes. The mechanism of the F1 assembly is preserved in all eukaryotic lineages that are capable of ATP synthesis via oxidative phosphorylation and requires Atp11p and Atp12p. Conversely, composition of the F(O) part as well as its assembly is more versatile. We found two distinct subtypes of the F(O)-a subunit, one of which seems to be dependent on the action of Atp10p while the other does not. Restricted occurrence of Fmc1p and Atp22p suggests the existence of lineage-specific assembly factors. Our phylogenetic data served as a source for comparative sequence analysis, which identified evolutionarily conserved residues, putative functional domains and their basic structural features for Atp10p, Atp11p, and Atp12p orthologs. These results provide the basis for detailed molecular analysis of the ATP synthase-specific chaperones.     

Identification of a nuclear gene (FMC1) required for the assembly/stability of yeast mitochondrial F(1)-ATPase in heat stress conditions

We have identified a yeast nuclear gene (FMC1) that is required at elevated temperatures (37 degrees C) for the formation/stability of the F(1) sector of the mitochondrial ATP synthase. Western blot analysis showed that Fmc1p is a soluble protein located in the mitochondrial matrix. At elevated temperatures in yeast cells lacking Fmc1p, the alpha-F(1) and beta-F(1) proteins are synthesized, transported, and processed to their mature size. However, instead of being incorporated into a functional F(1) oligomer, they form large aggregates in the mitochondrial matrix. Identical perturbations were reported previously for yeast cells lacking either Atp12p or Atp11p, two specific assembly factors of the F(1) sector (Ackerman, S. H., and Tzagoloff, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4986--4990), and we show that the absence of Fmc1p can be efficiently compensated for by increasing the expression of Atp12p. However, unlike Atp12p and Atp11p, Fmc1p is not required in normal growth conditions (28--30 degrees C). We propose that Fmc1p is required for the proper folding/stability or functioning of Atp12p in heat stress conditions.     

Palmitoylation and p8-Mediated Human T-Cell Leukemia Virus Type 1 Transmission

The orf-I gene of human T-cell leukemia type 1 (HTLV-1) encodes p8 and p12 and has a conserved cysteine at position 39. p8 and p12 form disulfide-linked dimers, and only the monomeric forms of p8 and p12 are palmitoylated. Mutation of cysteine 39 to alanine (C39A) abrogated dimerization and palmitoylation of both proteins. However, the ability of p8 to localize to the cell surface and to increase cell adhesion and viral transmission was not affected by the C39A mutation.     

Apoptosis signal-regulating kinase 1 (ASK1) induces neuronal differentiation and survival of PC12 cells

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase that activates the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling cascades. We report here that expression of constitutively active ASK1 (ASK1DeltaN) induces neurite outgrowth in the rat pheochromocytoma cell line PC12. We found that p38 and to a lesser extent JNK, but not ERK, were activated by the expression of ASK1DeltaN in PC12 cells. ASK1DeltaN-induced neurite outgrowth was strongly inhibited by treatment with the p38 inhibitor SB203580 but not with the MEK inhibitors, suggesting that activation of p38, rather than of ERK, is required for the neurite-inducing activity of ASK1 in PC12 cells. We also observed that ASK1DeltaN induced expression of several neuron-specific proteins and phosphorylation of neurofilament proteins, confirming that PC12 cells differentiated into mature neuronal cells by ASK1. Moreover, ASK1DeltaN-expressing PC12 cells survived in serum-starved condition. ASK1 thus appears to mediate signals leading to both differentiation and survival of PC12 cells. Together with previous reports indicating that ASK1 functions as a pro-apoptotic signaling intermediate, these results suggest that ASK1 has a broad range of biological activities depending on cell types and/or cellular context.     

The regulation of Th1 responses by the p38 MAPK

IL-12 is a dimeric cytokine that is produced primarily by APCs. In this study we examined the role that the p38 MAPKs (MAPK/p38) play in regulating IL-12 production. We show that inhibition of p38 dramatically increased IL-12 production upon stimulation, while decreasing TNF-α. This reciprocal effect on these two cytokines following MAPK/p38 inhibition occurred in many different APCs, following a variety of different stimuli. IL-12 production was also increased in macrophages treated with small interfering RNA to limit p38α expression, and in macrophages deficient in MKK3, a kinase upstream of p38. The increase in IL-12 production following MAPK/p38 inhibition appears to be due to enhanced IL-12 (p40) mRNA stability. We show that MAPK/p38 inhibition can promote Th1 immune responses and thereby enhance vaccine efficacy against leishmaniasis. In a mouse model of Leishmania major infection, vaccination with heat-killed L. major plus CpG and SB203580 elicited complete protection against infection compared with heat-killed L. major plus CpG without SB203580. Thus, this work suggests that MAPK/p38 inhibitors may be applied as adjuvants to bias immune responses and improve vaccinations against intracellular pathogens.     

Endoplasmic Reticulum and cis-Golgi Localization of Human T-Lymphotropic Virus Type 1 p12I: Association with Calreticulin and Calnexin

Human T-lymphotropic virus type 1 (HTLV-1) is a complex retrovirus encoding regulatory and accessory genes in four open reading frames (ORF I to IV) of the pX region. We have demonstrated an important role of pX ORF I expression, which encodes p12(I), in establishment of HTLV-1 infection in a rabbit model and for optimal viral infectivity in quiescent primary lymphocytes. These data indicated that p12(I) may enhance lymphocyte activation and thereby promote virus infection. To further define the role of p12(I) in cell activation, we characterized the subcellular localization of p12(I) in transfected 293T cells and HeLa-Tat cells by multiple methods, including immunofluorescence confocal microscopy, electron microscopy, and subcellular fractionation. Herein, we demonstrate that p12(I) accumulates in the endoplasmic reticulum (ER) and cis-Golgi apparatus. The location of p12(I) was unchanged following treatments with both cycloheximide (blocking de novo protein synthesis) and brefeldin A (disrupting ER-to-Golgi protein transport), indicating that the protein is retained in the ER and cis-Golgi. Moreover, using coimmunoprecipitation assays, we identify the direct binding of p12(I) with both calreticulin and calnexin, resident ER proteins which regulate calcium storage. Our results indicate that p12(I) directly binds key regulatory proteins involved in calcium-mediated cell signaling and suggest a role of p12(I) in the establishment of HTLV-1 infection by activation of host cells.     

The p21(WAF1) cyclin-kinase inhibitor: in vivo interaction with E1A adenoviral Ad2 and Ad12 oncoproteins

The capability of adenoviral oncoproteins E1A Ad2 and Ad12 to form complexes in vivo with cyclin-kinase inhibitor p21Waf1 has been analysed. The published data confirming direct interaction between E1A and p21Waf1 are insufficient. In the present work, a yeast two-hybrid SRS system was used to investigate the binding of different fragments of E1A Ad2 and Ad12 polypeptides with p21Waf1. We have shown that the full length product of 12S mRNA E1A Ad2 interacts weekly with p21Waf1, whereas the protein corresponding to 13S mRNA E1A Ad12 does not bind to cyclin-kinase inhibitor protein. Moreover, fragments 1-80 (Ad2), 1-29 (Ad12), 1-79 (Ad12), and 105-194 (Ad12) were able to interact with p21Waf1 to some extent. The difference between interacting regions of adenoviral proteins E1A Ad2/5 and Ad12 gives a new information about the mechanism of p21Waf1 functional inactivation and different transforming activity of Ad2/5 and Ad12.     

Expression of glucose transporter 1 is associated with loss of heterozygosity of chromosome 1p in oligodendroglial tumors WHO grade II

Objective Oligodendroglial tumors with a loss of heterozygosity of 1p (LOH1p) appear to have a better prognosis than oligodendrogliomas without LOH. Previously, we reported glucose uptake in low-grade oligodendroglial tumors to be related to LOH 1p status. Here, we performed an immunohistochemical study of the common glucose transporters (GLUT) in relation to LOH1p. Material and methods We examined 17 oligodendrogliomas (O II, 11 with LOH1p), 16 oligoastrocytomas (OA II, 5 with LOH1p) and 7 astrocytomas (A II, none with LOH1p). Confocal microscopy was performed for p53, GLUT-1, −3 and −12. Immunoreaction was rated semiquantitatively by percentage of positive cells and staining intensity on immunohistological stainings. Results Confocal microscopy depicted immunoreaction for GLUT-1, −3 and −12 in the cytoplasm of the tumor cells. Oligodendrogliomas revealed a lower immunoreactivity for GLUT-1 than oligoastrocytomas and astrocytomas (P = 0.0263). No differences in immunoreactivity were found for GLUT-3 and GLUT-12. GLUT-1 expression in tumors with LOH 1p was significantly lower than in tumors with wild type 1p status (P = 0.0017). GLUT-3 and GLUT-12 immunoreactivity was not correlated with LOH 1p. Conclusion Expression of GLUT-1 is significantly reduced in low-grade oligodendroglial tumors harboring LOH 1p. Further studies should address the functional role of GLUT-1 in regard to chemosensitivity of oligodendrogliomas.     

Expression of cell-cycle regulator CDK2-associating protein 1 (p12CDK2AP1) in transgenic mice induces testicular and ovarian atrophy in vivo

The novel cell-cycle regulator p12(CDK2AP1) (p12) gene encodes a cyclin-dependent kinase 2 (CDK2) partner that participates in cell-cycle regulation, apoptosis, and proliferation. CDK2 has been implicated in maintenance of gonadal homeostasis, as knockout mice display reproductive abnormalities. To investigate the role of p12 in homeostasis of gonadal tissues in vivo, we generated a transgenic mouse model driven by the human keratin 14 promoter, reported to target transgene expression to gonadal tissues and also stratified epithelia. Overexpression of the transgene was associated with a gonadal atrophy phenotype in mice of both sexes, yet fertility was not impaired. Histological evaluation of testes showed seminiferous tubule degeneration and decreased tubule diameter. Female transgenic mice had small ovaries, with a higher number of atretic follicles/mm(2) as compared to control nontransgenic mice. Also observed was increased germ cell apoptosis in both sexes (TUNEL). These results suggest that overexpression of p12 leads to testicular and ovarian abnormalities, a phenotype closely related to that of cdk2-/- mice. In combination, these observations suggest that the p12/CDK2 signaling pathways are carefully orchestrated to maintain proper gonadal tissue homeostasis. We suggest that the mechanisms of this regulation may be through p12-mediated altered expression of gonadal-specific genes and apoptotic pathways.