Identification of five human novel genes associated with cell proliferation by cell-based screening from an expressed cDNA ORF library

The development of functional profiling technologies provides opportunity for high-throughput functional genomics studies. We describe a cell-based screening system to identify novel human genes associated with cell proliferation. The method integrates luciferase reporter gene activity, fluorescence stain, automated microscopy and cellular phenotype assays. We successfully used the system to screen 409 novel human genes cloned by our lab and found that 27 genes significantly up-regulated promoter-Renilla luciferase reporter plasmid (pRL) activity. Among them, five genes, TRAF3IP3, ZNF306, ZNF250, SGOL1, and ZNF434, were determined through morphological observation, calcein AM fluorescence stain, MTT assay and cell cycle analysis to be associated with cell proliferation. Furthermore, we showed that the gene TRAF3IP3, which initially was identified to specifically interact with TRAF3, stimulated cell growth by modulating the c-Jun N-terminal kinase (JNK) pathway, and RNAi of TRAF3IP3 confirmed that the effect was physiological and necessary. In summary, we integrated a rapid and efficient system for screening novel growth regulatory genes. Using the new screening system we identified five genes associated with cell proliferation for the first time.     

Inhibitory effect of curcumin on oral carcinoma CAL-27 cells via suppression of Notch-1 and NF-κB signaling pathways

Curcumin has been reported to inhibit cell growth and induce apoptosis in oral cancer cells. Although many studies have been done to uncover the mechanisms by which curcumin exerts its antitumor activity, the precise molecular mechanisms remain to be unclear. In the present study, we assessed the effects of curcumin on cell viability and apoptosis in oral cancer. For mechanistic studies, we used multiple cellular and molecular approaches such as gene transfection, real-time RT-PCR, Western blotting, invasion assay, and ELISA. For the first time, we found a significant reduction in cell viability in curcumin-treated cells, which was consistent with induction of apoptosis and also associated with down-regulation of Notch-1 and nuclear factor-κB (NF-κB). Taken together, we conclude that the down-regulation of Notch-1 by curcumin could be an effective approach, which will cause down-regulation of NF-κB, resulting in the inhibition of cell growth and invasion. These results suggest that antitumor activity of curcumin is mediated through a novel mechanism involving inactivation of Notch-1 and NF-κB signaling pathways.     

Transcriptome analysis of signaling pathways targeted by Ellagic acid in hepatocellular carcinoma cells

BackgroundEllagic acid (EA) possesses prominent inhibitory activities against various cancers, including hepatocellular carcinoma (HCC). Our recent study demonstrated EA's activities in reducing HCC cell proliferation and tumor formation. However, the mechanisms of EA to exert its anticancer activities and its primary targets in cancer cells have not been systematically explored.MethodsCell proliferation assay and flow cytometric analysis were used to examine the effects of EA treatment on viability and apoptosis, respectively, of HepG2 cells. RNA-seq studies and associated pathway analyses by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were employed to determine EA's primary targets. Differentially expressed genes (DEG) in EA-treated HepG2 cells were verified by RT-qPCR and Western blot. Integrative analyses of the RNA-seq dataset with a TCGA dataset derived from HCC patients were conducted to verify EA-targeted genes and signaling pathways. Interaction network analysis of the DEGs, shRNA-mediated knockdown, cell viability assay, and colony formation assay were used to validate EA's primary targets.ResultsEA reduced cell viability, caused DNA damage, and induced cell cycle arrest at G1 phase of HepG2 cells. We identified 5765 DEGs encoding proteins with over 2.0-fold changes in EA-treated HepG2 cells by DESeq2. These DEGs showed significant enrichment in the pathways regulating DNA replication and cell cycle progression. As primary targets, p21 was significantly upregulated, while MCM2–7 were uniformly downregulated in response to EA treatment. Consistently, p21 knockdown desensitized liver cells to EA in cell viability and colony formation assays.ConclusionEA induced G1 phase arrest and promoted apoptosis of HCC cells through activating the p21 gene and downregulating the MCM2–7 genes, respectively.General significanceThe discoveries in this study provide helpful insights into developing novel strategies in the therapeutic treatment of HCC patients.     

Sarcophine-diol,a Chemopreventive Agent of Skin Cancer,Inhibits Cell Growth and Induces Apoptosis through Extrinsic Pathway in Human Epidermoid Carcinoma A431 Cells

Sarcophine-diol (SD), a structural modifications of sarcophine, has shown chemopreventive effects on 7,12-dimethylbenz(a)anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted skin tumor developments in mice. Tumorigenesis is associated with uncontrolled cell growth and loss of apoptosis. In the present study, the effects of SD on cell growth and apoptosis in human epidermoid carcinoma A431 cells were determined to assess whether SD could inhibit cell growth and/or induce apoptosis, thus elucidating possible mechanism of action. MTT assay was used for cell viability; bromodeoxyuridine incorporation assay was used for cell proliferation; fluorescence-activated cell sorting analysis of annexin V/propidium iodide staining and TUNEL assay were used for determining apoptotic cells; Western blot analysis was used for determining the expression of caspase-3 and colorimetric caspase activity assays were used for determination of caspase-3, -8, and -9 activity. The results showed that SD treatment at concentration of 200 to 600 µM resulted in a concentration-dependent decrease in cell viability and cell proliferation in A431 cells, which largely inhibited cell growth. Sarcophine-diol treatment induced a strong apoptosis and significantly (P < .05) increased DNA fragmentation in A431 cells. Furthermore, SD treatment significantly (P < .05) increased the activity and expression of caspase-3 through activation of upstream caspase-8 in A431 cells rather than the activation of caspase 9. Sarcophine-diol treatment is relatively much less cytotoxic in monkey kidney normal CV-1 cells. These results suggest that SD decreases cell growth and induces apoptosis through caspase-dependent extrinsic pathway in A431 cells, and this may contribute to its overall chemopreventive effects in mouse skin cancer models.     

Multiplexing siRNAs to compress RNAi-based screen size in human cells

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (~800 siRNAs, ~400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.     

cDNA expression array analysis of gene expression in human hepatocarcinoma Hep3B cells induced by BNIPL-1

Bcl-2/adenovirus E1B 19 kDa interacting protein 2 like-1 （BNIPL-1） is a novel human protein identified in our laboratory, which can interact with Bcl-2 and Cdc42GAP and induce apoptosis via the BNIP-2 and Cdc42GAP homology （BCH） domain. In the present study, we established the Hep3B-Tet-on stable cell line in which expression of BNIPL-1 can be induced by doxycycline. The cell proliferation activity assay showed that the overexpression of BNIPL-1 suppresses Hep3B cell growth in vitro. The differential expression profiles of 588 known genes from BNIPL-1-transfected Hep3B-Tet-on and vector control cells were determined using the Atlas human cDNA expression array. Fifteen genes were differentially expressed between these two cell lines, among which seven genes were up-regulated and eight genes were down-regulated by BINPL-1. Furthermore, the differential expression result was confirmed by semiquantitative RT-PCR. Among these differentially expressed genes, p16^INK4, IL-12, TRAIL and the lymphotoxin β gene involved in growth suppression or cell apoptosis were up-regulated, and PTEN involved in cell proliferation was down-regulated by BNIPL-1. These results suggest that BNIPL-1 might inhibit cell growth though cell cycle arrest and/or apoptotic cell death pathway（s）.     

GMI, an immunomodulatory protein from Ganoderma microsporum, induces autophagy in non-small cell lung cancer cells

Autophagy is a self-digestive process that degrades the cytoplasmic constituents. Immunomodulatory protein, one major bioactive component of Ganoderma, has antitumor activity. In this study, recombinant fungal immunomodulatory protein, GMI, was cloned from Ganoderma microsporum and purified. We demonstrated that GMI induces lung cancer cell death by activating autophagy, but does not induce apoptotic cell death. On western blot, GMI increased LC3 conversion and decreased p53 expression in a time- and concentration-dependent manner. Cytoplasmic calcium chelator BAPTA-AM was used to prove that GMI promotes autophagy via a calcium-mediated signaling pathway. 3-methyladenine (3-MA), an autophagy inhibitor, enhanced the cytotoxicity of GMI on cell viability assay. Using VZV-G pseudotyped lentivirus-shRNA system for autophagy-related genes silencing, the capabilities of GMI to reduce cell viability and colony formation were abolished in autophagy-defective cells. Furthermore, GMI did not stimulate apoptosis after blocking of autophagy by 3-MA or shRNA knockdown system. In xenograft studies, oral administration of GMI inhibited the tumor growth and induced autophagy significantly in nude mice that had received a subcutaneous injection of A549 cells. This is the first study to reveal the novel function of GMI in activating autophagy. GMI may be a potential chemopreventive agent against non-small cell lung cancer.     

Recipient-induced transfer of the symbiotic plasmid pRL1JI in Rhizobium leguminosarum bv. viciae is regulated by a quorum-sensing relay

Analysis of the regulation of plasmid transfer genes on the symbiotic plasmid pRL1JI in Rhizobium leguminosarum bv. viciae has revealed a novel regulatory relay that is specifically poised to detect an N-acyl-homoserine lactone (AHL) made by different cells (potential recipients of pRL1JI). Adjacent to the traI-trbBCDEJKLFGHI plasmid transfer operon on pRL1JI are two regulatory genes, bisR and traR, which encode LuxR-type quorum-sensing regulators required for conjugation. Potential recipients of pRL1JI induce the traI-trb operon and plasmid transfer via a quorum-sensing relay involving BisR, TraR and the traI-trb operon in donor cells. BisR induces expression of traR in response to N-(3-hydroxy-7-cis-tetradecenoyl)-l-homoserine lactone (3-OH-C14:1-HSL), which is produced by CinI in potential recipient strains. In donor strains (carrying pRL1JI), BisR represses the expression of the chromosomal gene cinI; this repression results in a very low level of formation of 3-OH-C14:1-HSL and hence relatively low levels of expression of traR and the traI-trb operon in strains carrying pRL1JI. However, if 3-OH-C14:1-HSL from potential recipients is present, then traR and plasmid transfer are induced. The induction of traR occurs at very low concentrations of 3-OH-C14:1-HSL (around 1 nm). TraR then induces the traI-trb operon in a quorum-sensing dependent manner in re-sponse to the TraI-made AHLs, N-(3-oxo-octanoyl)-l-homoserine lactone and N-(octanoyl)-l-homoserine lactone. The resulting autoinduction results in high levels of expression of the traI-trb operon. Premature expression of the traI-trb operon is reduced by TraM, which probably titres out TraR preventing expression of traI when there are low levels of traR expression. Expression of traR in stationary phase cells is limited by feedback inhibition mediated by TraI-made AHLs.     

Thio-functionalized carbohydrate thiosemicarbazones and evaluation of their anticancer activity

Thiosemicarbazides and their analogs have shown potential medical applications as antiviral, antibacterial and anticancer drugs. We designed, synthesized and evaluated in vitro anticancer activity against ovarian (A2780), cervix (HeLa), colon (LoVo), breast (MCF-7) and brain (MO59J) human cancer cell lines of seven novel compounds –S-glycosylated thiosemicarbazones. We assessed the cyto- and genotoxic properties of all novel compounds using a variety of methods including comet assay, XTT assay, various fluorescent assays and toxicology PathwayFinder expression array. We tried to evaluate their possible mechanism of action with particular attention to induction of DNA damage and repair, apoptosis, oxidative stress analysis and cellular response in terms of changes in gene expression. The most sensitive cell line was human ovarian cancer. The results revealed that the major activity against A2780 cancer cell line displayed by our compounds is induction of DNA damage. This effect is not associated with apoptosis or oxidative stress induction and the resulting damage will not lead to cell cycle arrest. We also observed up-expression of heat shock related genes and NQO1 gene in response to our compounds. The second effect seems to be specific to glycosylated S-bond compounds as we observed it earlier. Upregulation of heat shock protein encoding genes suggest that our compounds induce stressful conditions. The nature of this phenomena (heat shock, pH shift or hypoxia) needs further study.     

苏云金芽胞杆菌杀蚊基因在鱼腥藻中克隆和表达的初步研究

将苏云金芽胞杆菌以色列亚种的杀蚊晶体蛋白基因cry11A亚克隆到大肠杆菌－蓝藻的穿梭质粒载体pRL25C,然后用三亲本杂交的方法将重组质粒转移到一种具有固氮能力且可被蚊幼虫吞食的鱼腥藻（Anabaena）PCC7120中。Southernblot及Westernblot分析表明cry11A基因在鱼腥藻PCC7120中得以克隆和表达,但生物测定未能检测到转基因鱼腥藻对库蚊（Culex）的毒性,可能是因为带有苏云金芽胞杆菌自身启动子的Cry11A基因在鱼腥藻PCC7120中表达量不够高的缘故。     

Saponins do not affect the ecdysteroid receptor complex but cause membrane permeation in insect culture cell lines

This project studied the effects of four saponins with a triterpenoid (Quillajasaponaria saponin and aescin) or steroid structure (digitonin and diosgenin which is the deglycosylated form of dioscin) on insect cells, namely Schneider S2 cells of Drosophila melanogaster (Diptera). A series of different experiments were performed to investigate potential mechanisms of action by saponins with regard to ecdysteroid receptor (EcR) responsiveness, cell viability, cell membrane permeation, and induction of apoptosis with DNA fragmentation and caspase-3 like activity. Major results were that (1) exposure of S2 cells containing an EcR-based reporter construct to a concentration series of each saponin scored no EcR activation, while (2) a loss of ecdysteroid signaling was observed with median inhibitory concentrations (IC(50)'s) of 3-50 μM, and in parallel (3) a concentration-dependent change in loss of cell numbers in an cell viability assay with median effective concentrations (EC(50)'s) of 8-699 μM. In continuation, it was of interest that (4) a trypan blue assay with Q. saponaria saponin confirmed the cell membrane permeation effect leading to cell toxicity with a median lethal concentration (LC(50)) value of 44 μM, and interestingly this effect was very rapid. Another three interesting observations were that (5) exposure to 20E at 500 nM as used in the EcR-based report assay induced caspase-3 like activities which may help to explain the discrepancies between loss of EcR-responsiveness and cell viability, (6) low concentrations of saponins induced DNA fragmentation and caspase-3 like activities, confirming their potential to induce apoptosis, and (7) the saponin effects were counteracted with addition of cholesterol to the culture medium. In general the data obtained provide evidence that the anti-ecdysteroid action by saponins is not based on a true antagonistic interaction with EcR signaling, but can be explained by a cytotoxic action due to permeation of the insect cell membrane.     

A high-capacity assay for PPARgamma ligand regulation of endogenous aP2 expression in 3T3-L1 cells

A novel class of insulin-sensitizing agents, the thiazolidinedines (TZDs), has proven effective in the treatment of type 2 diabetes. These compounds, as well as a subclass of non-TZD insulin-sensitizing agents, have been shown to be peroxisome proliferator-activated receptor (PPAR) gamma agonists. PPARgamma plays a critical role in adipogenesis and PPARgamma agonists have been shown to induce adipocyte differentiation. Here, PPARgamma ligand activity has been assessed in murine 3T3-L1 cells, a commonly used in vitro model of adipogenesis, by measuring their ability to induce adipocyte fatty acid-binding protein (aP2) mRNA expression. In order to perform this task, we have developed a novel, multiwell assay for the direct detection of aP2 mRNA in cell lysates that is based on hybridization of mRNA to target-specific oligonucleotides. These oligonucleotide probes are conjugated to enzymes that efficiently process unique chemical substrates into robust fluorescent products. Ribosomal protein 36B4 mRNA, a gene whose expression is unaffected by adipogenesis, serves as the control in the assay. Two assay formats have been developed, a single analyte assay in which aP2 and 36B4 mRNA expression are assayed in separate lysate aliquots and a dual analyte assay which can measure aP2 and 36B4 mRNA simultaneously. Both forms of the assay have been used to quantify attomole levels of aP2 and 36B4 mRNAs in differentiating 3T3-L1 preadipocytes treated with PPARgamma agonists. The potencies of PPARgamma agonists determined by this novel methodology showed good correlation with those derived from aP2 mRNA slot-blot analysis and PPARgamma transactivation assays. We conclude that the aP2 single and dual analyte assays both provide specific and sensitive measurements of endogenous aP2 mRNA levels that can be used to assess the activity of PPARgamma ligands in 3T3-L1 cells. Since the assay obviates the need for RNA isolation and is performed in an automatable multiwell format, it can serve as a high-throughput, cell-based screen for the identification and characterization of PPARgamma modulators.     

Severity of oxidative stress generates different mechanisms of endothelial cell death

The role of reactive oxygen species (ROS) in the pathogenesis of vascular diseases is well established, but few data exist on the mechanisms by which ROS induce endothelial cell (EC) death. We examined the conditions and the mechanisms by which oxidative stress induces EC death, using cultured confluent bovine aortic ECs exposed for 30 min to different concentrations of hydroxyl radicals (HO*) generated by hydrogen peroxide (H(2)O(2)) in the presence of 100 microM ferrous sulfate (FeSO(4)). Cell viability assays, Hoechst DNA staining, TUNEL (TDT-mediated dUTP-biotin nick end-labeling) analysis, agarose gel electrophoresis and annexin V assay were used to determine the effect of HO* on the viability of ECs, and to distinguish between apoptosis and necrosis. The results showed that at concentrations of up to 0.1 mM H(2)O(2)/FeSO(4), the large majority of cells are viable, except for approximately 12.5% death, which occurs by apoptosis. At a concentration of 0.2 mM H(2)O(2), the cell viability is reduced to 66%, while EC apoptosis remained at comparable values (14%). At high oxidative stress (0.5 mM H(2)O(2)), the cell viability was drastically reduced (approximately 39%), and the prevalent form of death was necrosis; apoptosis accounted for only approximately 17%. Together, these data indicate that: (1) HO* induce EC death either by apoptosis or necrosis and (2) the mechanisms of EC death differ as a function of the concentration of HO. Thus, the same insult can cause apoptosis and/or necrosis, as a function of the intensity rather than the nature of the insult.     

Isolation, identification, and biological evaluation of HIF-1-modulating compounds from Brazilian green propolis

The tumor microenvironment is characterized by hypoxia, low-nutrient levels, and acidosis. A natural product chemistry-based approach was used to discover small molecules that modulate adaptive responses to a hypoxic microenvironment through the hypoxia-inducible factor (HIF)-1 signaling pathways. Five compounds, such as baccharin (3), beturetol (4), kaempferide (5), isosakuranetin (6), and drupanin (9), that modulate HIF-1-dependent luciferase activity were identified from Brazilian green propolis using reporter assay. Compounds 3, 9 and 5 reduced HIF-1-dependent luciferase activity. The cinnamic acid derivatives 3 and 9 significantly inhibited expression of the HIF-1α protein and HIF-1 downstream target genes such as glucose transporter 1, hexokinase 2, and vascular endothelial growth factor A. They also exhibited significant anti-angiogenic effects in the chick chorioallantoic membrane (CAM) assay at doses of 300 ng/CAM. On the other hand, flavonoids 4 and 6 induced HIF-1-dependent luciferase activity and expression of HIF-1 target genes under hypoxia. The contents (g/100g extract) of the HIF-1-modulating compounds in whole propolis ethanol extracts were also determined based on liquid chromatography-electrospray ionization mass spectrometry as 1.6 (3), 14.2 (4), 4.0 (5), 0.7 (6), and 0.7 (9), respectively. These small molecules screened from Brazilian green propolis may be useful as lead compounds for the development of novel therapies against ischemic cardiovascular disease and cancer based on their ability to induce or inhibit HIF-1 activity, respectively.     

Dihydromyricetin Reduced Bcl-2 Expression via p53 in Human Hepatoma HepG2 Cells

Dihydromyricetin (DHM) is a major active ingredient of flavonoids compounds. It exhibited anticancer activity and induced apoptosis in human hepatocellular carcinoma HepG2 cells according to our previous data. In this study, we investigated whether p53 is involved in DHM-triggered viability inhibition and apoptosis induction in cancer cells. MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay was employed to evaluate the viability of HepG2 cells after DHM treatment. Meanwhile, p53 small interfering RNA (siRNA) was adopted to silence p53 expression. Protein level of p53 and Bax/Bcl-2 were evaluated by western blot analysis. Cell counting assay showed that DHM inhibited HepG2 cell growth effectively in a time- and dose-dependent manner. P53 expression was significantly increased after DHM treatment, whereas Bcl-2 was reduced potently. Furthermore, after co-treatment with Pifithrin-α (PFT-α, p53 inhibitor), Bcl-2 expression was reversed. The expression of Bax was no significant change, which was also observed after p53 silence. These findings defined and supported a novel function that DHM could induce human hepatocellular carcinoma HepG2 cells apoptosis by up-regulating Bax/Bcl-2 expression via p53 signal pathway.     

COMP-Ang1 inhibits apoptosis as well as improves the attenuated osteogenic differentiation of mesenchymal stem cells induced by advanced glycation end products

Background

In the present study, we have investigated the possibility that cartilage oligomeric matrix protein angiopoietin1 (COMP-Ang1), important factor in angiogenesis, osteogenesis and the survival of mesenchymal stem cells (MSCs) through the Ang1/Tie2 pathway has beneficial effects on osteogenic differentiated cells (ODCs) from MSCs treated by advanced glycation end products (AGE), which are pathological factors of diabetes.

Methods

Primary culture of MSCs was used. For comparison analysis of AGE and COMP-Ang1 effects, we performed cell viability assay with each treated variety concentration for 24 h. Apoptosis rate and Caspase-3 activity were measured by each ELISA assay. To make sure with Ang1/Tie2 pathway, we performed small interfering RNA transfected to MSCs. Real-time RT-PCR was performed to identify ODCs marker genes. Immunoblotting was used to evaluate the expression of Tie2, AKT, p38 and ERK.

Results

Our results clearly demonstrate that COMP-Ang1 upregulates the phosphorylation of AKT and p38 by activating the Ang1/Tie2 signaling pathway, indicating that COMP-Ang1 affects both AGE-induced apoptosis and the attenuated osteogenic differentiation of MSCs through the p38/MAPK and PI3K/AKT pathways.

Conclusions

COMP-Ang1 improves cell viability and differentiation function of ODCs against AGE via Ang/Tie2 signaling pathway.

General significance

Our results suggest the potential importance of COMP-Ang1 as a new therapy for impaired bone formation that is associated with diabetes and advanced age.     

Expression of multiple unique rejection antigens on murine leukemia BALB/c RLmale symbol1 and the role of dominant Akt antigen for tumor escape.

Using the pRL1a Ag-loss RLmale symbol1 tumor variant cell line RM2-1, we demonstrated the presence of tumor Ags other than pRL1a that were recognized by CTLs on RLmale symbol1 cells. Semiallogeneic CB6F1 or syngeneic BALB/c CTLs generated against RM2-1 lysed RM2-1 and RLmale symbol1 cells to a similar extent, but no killing was observed with any other tumor or normal cells examined. Clonal analysis and sensitization with reversed phase-HPLC fractions revealed that there were Dd- and Ld-binding peptides recognized by RM2-1 CTLs. Lysis by bulk CTLs stimulated against RLmale symbol1 and limiting dilution analysis suggested that the pRL1a peptide was dominantly recognized to the RM2-1 peptides by CTLs on RLmale symbol1 cells. The rejection response against the parental RLmale symbol1 tumor was much less than that against RM2-1 cells in either CB6F1 or BALB/c mice, suggesting that the presence of altered Akt molecules from which the dominant pRL1a peptide was derived inhibited the rejection response against RLmale symbol1. Depletion of CD4 T cells caused the regression of RLmale symbol1 at the doses in which the tumor grew in untreated mice. The generation of pRL1a CTLs was inhibited in RLmale symbol1-bearing mice. Thus, immunoregulatory CD4 T cells were most likely activated by the altered Akt molecules and inhibited the efficient generation of CTLs against the dominant pRL1a Ag in RLmale symbol1.