Sphingosine kinase 2 deficient mice exhibit reduced experimental autoimmune encephalomyelitis: Resistance to FTY720 but not ST-968 treatments

The immunomodulatory drug FTY720 is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that requires activation by sphingosine kinase 2 (SK-2) to induce T cell homing to secondary lymphoid tissue. In this study, we have investigated the role of SK-2 in experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. We show that SK-2 deficiency reduced clinical symptoms of EAE. Furthermore, in SK-2-deficient mice, the protective effect of FTY720 on EAE was abolished, while the non-prodrug FTY720-derivative ST-968 was still fully active. Protection was paralleled by reduced numbers of T-lymphocytes in blood and a reduced blood-brain-barrier leakage. This correlated with reduced mRNA expression of ICAM-1, VCAM-1, but enhanced expression of PECAM-1. A similar regulation of permeability and of PECAM-1 was seen in primary cultures of isolated mouse brain vascular endothelial cells and in a human immortalized cell line upon SK-2 knockdown. In summary, these data demonstrated that deletion of SK-2 exerts a protective effect on the pathogenesis of EAE in C57BL/6 mice and that SK-2 is essential for the protective effect of FTY720 but not of ST-968. Thus, ST-968 is a promising novel immunomodulatory compound that may be a valuable alternative to FTY720 under conditions where SK-2 activity is limited.

Dentinhaftkraft zweier neuer CAD/CAM-Materialien in Abhängigkeit verschiedener adhäsiver Zementsysteme

Zielsetzung: Ziel der Studie war die Bestimmung der Dentinhaftkraft von zwei so-genannten Hybridmaterialien für computer-aided design/computer-aided manufacturing (CAD/CAM) Restaurationen unter Anwendung von fünf verschiedenen Zementen vor und nach sechsmonatiger Lagerung. Materialien und Methoden: Aus extrahierten menschlichen Molaren wurden 300 Dentinprobekörper hergestellt (n=15 pro Gruppe; 10 Gruppen (2 Hybridkeramiken, 5 Zemente) je nach 24 h/nach sechsmonatiger Lagerung). Aus Hybridkeramikblöcken von Lava Ultimate (3M ESPE) und VITA ENAMIC (VITA Zahnfabrik) wurden Zylinder hergestellt, welche standardisiert aufgeraut wurden. Anschliessend wurden die Hybrid-keramikzylinder mit einem der folgenden fünf Zemente auf die Dentinprobekörper zementiert: mit den Kompositzementen RelyX Ultimate (3M ESPE), PANAVIA F2.0 (Kuraray), Variolink II (Ivoclar Vivadent), els cem (Saremco Dental AG) oder als Negativkontrollgruppe mit dem kunststoffmodifizierten Glasionomerzement Ketac Cem Plus (3M ESPE). Die Dentinhaftkraft der Hybridkeramikzylinder wurde einerseits nach 24 h und andererseits nach sechsmonatiger Lagerung via Scherkrafttest bestimmt. Nach dem Scherkrafttest wurde das Bruchmuster unter einem Lichtmikroskop bei 40-facher Vergrösserung beurteilt. Die Dentinhaftkraftwerte wurden mittels nichtparametrischer ANOVA gefolgt von exakten Wilcoxon Rangsummen-Tests statistisch analysiert (α=0,05). Die Beurteilung des Bruchmusters wurde deskriptiv ausgewertet. Resultate: Für die Hybridkeramik Lava Ultimate und nach 24 h erzielten die Kompositzemente RelyX Ultimate und Variolink II die höchsten Dentinhaftkraftwerte. Die Dentinhaftkraftwerte von RelyX Ultimate und Variolink II unterschieden sich nicht signifikant. Die Dentinhaftkraftwerte von PANAVIA F2.0 unterschieden sich ebenfalls nicht signifikant von denjenigen von RelyX Ultimate, waren jedoch signifikant tiefer als diejenigen von Variolink II. Unter allen Kompositzementen erzielte els cem die tiefsten Dentinhaftkraftwerte. Nach sechsmonatiger Lagerung waren die Dentinhaftkraftwerte für RelyX Ultimate die höchsten, gefolgt von Variolink II, von els cem und anschliessend von PANAVIA F2.0, welcher nach sechsmonatiger Lagerung die tiefsten Dentinhaftkraftwerte der Kompositzemente zeigte. Der kunststoffmodifizierte Glasionomerzement Ketac Cem Plus zeigte sowohl nach 24 h als auch nach sechsmonatiger Lagerung die tiefsten Dentinhaftkraftwerte. Für VITA ENAMIC war die Reihenfolge der Zemente nach Dentinhaftkraft nach 24 h ähnlich wie diejenige nach sechsmonatiger Lagerung: Die Dentinhaftkraft war für RelyX Ultimate und Variolink II am höchsten, gefolgt von PANAVIA F2.0, von els cem und schlussendlich von Ketac Cem Plus mit den tiefsten Dentinhaftkraftwerten. Nach 24 h und für alle fünf Zemente unterschieden sich die Dentinhaftkraftwerte zwischen Lava Ultimate und VITA ENAMIC nicht signifikant. Nach sechsmonatiger Lagerung unterschieden sich die Dentinhaftkraftwerte zwischen Lava Ultimate und VITA ENAMIC ebenfalls nicht signifikant für RelyX Ultimate und els cem im Gegensatz zu den Dentinhaftkraftwerten von PANAVIA F2.0, Variolink II und Ketac Cem Plus, welche signifikant tiefer waren für Lava Ultimate als für VITA ENAMIC. Das häufigste Bruch-muster war für Lava Ultimate nach 24 h und für VITA ENAMIC sowohl nach 24 h als auch nach sechsmonatiger Lagerung adhäsiv zwischen Dentin und Zement. Nach sechs-monatiger Lagerung war für Lava Ultimate das häufigste Bruchmuster tendenziell gemischte Brüche. Schlussfolgerung: Basierend auf den Resultaten kann gesagt werden, dass für beide Hybridkeramiken sowohl RelyX Ultimate als auch Variolink II empfohlen werden können. PANAVIA F2.0 kann für VITA ENAMIC empfohlen werden, für Lava Ultimate allerdings weniger, da die Dentinhaftkraft nach sechsmonatiger Lagerung abnahm. Von einer konventionellen (allerdings nicht indizierten und in dieser Studie experimentellen) Zemen-tierung der beiden Hybridkeramiken mit dem kunststoffmodifizierten Glasionomerzement Ketac Cem Plus muss abgeraten werden.

Einfluss von Politursystemen auf Oberflächenrauigkeit und mechanische Eigenschaften von CAD/CAM-Materialien

Zielsetzung: Das Ziel dieser Studie war, den Einfluss von drei Politursystemen auf die Oberflächenrauigkeit von verschiedenen Materialien für computer-aided design/computer-aided manufacturing (CAD/CAM) Restaurationen mittels Profilometrie sowie die mikromechanischen Eigenschaften der Materialien mittels Mikrohärtemessgerät zu analysieren. Materialien und Methoden: Von dem CAD/CAM-Kompositmaterial Paradigm MZ100 (3M ESPE), der CAD/CAM-Feldspatkeramik VITABLOCS Mark II (VITA Zahnfabrik) und den CAD/CAM-Hybridmaterialien Lava Ultimate (3M ESPE), VITA ENAMIC (VITA Zahnfabrik) und AMBARINO High-Class (Creamed) wurden je 60 Prüfkörper zugeschnitten, gekennzeichnet und standardisiert aufgerauht. Die standardisierte Aufrauhung wurde mit Baseline-Rauigkeitsmessungen überprüft (Ra und Rz; µm). Die Prüfkörper wurden mit einem von drei Politursystemen poliert (n=20 pro CAD/CAM-Material): 1) Sof-Lex Scheiben (Disc-System, 3 Politurschritte: medium, fein und superfein; 3M ESPE), 2) VITA Polishing Set Clinical (Silikonpolitursystem, 2 Politurschritte: medium und fein; VITA Zahnfabrik) oder 3) KENDA Nobilis (Silikonpolierer, 1 Politurschritt (universal); KENDA Dental). Nach Politur der Prüfkörper wurden Ra und Rz sowie die mikromechanischen Eigenschaften Oberflächenhärte (VHN; Vickers Härte) und Elastizitätsmodul (EM; GPa) gemessen. In den darauf folgenden sechs Monaten wurden die Prüfkörper in Leitungswasser gelagert und insgesamt sechs Mal einem maschinellem Zahnbürsten zugeführt. Anschliessend wurden erneut Ra und Rz sowie VHN und EM gemessen. Ra-, Rz-, VHN- und EM-Werte wurden mittels nichtparametrischer ANOVA global analysiert und die p-Werte mittels Bonferroni-Holm Korrektur für multiples Testen korrigiert. Als post-hoc Tests wurden Kruskal-Wallis-Tests sowie exakte Wilcoxon Rangsummen-Tests verwendet und die p-Werte wurden nicht korrigiert. Das Signifikanzniveau wurde auf α=0,05 festgelegt. Resultate: Für alle drei CAD/CAM-Hybridmaterialien ergaben Sof-Lex Scheiben nach der Politur die tiefste Oberflächenrauigkeit (d. h. die tiefsten Ra- und Rz-Werte), gefolgt von KENDA Nobilis und von dem VITA Polishing Set Clinical. Bei dem CAD/CAM-Kompositmaterial sowie bei der CAD/CAM-Feldspatkeramik ergaben Sof-Lex Scheiben und KENDA Nobilis ähnliche Resultate, gefolgt von dem VITA Polishing Set Clinical. Bei einigen CAD/CAM-Materialien zeigten sich – zum Teil in Abhängigkeit des Politursystems – nach maschinellem Zahnbürsten und Lagerung signifikant höhere Ra- und Rz-Werte. Die CAD/CAM-Materialien zeigten unabhängig des Politursystems und der Lagerung signifikant verschiedene VHN- und EM-Werte. Bei einigen CAD/CAM-Materialien zeigten sich – zum Teil ebenfalls in Abhängigkeit des Politursystems – nach maschinellem Zahn-bürsten und Lagerung signifikant tiefere VHN- und EM-Werte. Schlussfolgerungen: Die Wahl des Politursystems beeinflusste die Oberflächenrauigkeit der CAD/CAM-Materialien markant, wobei Sof-Lex Scheiben insgesamt die besten Politurresultate zeigten, gefolgt von dem Silikonpolierer KENDA Nobilis. Von der Verwendung des Silikonpolitursystems VITA Polishing Set Clinical muss eher abgeraten werden. Das CAD/CAM-Kompositmaterial Paradigm MZ100 und die CAD/CAM-Hybridmaterialien Lava Ultimate und AMBARINO High-Class als weichere und elastischere Materialien liessen sich insgesamt besser polieren, waren aber bezüglich mechanischer Eigenschaften anfälliger auf Lagerung als die härtere CAD/CAM-Feldspatkeramik VITABLOCS Mark II und das CAD/CAM-Hybridmaterial VITA ENAMIC.

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum.

We report on the in vitro effects of the bumped kinase inhibitor 1294 (BKI-1294) in cultures of virulent Neospora caninum isolates Nc-Liverpool (Nc-Liv) and Nc-Spain7 and in two strains of Toxoplasma gondii (RH and ME49), all grown in human foreskin fibroblasts. In these parasites, BKI-1294 acted with 50% inhibitory concentrations (IC50s) ranging from 20 nM (T. gondii RH) to 360 nM (N. caninum Nc-Liv), and exposure of intracellular stages to 1294 led to the nondisjunction of newly formed tachyzoites, resulting in the formation of multinucleated complexes similar to complexes previously observed in BKI-1294-treated N. caninum beta-galactosidase-expressing parasites. However, such complexes were not seen in a transgenic T. gondii strain that expressed CDPK1 harboring a mutation (G to M) in the gatekeeper residue. In T. gondii ME49 and N. caninum Nc-Liv, exposure of cultures to BKI-1294 resulted in the elevated expression of mRNA coding for the bradyzoite marker BAG1. Unlike in bradyzoites, SAG1 expression was not repressed. Immunofluorescence also showed that these multinucleated complexes expressed SAG1 and BAG1 and the monoclonal antibody CC2, which binds to a yet unidentified bradyzoite antigen, also exhibited increased labeling. In a pregnant mouse model, BKI-1294 efficiently inhibited vertical transmission in BALB/c mice experimentally infected with one of the two virulent isolates Nc-Liv or Nc-Spain7, demonstrating proof of concept that this compound protected offspring from vertical transmission and disease. The observed deregulated antigen expression effect may enhance the immune response during BKI-1294 therapy and will be the subject of future studies.

CHARACTERIZATION OF SERINE KINASE ACTIVITY ASSOCIATED WITH MOLONEY MURINE SARCOMA VIRUS V-MOS GENE PRODUCTS

Various Moloney murine sarcoma virus (Mo-MuSV) isolates contain a cellular sequence, termed mos, which is responsible for the transforming ability of Mo-MuSV. A serine kinase activity has been found to be associated with mos gene products of several isolates of Mo-MuSV. A mutant of Mo-MuSV strain 124 (designated MuSV ts110) is temperature-sensitive (ts) for transformation and encodes two proteins, P85('gag-mos) (an 85,000 M(,r) protein encoded by the gag and mos genes) and P58('gag), at the permissive temperature (28(DEGREES)C). At the nonpermissive temperature (39(DEGREES)C), only P58('gag) is found in MuSV ts110-infected NRK cells (6m2 cells). Both P85('gag-mos) and P58('gag) were phosphorylated when anti-gag immune complexes containing these proteins were incubated at 22(DEGREES)C with (lamda)-('32)P -ATP and MnCl(,2). The kinase detected in anti-gag complexes from 6m2 cells at permissive temperature was associated with P85('gag-mos) since immune complexes from 39(DEGREES)C 6m2 cells, which lack P85('gag-mos), produced no phosphorylated P58('gag) molecules. In addition, an anti-mos complex (anti-mos 37-55 complexes) allowed in vitro phosphorylation of P85('gag-mos) in the absence of P58('gag). No kinase activity was detectable with other gag gene products (e.g., Mo-MuSV-124 P62('gag)), suggesting that the P85('gag-mos) kinase activity was present within the mos portion of the protein. The P85('gag-mos) kinase activity was very thermolabile upon shifting 6m2 cells from permissive to nonpermissive temperatures (t(, 1/2) for inactivation = 5 min). In contrast, a spontaneous revertant of MuSV ts110 encodes a larger gag-mos protein (termed P100('gag-mos)) which contained a kinase activity stable to 39(DEGREES)C. Using the optimal conditions developed for the P85('gag-mos) kinase, Mo-MuSV-encoded p37('mos) was also found to be associated with a serine kinase activity. Phosphorylation of p37('mos) and a 43 Kd protein (super-phosphorylated p37('mos)) occurred in anti-mos(37-55) complexes from Mo-MuSV-124 acutely-infected NIH 3T3 cells, but neither in mos 37-55 peptide-blocked anti-mos(37-55) complexes nor in immune complexes from uninfected NIH 3T3 cells. Antibodies directed against the C-terminus of v-mos were found to inhibit the in vitro phosphorylation of p37('mos), suggesting that the extreme C-terminal sequence of v-mos may be important for an intrinsic kinase activity. This inhibitory action by antibodies to the C-terminus of p37('mos), when considered with all the other data reported here, provides convincing evidence that the v-mos gene encodes a serine protein kinase activity. ^

THE EFFECT OF RELAXIN ON BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS OF RAT MYOMETRIAL CELLS IN CULTURE (CYCLIC-AMP, CELL SHAPE CHANGE)

Relaxin is able to inhibit spontaneous, oxytocin-and prostaglandin-driven uterine contractions. The intracellular mechanism of action of relaxin on uterine relaxation had previously been studied using isometrically suspended uterine strips. Since uterine strips contain stroma as well as myometrium, the changes in biochemical parameters induced by relaxin treatment may not occur in the same cell types responsible for the physical changes. In these studies, cultures of enriched populations of rat myometrial cells were used to investigate the effect of relaxin on biochemical and morphological parameters which are related to relaxation.^ Under optimal culture conditions (initial plating density 1 - 1.5 x 10('6)cells/ml, 3 ml/35 mm dish, 2 days culture), enzymatically isolated rat myometrial cells were able to respond to relaxin with cAMP elevation. Relaxin elevated cAMP levels in the presence but not the absence of 0.1 mM methylisobutylxanthine or 0.4 um forskolin in a time- and concentration-dependent manner. In contrast, isoproterenol was able to elevate cAMP levels in the presence and absence of 0.1 mM methylisobutylxanthine.^ Oxytocin treatment caused a decrease in mean cell length and area of myometrial cells in culture which could be considered analogous to contraction. Under optimal culture conditions, relaxin increased myometrial cell length and area (i.e. analogous to relaxation) of oxytocin-treated cells in a time- and concentration-dependent manner. Other relaxants such as isoproterenol and dibutyryl cAMP also increased cell length and area of oxytocin - treated myometrial cells in culture.^ Under optimal culture conditions, relaxin decreased myosin light chain kinase activity in a time-and concentration-dependent manner by increasing the K(,50) of the enzyme for calmodulin (CaM), i.e. decreasing the affinity of the enzyme for CaM. The decrease in the affinity of myosin light chain kinase for CaM may be due to the phosphorylation of the enzyme by cAMP-dependent protein kinase. Relaxin also decreased the Ca('2+)(.)CaM-independent myosin light chain kinase activity to a lesser extent than that of the Ca('2+)(.)CaM-dependent enzyme activity. This was not attributable to a decrease in the affinity of the enzyme for myosin in myometrial cells in culture, in contrast to the finding of such a change following relaxin treatment of uterine strips. Further studies are required to clarify this point.^ There was a temporal association between the effects of relaxin on elevation of cAMP levels in the presence of 0.4 uM forskolin, increase in cell length and decrease in myosin light chain kinase activity. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Purification and cloning of the novel phosphoprotein copine III and characterization of its associated kinase activity

The copines, named and first described by Creutz et al. (1998), comprise a two C2 domain-containing protein family that can aggregate phosphatidylserine membranes in a calcium-dependent manner. Although no enzymatic function has been attributed to copines, their carboxyl terminus shows homology to the A domain found in integrins that allows binding of magnesium ions. The secondary structure of A domains resembles a Rossmann fold, which can bind dinucleotides and is present in a number of intracellular enzymes. Due to a crossreacting activity of Mik b 1, an antibody to the IL-2R b chain, we were able to serendipitously clone human copine III (CIII). CIII is 65% identical to copine I (CI) and the 5 kb CIII transcript is expressed ubiquitously as determined by a multitissue Northern blot. A polyclonal antibody generated against the carboxyl terminus of CIII recognized CIII in immunoblots and immunoprecipitations. Phosphorylation of CIII was observed on serine and threonine residues, as determined by phosphoamino acid analysis. ^ Experiments were designed to determine whether or not any enzymatic activity, specifically kinase activity, was intrinsic to or associated with CIII. In vitro and in gel kinase assays were performed using transfected HA-tagged CI and CIII, immunoprecipitated endogenous CIII and purified endogenous CIII. The exogenous substrate MBP was phosphorylated in all in vitro kinase assays containing CIII protein purification and column chromatography expertise with me. ^

The regulation of protein kinase C by thiol oxidation

The Ser/Thr protein kinase C (PKC) isozyme family plays an important role in cell growth and differentiation and also contributes to key events in the development and progression of cancer. PKC isozymes are activated by phospholipid-dependent mechanisms, and they are also subject to oxidative activation and inactivation. Oxidative regulatory mechanisms are important in the governance of PKC isozyme action. While oxidative PKC activation involves phospho-tyrosine (P-Y) stabilization, the molecular mechanism(s) for oxidative PKC inactivation have not been defined. We previously reported that Thr → Cys peptide-substrate analogs inactivate several PKC isozymes including PKC-α via S-thiolation, i.e., by forming disulfides with PKC thiols. This inactivation mechanism is chemically analogous to protein S-glutathiolation, a post-translational modification that has been shown to oxidatively regulate several enzymes. To determine if PKC-α could be inactivated by S-glutathiolation, we employed the thiol-specific oxidant diamide (0.01–10mM) and 100μM glutathione (GSH). Diamide alone (0.1–5.0 mM) weakly inactivated PKC-α (<20%), and GSH alone had no effect on the isozyme activity. Marked potentiation of diamide-induced PKC-α inactivation (>90%) was achieved by 100μM GSH, resulting in full inactivation of the isozyme. Inactivation was reversed by DTT, consistent with a mechanism involving PKC-α S-glutathiolation. S-glutathiolation was demonstrated as DTT-reversible incorporation of [35S] GSH into PKC-α isozyme structure. These results indicate that a mild oxidative stimulus can inactivate purified PKC-α via S-glutathiolation. In addition, diamide treatment of metabolically labeled NIH3T3 cells induced potent PKC-α inactivation via isozyme [35S] S-thiolation. These results indicate that cellular PKC-α can be regulated via S-glutathiolation. ^

Emodin, a tyrosine kinase inhibitor, and human cancer

Many human diseases, including cancers, result from aberrations of signal transduction pathways. The recent understanding of the molecular biochemistry of signal transduction in normal and transformed cells enable us to have a better insight about cancer and design new drugs to target this abnormal signaling in the cancer cells. Tyrosine kinase pathway plays a very important role in normal and cancer cells. Enhanced activity of tyrosine kinases has been associated with many human cancer types. Therefore, identifying the type of tyrosine kinases involved in a particular cancer type and blocking these tyrosine kinase pathways may provide a way to treat cancer. Receptor tyrosine kinase expression, namely epidermal growth factor receptor (EGFR) family, was examined in the oral squamous cell carcinoma patients. The expression levels of different members of the EGFR family were found to be significantly associated with shorter patients' survival. Combining EGFR, HER-2/neu, and HER-3 expression can significantly improve the predicting power. The effect of emodin, a tyrosine kinase inhibitor, on these receptors in head and neck squamous cell carcinoma cell lines was examined. Emodin was found to suppress the tyrosine phosphorylation of HER-2/neu and EGF-induced tyrosine phosphorylation of EGFR. Emodin also induced apoptosis and downregulated the expression of anti-apoptotic protein bcl-2 in oral squamous cell carcinoma cells. It is known that tyrosine kinase pathways are involved in estrogen receptor signaling pathway. Therefore, the effects of inhibiting the tyrosine kinase pathway in estrogen receptor-positive breast cancers was studied. Emodin was found to act similarly to antiestrogens, capable of inhibiting estrogen-stimulated growth and DNA synthesis, and the phosphorylation of Rb protein. Interestingly, emodin, and other tyrosine kinase inhibitors, such as RG 13022 and genistein, depleted cellular levels of estrogen receptor protein. Emodin-induced depletion of estrogen receptor was mediated by the proteasome degradation pathway. In summary, we have demonstrated that tyrosine kinase pathways play an important role in oral squamous cell carcinoma and estrogen receptor-positive breast cancer. Targeting the tyrosine kinases by inhibitors, such as emodin, may provide a potential way to treat the cancer patients. ^

Receptor tyrosine kinase HER2-mediated signaling pathways in metastasis and angiogenesis

Metastasis, the major cause of morbidity and mortality in most cancers, is a highly organized and organ-selective process. The receptor tyrosine kinase HER2 enhances tumor metastasis, however, its role in homing to metastatic organs is poorly understood. The chemokine receptor CXCR4 has recently been shown to mediate the malignant cancer cells to specific organs. Here we show that HER2 enhances the expression of CXCR4 by increasing CXCR4 protein synthesis and inhibiting its degradation. We also observed significant correlation between HER2 and CXCR4 expression in human breast tumor tissues, and an association between CXCR4 expression and a poor overall survival rate in patients with breast cancer. Furthermore, we found that CXCR4 is required for HER2-induced invasion, migration, and adhesion activities in vitro . Finally we established stable transfectants using retroviral RNA interference to inhibit CXCR4 expression and showed that the CXCR4 is required for HER2-mediated lung metastasis in vivo. These results provide a plausible mechanism for HER2-mediated breast tumor metastasis and homing to metastatic organs, and establish a functional link between the receptor tyrosine kinase HER2 and the chemokine receptor CXCR4 signaling pathways. ^ The HER2 overexpression activates PI-3K/Akt pathways and plays an important role in mediating cell survival and tumor development. Hypoxia inducible factors (HIF) are the key regulator for angiogenesis and energy metabolism, and thereby enhance tumor growth and metastasis. HIF activation occurs in the majority of human cancers, including the HER2 overexpressing cancer cells. Previous reports suggested that increased PI-3K/Akt may activate HIF pathway in various tumors, but the detail mechanism is still not completely understood. Here we found that HER2/PI-3K/Akt pathway induces HIF-1α activation, which is independent of hypoxia, but relatively weaker than hypoxic stimulation. This phenomenon was further observed in Akt knock out mouse embryonic fibroblast cells. The PI-3K/Akt pathway does not affect HIF-1α binding with its E3 ligase VHL, but enhances the binding affinity between HIF-1α and β unit. Furthermore, we found Akt phosphorylates HIF-1β at serine 271 and further regulated HIF transcriptional activity. Our findings provided one mechanism that HER2 induce HIF activation via Akt to promote angiogenesis, and this process is independent on hypoxia, which may have implications in the oncogenic activity of HER2 and PI-3K/Akt pathway. ^

Role of the cyclin dependent kinase-4 in normal and neoplastic proliferation

In the last few years, our laboratory has studied the regulatory mechanisms of proliferation and differentiation in epidermal tissues. Our results showed differences in the roles of cyclin dependent-kinases 4 and 6, and the three D-type cyclins, during normal epidermal proliferation and neoplastic development. Thus, to elucidate the role of the different cell cycle regulators, we developed transgenic mice that overexpress CDK4 (K5-CDK4), or their cognate D-type cyclins, in epithelial tissues. The most severe phenotype was observed in K5-CDK4 animals that developed dermal fibrosis, epidermal hyperplasia and hypertrophy. Forced expression of CDK4 in the epidermal basal cell layer increased the malignant conversion of skin papillomas to squamous cell carcinomas (SCC). Contrastingly, lack of CDK4 completely inhibited tumor development, suggesting that CDK4 is required in this process. Biochemical studies demonstrated that p21 Cip1 and p27Kip1 inhibitors are sequestered by CDK4 resulting in indirect activation of Cyclin E/CDK2, implicating the non-catalytic activity of CDK4 in deregulation of the cell cycle progression. ^ It has been proposed that the proliferative and oncogenic role of Myc is linked to its ability to induce the transcription of CDK4, cyclin D1, and cyclin D2 in vitro. Deregulation of Myc oncogene has been found in several human cancers. Also it has been demonstrated that CDK4 has the ability to functionally inactivate the product of the tumor suppressor gene Rb, providing a link between Myc and the CDK4/cyclin D1/pRb/p16 pathway in some malignant tumors. Here, we sought to determine the role of CDK4 as a mediator of Myc activities by developing a Myc overexpressing mouse nullizygous for CDK4. We demonstrated that lack of CDK4 results in reduced keratinocyte proliferation and epidermal thickness in K5-Myc/CDK4-null mice. In addition, complete reversion of tumor development was observed. All together, this work demonstrates that CDK4 acts as an oncogene independent of the D-type cyclin levels and it is an important mediator of the tumorigenesis induced by Myc. In addition, we showed that the sequestering activity of CDK4 is critical for the development of epidermal hyperplasia during normal proliferation, malignant progression from papillomas to squamous cell carcinomas, and tumorigenesis induced by Myc. ^

The Role of Raf Kinase Inhibitor Protein in Cell Motility

Raf Kinase Inhibitor Protein (RKIP) has been identified as a phosphatidylethanolamine-binding protein capable of inhibiting Raf-1 kinase, an enzyme significant in cell proliferation and cancer development. When properly functioning, RKIP can mediate the expression of Raf-1 kinase and help prevent uncontrolled cell division. RKIP also has suggested, but unclear, roles in spindle fiber formation during mitosis, regulation of apoptosis, and cell motility. The Fenteany laboratory in the Chemistry Department identified a new small molecule, named Locostatin, as a cell migration inhibitor in mammalian cells, with RKIP as its primary molecular target. Dictyostelium discoideum possess two RKIP proteins, RKIP-A and RKIP-B. In order to begin to study the function of RKIP in D. discoideum and its role in cell motility, I created a mutant cell line which lacks a functional RKIP-A gene. In this paper, we show that removal of RKIP-A does not affect vegetative motility, but impairs chemotaxis and development in the presence of drug. Interestingly, RKIP-A knockout mutants appear more resistant to drug effects on vegetative motility than wild-type cells. More research is needed to reconcile these seemingly contrasting results, and to better develop a model for RKIP-A’s role in cell motility.

RNA-Activated Protein Kinase, PKR

The double-stranded RNA (dsRNA) activated protein kinase, PKR, is one of the several enzymes induced by interferons and a key molecule mediating the antiviral effects of interferons. PKR contain an N-terminal, double-stranded RNA binding domain (dsRBD), which has two tandem copies of the motifs (dsRBM I and dsRBM II). Upon binding to viral dsRNA, PKR is activated via autophosphorylation. Activated PKR has several substrates; one of the examples is eukaryotic translation initiation factor 2 (eIF2a). The phosphorylation of eIF2a leads to the termination of cell growth by inhibiting protein synthesis in response to viral infection. The objective of this project was to characterize the dsRBM I and define the dsRNA binding using biophysical methods. First, the dsRBM I gene was cloned from a pET-28b to a pET-11a expression plasmid. N-terminal poly-histidine tags on pET-28b are for affinity purification; however, these tags can alter the structure and function of proteins, thus the gene of dsRBM I was transferred into the plasmid without tags (pET-11a) and expressed as a native protein. The dsRBM I was transformed into and expressed by Rosetta DE3plyS expression cells. Purification was done by FPLC using a Sepharose IEX ion exchange followed by Heparin affinity column; yielding pure protein was assayed by PAGE. Analytical Ultracentrifugation, Sedimentation Velocity, was used to characterize free solution association state and hydrodynamic properties of the protein. The slight decrease in S-value with concentration is due to the hydrodynamic non-ideality. No self association was observed. The obtained molecule weight was 10,079 Da. The calculated sedimentation constant at zero concentration at 20°C in water was 1.23 and its friction coefficient was 3.575 ´ 10-8. The frictional ratio of sphere and dsRBM I became 1.30. Therefore, dsRBM I must be non-globular and more asymmetric shape. Isolated dsRBM I exhibits the same tertiary fold as compared to context in the full domain but it exhibited weaker binding affinity than full domain to a 20 bp dsRNA. However, when the conditions allowed for its saturation, dsRBM I to 20 bp dsRNA has similar stoichiometry as full dsRBD.