Regulation of blood-testis barrier dynamics by the mTORC1/rpS6 signaling complex: An in vitro study / 亚洲男科学杂志(英文版)

During spermatogenesis, developing germ cells that lack the cellular ultrastructures of filopodia and lamellipodia generally found in migrating cells, such as macrophages and fibroblasts, rely on Sertoli cells to support their transport across the seminiferous epithelium. These include the transport of preleptotene spermatocytes across the blood-testis barrier (BTB), but also the transport of germ cells, in particular developing haploid spermatids, across the seminiferous epithelium, that is to and away from the tubule lumen, depending on the stages of the epithelial cycle. On the other hand, cell junctions at the Sertoli cell-cell and Sertoli-germ cell interface also undergo rapid remodeling, involving disassembly and reassembly of cell junctions, which, in turn, are supported by actin- and microtubule-based cytoskeletal remodeling. Interestingly, the underlying mechanism(s) and the involving biomolecule(s) that regulate or support cytoskeletal remodeling remain largely unknown. Herein, we used an in vitro model of primary Sertoli cell cultures that mimicked the Sertoli BTB in vivo overexpressed with the ribosomal protein S6 (rpS6, the downstream signaling protein of mammalian target of rapamycin complex 1 [mTORC1]) cloned into the mammalian expression vector pCI-neo, namely, quadruple phosphomimetic and constitutively active mutant of rpS6 (pCI-neo/p-rpS6-MT) versus pCI-neo/rpS6-WT (wild-type) and empty vector (pCI-neo/Ctrl) for studies. These findings provide compelling evidence that the mTORC1/rpS6 signal pathway exerted its effects to promote Sertoli cell BTB remodeling. This was mediated through changes in the organization of actin- and microtubule-based cytoskeletons, involving changes in the distribution and/or spatial expression of actin- and microtubule-regulatory proteins.

Phosphorylated S6 Kinase-1 as Predictive Marker of Lapatinib Efficacy in Human Epidermal Growth Factor Receptor 2-Positive Metastatic Breast Cancer Patients

PURPOSE: The 40S ribosomal protein S6 kinase-1 (S6K1) is a crucial downstream effector of the PI3K/AKT/mTOR pathway. S6K1 overexpression is found in 10% to 30% of breast cancers and is associated with aggressive disease and poor prognosis. Herein, we investigated the relationship between the expression of phosphorylated S6K1 (p-S6K1) and efficacy of lapatinib in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. METHODS: We retrospectively analyzed the data of 36 patients with HER2-positive metastatic breast cancer treated with lapatinib between January 2010 and September 2014. The p-S6K1 expression status of the primary tumor was assessed via immunohistochemistry using a mouse monoclonal antibody. RESULTS: Fourteen of the 36 patients (38.9%) had p-S6K1-positive tumors. The median progression-free survival (PFS) of patients with p-S6K1-positive tumors was significantly longer than that of patients with p-S6K1-negative tumors (13.4 months vs. 7.1 months, p=0.025). In multivariate analysis, p-S6K1 positivity remained an independent, favorable predictive factor for PFS (hazard ratio, 0.32; 95% confidence interval, 0.11–0.97; p=0.044). CONCLUSION: The high expression of p-S6K1 was significantly associated with prolonged PFS, suggesting that p-S6K1 can be a potential biomarker for predicting the efficacy of lapatinib in patients with HER2-positive metastatic breast cancer.

Molecular analysis of TP53, D9S1747, D9S162 and RPS6 in oral squamous cell carcinoma / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the correlation between the frequency of molecular abnormalities of 4 loci at chromosomal 9p21 (D9S1747, D9S162, RPS6) and 17p13 (TP53) and the clinical characteristics and prognosis.</p><p><b>METHODS</b>The oral squamous cell carcinoma (OSCC) lesions in 71 patients were manually microdessected. Genomic DNA from these lesions and normal lymphnode tissu or peripheral blood of the same patients were extracted using the Watson's tissue kit. The loss of heterozygosity (LOH) and microsatellite instability (MI) of 17p13 and 9p21 were analyzed by PCR-page electrophoresis after DNA extraction.</p><p><b>RESULTS</b>LOH and MI were detected in the OSCC of 48 patients (68%). The LOH and MI frequency at chromosomes 17p13 and 9P21 were 56% (35/63) and 59% (40/68) respectively. The LOH and MI frequency at 9p21 was significantly associated with WHO grading (P < 0.01) and lymphonode metastasis (P < 0.01). The LOH and MI frequency at 17p13 was significantly associated with clinical stage (P < 0.05). TP53 genetic aberration and 9p21 genetic aberration were significant prognostic factors for OSCC. The prognosis was poor in the LOH and MI positive group of chromosome 17p13 and 9p21. The frequency of LOH and MI at TP53 was the only independent factor for overall survival (P < 0.05).</p><p><b>CONCLUSIONS</b>The LOH and MI of 17p13 and 9p21 were related to clinical stage and lymphonode metastasis. LOH of TP53 was an independent prognostic factor for OSCC.</p>

The role of intracellular signal pathway of mTOR/S6 in CD3+ T lymphocytes of refractory/relapsed aplastic anemia patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the activation status of signal pathway of mTOR/S6 in bone marrow (BM) T lymphocytes of refractory/relapsed aplastic anemia patients (AA), and the effects of rapamycin (RAPA) and CTLA-4 immunoglobulin (CTLA-4Ig) on this pathway.</p><p><b>METHODS</b>BM was collected from 13 refractory/relapsed AA patients, 8 newly diagnosed severe AA (SAA) patients and 10 iron deficiency anemia (IDA) (as controls) patients, and cocultured with RAPA and CTLA-4 Ig. The expression of p-mTOR, p-S6 and Interferon gamma (IFN-gamma) in CD3(+)T cells was measured by flow cytometry (FCM).</p><p><b>RESULTS</b>(1) The expression of p-mTOR, p-S6 and IFN-gamma in CD3(+)T cells in refractory/relapsed AA group were significantly higher than those in controls (P < 0.01). (2) The expression of p-mTOR and p-S6 in T cells in newly diagnosed SAA group, was similar to those in controls (P > 0.05), but significantly lower than those in refractory/relapsed AA group (P < 0.01). The expression level of IFN-gamma in T cells were significantly higher than that in controls (P < 0.01). (3) On exposure to RAPA, the levels of p-mTOR, p-S6 and IFN-gamma in T cells in refractory/relapsed AA patients were significantly lower than those before the exposure (all P < 0.05). And so were when exposed to CTLA-4 Ig (all P < 0.01).</p><p><b>CONCLUSION</b>(1) The mTOR/S6 signal pathway is activated in refractory/relapsed AA. (2) The expression of p-mTOR, p-S6 and IFN-gamma in refractory/relapsed AA can be suppressed by RAPA or CTLA-4Ig. (3) The signal pathway of CD28/mTOR/S6/IFN-gamma might take part in immune pathogenesis of refractory/relapsed AA.</p>

The TP53 and RPS6 alterations at the invasive tumor front, center and stroma of oral squamous cell carcinoma / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To assess the difference of genetic alteration patterns among different areas in the same oral squamous cell carcinoma (OSCC).</p><p><b>METHODS</b>Studied the loss of heterozygosity (LOH) and microsatellite instability (MI) at chromosomal loci TP53 and RPS6 on the invasive tumor front (ITF), the center/superficial part and stroma cells by combining laser capture microdissection (LCM) and PCR technique.</p><p><b>RESULTS</b>There existed a high frequency of LOH and MI on chromosomes loci TP53 and RPS6. The frequency of RPS6 and TP53 aberration at the stroma was 23.5% (4/17) and 43.8% (7/16), respectively. While in epithelial part (both ITF and center), it reached up to 64.7% (11/17) and 70.6% (12/17) respectively, and the difference was significant (P < 0.05). The overall frequency of the two markers was statistically higher at the ITF (20/32) than at the center/superficial part (15/34) (P < 0.05).</p><p><b>CONCLUSIONS</b>The current study revealed that genetic alterations were different in different areas of the same tumor and there existed a relationship between the histological grading and genotypes of OSCC.</p>

Proteomic Analysis of Helicobacter pylori Whole Cell Proteins using the Narrow Range IPG Strips

It has been reported that most of Helicobacter pylori proteome components appear so crowded in the region of pH 4.5~8.0 that a lot of them were inseparable in 2-DE using the broad range IPG strip. Therefore, inseparable protein spots in 2-DE profiles have to be apart from each other for improving the protein identification. Here, we attempt to examine the usability of the narrow range IPG strips for separating close spots in the broad range IPG strip at proteomic analysis of H. pylori. The whole cell proteins of H. pylori strain 26695 were separated by narrow range IPG strips (pI 3.9~5.1, 4.7~5.9, 5.5~6.7, and 6.3~8.3, respectively), followed by SDS-PAGE, and visualized by silver staining, showing that the distances between spots were widened and the total number of detectable spots was increased. Resolved protein spots were identified by the peptide fingerprinting using MALDI-TOF-MS. As a result, 87 expressed proteins were identified by the peptide fingerprinting. Of them, 23 proteins, including hydrogenase expression/formation protein, purine-binding chemotaxis protein, and ribosomal protein S6, have not been reported in the previous proteome studies of H. pylori. Thus, these results demonstrate that the high complexity proteome components could be effectively separated using the narrow range IPG strips, which might be helpful to strengthen the contents of the master protein map of the H. pylori reference strain.

Phosphorylation of ribosomal protein S6 and its regulation during differentiation of human leukemic cells

We attempted to study the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the cascade of phosphorylation of ribosomal protein S6 during differentiation of leukemic cells (HL-60, THP-1, and RWLeu-4). Neither activation nor inhibition of colony stimulating factor-1 (CSF-1) receptor's PTK activity with CSF-1 or genistein respectively affected the phosphorylation of S6. However, vanadate which is a protein tyrosine phosphatase (PTP) inhibitor showed enhancement of S6 phosphorylation. Dimethylsulfoxide which does not affect either PTK or PKC demonstrated no change in S6 phosphorylation. PKC activation by acute 12-0-tetradecanoyl phorbol-13-acetate (TPA) treatment induced monocytic differentiation and S6 phosphorylation. Surprisingly, the more prominent phosphorylation of S6 protein was observed in PKC-depleted cells by prolonged TPA treatment. Our results suggest that PTK/PTP play a lesser role in S6 phosphorylation of HL-60 cells than PKC does. In addition, two different mechanisms seem to be involved in TPA-induced S6 phosphorylation during HL-60 differentiation: PKC activation by acute TPA treatment and PKC depletion which may lead to the synthesis of some endogenous protein responsible for the differentiation by chronic TPA treatment.