[Effect of concurrent chemoradiotherapy and radiotherapy alone on peripheral myeloid-derived suppressor and T regulatory cells in patients with nasopharyngeal cancer].

Objective: To investigate the percentage of myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg) in peripheral blood of nasopharyngeal cancer (NPC) patients undergoing concurrent chemoradiotherapy or radiotherapy alone. Methods: Sixty NPC patients who received radiotherapy or concurrent chemoradiotherapy from September 2012 to November 2015 and 20 healthy individuals were included in this study. For the patients, the blood samples were collected at four time points: pre-radiation (Pre-RT), reaching a dose of 40 Gy (RT-40 Gy), finishing radiation (RT-finish) and three months after finishing radiation (3m-post-RT). Flow cytometry was used to evaluate the percentage of Treg (CD4(+) CD25(+) CD127(low/-)) and MDSC (HLA-DR(-)CD11b(+) CD33(+) ) cells in peripheral blood. Results: Treg and MDSC cells were present in peripheral blood lymphocytes of healthy individuals as a percentage of (7.50±1.62)% and (1.08±0.48)%, respectively. The proportions of peripheral Treg cells in patients at Pre-RT, RT-40 Gy, RT-finish and 3m-post-RT time points were (8.42± 1.52)%, (9.10±1.57)%, (8.87±1.56)% and (7.31±1.43)%, respectively, showing a statistically significant difference between Pre-RT and the other groups (P<0.05). At Pre-RT point, the percentage of Treg cells in Stage Ⅲ-Ⅳ patients [(8.63±1.39)%] was higher than that in Stage Ⅰ-Ⅱ [(7.65±1.94)%, P=0.042]. Moreover, the proportions of peripheral MDSC cells in patients at Pre-RT, RT-40 Gy, RT-finish and 3m-post-RT time points were (2.14±1.21)%, (4.08±1.90)%, (3.76±1.31)% and (1.52±0.88)%, respectively. The percentages of MDSC cells at RT-40 Gy and RT-finish points were significantly higher than those at Pre-RT, while the percentage of MDSC cells at 3m-post-RT was significantly lower than those at Pre-RT (P<0.05). At Pre-RT point, the percentage of MDSC cells in Stage Ⅲ-Ⅳ patients [(2.25±1.26)%] was higher than that in Stage Ⅰ-Ⅱ [(1.35±0.66)%, P=0.007]. At RT-finish point, the proportions of MDSC and Treg cells in patients with Ⅲ-Ⅳ grade of radiation induced oral mucositis [(4.41±1.27)% and (9.91±1.23)%] were significantly higher than those in Ⅰ-Ⅱ grade patients [(3.15±1.04)% and (8.41±1.52)%, both of P<0.05]. Conclusions: The proportions of MDSC and Treg cells in initial treated NPC patients are higher than healthy individuals, and they are also associated with the tumor stages. During the concurrent chemoradiotherapy and radiation, the percentage of MDSC and Treg cells is elevated, suggesting a decreased immune activity. The increase of MDSC and Treg cells is related to radiation induced oral mucositis.

Germline mutations of DICER1 in Chinese women with BRCA1/BRCA2-negative familial breast cancer.

Germline mutations in identified breast cancer susceptibility genes account for less than 20% of Chinese familial breast cancers. Dicer is an essential component of the microRNA-producing machinery; germline mutations of DICER1 have been confirmed in familial pleuropulmonary blastoma, ovarian sex cord-stromal tumors, and other cancers. Low expression of DICER1 is frequently detected in breast cancer. However, whether germline mutations of DICER1 occur in familial breast cancers remain unknown. Sixty-five breast cancer probands from BRCA1/BRCA2-negative Chinese breast cancer families were screened for germline mutations in DICER1. In addition, 100 unrelated healthy females were enrolled as controls. A polymerase chain reaction sequencing assay was used to screen for mutations in coding regions and at the exon-intron boundaries of DICER1. All variants in introns were evaluated using the NNSplice software to determine the potential splicing effect. A total of 12 germline variants were found, including 11 variants in introns and 1 variant in the 3'-non-coding region. Four variants (IVS8-205 C>T, IVS11+131 delGAAA, IVS16+42 delTA, and IVS19+160 T>C) were novel. Three variants (IVS11+105 C>T, IVS16+42 delTA, and 6095 T>A) may affect splice sites. None of the observed variants appeared to be disease-related, suggesting that germline mutations in DICER1 are rare or absent in familial breast cancer patients.

Interferon alpha decreases expression of human scavenger receptor class BI, a possible HCV receptor in hepatocytes.

BACKGROUND: Infection with the hepatitis C virus (HCV) causes acute hepatitis. This disease has a high probability of becoming chronic and leading to cirrhosis, but a more deadly consequence is hepatocellular carcinoma. Interferon alpha (IFN alpha)-based treatment combined with ribavirin is the major therapeutic choice available for the treatment of chronic HCV infection. AIMS: The scavenger receptor class B type I (SR-BI) or its human homologue CD36 and LIMPII Analogous-1 (hSR-BI/CLA-1) has recently been shown to interact with HCV envelope glycoprotein E2, thus suggesting that it might participate in entry of the virus into host cells. This rationale underlies current interest in the potential role of IFN alpha in hSR-BI/CLA-1 expression in HepG2 cells. RESULTS: It was shown that endogenous hepatocyte expression of hSR-BI/CLA-1 was suppressed by exposure to IFN alpha. Decreased hSR-BI/CLA-1 expression in IFN alpha-treated cells was due to lower transcriptional activity of the promoter. A potential pathway for the effect of IFN alpha on hSR-BI/CLA-1 promoter activity was identified when the inhibitory action of IFN was abrogated in signal transducer and activator of transcription 1 (STAT1)/STAT2 knocked-down cells. Exposure of HepG2 cells to IFN alpha elicited a rapid phosphorylation of STAT1/STAT2, a known target of IFN alpha signalling. In addition, the mutagenesis of a STAT1/STAT2 response element in the hSR-BI/CLA-1 promoter abolished the ability of IFN alpha to suppress promoter activity. CONCLUSIONS: Together, these results indicate that the STAT1/STAT2 pathway participates in IFN alpha inhibition of hSR-BI/CLA-1 expression, and raise the possibility that lowering the expression of this gene may be of therapeutic value for treating HCV infections.

Prolactin regulatory element binding protein as a potential transcriptional factor for the insulin gene in response to glucose stimulation.

AIMS/HYPOTHESIS: Prolactin regulatory element binding (PREB) protein has been identified as a factor that regulates prolactin promoter activity in rat anterior pituitary. PREB is located not only in the anterior pituitary but also in pancreas; however its role in the pancreas is not known. We therefore examined the role of PREB in insulin gene expression. MATERIALS AND METHODS: To analyse the effects of PREB on insulin gene transcription, we employed the luciferase reporter gene assay and electrophoretic mobility shift assay (EMSA). In cells expressing or knocked down for PREB, insulin expression and secretion were determined. RESULTS: PREB was located mainly in nuclei of rat pancreatic beta cells and its cell line, INS-1. A nuclear extract of INS-1 cells contained material that was recognised by PREB antiserum. This nuclear extract also showed insulin promoter binding activity that was super-shifted by PREB antiserum in EMSA studies. In the INS-1 cells, co-expression of PREB and the insulin promoter induced activity of the latter. The addition of glucose to the cells increased PREB expression. Deletional analysis of the insulin promoter showed that A3, a glucose-responsive cis-element in the insulin promoter, mediated the transcriptional effect of PREB. In addition, synthesised PREB bound the A3 element by EMSA, while a mutant of this motif in the insulin promoter abrogated the effect of PREB. Cells expressing or knocked down for PREB exhibited increased or decreased insulin expression, respectively. CONCLUSIONS/INTERPRETATION: These results demonstrate that PREB may contribute to the regulation of insulin gene transcription and insulin secretion in response to glucose stimulation.

Phosphatidylserine receptor cooperates with high-density lipoprotein receptor in recognition of apoptotic cells by thymic nurse cells.

The thymus contains many apoptotic cells that arise from the process of positive and negative selection. Both thymic macrophages and thymic nurse cells/nursing thymic epithelial cells (nursing TECs), non-professional phagocytes, recognize and ingest apoptotic cells without inflammation or tissue damage. Previously we reported that human scavenger receptor class B (SR-B1) is involved in recognition of apoptotic thymocytes by nursing TECs. In this study, we examined the expression and role of a phosphatidylserine receptor (PSR). This receptor is believed to participate in the clearance of apoptotic cells. PSR was strongly expressed in nursing TECs. Transforming growth factor-beta augmented the expression of PSR leading to enhanced binding of apoptotic cells to nursing TECs. In nursing TECs, suppressed expression of human SR-B1 with anti-PSR antibody decreased binding of apoptotic thymocytes to nursing TECs. Our results suggest that both PSR and SR-B1 are expressed in nursing TECs and these receptors appear to play a major role in the clearance of apoptotic cells from the thymus.

The multiple endocrine neoplasia type 1 gene product, menin, inhibits the human prolactin promoter activity.

Menin is a protein encoded by the gene mutated in multiple endocrine neoplasia type 1 (MEN1) characterized by multiple endocrine tumors of the parathyroid glands, pancreatic islets and the anterior pituitary, especially prolactinoma. In this study, we examined the effects of menin on human prolactin (hPRL) expression. In rat pituitary GH3 cells stably expressing menin, both PRL gene expression/secretion and thymidine incorporation into DNA were inhibited as compared with mock-transfected cells. The transcriptional activity of PRL promoter in GH3 cells co-transfected with menin was significantly decreased. A deletion mutation (569 delC), which we identified in a Japanese MEN1 family, was introduced into menin. When GH3 cells were transfected with a mutant menin expression vector, inhibition of hPRL promoter activity was partially reversed. These observations suggest that menin inhibits hPRL promoter activity and cell proliferation, raising the possibility that menin might play an important role in the tumorigenesis of prolactinoma.

Inhibition of monocyte chemoattractant protein-1 expression in cytokine-treated human lung epithelial cells by thiazolidinedione.

STUDY OBJECTIVE: Several lung diseases are characterized by the presence of increased numbers of activated macrophages. The recruitment and activation of peripheral blood monocytes are potentially critical regulatory events for the control of pulmonary inflammation. The chemokine monocyte chemoattractant protein (MCP)-1 is a potent chemoattractant for monocytes. MCP-1 is produced by lung epithelial cells during the course of inflammatory lung diseases. In the present study, we examined the effects of a thiazolidinedione (TZD), which is used to improve the insulin resistance of individuals with diabetes mellitus, on MCP-1 expression in a human lung epithelial cell line, A549. MEASUREMENTS AND RESULTS: In A549 cells, interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha induced endogenous MCP-1 protein secretion and messenger RNA expression. The TZD inhibited the increase of MCP-1 secretion by IL-1beta and TNF-alpha treatment. The TZD inhibited the expression of MCP-1 messenger RNA with IL-1beta treatment, but not with TNF-alpha treatment. This observation was confirmed by the results of a monocyte chemotactic assay. The transcriptional activity of human MCP-1 promoter in A549 cells paralleled the endogenous messenger RNA expression by cytokines and TZD treatment. CONCLUSIONS: Our findings indicated that the suppression of the expression of MCP-1 could be accomplished by TZD treatment, raising the possibility that TZD may be of therapeutic value in several lung diseases in which MCP-1 plays an important role.

Expression of HDL receptor, CLA-1 in human smooth-muscle cells and effect of interferon-gamma on its regulation.

High-density lipoprotein (HDL) exerts antiatherogenic effects by various mechanisms. The protective effect of HDL is thought to involve the reverse transport of cholesterol from cells in the arterial wall to the liver for disposal. We previously identified human scavenger receptor BI (hSR-BI/CLA-1) as a receptor for human HDL, but did not examine the expression of hSR-BI/CLA-1 in smooth-muscle cells. In this present study, a human aortic intima smooth-muscle cell line immortalized with SV 40 DNA was established, and the expression of hSR-BI/CLA-1 in this cell line analyzed by Western blot and RT-PCR. HSR-BI/CLA-1 mRNA and protein were detected in both this cell line and primary human aortic smooth-muscle cells. A cytokine, interferon-gamma (IFN-gamma) inhibited the hSR-BI/CLA-1 protein expression, but not mRNA expression. This observation confirmed that selective cholesterol ester uptake from HDL was inhibited by IFN-gamma. These results indicated that hSR-BI/CLA-1 may be expressed in human smooth-muscle cells, and the expression may be modulated by IFN-gamma. HSR-BI/CLA-1 on smooth-muscle cells could play an important role in atherogenesis.

TNF-alpha stimulation of MCP-1 expression is mediated by the Akt/PKB signal transduction pathway in vascular endothelial cells.

MCP-1 is expressed in a variety of cell types including vascular endothelial cells following induction by different stimuli such as tumor necrosis factor (TNF)-alpha. Although TNF-alpha stimulates MCP-1 expression and secretion, the mechanism by which TNF-alpha stimulates expression of the MCP-1 gene is not known. In this study, we examine the involvement of the phosphatidylinositol-3-OH kinase (PI3-kinase)-Akt/PKB pathway. Exposure of human umbilical vein endothelial cells (HUVECs) to TNF-alpha elicited the rapid phosphorylation of Akt/PKB. In HUVECs, wortmannin, a PI3-kinase inhibitor, inhibits TNF-alpha-mediated MCP-1 secretion at a dose-dependent manner. Constitutively active form of Akt/PKB induces transcription of the MCP-1 gene, and cotransfection of dominant negative Akt/PKB suppressed the activation of the MCP-1 promoter induced by TNF-alpha. These findings show that Akt/PKB participates in the TNF-alpha induction of MCP-1 gene transcription in endothelial cells.