Mullerian inhibiting substance induces apoptosis of human endometrial stromal cells in endometriosis.

CONTEXT: Müllerian inhibiting substance (MIS) is produced in Sertoli cells of fetal testis and causes regression of müllerian ducts in male embryos. MIS also can induce the cell cycle arrest and apoptosis in müllerian duct-derived tumors in vivo and in vitro. OBJECTIVE: Our objective was to investigate the expression of MIS type II receptor (MISR II) and whether MIS can inhibit the proliferation and induce apoptosis in primary cultures of endometrial stromal cells (ESC) of endometriosis. DESIGN AND SETTINGS: In vitro experiments were performed in the university research laboratory. PARTICIPANTS: Tissue samples from 12 patients who had undergone evisceration for ovarian endometrial cysts were included in this study. INTERVENTIONS AND MAIN OUTCOME MEASURES: The expression of MISR II in ESC was investigated by immunohistochemistry. The cell viability and apoptosis in ESC treated with MIS was measured by methylthiazoletetrazolium assay and annexin V analysis. The expression of regulatory proteins in ESC treated with MIS was shown by Western blotting. RESULTS: ESC showed specific immunostaining for the MISR II. ESC treated with MIS exhibited 32% growth inhibition (P = 0.0001). The changes in cell cycle distribution after MIS exposure at 72 h demonstrated that S and G(2)M phases were decreased; G(0)G(1) and sub-G(0)G(1) phases were increased. ESC treated with MIS showed 13.72% annexin V-fluorescein isothiocyanate positivity. In the ESCs, which contain defective p16, MIS increased the expression of pocket proteins p107 and p130 and decreased E2F transcription factor 1. CONCLUSIONS: The results support a central role for MIS in endometriosis. Although the precise mechanism of MIS-mediated inhibition of ESC growth has not been fully defined, these data suggest that MIS has activity against ESC in vitro and may also be an effective targeted therapy for endometriosis.

In vitro senescence of rat mesenchymal stem cells is accompanied by downregulation of stemness-related and DNA damage repair genes.

Mesenchymal stem cells (MSCs) are of particular interest because they are being tested using cell and gene therapies for a number of human diseases. MSCs represent a rare population in tissues. Therefore, it is essential to grow MSCs in vitro before putting them into therapeutic use. This is compromised by senescence, limiting the proliferative capacity of MSCs. We analyzed the in vitro senescence of rat MSCs, because this animal is a widespread model for preclinical cell therapy studies. After initial expansion, MSCs showed an increased growth doubling time, lost telomerase activity, and expressed senescence-associated beta-galactosidase. Senescence was accompanied by downregulation of several genes involved in stem cell self-renewal. Of interest, several genes involved in DNA repair also showed a significant downregulation. Entry into senescence occurred with characteristic changes in Retinoblastoma (RB) expression patterns. Rb1 and p107 genes expression decreased during in vitro cultivation. In contrast, pRb2/p130 became the prominent RB protein. This suggests that RB2/P130 could be a marker of senescence or that it even plays a role in triggering the process in MSCs.

Glutathione is recruited into the nucleus in early phases of cell proliferation.

We have studied the possible correlation between nuclear glutathione distribution and the progression of the cell cycle. The former was studied by confocal microscopy using 5-chloromethyl fluorescein diacetate and the latter by flow cytometry and protein expression of Id2 and p107. In proliferating cells, when 41% of them were in the S+G(2)/M phase of the cell cycle GSH was located mainly in the nucleus. When cells reached confluence (G(0)/G(1)) GSH was localized in the cytoplasm with a perinuclear distribution. The nucleus/cytoplasm fluorescence ratio for GSH reached a maximal mean value of 4.2 +/- 0.8 at 6 h after cell plating. A ratio higher than 2 was maintained during exponential cell growth. In the G(0)/G(1) phase of the cell cycle, the nucleus/cytoplasm GSH ratio decreased to values close to 1. We report here that cells concentrate GSH in the nucleus in the early phases of cell growth, when most of the cells are in an active division phase, and that GSH redistributes uniformly between the nucleus and the cytoplasm when cells reach confluence.

Pocket proteins p107 and p130 exhibit increased expression in macrophages during SIV encephalitis.

Progression of HIV encephalitis (HIVE) is associated with neuronal damage and loss because of infiltration of infected and/or activated macrophages into the CNS. We have previously observed increased inactivation of the retinoblastoma susceptibility gene product (pRb) by phosphorylation in neurons and glia of HIVE and the simian model of HIVE (SIVE). To determine if other pRb family members are altered in response to increased macrophage-secreted factors, we investigated expression of pRb family members p107 and p130 in SIVE. Both p130 and p107 exhibited increased staining in macrophages, but not neurons, astrocytes or T-cells in SIVE. Increased p130 and p107 immunostaining was not limited to virally infected or PCNA-expressing macrophages. Most p107-positive staining was observed in perivascular macrophages, suggesting p107 may indicate macrophages at a specific stage of differentiation soon after migration. In contrast, cytoplasmic p130 was found in the majority of macrophages present in SIVE cases and may indicate activation as it was not seen in microglia in control CNS. These findings suggest that p107 and p130 are differentially expressed in CNS macrophage populations which may have multiple derivations and/or roles in lentiviral encephalitis.

[A primary study on the gene expression profiling of human brain contusion by cDNA microarray].

OBJECTIVE: To screen the differential expression of oncogenes and tumors suppressed genes(OTS genes) after human brain contusion by cDNA microrarray. METHODS: The total RNAs isolated from normal and contusion human brain tissues were purified by Oligotex to obtain mRNAs. Both sources of mRNAs were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP to prepare the hybridization probes. The probe from normal tissue and the contusion brain tissue were labeled with Cy3-dUTP and Cy5-dUTP respectively. The mixed probes were hybridized to the BioDoor Chip OTS-2.2S, a cDNA microarray which contains 227 oncogenes and tumors suppressed genes. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and showed differences between two tissues. RESULTS: Among the 227 target genes, 3 genes including Human carcinoma associated HOJ-1 (HoJ-1), Human KIAAOO65 gene,Human retinoblastoma related protein (p107) gene, showed distinct deference in expression level between the human brain contusion tissue and normal tissue. CONCLUSION: The 3 genes in the brain contusion was significantly the differential expression by OTS 2.2S cDNA microarray. Further analysis of these genes will be helpful to understand the molecular mechanism of brain injury and utilization in forensic medicine.