Purification of Recombinant DHHC Proteins Using an Insect Cell Expression System.

DHHC enzymes are a family of integral membrane proteins that catalyze the posttranslational addition of palmitate, a 16-carbon fatty acid, onto a cysteine residue of a protein. While the library of identified palmitoylated proteins has grown tremendously over the years, biochemical and mechanistic studies on DHHC proteins are challenged by the innate difficulty of purifying the enzyme in large amounts. Here we describe our protocol for preparing recombinant DHHC proteins tagged with a hexahistidine sequence and a FLAG epitope that aid in the purification. This procedure has been tested successfully in purifying several members of the enzyme family; DHHC3 and its catalytically inactive cysteine mutant, DHHS3 are used as examples. The recombinant protein is extracted from whole cell lysates using the detergent dodecylmaltoside (DDM) and is subjected to a two-column purification. Homogeneity and monodispersity of the purified protein are checked by size exclusion chromatography (SEC). A preparation from a 400-mL infection of Sf9 insect cell culture typically yields 0.5 mg of DHHC3 and 1.0 mg of catalytically inactive DHHS3. Both forms appear monodisperse up to a concentration of 1 mg/mL by SEC.

Rewiring of cisplatin-resistant bladder cancer cells through epigenetic regulation of genes involved in amino acid metabolism.

Alterations in DNA methylation are important epigenetic markers in bladder cancer (BC). These epigenome modifications may drive the mechanisms of aggressive chemo-resistant BC. Clinicopathological biomarkers that indicate chemotherapeutic resistance are critical for better assessing treatment strategies for individual patients. Thus, in this study, we aimed to determine whether DNA methylation of certain metabolic enzymes is significantly altered in cisplatin-resistant BC cells. Methods: To characterize CpG methylation and nucleosome accessibility in cisplatin-resistant BC cells, the Illumina Infinium HM450 DNA methylation assay was performed. Perturbed gene expression was found to be associated with cisplatin resistance, and the biological roles of spermidine/spermine N1-acetyltransferase (SAT1) and argininosuccinate synthase 1 (ASS1) were further studied using qRT-PCR analysis and various cell biology assays, including western blot. Results:ASS1 and SAT1, genes for amino acid and polyamine metabolism catalysts, respectively, were found to be vastly hypermethylated, resulting in greatly downregulated expression. ASS1 expression is of particular interest because prior studies have demonstrated its potential association with BC stage and recurrence. In regard to chemoresistance, we found that aberrant expression or induced stimulation of SAT1 restored cisplatin sensitivity in the cell culture system. We also found that the addition of exogenous arginine deiminase through administration of ADI-PEG 20 (pegylated arginine deiminase) increased ASS1 expression and enhanced cisplatin's apoptotic effects. Conclusions: Our study demonstrates a novel mechanistic link between the epigenetic perturbation of SAT1 and ASS1 and cancer metabolism in cisplatin-resistant bladder cancer cells. These findings suggest potential utility of SAT1 and ASS1 as predictive biomarkers in re-sensitizing bladder cancer to chemotherapy and personalizing therapy.

Extracellular polyamines-induced proliferation and migration of cancer cells by ODC, SSAT, and Akt1-mediated pathway.

High levels of polyamines were observed and were related to a poor prognosis in cancer patients. However, the mechanism is not obvious. The aim of this study is to mimic the extracellular polyamines in a tumor microenviroment and to explore the role of extracellular polyamines in the proliferation and migration of cancer cells. Three different concentrations of polyamines composed of putrescine, spermidine, and spermine were used. Colony formation assay, wound healing assay, and transwell migration assay were performed. Akt1-overexpression cells were constructed. The related protein expression was examined using a western blot. In this study, polyamines promoted colony formation and cell migration in a concentration-dependent and time-dependent manner. Polyamines upregulated the expression of ornithine decarboxylase (ODC), SSAT, Akt1, Akt, hypoxia-inducible factors-1α, vascular endothelial growth factor, and matrix metalloproteinases, and downregulated p27 expression. The effects of combination of polyamines and Akt1 overexpression on colony formation and migration were more obvious than the effects of Akt1 overexpression alone. In Akt1-overexpression cells, polyamines also upregulated the expression of ODC, SSAT, hypoxia-inducible factors-1α, vascular endothelial growth factor, and matrix metalloproteinases and downregulated p27 expression. In conclusion, extracellular polyamines induced proliferation and cancer cell migration by inducing ODC and SSAT expression, and the Akt1-mediated pathway.

MCM2 and TIP30 are prognostic markers in squamous cell/adenosquamous carcinoma and adenocarcinoma of the gallbladder.

The clinicopathological and biological characteristics of squamous cell/adenosquamous carcinoma (SC/ASC) of the gallbladder remain to be fully elucidated, due to the fact that it is a rare gallbladder cancer subtype. In the current study, the expression of minichromosome maintenance complex component 2 (MCM2) and HIV­1 tat interactive protein 2 (TIP30) was measured in 46 cases of SC/ASC and 80 adenocarcinomas (AC) using immunohistochemistry. Positive MCM2 and negative TIP30 expression were significantly associated with large tumor size, high TNM stage, invasion, lymph node metastasis and lack of surgical curability in SC/ASC and AC. Positive MCM2 and negative TIP30 expression were significantly associated with poor differentiation in AC, whereas only MCM2 was correlated with differentiation in SC/ASC. Univariate Kaplan­Meier analysis demonstrated that positive MCM2 and negative TIP30 expression, the degree of differentiation, tumor size, TNM stage, invasion, lymph node metastasis and surgical curability were significantly associated with post­operative survival in patients with SC/ASC and AC. Multivariate Cox regression analysis demonstrated that positive MCM2 and negative TIP30 expression, the degree of differentiation, tumor size, TNM stage, invasion, lymph node metastasis and lack of surgical curability were also independent predictors of poor prognosis in patients with SC/ASC and AC. These data suggest that positive MCM2 and negative TIP30 expression are closely correlated with the clinical, pathological and biological parameters, in addition to poor prognosis in patients with gallbladder cancer.

Metformin inhibits the prometastatic effect of sorafenib in hepatocellular carcinoma by upregulating the expression of TIP30.

We previously found that a low dose of sorafenib had a prometastatic effect on hepatocellular carcinoma (HCC), which was caused by downregulation of TIP30 expression. More recently, metformin has been shown to have potential as a preventive and therapeutic agent for different cancers, including HCC. This study evaluated whether the combination of sorafenib and metformin is sufficient to revert the expression of TIP30, thereby simultaneously reducing lung metastasis and improving survival. Our data show that the combination of sorafenib and metformin inhibits proliferation and invasion in vitro, prolongs median survival, and reduces lung metastasis of HCC in vivo. This effect is closely associated with the upregulation of TIP30, partly through activating AMP-activated protein kinase. Thioredoxin, a prometastasis factor, is negatively regulated by TIP30 and plays an essential role during the process of HCC metastasis. Overall, our results suggest that metformin might be a potent enhancer for the treatment of HCC by using sorafenib.

The inhibition of PI3K and NFκB promoted curcumin-induced cell cycle arrest at G2/M via altering polyamine metabolism in Bcl-2 overexpressing MCF-7 breast cancer cells.

Bcl-2 protein has been contributed with number of genes which are involved in oncogenesis. Among the many targets of Bcl-2, NFκB have potential role in induction of cell cycle arrest. Curcumin has potential therapeutic effects against breast cancer through multiple signaling pathways. In this study, we investigated the role of curcumin in induction of cell cycle arrest via regulating of NFκB and polyamine biosynthesis in wt and Bcl-2+ MCF-7 cells. To examine the effect of curcumin on cell cycle regulatory proteins, PI3K/Akt, NFκB pathways and polyamine catabolism, we performed immunoblotting assay. In addition, cell cycle analysis was performed by flow cytometry. The results indicated that curcumin induced cell cycle arrest at G2/M phase by downregulation of cyclin B1 and Cdc2 and inhibited colony formation in MCF-7wt cells. However, Bcl-2 overexpression prevented the inhibition of cell cycle associated proteins after curcumin treatment. The combination of LY294002, PI3K inhibitor, and curcumin induced cell cycle arrest by decreasing CDK4, CDK2 and cyclin E2 in Bcl-2+ MCF-7 cells. Moreover, LY294002 further inhibited the phosphorylation of Akt in Bcl-2+ MCF-7 cells. Curcumin could suppress the nuclear transport of NFκB through decreasing the interaction of P-IκB-NFκB. The combination of wedelolactone, NFκB inhibitor, and curcumin acted different on SSAT expression in wt MCF-7 and Bcl-2+ MCF-7 cells. NFκB inhibition increased the SSAT after curcumin treatment in Bcl-2 overexpressed MCF-7 cells. Inhibition of NFκB activity as well as suppression of ROS generation with NAC resulted in the partial relief of cells from G2/M checkpoint after curcumin treatment in wt MCF-7 cells. In conclusion, the potential role of curcumin in induction of cell cycle arrest is related with NFκB-regulated polyamine biosynthesis.

GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development.

Human chromosome 21 is known to be associated with the high risk of hematological malignancy but with resistance to breast cancer in the study of Down syndrome. In human cancers, we previously observed the significant alterations of the protein expression encoded by the ganp/MCM3AP gene on human chromosome 21q22.3. Here, we investigated GANP protein alterations in human breast cancer samples (416 cases) at various stages by immunohistochemical analysis. This cohort study clearly showed that expression of GANP is significantly decreased in human breast cancer cases with poor prognosis as an independent risk factor (relapse-free survival, hazard ratio = 2.37, 95% confidence interval, 1.27-4.42, P = 0.007 [univariate analysis]; hazard ratio = 2.70, 95% confidence interval, 1.42-5.13, P = 0.002 [multivariate analysis]). To investigate whether the altered GANP expression is associated with mammary tumorigenesis, we created mutant mice that were conditionally deficient in the ganp/MCM3AP gene using wap-cre recombinase transgenic mice. Mammary gland tumors occurred at a very high incidence in female mammary gland-specific GANP-deficient mice after severe impairment of mammary gland development during pregnancy. Moreover, tumor development also occurred in female post parous GANP-heterodeficient mice. GANP has a significant role in the suppression of DNA damage caused by estrogen in human breast cancer cell lines. These results indicated that the GANP protein is associated with breast cancer resistance.

Increased expression of ESCO1 is correlated with poor patient survival and its role in human bladder cancer.

There is increasing evidence suggesting that establishment of sister chromatid cohesion N-acetyltransferase 1 (ESCO1) was involved in tumorigenesis. However, its role in bladder cancer remains unclear. In this study, we aimed to study the clinical correlation and biological significance of ESCO1 in bladder cancer. Our results showed that ESCO1 was significantly over-expressed in bladder cancer tissues compared with that in adjacent normal tissues. And, increased ESCO1 expression was significantly associated with higher grade (P < 0.001), higher tumor stage (P = 0.014), and multifocality (P = 0.042). Kaplan-Meier analysis and Cox proportional hazards model were performed to determine the prognostic significance of ESCO1, and the results showed that ESCO1 is a useful prognostic marker for bladder cancer patients. Moreover, we found that ESCO1 knockdown inhibited the growth, migration, and invasion of bladder cancer cells. In conclusion, our findings indicated that ESCO1 may play an important role in human bladder cancer, and ESCO1 might serve as a novel target and prognosis factor for human bladder cancer.

Downregulation of HTATIP2 expression is associated with promoter methylation and poor prognosis in glioma.

Glioma is an aggressive tumor with poor prognosis. Identification of precise prognostic marker and effective therapeutic target is important in the treatment of glioma. HTATIP2 is a novel tumor suppressor gene, which is frequently silenced by epigenetic mechanisms in many caners. However, the expression of HTATIP2 and how it is regulated in glioma are unknown. Hence, we assessed whether loss of HTATIP2 expression occurs in glioma, and, if so, what is the mechanism of such loss. We found that HTATIP2 expression was absent or diminished in primary gliomas compared with normal brain tissue. In vitro experiments showed that HTATIP2 expression could be restored via 5-aza-2'deoxycytidine treatment in U87 and U251 cell lines. Methyl-specific PCR indicated that the two cell lines and 60% primary gliomas carried aberrant methylated HTATIP2 alleles while normal brain tissue did not. Pyrosequencing confirmed these results and showed a higher density of methylation in the minimal promoter element, which contains four Sp1 binding sites in primary gliomas, than in normal brain tissue. Finally, we found that the overall survival was significantly higher in patients with positive HTATIP2 expression than those with loss of HTATIP2 expression. Overexpression of HTATIP2 inhibited glioma proliferation and growth in vitro. Taken together, the present study showed that loss of HTATIP2 expression was a frequent event in glioma and is associated with poor prognosis. Promoter methylation may be an underlying mechanism.

Overexpression of spermidine/spermine N1-acetyltransferase impairs osteoblastogenesis and alters mouse bone phenotype.

Spermidine/spermine N (1)-acetyltransferase (SSAT) is a catabolic regulator of polyamines, ubiquitous molecules essential for cell proliferation and differentiation. In pathological conditions, the increased polyamine catabolism has been shown to mediate its cellular functions not only by changed polyamine levels but also by the availability of metabolites shared with other metabolic pathways or by production of toxic compounds. Our previous results showed that mice overexpressing SSAT (SSAT mice) developed a myeloproliferative disease and the bone marrow microenvironment partly contributed to its development. In this study, the physiological role of SSAT and polyamines in bone remodeling was characterized. Skeletal development of the SSAT mice appeared outwardly similar to wild-type mice until maturity, after which the SSAT mice developed kyphosis. With aging, the SSAT overexpression elicited increased bone perimeter with strikingly thinned cortical bone, decreased trabecular thickness and increased trabecular number in mice. In vitro studies showed that the maturation of SSAT overexpressing osteoblasts was impaired and the expression of bone formation marker genes was dramatically decreased. The polyamine pattern in osteoblasts of SSAT mice was distorted in comparison with wild-type mice. However, treatment of osteoblasts with a SSAT-inducing functional polyamine analogue suggested that defective osteoblastogenesis resulted rather from other consequences of enhanced SSAT activity than lowered levels of the higher polyamines. In comparison to SSAT overexpressing mice, SSAT deficiency led to opposite changes in osteoblastogenesis and differences in bone phenotype in mice. In conclusion, the level of SSAT enzyme activity affected osteoblastogenesis and hence influenced bone remodeling and the bone phenotype in mice. Furthermore, our results suggest the contribution of the catabolic part of the polyamine cycle, other than polyamine depletion, in pathophysiological processes of bone remodeling.

Decreased TIP30 promotes Snail-mediated epithelial-mesenchymal transition and tumor-initiating properties in hepatocellular carcinoma.

The poor prognosis of hepatocellular carcinoma (HCC) is mainly due to tumor recurrence and metastases. Recently, epithelial-mesenchymal transition (EMT) has been implicated in tumor invasion and metastasis. However, the underlying molecular mechanisms are yet to be elucidated. Here, we show that 30-kDa Tat-interacting protein (TIP30), also called CC3, is significantly downregulated during transforming growth factor-ß-induced EMT. In our in vitro and in vivo studies, we show that decreased TIP30 expression leads to EMT, as well as enhanced motility and invasion of HCC cells. Also, increased self-renewal ability and chemotherapeutic resistance are observed with TIP30 depletion. Moreover, Snail is one of the key transcription factors promoting EMT, and overexpression of TIP30 greatly decreased nucleic accumulation in Snail through the regulation of intracellular localization. Small interfering RNAs targeting Snail attenuated EMT and tumor-initiating properties induced by TIP30 deficiency. We further confirmed that TIP30 competitively interrupted the interaction of Snail with importin-ß2 to block the nuclear import of Snail. Consistently, TIP30 expression significantly correlates with E-cadherin expression in HCC patients. TIP30 or combination of E-cadherin is a powerful marker in predicting the prognosis of HCC. Taken together, our results suggest a novel and critical role of TIP30 involved in HCC progression and aggressiveness.

The combination of HTATIP2 expression and microvessel density predicts converse survival of hepatocellular carcinoma with or without sorafenib.

Our previous studies have demonstrated that sorafenib can promote the dissemination of hepatocellular carcinoma (HCC) through downregulation of HTATIP2, a suppressor of tumor growth and metastasis that is associated with inhibition of angiogenesis. Here, we investigated the predictive values of the HTATIP2 level and microvessel density (MVD) with or without sorafenib administration for HCC. Three independent cohorts were included. Using tissue microarray, we assessed the relationship between HTATIP2 expression/MVD and overall survival. The results showed that high HTATIP2 expression and a low MVD value were independent protective prognostic factors after curative HCC resection (297 cases/cohort 1); however, both parameters were converted to independent negative prognostic indicators for patients with postsurgical sorafenib treatment (69/143 cases/cohort 2; P<0.05 for all). This same relationship was observed in patients that received sorafenib treatment for advanced HCC (83 cases/cohort 3; efficacy measures and survival analyses, P<0.05 for all). Moreover, the combination of HTATIP2 and MVD had better power to predict patient death and disease recurrence (P<0.001 for both). We conclude that the combination of HTATIP2 and MVD predicts the converse survival of HCC with or without sorafenib intervention. Our findings can assist in the selection of candidates for personalized treatment with sorafenib.

Fatty acid elongation in non-alcoholic steatohepatitis and hepatocellular carcinoma.

Non-alcoholic steatohepatitis (NASH) represents a risk factor for the development of hepatocellular carcinoma (HCC) and is characterized by quantitative and qualitative changes in hepatic lipids. Since elongation of fatty acids from C16 to C18 has recently been reported to promote both hepatic lipid accumulation and inflammation we aimed to investigate whether a frequently used mouse NASH model reflects this clinically relevant feature and whether C16 to C18 elongation can be observed in HCC development. Feeding mice a methionine and choline deficient diet to model NASH not only increased total hepatic fatty acids and cholesterol, but also distinctly elevated the C18/C16 ratio, which was not changed in a model of simple steatosis (ob/ob mice). Depletion of Kupffer cells abrogated both quantitative and qualitative methionine-and-choline deficient (MCD)-induced alterations in hepatic lipids. Interestingly, mimicking inflammatory events in early hepatocarcinogenesis by diethylnitrosamine-induced carcinogenesis (48 h) increased hepatic lipids and the C18/C16 ratio. Analyses of human liver samples from patients with NASH or NASH-related HCC showed an elevated expression of the elongase ELOVL6, which is responsible for the elongation of C16 fatty acids. Taken together, our findings suggest a detrimental role of an altered fatty acid pattern in the progression of NASH-related liver disease.

TIP30: A Novel Tumor-Suppressor Gene.

TIP30/CC3 was first identified and characterized as a "candidate" tumor-suppressor gene in 1997. Recently, the TIP30 tumor-suppressor status has been fully established since several studies have described that TIP30 protein expression is frequently downregulated in diverse types of human tumors, and the downregulation is often associated with tumor progression. TIP30 is involved in the control of cell apoptosis, growth, metastasis, angiogenesis, DNA repair, and tumor cell metabolism. Moreover, TIP30(-/-) mice spontaneously develop hepatocellular carcinoma and other tumors at a higher incidence than that of wild-type mice. In this review, we provide an overview of current knowledge concerning the role of TIP30 in tumor development and progression. To our knowledge, this is the first review about the role of novel tumor-suppressor gene TIP30 in tumor development and progression.

The importance of tissue environment surrounding the tumor on the development of cancer cachexia.

The relationship between host factors and cancer cachexia was investigated. A single cell clone (clone 5 tumor) established from colon 26 adenocarcinoma by limiting dilution cell cloning methods was employed to eliminate the inoculation site-dependent differences in the composition of cell clones. Clone 5 tumor did not provoke manifestations of cancer cachexia when inoculated in subcutaneous tissue. However, when inoculated in the gastrocnemius muscle, the peritoneal cavity or the thoracic cavity of CD2F1 male mice, typical manifestations of cancer cachexia were observed in all groups of mice with intergroup variations. The blood levels of various cytokines, chemokines and hormones were increased but with wide intergroup variations. Analyses by stepwise multiple regression models revealed that serum interleukin-10 was the most significant factor associated with manifestations of cancer cachexia, suggesting the possible involvement of mechanisms similar to cancer patients suffering cancer cachexia. White blood cells, especially neutrophils, seemed to have some roles on the induction of cancer cachexia, because massive infiltrations and an increase in peripheral blood were observed in cachectic mice bearing clone 5 tumors. The amount of malonyl-CoA in liver correlated with manifestations of cancer cachexia, however the mRNA levels of spermidine/spermine N-1 acetyl transferase (SSAT) (of which overexpression has been shown to provoke manifestations similar to cancer cachexia) were not necessarily associated with cancer cachexia. These data suggest that the induction of cancer cachexia depends on the environment in which the tumor grows and that the infiltration of host immune cells into the tumor and the resultant increase in inflammation result in the production of cachectic factors, such as cytokines, leading to SSAT activation. Further, multiple factors likely mediate the mechanisms of cancer cachexia. Finally, this animal model was suitable for the investigation of the mechanisms involved in cachexia of cancer patients.

GANP interacts with APOBEC3G and facilitates its encapsidation into the virions to reduce HIV-1 infectivity.

The ssDNA-dependent deoxycytidine deaminase apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (A3G) is a potent restrictive factor against HIV-1 virus lacking viral-encoded infectivity factor (Vif) in CD4(+) T cells. A3G antiretroviral activity requires its encapsulation into HIV-1 virions. In this study, we show that germinal center-associated nuclear protein (GANP) is induced in activated CD4(+) T cells and physically interacts with A3G. Overexpression of GANP augments the A3G encapsidation into the virion-like particles and ΔVif HIV-1 virions. GANP is encapsidated in HIV-1 virion and modulates A3G packaging into the cores together with cellular RNAs, including 7SL RNA, and with unspliced HIV-1 genomic RNA. GANP upregulation leads to a significant increase in A3G-catalyzed G→A hypermutation in the viral genome and suppression of HIV-1 infectivity in a single-round viral infection assay. Conversely, GANP knockdown caused a marked increase in HIV-1 infectivity in a multiple-round infection assay. The data suggest that GANP is a cellular factor that facilitates A3G encapsidation into HIV-1 virions to inhibit viral infectivity.

Histone deacetylase inhibition overcomes drug resistance through a miRNA-dependent mechanism.

The treatment of specific tumor cell lines with poly- and oligoamine analogs results in a superinduction of polyamine catabolism that is associated with cytotoxicity; however, other tumor cells show resistance to analog treatment. Recent data indicate that some of these analogs also have direct epigenetic effects. We, therefore, sought to determine the effects of combining specific analogs with an epigenetic targeting agent in phenotypically resistant human lung cancer cell lines. We show that the histone deacetylase inhibitor MS-275, when combined with (N(1), N(11))-bisethylnorspermine (BENSpm) or (N(1), N(12))-bis(ethyl)-cis-6,7-dehydrospermine tetrahydrochloride (PG-11047), synergistically induces the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT), a major determinant of sensitivity to the antitumor analogs. Evidence indicates that the mechanism of this synergy includes reactivation of miR-200a, which targets and destabilizes kelch-like ECH-associated protein 1 (KEAP1) mRNA, resulting in the translocation and binding of nuclear factor (erythroid-derived 2)-like 2 (NRF2) to the polyamine-responsive element of the SSAT promoter. This transcriptional stimulation, combined with positive regulation of SSAT mRNA and protein by the analogs, results in decreased intracellular concentrations of natural polyamines and growth inhibition. The finding that an epigenetic targeting agent is capable of inducing a rate-limiting step in polyamine catabolism to overcome resistance to the antitumor analogs represents a completely novel chemotherapeutic approach. In addition, this is the first demonstration of miRNA-mediated regulation of the polyamine catabolic pathway. Furthermore, the individual agents used in this study have been investigated clinically; therefore, translation of these combinations into the clinical setting holds promise.

Modulation of the expression of folate cycle enzymes and polyamine metabolism by berberine in cisplatin-sensitive and -resistant human ovarian cancer cells.

Berberine is a natural isoquinoline alkaloid with significant antitumor activity against many types of cancer cells, including ovarian tumors. This study investigated the molecular mechanisms by which berberine differently affects cell growth of cisplatin (cDDP)-sensitive and -resistant and polyamine analogue cross-resistant human ovarian cancer cells. The results show that berberine suppresses the growth of cDDP-resistant cells more than the sensitive counterparts, by interfering with the expression of folate cycle enzymes, dihydrofolate reductase (DHFR) and thymidylate synthase (TS). In addition, the impairment of the folate cycle also seems partly ascribable to a reduced accumulation of folate, a vitamin which plays an essential role in the biosynthesis of nucleic acids and amino acids. This effect was observed in both lines, but especially in the resistant cells, correlating again with the reduced tolerance to this isoquinoline alkaloid. The data also indicate that berberine inhibits cellular growth by affecting polyamine metabolism, in particular through the upregulation of the key catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT). In this regard, berberine is shown to stimulate the SSAT induction by the spermine analogue N1, N12 bisethylspermine (BESpm), which alone was also able to downregulate DHFR mRNA more than TS mRNA. We report that the sensitivity of resistant cells to cisplatin or to BESpm is reverted to the levels of sensitive cells by the co-treatment with berberine. These data confirm the intimate inter-relationships between folate cycle and polyamine pathways and suggest that this isoquinoline plant alkaloid could be a useful adjuvant therapeutic agent in the treatment of ovarian carcinoma.

A dual role for SAGA-associated factor 29 (SGF29) in ER stress survival by coordination of both histone H3 acetylation and histone H3 lysine-4 trimethylation.

The SGF29 protein binds to tri-methylated lysine-4 of histone H3 (H3K4me3), which is a histone modification associated with active promoters. Human SGF29 is a subunit of the histone acetyltransferase module of the SAGA (Spt-Ada-Gcn5 acetyltransferase) and ATAC (Ada-Two-A-containing 2A) co-activator complexes. Previous work revealed that the SAGA complex is recruited to endoplasmic reticulum (ER) stress target genes and required for their induction. Here, we report the involvement of SGF29 in the survival of human cells from ER stress. SGF29 knockdown results in impaired transcription of the ER stress genes GRP78 and CHOP. Besides histone H3K14 acetylation, we find that SGF29 is also required for the maintenance of H3K4me3 at these genes, which is already present prior to ER stress. Reduced levels of H3K4me3 in the absence of SGF29 correlate with a decreased association of ASH2L, which is a core component of the SET1/MLL complexes, to GFP78 and CHOP. In conclusion, our results suggest that the H3K4me3-binding protein SGF29 plays a central and dual role in the ER stress response. Prior to ER stress, the protein coordinates H3K4me3 levels, thereby maintaining a 'poised' chromatin state on ER stress target gene promoters. Following ER stress induction, SGF29 is required for increased H3K14 acetylation on these genes, which then results in full transcriptional activation, thereby promoting cell survival.

Identification of endometriosis-related genes by representational difference analysis of cDNA.

BACKGROUND: Accumulated evidence reveals that abnormally expressed genes in eutopic endometrium of endometriosis play a critical role in the pathogenesis of endometriosis. AIMS: Identification of endometriosis-related genes for further revealing the pathogenesis of endometriosis and offering the basis for developing the molecular-targeted diagnosis and therapy of endometriosis. METHODS: Forty women with endometriosis and forty control women without endometriosis during their secretory phase were selected for this study. cDNA representational difference analysis (cDNA-RDA) was performed to screen the up-regulated genes in eutopic endometrium samples of endometriosis (n = 10) compared with the controls (n = 10). To validate the results, MAT2A, the most abundantly expressed gene, was selected to detect mRNA and protein levels between eutopic endometrium of endometriosis (n = 40) and controls (n = 40) using immunohistochemistry, real-time fluorescent quantitative PCR and Western blotting. RESULTS: Ten up-regulated genes were identified in eutopic endometria of endometriosis compared with controls. Among these genes, COX-2, BRAF, NRAS and CFL1 have already been reported to be associated with the endometriosis in previous studies. MAT2A, SEPT9, ATAD3A and CADM2 have been reported to be involved in other diseases but not in endometriosis. NAA15 and CCDC21 have not reported in any diseases. Further study showed that MAT2A protein was localised in both endometrial glandular and stromal cells. Compared with controls, the mRNA and protein levels of MAT2A were significantly higher in eutopic endometrium of endometriosis (P < 0.05). CONCLUSIONS: cDNA-RDA can be used to effectively identify the endometriosis-related genes, which can provide novel experimental data and further understand the pathogenesis of endometriosis.