Actions of TGF-β as tumor suppressor and pro-metastatic factor in human cancer

Transforming growth factor-β (TGF-β) is a secreted polypeptide that signals via receptor serine/threonine kinases and intracellular Smad effectors. TGF-β inhibits proliferation and induces apoptosis in various cell types, and accumulation of loss-of-function mutations in the TGF-β receptor or Smad genes classify the pathway as a tumor suppressor in humans. In addition, various oncogenic pathways directly inactivate the TGF-β receptor-Smad pathway, thus favoring tumor growth. On the other hand, all human tumors overproduce TGF-β whose autocrine and paracrine actions promote tumor cell invasiveness and metastasis. Accordingly, TGF-β induces epithelial–mesenchymal transition, a differentiation switch that is required for transitory invasiveness of carcinoma cells. Tumor-derived TGF-β acting on stromal fibroblasts remodels the tumor matrix and induces expression of mitogenic signals towards the carcinoma cells, and upon acting on endothelial cells and pericytes, TGF-β regulates angiogenesis. Finally, TGF-β suppresses proliferation and differentiation of lymphocytes including cytolytic T cells, natural killer cells and macrophages, thus preventing immune surveillance of the developing tumor. Current clinical approaches aim at establishing novel cancer drugs whose mechanisms target the TGF-β pathway. In conclusion, TGF-β signaling is intimately implicated in tumor development and contributes to all cardinal features of tumor cell biology.     

Global DNA methylation profile at LINE-1 repeats and promoter methylation of genes involved in DNA damage response and repair pathways in human peripheral blood mononuclear cells in response to γ-radiation

Molecular and Cellular Biochemistry - DNA methylation is an epigenetic mechanism, which plays an important role in gene regulation. The present study evaluated DNA methylation profile of LINE1...     

Estrogen receptor alpha (ERα) promoter methylation status in tumor and serum DNA in Egyptian breast cancer patients

Background

Status of DNA methylation is one of the most common molecular alterations in human neoplasia. Because it is possible to detect these epigenetic alterations in the bloodstream of patients, we investigated the aberrant DNA methylation status of estrogen receptor alpha (ERα) in patient pretherapeutic sera and tissue.

Materials and methods

In this case control study the patient series consisted of 120 sporadic primary breast cancer cases and 100 patients with benign breast lesion. ER3, ER4, and ER5 primers were used for methylation-specific polymerase chain reaction (MSP) to analyze the CpG methylation of promoter region of ERα gene. Correlation between ER3, ER4, and ER5 methylation and clinicopathological characteristics of the patients was investigated.

Result

The methylation status of ER3, ER4 and ER5 was 65%, 26.7% and 61.7% in tissue respectively and 57.5%, 21.7% and 55.8% in serum respectively. The concordance between tumor and serum DNA methylation was 80%, 72% and 92% for ER3, ER4 and ER5 respectively.

Conclusions

This study demonstrated the potential utility of serum DNA methylation of ERα gene promoter as a non-invasive diagnostic and/or prognostic marker in patients with breast cancer.     

Sequential metabolism of 5′-isobutylthioadenosine by methylthioadenosine phosphorylase and purine-nucleoside phosphorylase in viable human cells

The exact route of metabolism of 5′-isobutylthioadenosine is controversial. Using human cell lines deficient in methylthioadenosine phosphorylase, purine-nucleoside phosphorylase, or adenosine deaminase, we have ascertained the relative roles of the three enzymes in isobutylthioadenosine metabolism. The results showed that viable human cells progressively converted isobutylthioadenosine to 5′-isobutylthioinosine via sequential metabolism by methylthioadenosine phosphorylase and purine nucleoside phosphorylase acting in opposite directions, rather than through direct deamination. An identical pathway converted 5′-methylthioadenosine to 5′-methylthioinosine.     

Purine 5′,8-cyclo-2′-deoxynucleoside lesions: formation by radical stress and repair in human breast epithelial cancer cells

5′,8-Cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) in their two diastereomeric forms, 5′S and 5′R, are tandem lesions produced by the attack of hydroxyl radicals to the purine moieties of DNA. Their formation has been found to challenge the cells’ repair machinery, initiating the nucleotide excision repair (NER) for restoring the genome integrity. The involvement of oxidatively induced DNA damage in carcinogenesis and the reduced capacity of some cancer cell lines to repair oxidised DNA base lesions, intrigued us to investigate the implication of these lesions in breast cancer, the most frequently occurring cancer in women. Using liquid chromatography tandem mass spectrometry (LC-MS/MS), we measured the levels of diastereomeric cdA’s and cdG’s in estrogen receptor-alpha positive (ER-α) MCF-7 and triple negative MDA-MB-231 breast cancer cell lines before and after exposure to two different conditions: ionising radiations and hydrogen peroxide, followed by an interval period to allow DNA repair. An increase at the measured levels of all four lesions, i.e. 5′S-cdA, 5′R-cdA, 5′S-cdG and 5′R-cdG, was observed either after γ-irradiation (5?Gy dose) or hydrogen peroxide treatment (300?μM) compared to the untreated cells (control), independently from the length of the interval period for repair. For comparison reasons, we also measured the levels of 8-oxo-2′-deoxyadenosine (8-oxo-dA), a well-known oxidatively induced DNA damage lesion and base excision repair (BER) substrate. The collected data indicate that MCF-7 and MDA-MB-231 breast cancer cells are highly susceptible to radiation-induced DNA damage, being mainly defective in the repair of these lesions.     

Destabilization of microRNAs in human cells by 3′ deadenylation mediated by PARN and CUGBP1

MicroRNA-122 (miR-122), which is expressed at high levels in hepatocytes, is selectively stabilized by 3′-adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2. Here, we report that poly(A)-specific ribonuclease (PARN) is responsible for the deadenylation and destabilization of miR-122. The 3′-oligoadenylated variant of miR-122 was detected in Huh7 cells when PARN was down-regulated. In addition, both the steady-state level and stability of miR-122 were increased in PARN knockdown cells. We also demonstrate that CUG-binding protein 1 (CUGBP1) specifically interacts with miR-122 and other UG-rich miRNAs, and promotes their destabilization. Overexpression of CUGBP1 or PARN in Huh7 cells reduced the steady-state levels of these miRNAs. Because CUGBP1 interacts directly with PARN, we hypothesized that it specifically recruits PARN to miR-122. In fact, CUGBP1 enhanced PARN-mediated deadenylation and degradation of miR-122 in a dose-dependent manner in vitro. These results indicate that the cellular level of miR-122 is determined by the balance between the opposing effects of GLD-2 and PARN/CUGBP1 on the metabolism of its 3′-terminus.     

α1,3 Fucosyltransferase-VII modifies the susceptibility of apoptosis induced by ultraviolet and retinoic acid in human hepatocarcinoma cells

The role of α1,3fucosyltransferase-VII (α1,3 FucT-VII) in cell apoptosis was studied in human hepatocellular carcinoma H7721 cells. After the cells were transfected with α1,3 FucT-VII cDNA, the expression of apoptotic protease, procaspase-3, was decreased, while the anti-apoptotic proteins, phospho-PKB and phospho-Bad were increased as compared with mock (vector) transfected cells, indicating that α1,3FucT-VII is a potential anti-apoptotic factor in H7721 cells. After “α1,3FucT-VII” cells were irradiated by UV to induce apoptosis, the anti-apoptotic potential of α1,3FucT-VII became more apparent, as evidenced by the less apoptotic cell % and active cleaved caspase-3, more phospho-p38 MAPK and JNK (two anti-apoptotic signaling molecules in H7721 cells responsible to UV stress) when compared with the “Mock” cells. In contrast, “α1,3FucT-VII” cells facilitated the apoptosis induced by all-trans retinoic acid (ATRA), which was verified by the greater sub-G1 (apoptotic cells) peak in flow cytometry analysis, more expressions of active caspase-3 and pro-apoptotic protein Bax, as well as less expressions of anti-apoptotic proteins, Bcl-2 and Bcl-X_L. The up regulation of α1,3FucT-VII mRNA and cell surface SLe^x (α1,3FucT-VII product) by UV and down regulation of them by ATRA was speculated to be one of the mechanisms that α1,3FucT-VII decreased and increased the susceptibility of apoptosis induced by UV and ATRA respectively. Hao Wang and Qiu-Yan Wang contributed to this article equally.     

γ-Glutamyl hydrolase modulation significantly influences global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells

γ-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0444-0) contains supplementary material, which is available to authorized users.     

Localization of adenylate cyclase and 5′-nucleotidase activities in human thyroid follicular cells

Summary The localization of adenylate cyclase and 5-nucleotidase activities in the follicular cells of adenomatous goiter and normal thyroid was studied by light and electron microscopy. Simultanous biochemical measurement for both activities was carried out to confirm the histochemical findings. Adenylyl-imidodiphosphate (AMP-PNP) was used as an effective substrate for adenylate cyclase. The specificity of the adenylate cyclase reaction was also examined by adding oxalacetic acid or PCMB as an adenylate cyclase inhibitor, and by adding sodium fluoride or TSH as an adenylate cyclase stimulator to the reaction mixture. In the case of tissue from adenomatous goiter, a large amount of the reaction product of the adenylate cyclase activity was found uniformly in the apical and lateral plasma membrane and not in the basal plasma membrane. In the cases of normal thyroid, a small amount of the reaction product of adenylate cyclase activity was demonstrated, and only in the lateral plasma membrane of the follicular cells. On the other hand, the histochemical localization of 5-nucleotidase activity was the same in adenomatous goiter and normal thyroid. The reaction product of 5-nucleotidase activity was found predominantly in the apical plasma membrane of the follicular cells. The biochemical findings indicated that the activity of adenylate cyclase per gram tissue was approximately 2 times higher in the case of adenomatous goiter than that in the case of normal thyroid, while the 5-nucleotidase activity in adenomatous goiter was in slightly higher level than in normal thyroid. Thus the histochemically demonstrable amount of adenylate cyclase and 5-nucleotidase reflected the activity levels measured biochemically. The lack of demonstrable adenylate cyclase activity in the basal plasma membrane suggests the possibility that this structure may not play any important role in TSH reception.     

Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.