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.

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.

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 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.

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.

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.

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.

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.

Hydroxyl-HIF2-alpha is potential therapeutic target for renal cell carcinomas.

Dormant cancer cells are deprivation-resistant, and cause a number of problems for therapeutic approaches for cancers. Renal cell carcinomas (RCCs) include deprivation-resistant cells that are resistant to various treatments. In this study, the specific characteristics of deprivation-resistant cells were transcriptionally identified by next generation sequencing. The hypoxia-inducible factors (HIF) transcription factor network was significantly enhanced in deprivation-resistant RCCs compared to the sensitive RCCs. Deprivation-resistant RCCs, that had lost Von Hippel-Lindau tumor suppressor expression, expressed hydroxyl-HIF2-alpha in the nucleus, but not sensitive-RCCs. Hydroxyl-HIF-alpha was also expressed in nuclei of RCC tissue samples. Knockdown for HIF2-alpha, but not HIF1-alpha, induced cell death related to a reduction in HIF-related gene expression in deprivation-resistant RCC cells. Chetomin, a nuclear HIF-inhibitor, induced marked level of cytotoxicity in deprivation-resistant cells, similar to the knockdown of HIF2-alpha. Therefore, hydroxyl-HIF2-alpha might be a potential therapeutic target for RCCs.

Therapeutic inhibition of mitochondrial function induces cell death in starvation-resistant renal cell carcinomas.

Renal cell carcinomas (RCC) have two types of cells for carbon metabolism and for cell signaling under nutrient-deprivation conditions, namely starvation-resistant and starvation-sensitive cells. Here, we evaluated the mitochondrial characteristics of these cell types and found that the resistant type possessed higher activities for both mitochondrial oxidative phosphorylation and glycolysis than the sensitive types. These higher activities were supported by the stored carbon, lipid and carbohydrate sources, and by a low level of mitochondrial reactive oxygen species (ROS) due to sustained SOD2 expression in the resistant RCC cells. In metastatic RCC cases, higher SOD2 expression was associated with a significantly shorter survival period. We found that treatment with the drugs etomoxir and buformin significantly reduced mitochondrial oxidative phosphorylation and induced cell death under glucose-deprivation conditions in starvation-resistant RCC cells. Our data suggest that inhibitory targeting of mitochondria might offer an effective therapeutic option for metastatic RCC that is resistant to current treatments.

Metastatic Prostatic Ductal Adenocarcinoma Successfully Treated with Docetaxel Chemotherapy: A Case Report.

A 68-year-old man presented with gross hematuria. A papillary urethral tumor adjacent to the verumontanum was found by cystourethroscopy. Serum prostate-specific antigen (PSA) was 3.246 ng/ml. A transurethral biopsy specimen was most suggestive of a primary urothelial carcinoma of the prostate, for which a radical cystoprostatectomy was performed. The final pathology was prostatic ductal adenocarcinoma with very focal acinar features (Gleason score 5 %plus; 4 = 9, pT3bN0M0). Local recurrence and pelvic bone metastases developed 17 months later, and his PSA rose to 10.806 ng/ml. He was treated with combined androgen blockade and radiation. Two years later, the lesion showed progressive growth. Treatment followed with docetaxel (70 mg/m(2) every 3 weeks) and prednisolone 5 mg twice daily. After 10 cycles of chemotherapy, all lesions disappeared and PSA decreased to <0.005 ng/ml. Three years after chemotherapy, he maintains a complete response without any additional treatments. Docetaxel chemotherapy can be an effective treatment for patients with recurrent prostatic ductal adenocarcinoma.

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.

Glucose deprivation induces G2/M transition-arrest and cell death in N-GlcNAc2-modified protein-producing renal carcinoma cells.

Some cancer cells can survive under glucose deprivation within the microenvironment of a tumor. Recently, we reported that N-linked (ß-N-acetylglucosamine)2 [N-GlcNAc2]-modified proteins induce G2/M arrest and cell death under glucose deprivation. Here, we investigated whether such a response to glucose deprivation contributes to the survival of renal cell carcinomas, which are sensitive to nutritional stress. Specifically, we analyzed seven renal carcinoma cell lines. Four of these cell lines produced N-GlcNAc2-modified proteins and led G2/M-phase arrest under glucose deprivation, leading to cell death. The remaining three cell lines did not produce N-GlcNAc2-modified proteins and undergo G1/S-phase arrest under glucose deprivation, leading to survival. The four dead cell lines displayed significant up-regulation in the UDP-GlcNAc biosynthesis pathway as well as increased phosphorylation of p53, which was not observed in the surviving three cell lines. In addition, the four dead cell lines showed prolonged up-regulated expression of ATF3, which is related to unfolded protein response (UPR), while the surviving three cell lines showed only transient up-regulation of ATF3. In this study, we demonstrated that the renal carcinoma cells which accumulate N-GlcNAc2-modified proteins under glucose deprivation do not survive with abnormaly prolonged UPR pathway. By contrast, renal carcinoma cells that do not accumulate N-GlcNAc2-modified proteins under these conditions survive. Morover, we demonstrated that buformin, a UPR inhibitor, efficiently reduced cell survival under conditions of glucose deprivation for both sensitive and resistant phenotypes. Further studies to clarify these findings will lead to the development of novel chemotherapeutic treatments for renal cancer.

Suppression of cerebral aneurysm formation in rats by a tumor necrosis factor-α inhibitor.

OBJECT: Although cerebral aneurysmal subarachnoid hemorrhage is a devastating disease for humans, effective medical treatments have not yet been established. Recent reports have shown that regression of some inflammatory-related mediators has protective effects in experimental cerebral aneurysm models. This study corroborated the effectiveness of tumor necrosis factor-α (TNF-α) inhibitor for experimentally induced cerebral aneurysms in rats. METHODS: Five-week-old male rats were prepared for induction of cerebral aneurysms and divided into 3 groups, 2 groups administered different concentrations of a TNF-α inhibitor (etanercept), and 1 control group. One month after aneurysm induction, 7-T MRI was performed. The TNF-α inhibitor groups received subcutaneous injection of 25 µg or 2.5 µg of etanercept, and the control group received subcutaneous injection of normal saline every week. The TNF-α inhibitor administrations were started at 1 month after aneurysm induction to evaluate its suppressive effects on preexisting cerebral aneurysms. Arterial circles of Willis were obtained and evaluated 3 months after aneurysm induction. RESULTS: Rats administered a TNF-α inhibitor experienced significant increases in media thickness and reductions in aneurysmal size compared with the control group. Immunohistochemical staining showed that treatment with a TNF-α inhibitor suppressed matrix metalloproteinase (MMP)-9 and inducible nitric oxide synthase (iNOS) expression through the luminal surface of the endothelial cell layer, the media and the adventitia at the site of aneurysmal formation, and the anterior cerebral artery-olfactory artery bifurcation. Quantitative polymerase chain reaction also showed suppression of MMP-9 and iNOS by TNF-α inhibitor administration. CONCLUSIONS: Therapeutic administration of a TNF-α inhibitor significantly reduced the formation of aneurysms in rats. These data also suggest that TNF-α suppression reduced some inflammatory-related mediators that are in the downstream pathway of nuclear factor-κB.

Analysis of new biomarkers for cholangiocarcinoma.

Cholangiocarcinoma is one of the most serious diseases in northeast Thailand, where its incidence is reported to be the highest in the world. We tried to develop a new method to detect cholangiocarcinoma in the early stages using serum proteins. We found that after fluorescent labeling of the sugar moiety of serum proteins, a new peak was identified, which might be a promising marker for cholangiocarcinoma.

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.

Study of global transcriptional changes of N-GlcNAc2 proteins-producing T24 bladder carcinoma cells under glucose deprivation.

Increased levels of N-linked (ß-N- acetylglucosamine)2 [N-GlcNAc2]-modified proteins have been recognized to be an effective response to glucose deprivation. In the first step of this study, using a next generation sequencer, we investigated the global transcriptional changes induced by glucose deprivation in a T24 bladder carcinoma cell line, producing N-GlcNAc2-modified proteins under glucose deprivation. Our transcriptome analysis revealed significant up-regulation of the UDP-GlcNAc biosynthesis pathway and unfolded protein response genes, and down-regulation of G2/M transition-related genes containing mitotic kinases. Our biological analysis confirmed that N-GlcNAc2-modified proteins were localized with BiP proteins in the ER. G2/M arrest was caused by glucose deprivation in T24 cells. Moreover, the knockdown of unfolded protein response genes induced the expressional recovery of mitotic kinases under glucose deprivation. Taken together, our results suggest N-GlcNAc2-modified proteins produced under glucose deprivation caused unfolded protein response in the ER, and that this response induced G2/M arrest.

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.

PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells.

We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation) of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca(2+) and PKC (protein kinase C) have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1) was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species.