A risk stratification model based on four novel biomarkers predicts prognosis for patients with renal cell carcinoma.

BACKGROUND: Accurate prediction of the prognosis of RCC using a single biomarker is challenging due to the genetic heterogeneity of the disease. However, it is essential to develop an accurate system to allow better patient selection for optimal treatment strategies. ARL4C, ECT2, SOD2, and STEAP3 are novel molecular biomarkers identified in earlier studies as survival-related genes by comprehensive analyses of 43 primary RCC tissues and RCC cell lines. METHODS: To develop a prognostic model based on these multiple biomarkers, the expression of four biomarkers ARL4C, ECT2, SOD2, and STEAP3 in primary RCC tissue were semi-quantitatively investigated by immunohistochemical analysis in an independent cohort of 97 patients who underwent nephrectomy, and the clinical significance of these biomarkers were analyzed by survival analysis using Kaplan-Meier curves. The prognostic model was constructed by calculation of the contribution score to prognosis of each biomarker on Cox regression analysis, and its prognostic performance was validated. RESULTS: Patients whose tumors had high expression of the individual biomarkers had shorter cancer-specific survival (CSS) from the time of primary nephrectomy. The prognostic model based on four biomarkers segregated the patients into a high- and low-risk scored group according to defined cut-off value. This approach was more robust in predicting CSS compared to each single biomarker alone in the total of 97 patients with RCC. Especially in the 36 metastatic RCC patients, our prognostic model could more accurately predict early events within 2 years of diagnosis of metastasis. In addition, high risk-scored patients with particular strong SOD2 expression had a much worse prognosis in 25 patients with metastatic RCC who were treated with molecular targeting agents. CONCLUSIONS: Our findings indicate that a prognostic model based on four novel biomarkers provides valuable data for prediction of clinical prognosis and useful information for considering the follow-up conditions and therapeutic strategies for patients with primary and metastatic RCC.

Abundance of mitochondrial superoxide dismutase is a negative predictive biomarker for endometriosis-associated ovarian cancers.

BACKGROUND: Endometrioid ovarian carcinoma and clear cell ovarian carcinoma are both classified as endometriosis-associated ovarian cancers (EAOCs). Despite the high rates of recurrence and mortality of EAOC, only a few prognostic biomarkers have been reported. Mitochondrial superoxide dismutase (SOD2) plays an important role in maintaining mitochondrial function through oxidative stress tolerance and contributes to chemotherapeutic resistance. METHODS: To clarify the clinical significance of SOD2 in EAOC, SOD2 expression was semi-quantitatively investigated by immunohistochemical analysis in 61 primary EAOC cases, and the correlations between SOD2 expression and clinicopathological data and survival were analyzed. RESULTS: Forty-six (75%) cases expressed high levels of SOD2. High SOD2 expression was associated with a poor prognosis on both univariate and multivariate analyses after adjusting for variables such as age, International Federation of Gynecology and Obstetrics (FIGO) stage, blood markers, histological type, and completion of treatment. There were 14 fatalities from 15 recurrences among 46 cases with high SOD2 expression. In contrast, only one recurrence and no fatalities were seen among 15 cases with low SOD2 expression. CONCLUSION: Increased SOD2 expression is a predictive biomarker for worse prognosis in EAOC. The therapeutic efficacy of the current standard therapeutic protocol for EAOC is limited; thus, mitochondrial SOD2 should be a therapeutic target for SOD2-abundant EAOC.

Mechanisms of Tumor Growth Inhibition by Depletion of γ-Glutamylcyclotransferase (GGCT): A Novel Molecular Target for Anticancer Therapy.

γ-Glutamylcyclotransferase (GGCT), which is one of the major enzymes involved in glutathione metabolism, is upregulated in a wide range of cancers-glioma, breast, lung, esophageal, gastric, colorectal, urinary bladder, prostate, cervical, ovarian cancers and osteosarcoma-and promotes cancer progression; its depletion leads to the suppression of proliferation, invasion, and migration of cancer cells. It has been demonstrated that the suppression or inhibition of GGCT has an antitumor effect in cancer-bearing xenograft mice. Based on these observations, GGCT is now recognized as a promising therapeutic target in various cancers. This review summarizes recent advances on the mechanisms of the antitumor activity of GGCT inhibition.

Proteome research in urothelial carcinoma.

In all creatures including humans, the molecules that function in accordance with the genetic code are mainly proteins. After completing the sequencing of the human genome, rapid progress has been made in proteome analysis. The primary structures of almost all proteins were determined by the human genome sequence. However, the whole picture of proteins cannot be elucidated because of alternative splicing and post-translational modifications. Therefore, genomic as well as systematic and comprehensive information of proteins is required. Modern methods of proteomics have dramatically improved the quality and speed of protein analysis. Developments in both bioinformatics and mass spectrometry have contributed to the technical improvement, making it possible to identify proteins in a short time with high accuracy even from a very small sample. In the field of cancer research, many studies of useful diagnostic and prognostic biomarkers using these proteomic technologies have been reported, and target molecules for treatment have been explored. The aim of the present review was to summarize the basic technologies of proteomics and recent research in the field of urothelial cancer obtained using proteomic methods.

Female-specific rectal carcinogenesis in cyclin D1b transgenic mice.

Human cyclin D1 generates two major isoforms via alternative splicing: cyclin D1a and cyclin D1b. Cyclin D1b is hardly expressed in normal tissues but is frequently expressed in certain types of cancer tissues. To clarify the oncogenic potential of cyclin D1b variant, we developed cyclin D1b transgenic (Tg) mice and analyzed their phenotypes. We detected rectal tumors in 63% (15/24) of the female Tg mice. All rectal tumors had the histological characteristics similar to human sessile serrated adenoma/polyps (SSA/Ps). Adenocarcinomas were also found in 53% (8/15) of the rectal tumors, suggesting that these adenocarcinomas originated from the SSA/P-like lesions. No rectal tumors were found in the ovariectomized female cyclin D1b Tg mice (0/10), indicating that ovarian hormones played a critical role in rectal carcinogenesis in these Tg mice. Both phosphorylation of Erk, without activating MEK, and expression of estrogen receptor ß were elevated in the rectal tumors of female cyclin D1b Tg mice compared with normal rectums of female wild-type mice. In addition, we established a cell line, D1bTgRT, derived from a rectal cancer of female Tg mouse. Small interfering RNA-induced cyclin D1b knockdown in this cell line suppressed Erk phosphorylation, anchorage-independent growth, cell invasiveness and tumorigenicity in nude mice. In humans, expression of cyclin D1b messenger RNA was detected in 17% (1/6) of colorectal cancer cell lines and 9.7% (3/31) of colorectal cancer tissues. Taken together, these results indicate that cyclin D1b expression contributes to the female- specific rectal carcinogenesis in mouse model.

A global transcriptome analysis of a dog model of congestive heart failure with the human genome as a reference.

BACKGROUND: The global molecular changes in cardiac tissue during congestive heart failure (CHF) have not been fully examined. Transcriptome analysis with the use of next-generation sequencers is a useful tool for elucidating the pathogenesis of CHF. Although there are some advantages in a dog CHF model, transcriptome analyses in dogs are limited by the relative lack of genomic information. METHODS AND RESULTS: The transcriptome analysis of hearts from dogs with CHF was conducted with the use of a genome analyzer and the Casava software. The mRNA sequence reads showed alignments with ∼800 of 1,019 genes from the dog reference database. On the other hand, the reads aligned with ∼15,000 of the 21,407 genes in the hg19 human reference database. The correlation of expressed genes was extremely high (r = 0.93; P < .0001) between the dog and human databases. A pathway analysis using the hg19 reference revealed increased expression of p53 pathway-related (P < 10(-10)) and inflammatory interleukin-related (P < 10(-10)) genes in the CHF model. CONCLUSIONS: The use of the human genome as a reference in global transcriptome analyses of dogs is a useful approach for investigating diseases such as CHF. Such an approach would also be useful for analyzing disease models in other experimental animals.

Transcriptome analysis of a dog model of congestive heart failure shows that collagen-related 2-oxoglutarate-dependent dioxygenases contribute to heart failure.

Fibrosis is an important pathological mechanism in heart failure (HF) and is associated with poor prognosis. We analyzed fibrosis in HF patients using transcriptomic data. Genes differentially expressed between normal control and congestive HF (CHF) dogs included P3H1, P3H2, P3H4, P4HA2, PLOD1 and PLOD3, which belong to the 2-oxoglutarate-dependent dioxygenases (2OGD) superfamily that stabilizes collagen during fibrosis. Quantitative polymerase chain reaction analysis demonstrated 2OGD gene expression was increased in CHF samples compared with normal left ventricle (LV) samples. 2OGD gene expression was repressed in angiotensin converting enzyme inhibitor-treated samples. These genes, activated the hydroxylation of proline or lysin residues of procollagen mediated by 2-oxoglutaric acid and O2, produce succinic acid and CO2. Metabolic analysis demonstrated the concentration of succinic acid was significantly increased in CHF samples compared with normal LV samples. Fibrosis was induced in human cardiac fibroblasts by TGF-ß1 treatment. After treatment, the gene and protein expressions of 2OGD, the concentration of succinic acid, and the oxygen consumption rate were increased compared with no treatment. This is the first study to show that collagen-related 2OGD genes contribute to HF during the induction of fibrosis and might be potential therapeutic targets for fibrosis and HF.

Characterization of the rapidly activating delayed rectifier potassium current, I (Kr), in HL-1 mouse atrial myocytes.

HL-1 is the adult murine cardiac cell line that can be passaged repeatedly in vitro without losing differentiated phenotype. The present study was designed to characterize the rapidly activating delayed rectifier potassium current, I (Kr), endogenously expressed in HL-1 cells using the whole-cell patch-clamp technique. In the presence of nisoldipine, depolarizing voltage steps applied from a holding potential of -50 mV evoked the time-dependent outward current, followed by slowly decaying outward tail current upon return to the holding potential. The amplitude of the current increased with depolarizations up to 0 mV but then progressively decreased with further depolarizations. The time-dependent outward current as well as the tail current were highly sensitive to block by E-4031 and dofetilide (IC(50) of 21.1 and 15.1 nM, respectively) and almost totally abolished by micromolar concentrations of each drug, suggesting that most of the outward current in HL-1 cells was attributable to I (Kr). The magnitude of I (Kr) available from HL-1 cells (18.1 +/- 1.5 pA pF(-1)) was sufficient for reliable measurements of various gating parameters. RT-PCR and Western blot analysis revealed the expression of alternatively spliced forms of mouse ether-a-go-go-related genes (mERG1), the full-length mERG1a and the N-terminally truncated mERG1b isoforms. Knockdown of mERG1 transcripts with small interfering RNA (siRNA) dramatically reduced I (Kr) amplitude, confirming the molecular link of mERG1 and I (Kr) in HL-1 cells. These findings demonstrate that HL-1 cells possess I (Kr) with properties comparable to those in native cardiac I (Kr) and provide an experimental model suitable for studies of I (Kr) channels.

Cyclin D1b variant promotes cell invasiveness independent of binding to CDK4 in human bladder cancer cells.

Alternative splicing in the cyclin D1 gene produces cyclin D1b variant which lacks a C-terminal region containing the threonine-286 (T286) phosphorylation site required for nuclear export. We have shown that the expression of the cyclin D1b variant is detected in about 60% of human bladder cancer tissues (15/26) and cell lines (3/5). To examine the role of the cyclin D1b variant in bladder carcinogenesis, we introduced wild-type cyclin D1a, cyclin D1b variant or mutant cyclin D1-T286A cDNAs into a human bladder cancer cell line, SBT991, in which cyclin D1b transcript was not expressed, and compared their oncogenic activities. Ectopic expression of cyclin D1b promoted cell invasiveness and anchorage-independent growth of the cancer cells. On the other hand, cyclin D1-T286A enhanced anchorage-independent growth, but did not promote cell invasiveness. The amount of nuclear-localized cyclin D1b and cyclin D1-T286A was higher than that of nuclear-localized cyclin D1a. In addition, introduction of siRNA specific for cyclin D1b into cells of the T24 bladder cancer cell line, in which cyclin D1b transcript was expressed, significantly suppressed cell invasiveness. Immunoprecipitation analysis revealed that cyclin D1a and cyclin D1-T286A could bind to cyclin-dependent kinase 4 (CDK4) but cyclin D1b has lost its capacity to associate with CDK4. Unlike cyclin D1a and cyclin D1-T286A, expression of cyclin D1b did not enhance phosphorylation of Rb protein in SBT991 cells. These results indicate that cyclin D1b promotes cell invasiveness independent of binding to CDK4 to enhance Rb phosphorylation.

Suppression of cell invasiveness by periostin via TAB1/TAK1.

We have previously shown that the expression of periostin is significantly downregulated in human bladder cancer tissues and that periostin suppresses cell invasiveness and metastasis of cancer cells. To clarify the molecular mechanism of this suppression by periostin, we searched for periostin-binding proteins and identified TAB1, which interacts with and activates TAK1, by mass analysis of proteins co-precipitated with periostin in 293T cells expressing periostin. The association between periostin and TAB1 was confirmed by a pulldown assay in 293T cells co-tranfected with expression plasmids of periostin, TAB1 and TAK1. TAK1 was also co-precipitated with periostin in this assay. Co-transfection experiments in 293T also showed that periostin could activate TAK1. Introduction of siRNA for TAB1 suppressed TAK1 activation by periostin. Analyses with deletion mutants of periostin revealed that the C-terminal region of periostin was necessary and sufficient for the association with TAB1 and the TAK1 activation. The suppression of invasiveness by periostin was attenuated by siRNA targeting TAK1 or TAB1 in 293T (human embryonic kidney) and T24 (human bladder carcinoma) cell lines. These findings indicate that periostin is involved in the suppression of cell invasiveness via the TAB1/TAK1 signaling pathway.

Urinary calreticulin in the diagnosis of bladder urothelial carcinoma.

OBJECTIVES: To evaluate the potential suitability of calreticulin (CRT) as a urinary marker for bladder cancer. METHODS: Urine specimens were collected from patients with histologically confirmed bladder urothelial carcinoma (Group 1; n = 109), urological patients without urothelial carcinoma (Group 2; n = 60), and non-urological patients (Group 3; n = 40). We developed an enzyme-linked immunosorbent assay (ELISA) procedure using commercially available anti-CRT mono/polyclonal antibodies, and then measured the concentration of urinary CRT. RESULTS: Urinary CRT concentration of group 1 was significantly higher than group 2 and 3 (Mann-Whitney U-test, P < 0.001). Groups 2 and 3 were joined together and considered as a non-bladder cancer group (n = 100), and a cutoff value (2.85 ng/mL) was determined using receiver operating characteristic (ROC) analysis. The sensitivity, specificity, and the area under the curve were 67.9%, 80.0%, and 0.742, respectively. The overall sensitivity of voided urine cytology (VUC) was 39.0% (n = 105), and the sensitivity of urinary CRT was significantly superior to VUC (McNemar test, P < 0.001). Higher sensitivity was observed especially in Ta, G1-2, and

Analysis of the characteristics of chemotherapy-resistant renal cell carcinomas based on global transcriptional analysis of their tissues and cell lines.

Starvation-resistant renal cell carcinoma (RCC) cell lines are considered dormant-state cells that survive even under glucose starvation. The cellular biological and global transcriptional analysis using these cells identified potential markers of chemotherapy-resistant RCC and therapeutic agent candidates. Recently, we showed that ARL4C was a predictive biomarker for poor prognosis in patients with chemotherapy-resistant RCC by the global transcriptional analysis of patient primary tissues. The objective of this study was to identify the characteristics of chemotherapy-resistant RCC by the global transcriptional analysis of primary tissues of patients with RCC and RCC cell lines. The connective global transcriptional analysis showed that two starvation-resistant RCC cell lines, SW839 and KMRC-1, were strongly correlated to tissues of patients with chemotherapy-resistant RCC and showed high expressions of invasive- and proliferation-related genes. We found fibronectin (FN1) expression was a predictive biomarker in some patients with chemotherapy-resistant RCC, which especially correlated with two starvation-resistant RCC cell lines. These results indicate these cell lines emulate chemotherapy-resistant RCC and might be useful in the search for markers to predict poor prognosis and in the development of therapeutic agents and their index markers for chemotherapy-resistant RCCs.

ADP-ribosylation factor-like 4C predicts worse prognosis in endometriosis-associated ovarian cancers.

BACKGROUND: Endometrioid ovarian carcinoma and clear cell ovarian carcinoma are both classified as endometriosis-associated ovarian cancer (EAOC). Despite the high rates of recurrence and mortality of EAOC, no prognostic biomarkers have been determined. ADP-ribosylation factor-like protein 4C (ARL4C) has been reported to be involved in various tumor progression processes, but its clinical significance for predicting prognosis in EAOC cases has never been studied. OBJECTIVE: The present study aimed to determine the clinical significance of ARL4C expression in EAOC prognosis. METHODS: ARL4C expression was semi-quantitatively evaluated via immunohistochemistry in 61 EAOC patients, and the correlations between ARL4C expression and clinicopathological data and survival were statistically analyzed. RESULTS: Thirty-six (59%) cases had high levels of ARL4C, which was related to worse 5-year overall survival (OS) (log-rank test, p= 0.036). In multivariate Cox proportional hazard model, high ARL4C expression was a significantly independent predictive factor for worse 5-year OS (hazard ratio = 12.048, p= 0.0201) and 5-year PFS (hazard ratio = 8.130, p= 0.0036). CONCLUSIONS: ARL4C is a biomarker for worse prognosis and a novel therapeutic target in EAOC.

ADP-ribosylation factor-like 4C is a predictive biomarker of poor prognosis in patients with renal cell carcinoma.

Renal cell carcinoma (RCC) has the high mortality rate among urological malignancies. The development of RCC cannot be effectively reduced by molecular targeted therapies based on nutrient deprivation, such as inhibition of tumor angiogenesis. The objective of this study was to identify predictive biomarkers of poor prognosis and therapeutic molecular targets in patients with RCC. Two independent cohorts were analyzed in the present study. Global transcriptomics were used in the first cohort (43 patients with RCC) to identify biomarker genes. Each identified biomarker was subsequently analyzed using immunohistochemistry in the second cohort (97 patients with RCC). Following transcriptomics, biomarkers were evaluated using receiver operating characteristic curve analysis. Predictive accuracy for poor survivals was assessed using the log-rank test and Cox multivariate analysis. Global transcriptomic analysis in the first cohort focusing on cases with survival periods <2 years after initial diagnosis of metastasis detected seven overexpressed genes, which correlated with poor prognosis. The ADP-ribosylation factor-like 4C (ARL4C) exhibited the best accuracy in the receiver operating characteristic curve analysis and predicted poor survival in the first cohort (log-rank test, P<0.001; Cox multivariate analysis, hazard ratio =167, P=0.005). In the second cohort, the expression of ARL4C was semi-quantitatively evaluated through immunohistochemistry. Twenty-seven cases showed high levels of ARL4C, confirming a significant association with shorter survivals (log-rank test, P<0.001; Cox multivariate analysis, hazard ratio =9.41, P=0.004). ARL4C was shown to be a predictive biomarker for poor prognosis in patients with RCC and may be a novel target in the treatment of RCC.

Role of alternative splicing of periostin in human bladder carcinogenesis.

We have previously reported that the expression of periostin mRNA is significantly repressed in human bladder cancer tissues, and that periostin plays a role as a suppressive factor for invasion and metastasis in the progression of human bladder cancers. In this study, to clarify the role of alternative splicing of periostin in human bladder carcinogenesis, we examined the expression of wild-type (WT) and spliced variants of periostin mRNA in normal bladder and bladder cancer tissues. Although both WT and spliced periostin mRNA were expressed in all normal bladder tissues examined, no WT periostin mRNA was detected in the examined transitional cell carcinomas (TCCs) of the bladder (0/23) or in bladder cancer cell lines (0/6). Spliced variants of periostin were detected in 48% (11/23) of TCC tissues and 33% (2/6) of bladder cancer cell lines. Two types of spliced periostin (Variants I and II) were successfully isolated from bladder cancer tissues, but Variant I, which is predominantly expressed in bladder cancer tissues, did not show suppressor activity on in vitro invasiveness and in vivo metastasis of cancer cells. Immunohistochemical analysis indicated that strong belt-like expression of periostin protein was observed in the stroma just beneath the normal bladder epithelium, while it was mostly attenuated in bladder cancer tissues. These results indicate that the loss of WT periostin by down-regulation and/or alternative splicing, which produces Variant I, is closely correlated with the development of bladder cancer.

Superoxide dismutase 2 expression can predict prognosis of renal cell carcinoma patients.

BACKGROUND AND OBJECTIVE: Renal cell carcinoma (RCC) is the urological malignancy with the highest mortality rate and is increasing in incidence. The prognostic and predictive biomarkers are highly desired. This study aims to investigate the significance of superoxide dismutase 2 (SOD2) as a clinical biomarker in patients with renal cell carcinomas. METHODS: A cohort of 97 patients with RCC was analyzed retrospectively using various clinical parameters and SOD2 expression by immunohistochemistry. RESULTS: Cases with stronger SOD2 positivity of the tumor in comparison to the adjacent normal renal tubule by immunohistochemistry were categorized as high SOD2 and were associated with worse overall survivals (p= 0.005). In particular, in cases with metastatic RCC, high SOD2 expression in the tumors was significantly associated with a worse overall survival (p= 0.001), and the maximum critical risk. Treatment with current molecular targeting therapies did not improve the prognoses of cases with metastatic or recurrent RCC. CONCLUSIONS: High SOD2 expression can be predictive of a poor clinical outcome and be clinically useful in the follow-up of metastatic RCC. Therapeutics for metastatic RCCs require further improvement, such as supplementary administration of agents targeting mitochondrial SOD2.

Abundance of TRAIL attenuated by HIF2α and c-FLIP affects malignancy in renal cell carcinomas.

Dormant cancer cells are starvation-resistant leading to problems in the management of cancer. In renal cell carcinomas (RCCs), starvation-resistant cells are resistant to various currently available therapies. However, targeting hypoxia inducible factor 2-alpha (HIF2-alpha) induces cell death in dormant-like/starvation-resistant RCCs. This study showed that the apoptotic cell death caused by tumor necrosis factor (TNF)-related apoptosis-induced ligand (TNFSF10/TRAIL) was attenuated by CASP8 and FADD-like apoptosis regulator (CFLAR/c-FLIP) following HIF2-alpha activation, despite the high expression of TRAIL in such RCCs. Knockdowns of TRAIL averted apoptotic cell death caused by HIF2-alpha inhibition in starvation-resistant RCCs. Knockdowns of both HIF2-alpha and c-FLIP augmented apoptotic cell death, whereas overexpression of c-FLIP completely averted apoptosis. In addition, high abundance of TRAIL was correlated with poor prognosis in patients with RCC, suggesting that TRAIL, followed by HIF2-alpha and c-FLIP, play a role in the survival and/or progression of malignant RCCs.

Angiotensin II potentiates the slow component of delayed rectifier K+ current via the AT1 receptor in guinea pig atrial myocytes.

BACKGROUND: Angiotensin II (Ang II) has diverse actions on cardiac electrical activity. Little information is available, however, regarding immediate electrophysiological effects of Ang II on cardiac repolarization. METHODS AND RESULTS: The present study investigated the immediate effects of Ang II on the slow component of delayed rectifier K+ current (IKs) and action potentials in guinea pig atrial myocytes using the whole-cell patch-clamp technique. Bath application of Ang II increased the amplitude of IKs (EC50, 6.16 nmol/L) concentration dependently. The stable analogue Sar1-Ang II was also effective at increasing IKs. The voltage dependence of IKs activation and the kinetics of deactivation were not significantly affected by these agonists. The enhancement of IKs was blocked by the Ang II type 1 (AT1) receptor antagonist valsartan (1 micromol/L) and was markedly attenuated by inclusion of GDPbetaS (2 mmol/L) in the pipette, indicating an involvement of G protein-coupled AT(1) receptor. The stimulatory effect was also significantly reduced by the phospholipase C inhibitor compound 48/80 (100 micromol/L) and the protein kinase C inhibitors bisindolylmaleimide I (200 nmol/L) and H-7 (10 micromol/L), suggesting that AT1 receptor acts through phospholipase C-protein kinase C signaling cascade to potentiate I(Ks). As expected from its stimulatory action on IKs, Sar1-Ang II markedly shortened the action potential duration, which could be reversed by valsartan. CONCLUSIONS: The potentiation of IKs via AT1 stimulation in atrial myocytes, accompanied by a shortening of the action potential duration, suggests a potential mechanism by which elevated levels of Ang II may promote atrial fibrillation in heart failure and warrants further investigation.

GADD34 inhibits mammalian target of rapamycin signaling via tuberous sclerosis complex and controls cell survival under bioenergetic stress.

Cells regulate the rate of protein synthesis during conditions of cell stress to adapt to environmental changes. However, the molecular interactions between signaling pathways controlling translation and the cellular response to stress remain to be elucidated. Here, we show that the expression of growth arrest and DNA damage protein 34 (GADD34) is induced by energy depletion and that the expression of this protein protects cells from apoptotic cell death. During conditions of cell stress, GADD34 forms a stable complex with tuberous sclerosis complex (TSC) 1/2, causes TSC2 dephosphorylation, and inhibits signaling by mammalian target of the rapamycin (mTOR). These findings demonstrate that crosstalk between GADD34 and the mTOR signaling pathways contributes to the response of the protein synthetic machinery to environmental stress. GADD34 may find clinical potential as a target drug for the treatment of mTOR-associated diseases.

Effect of ATP on preadipocyte migration and adipocyte differentiation by activating P2Y receptors in 3T3-L1 cells.

The effect of extracellular ATP on adipogenesis was investigated using the mouse 3T3-L1 cell line. Incubation of cells with ATP (1-100 microM) for 5 min induced actin filament reorganization and membrane ruffling mediated through P2Y receptors. Enhancement of preadipocyte migration into fat cell clusters is one of the essential processes of adipose tissue development in vivo and cell migration assays revealed that stimulation of P2Y receptors enhanced chemokinesis (migration) in a concentration dependent manner. In this cell line, growth arrest is required before initiation of differentiation and growth-arrested post-confluent cells can be converted into adipocytes by the presence of the adipogenic hormones dexamethasone, 3-isobutyl-1-methylxanthine and insulin. On the other hand, those hormones alone do not trigger differentiation in proliferating cells. ATP did not induce differentiation when applied alone to either proliferating or postconfluent cells. By contrast, proliferating cells (density <50%) preincubated with ATP for 5 min and subsequently given the adipogenic hormones in the continued presence of ATP, underwent adipocyte differentiation mediated through phospholipase C-coupled P2Y receptors. These adipocytes were found to show very similar characteristics, including morphology and intracellular triacylglycerol accumulation compared with adipocytes differentiated from post-confluent preadipocytes with those adipogenic hormones. When proliferating cells were preincubated with ATP before the addition of the adipogenic hormones, gene expression of aP2 (adipose protein 2) was markedly increased within 6 days, whereas without ATP pretreatment the expression level stayed very low. These results suggest that extracellular ATP renders preadipocytes responsive to adipogenic hormones during the growth phase.