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List of the supported functional analysis software .

PMC3459790

Description : Complete list of the supported functional analysis software for the Gene List Output Utility Tool .

PMC3459790

Access information , methods , input file requirements , supported organisms , matching Microarray Я US output file , and other details are listed for each supported software .

PMC3459790

Click here for file Additional file 3 .

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List of the supported microarray data types .

PMC3459790

Complete list of the supported microarray chips .

PMC3459790

Click here for file

PMC3459790

HPV16 E7-Dependent Transformation Activates NHE1 through a PKA-RhoA-Iinduced Inhibition of p38alpha .

PMC2568952

Background .

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Neoplastic transformation originates from a large number of different genetic alterations .

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Despite this genetic variability , a common phenotype to transformed cells is cellular alkalinization .

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We have previously shown in human keratinocytes and a cell line in which transformation can be turned on and followed by the inducible expression of the E7 oncogene of human papillomavirus type 16 ( HPV16 ) , that intracellular alkalinization is an early and essential physiological event driven by the up-regulation of the Na / + H + exchanger isoform 1 ( NHE1 ) and is necessary for the development of other transformed phenotypes and the in vivo tumor formation in nude mice .

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Methodology .

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Here , we utilize these model systems to elucidate the dynamic sequence of alterations of the upstream signal transduction systems leading to the transformation-dependent activation of NHE1 .

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Principal Findings .

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We observe that a down-regulation of p38 MAPK activity is a fundamental step in the ability of the oncogene to transform the cell .

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Further , using pharmacological agents and transient transfections with dominant interfering , constitutively active , phosphorylation negative mutants and siRNA strategy to modify specific upstream signal transduction components that link HPV16 E7 oncogenic signals to up-regulation of the NHE1 , we demonstrate that the stimulation of NHE1 activity is driven by an early rise in cellular cAMP resulting in the down-stream inhibition of p38 MAPK via the PKA-dependent phosphorylation of the small G-protein , RhoA , and its subsequent inhibition .

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Conclusions .

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All together these data significantly improve our knowledge concerning the basic cellular alterations involved in oncogene-driven neoplastic transformation .

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Introduction .

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Neoplastic transformation is the first step of the carcinogenic process that involves the initial altered responses of the cells to normal regulatory influences and sets the stage for further alterations that result in carcinoma .

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A wide variety of altered phenotypes appear as a result of transformation .

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Hallmarks of epithelial transformation and carcinogenesis include loss of polarity , as well as uncontrolled , serum-independent and anchorage-independent proliferation and resistance to apoptosis [ 1 ] .

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Other fundamental hallmarks of epithelial carcinogenesis include an elevated intracellular pH ( pHi ) as well as their increased rate of glucose utilization over oxidative phosphorylation [ 2 ] , [ 3 ] .

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However , our understanding of the sequence of early events mediating the initiation , development and regulation of malignant transformation is still incomplete.One major group of cellular signal transduction components implicated in carcinogenesis are the mitogen-activated protein kinases ( MAPKs ) .

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Altered expression / activity of each of the MAPKs such as ERK ( extracellular signal-regulated kinase ) , JNK ( Jun N-terminal kinase ) and p38 has been linked to tumor progression in a wide variety of cellular contexts [ 4 ] – [ 6 ] .

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In particular , mounting evidence indicates a negative role for the p38alpha MAP Kinase in chemical - [ 7 ] and oncogene - [ 8] induced tumor formation and proliferation [ 9 ] , in tumor cell directed cell polarity [ 10 ] – [ 12 ] and in malignant invasion [ 11 ] , [ 12 ] .

PMC2568952

Conversely , a positive role of p38 has been shown in tumor suppression and delay of tumorigenesis [ 13 ] , [ 14 ] , in induction of apoptosis [ 15 ] , [ 13 ] , [ 16 ] , in a specific tumor-suppressing defense mechanism of normal non transformed cells known as oncogene induction of senescence , [ 17 ] , in dormancy [ 18 ] , [ 19 ] and in the increased cell viability and enhanced growth of HPV-induced recurrent respiratory papillomatosis [ 20 ] .

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The importance of p38 as tumor suppressor is highlighted by recent attempts to identify cancer-associated mutations in protein kinase genes , which revealed that several components of the p38 pathway , including p38alpha , are mutated in human tumors [ 17 ] .

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Further , p38 is activated in cancer cells during paclitaxel-driven [ 21 ] , cisplatin-driven [ 22 ] and ROS-driven [ 23 ] apoptosis .

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In liver cells chemically induced to form tumors , p38alpha negatively regulated tumor proliferation via a repression of the JNK-c-Jun pathway [ 24 ] .

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While the negative role for p38alpha in regulating carcinogenesis is well described , whether it plays a similar negative role in the initiation of neoplastic transformation and through which signaling pathways are still undetermined .

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In this context , important questions concern if p38alpha plays a role in the development of the initial transformed phenotype after oncogene expression , what is its pattern of involvement and what are its critical upstream and downstream components .

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Recent progress suggests possible candidate signal transduction pathways .

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As a regulator of gene expression , cell cycle progression and actin cytoskeleton organization , it is now clear that RhoA , a member of the Rho family of GTPases , plays a central role in carcinogenesis and tumor progression [ 25 ] – [ 27 ] .

PMC2568952

Recent studies indicate RhoA as a central upstream regulator of MAP kinase activity [ 26 ] , [ 28 ] – [ 31 ] and specifically in breast cancer [ 11 ] and pancreatic carcinoma [ 32 ] cells .

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Importantly , forced expression of the E7 oncogene of HPV16 in keratinocytes has recently been shown to inhibit RhoA activity although the mechanism is still not completely clear [ 33 ] .

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The cAMP / PKA system has been demonstrated to be involved in both transformation / tumor progression [ 11 ] , [ 34 ] , [ 35 ] proliferation [ 35 ] – [ 37 ] and apoptosis [ 38 ] – [ 40 ] .

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This system is also involved in regulating p38 activity [ 11 ] , [ 41 ] – [ 43 ] and there is evidence that direct PKA-dependent phosphorylation of RhoA at Ser 188 inhibits its activity in endothelial cells [ 44 ] , in smooth muscle cells [ 45 ] , in cytotoxic lymphocytes [ 46 ] and in tumor cells [ 11 ] suggesting that PKA and RhoA could regulate p38 activity through a common pathway.The goal of the present work is to determine which signal transduction systems are involved in transformation driven by a relevant oncogene .

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That little is known about the modifications occuring in the regulatory pathways during malignant transformation has been due , in part , to the lack of experimental models in which the transformation of normal cells by a relevant oncogene can be finely controlled and followed [ 17 ] .

PMC2568952

Since the human papilloma viruses , especially HPV16 , are the prime cause of the majority of virus-associated carcinomas and the E7 and E6 proteins are the viral proteins responsible for malignant transformation [ 47 ] , we chose the E7 oncogene of HPV16 to create an experimental cell model useful for the investigation of the alterations in signal transduction mechanisms underlying the initial phases of the shift from the normal to transformed state [ 48 ] .

PMC2568952

This cell model ( 2BN11 cells ) consists of normal NIH-3T3 fibroblasts stably transfected with the gene for the HPV16 E7 oncoprotein in a vector in which its expression is under the control of a tetracycline ( tet ) inducible promoter such that the level of E7 expression and , therefore , transformation can be tightly regulated .

PMC2568952

This inducible expression permits the repeated following of the time-dependent development of the events in which the same cell constitutes its own control .

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Using the 2BN11 model system , we previously observed that intracellular alkalinization driven by an up-regulation of the Na / H - exchanger ( NHE1 ) is a very early physiological event in transformation and is essential for the development of other transformed phenotypes such as increased glycolytic metabolism , increased growth rate and both serum-independent and anchorage-independent growth [ 48 ] .

PMC2568952

Here , we report that HPV16 E7-dependent transformation mediated by NHE1 is driven by the action of a RhoA-p38alpha module gated by the PKA dependent phosphorylation of RhoA .

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Materials and Methods .

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Cell culture , transfections and reagents .

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NIH3T3 cells were cultured in Dulbecco 's modified Eagle 's medium ( DMEM ) with 4.5 g of glucose per liter , supplemented with 10 % calf serum .

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Normal human foreskin keratinocytes ( HFKs ) were isolated from neonatal foreskin as described previously [ 49 ] and were maintained in keratinocyte growth medium supplemented with bovine pituitary extract ( Clonetics ) .

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Primary human keratinocytes infected with HPV16 [ 50 ] and cultured at low ( HPK1a early ) or high ( HPK1A late ) passage numbers were maintained as previously described [ 48 ] .

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These cells spontanously transform at high passages and high passage cells are tumorgenic in nude mice [ 48 ] .

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High-titer retroviral supernatants ( 10 virus particles / ml ) were generated by transient transfection of Bosc23 cells ( ecotropic viruses ) or Phoenix ( amphotropic viruses ) and used to infect the cells as described previously [ 48 ] .

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Cells were grown to about 80 % confluence in a 10-cm-diameter dish and infected with 5 ml of the recombinant retrovirus or parental virus in the presence of 8 µ g of Polybrene per ml in order to enhance infection efficacy .

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After 6 h infected cells were fed with complete medium and kept at 37 ° C with 5 % CO for 48 h.

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The cells were then transferred to four 10-cm-diameter dishes and selected with 2.0 µ g of puromycin / ml for 3 to 4 days .

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Selected cells were expanded and used for experiments , generally when at 80 % confluence .

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NIH3T3 or HFK cells were infected with recombinant retrovirus ( pLXSN vector , Clontech , BD , Le Pont Claix , France ) expressing non tagged HPV16 E7 proteins .

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In some experiments , NIH3T3 cells constitutively expressing E7 genes were generated by infection with pBabepuro vector expressing HPV16 HA-tagged E7 proteins , wild-type or mutant.The 2BN11 cell line was created by infecting NIH3T3 cells with recombinant retrovirus expressing the HPV16 E7 gene under the control of a tetracycline repressed promoter .

PMC2568952

The tetracycline-controlled expression system consisted of regulator and response elements in a one vector system [ 48 ] .

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Cells were cultured in DMEM high glucose ( 4500 mg / l ) supplemented with NaHCO ( 3700 mg / l ) , 10 % ( v / v ) heat inactivated fetal bovine serum , L-glutamine ( 2 mM ) , Na-Pyruvate ( 1 mg / ml ) and 1 µ M tetracycline .

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The clones , after selection in puromycin , were selected on the basis of having low basal E7 expression in the presence of tetracycline and high inducibility as determined by Western blot and RT-PCR analysis.Transient transfections with the various mutated cDNAs were performed with the FuGENE HD Transfection Reagent ( Roche ) according to the manufacturer 's instructions [ 12 ] .

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H89 , SB203580 and IBMX were purchased from Sigma whereas Forskolin was from Calbiochem ( Novabiochem Corp ., La Jolla , CA ) .

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The effect of these clones or pharmacological agents on E7 expression were tested in an RT-PCR assay as described below and none of them had any effect on E7 message ( Figure S1 ) RNA preparation and semiquantitative RT-PCR .

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Total RNA was extracted from cells using the RNeasy system ( Qiagen , Valencia , CA ) and treated with RNAase-free Dnase ( Qiagen ) for 15 min at room temperature .

PMC2568952

Spectrophotometric ratios of A260 to A280 were greater than 1.8 .

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Total RNA ( 500 ng ) was reverse transcribed in 20 µ l reaction system using Random Hexamers priming and MuLV Reverse Transcriptase ( RT ) with the RNA PCR Kit GeneAmp ( Applied Biosystems ) under conditions described by the supplier .

PMC2568952

Reverse transcription-PCRs ( RT-PCRs ) were carried out essentially as previously reported [ 12 ] .

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Quantification of HPV16 E7 expression levels was performed by comparison to the GAPDH ( glyceraldehyde-3-phosphate dehydrogenase ) housekeeping gene .

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Primer sequences were as follows : HPV16 E7 gene , 5 ′ - ATG CAT GGA GAT ACA CCT AC-3 ′ ( forward ) and 5 ′ - TAT GGT TTC TGA GAA CAG ATG-3 ′ ( reverse ) ; GAPDH gene , 5 ′ - ACC ACA GTC CAT GCC ATC AC-3 ′ ( forward ) and 5 ′ - TCC ACC ACC CTG TTG CTG TA-3 ′ ( reverse ) .

PMC2568952

PCRs for HPV16 E7 and the GAPDH gene were performed by a touchdown protocol with the following cycling conditions : 10 min at 95 ° C ( initial denaturation ) , 6 cycles of step-down PCR consisting of 45 s at 95 ° C ( denaturation ) , 60 s at 58 ° C ( annealing ) – decrease 1 ° C each cycle until 53 ° C ; and 120 s at 72 ° C ( extension ) .

PMC2568952

Amplification of the final product was completed for 26 cycles of 45 s at 95 ° C , 60 s at 53 ° C , and 120 s at 72 ° C , with a final extension of 10 min at 72 ° C.

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In the negative control , RNase free water ( Gibco ) was used instead of template RNA .

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The positive control included HPV16 E7 cDNA .

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Amplicons were separated on 1.5 % agarose gel and visualized by ethidium bromide staining .

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NHE1 activity .

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Intracellular pH was determined spectrofluorimetrically in cells loaded with the acetoxy-methyl ester derivative of the pH-sensitive dye 2,7 - biscarboxyethyl-5 ( 6 ) - carboxyfluorescein ( AM-BCECF , Invitrogen ) .

PMC2568952

NHE1 activity was determined by measuring the rate of pHi recovery from an acid load produced with the NH4Cl prepulse technique by evaluating the derivative of the slope of the time-dependent pHi recovery ( dpHi / dt ) as previously described [ 40 ] , [ 48 ] .

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The use of CO / HCO free solutions minimizes the likelihood that Na - dependent HCO transport was responsible for the observed pHi changes.After each experiment trypan blue exclusion was also measured for each cover slip and when was more than 5 % the experiment was not used .

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Construction of expression vectors containing RhoA mutants .

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Site-directed mutagenesis of RhoA to create RhoAS188A was performed by PCR overlap extension as previously described [ 11 ] .

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The successful construction of the mutants was confirmed by DNA sequence analysis .

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The cDNA were cloned into the pBabe puro expression vector containing a hemagglutinin ( HA ) tag .

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RhoA RNA Interference .

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Small interfering RNA ( siRNA ) duplex against RhoA ( sc-29471 ) was obtained from Santa Cruz Biotechnology .

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The specificity of the silencing tecnique was verified by using a control , non targeting 20 – 25 nucleotide siRNA designed as a negative control ( Control siRNA-A , sc-37007 , Santa Cruz Biotechnology ) .

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siRNAs were used at 100 nM concentration and transfection with siRNA was performed using Lipofectamine 2000 ( Invitrogen ) 24 hr after plating cells .

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The efficiency of RhoA silencing was measured in immunofluroescence microscopy using a monoclonal anti-RhoA antibody ( sc-418 , Santa Cruz Biotechnology ) and was found to be ∼ 75 % after 72 hrs siRNA treatment ( Fig. S2 ) .

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RhoA expression levels were measured via pixel density analysis using the WCIF Image J 1.37c software ( Wayne Rasband , NIH , USA ) .

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Therefore , NHE1 activity was analyzed 72 hrs post-transfection with either RhoA siRNA or control siRNA-A in the presence of tet or 24 hrs after tet removal .

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Adherent cell cAMP assay .

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Cells ( 10 cells in 100 µ l of media ) were plated into each well of a Greiner Bio-one tissue culture grade 96-well white clear bottom microassay plate ( PBI S.p.a ., Italy ) and incubated in 5 % CO atmosphere at 37 ° C for 24 hr .

PMC2568952

Cells were washed once with 100 µ l of DMEM serum free medium plus 100 µ M IBMX and then treated with 20 µ M of FSK and 1 mM of IBMX in 20 µ l of reaction media at 37 ° C for 30 min .

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Levels of cytosolic cAMP were then measured with the cAMP-Glo ™ assay ( Promega , Italy ) as per manufacturers instructions .

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Briefly , the medium was aspirated , 20 µ l of lysis buffer and 40 µ l of cAMP-Detection Solution was added and the plate was incubated with gentle rocking at room temperature for 20 min .

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Next , 80 µ l of Kinase-Reagent were added , and luminescence was read after incubation for 10 minutes in a Fluorescence Spectophotometer Cary Eclipse VARIAN in its chemiluminescence mode where each well is read for 20 seconds .

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Increases in cAMP levels result in decreases in chemiluminescence .

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cAMP FRET measurements and cell imaging .

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For time-lapse FRET experiments , cells were cotransfected with 1.5 µ g each of pcDNA3Cat-YFP and pcDNA3RII-CFP plasmids using Fugene 6 ( Roche , Switzerland ) for 24 hr as described [ 51 ] .

PMC2568952

Twenty-four hours after transfection , monolayers of cells were cultured an additional 3 or 24 h in the presence or absence of tetracycline and imaged on a Nikon ECLIPSE TE 2000-S equipped with a charge-coupled device camera , a controlled DeltaRAM monochromator on the excitation side and a beam splitter ( Optical Insight , St. Cloud , MN ) on the emission side fitted with a 505DCRX dichroic and two emission filters , D480 / 30 and D535 / 40 .

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Excitation was at 430 nm and the dichroic mirror was a 455DRLP .

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