Engaging adolescent girls in transactional sex through compensated dating.

Transactional sex through so-called compensated dating in adolescent girls is a problem in need of public concern. Compensated dating typically involves the use of information communication technology to advertise, search, bargain, and eventually arrange for transactional sex. The technology enables the sexual partners to maintain privacy and secrecy in transactional sex. Such secrecy necessitates the girls' disclosure about their life experiences in order to address the concern. The disclosure is the focus of the present qualitative study of 27 girls practicing the dating in Hong Kong, China. Based on the disclosure, the study presents a grounded theory that epitomizes engagement in compensated dating by referential choice. Such a referential choice theory unravels that choice with reference to the family push and social norms sustains the engagement. Meanwhile, the choice rests on expectancy and reinforcement from experiential learning about compensated dating. The theory thus implies ways to undercut the engagement through diverting the referential choice of the dating.

Dormancy activation mechanism of oral cavity cancer stem cells.

Radiotherapy and chemotherapy are targeted primarily at rapidly proliferating cancer cells and are unable to eliminate cancer stem cells in the G0 phase. Thus, these treatments cannot prevent the recurrence and metastasis of cancer. Understanding the mechanisms by which cancer stem cells are maintained in the dormant G0 phase, and how they become active is key to developing new cancer therapies. The current study found that the anti-cancer drug 5-fluorouracil, acting on the oral squamous cell carcinoma KB cell line, selectively killed proliferating cells while sparing cells in the G0 phase. Bisulfite sequencing PCR showed that demethylation of the Sox2 promoter led to the expression of Sox2. This then resulted in the transformation of cancer stem cells from the G0 phase to the division stage and suggested that the transformation of cancer stem cells from the G0 phase to the division stage is closely related to an epigenetic modification of the cell.

Downregulation of KPNA2 in non-small-cell lung cancer is associated with Oct4 expression.

BACKGROUND: Oct4 is a major transcription factor related to stem cell self-renewal and differentiation. To fulfill its functions, it must be able to enter the nucleus and remain there to affect transcription. KPNA2, a member of the karyopherin family, plays a central role in nucleocytoplasmic transport. The objective of the current study was to examine the association between Oct4 and KPNA2 expression levels with regard to both the clinicopathological characteristics and prognoses of patients with non-small-cell lung cancer (NSCLC). METHODS: Immunohistochemistry was used to detect the expression profile of Oct4 and KPNA2 in NSCLC tissues and adjacent noncancerous lung tissues. Real-time polymerase chain reaction and western blotting were used to detect the mRNA and protein expression profiles of Oct4 and KPNA2 in lung cancer cell lines. Small interfering RNAs were used to deplete Oct4 and KPNA2 expressions. Double immunofluorescence was used to detect Oct4 expression in KPNA2 knockdown cells. Co-immunoprecipitation was used to detect the interaction of Oct4 and KPNA2. RESULTS: Oct4 was overexpressed in 29 of 102 (28.4%) human lung cancer samples and correlated with differentiation (P = 0.002) and TNM stage (P = 0.003). KPNA2 was overexpressed in 56 of 102 (54.9%) human lung cancer samples and correlated with histology (P = 0.001) and differentiation (P = 0.045). Importantly, Oct4 and KPNA2 expression levels correlated significantly (P < 0.01). Expression of Oct4 and KPNA2 was associated with short overall survival. In addition, depleting Oct4 and KPNA2 expression using small interfering RNAs inhibited proliferation in lung cancer cell lines. Real-time polymerase chain reaction and western blotting analysis indicated that reduction of KPNA2 expression significantly reduced mRNA and nucleoprotein levels of Oct4. Double immunofluorescence analysis revealed that nuclear Oct4 signals were reduced significantly in KPNA2 knockdown cells. Co-immunoprecipitation experiments revealed that KPNA2 interacts with Oct4 in lung cancer cell lines. CONCLUSION: Oct4 and KPNA2 play an important role in NSCLC progression. Oct4 nuclear localization may be mediated by its interaction with KPNA2.

Fluorouracil selectively enriches stem-like cells in the lung adenocarcinoma cell line SPC.

Most adult stem cells are in the G0 or quiescent phase of the cell cycle and account for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. This study sought to enrich cancer stem cells and explore cancer stem-like cell clones using 5-fluorouracil (5-FU) in the lung adenocarcinoma cell line, SPC. Proliferation inhibition was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, according to which half maximal inhibitory concentration values were calculated. Expression levels of stem cell markers after treatment with 5-FU were examined using immunofluorescence and Western blotting. Additionally, side population (SP) cells were sorted using FACS. Properties of SP cells were evaluated by using Transwell, colony-forming assays, and tumor formation experiments. 5-FU greatly inhibits proliferation, especially of cells in S phase. SP cells possess greater invasive potential, higher clone-forming potential, and greater tumor-forming ability than non-SP cells. Treatment with 5-FU enriches the SP cells with stem cell properties in human lung adenocarcinoma cell lines.

Enhanced Autophagy and Reduced Expression of Cathepsin D Are Related to Autophagic Cell Death in Epstein-Barr Virus-Associated Nasal Natural Killer/T-Cell Lymphomas: An Immunohistochemical Analysis of Beclin-1, LC3, Mitochondria (AE-1), and Cathepsin D in Nasopharyngeal Lymphomas.

This study investigated autophagy in 37 cases of nasopharyngeal lymphomas including 23 nasal natural killer (NK)/T-cell lymphomas (NKTCL), 3 cytotoxic T-cell lymphomas (cytotoxic-TML) and 9 B-cell lymphomas (BML) by means of antigen-retrieval immunohistochemistry of beclin-1, LC3, mitochondria (AE-1) and cathepsin D. Peculiar necrosis was noted in EBV(+) lymphomas comprising 21 NKTCL, 2 cytotoxic-TML and 1 BML. Lymphomas without peculiar necrosis showed high expression of beclin-1, macrogranular cytoplasmal stain of LC3 with sporadic nuclear stain, a hallmark of autophagic cell death (ACD), some aggregated mitochondria and high expression of cathepsin D, suggesting a state of growth with enhanced autophagy with sporadic ACD. EBV(+) NKTCL with the peculiar necrosis, showed significantly low level of macrogranular staining of LC3, aggregated mitochondria and low expression of cathepsin D in the cellular areas when degenerative lymphoma cells showed decreased beclin-1, significantly advanced LC3-labeled autophagy, residual aggregated mitochondria and significantly reduced expression of cathepsin D, suggesting advanced autophagy with regional ACD. Consequently it was suggested that enhanced autophagy and reduced expression of lysosomal enzymes induced regional ACD under EBV infection in NKTCL.

Epigenetic Mechanism of Enrichment of A549 Lung Cancer Stem Cells with 5-Fu.

BACKGROUND: The influence of 5-fluorouracil (5-Fu) and cisplatin (CDDP) on the A549 and NCI-H226 cells was studied, and the epigenetic mechanism of enrichment of A549 lung cancer stem cells with 5-Fu was explored. MATERIALS AND METHODS: The cell proliferation of both A549 and NCI-H226 was detected by BrdU assay, and apoptosis condition was measured by flow cytometric analysis. The expressions of OCT3/4 and Nanog in cells treated with 5-Fu or CDDP were measured by immunofluorescence, Western blot and qPCR. qPCR was also performed to determine the relative expression of methyltransferase genes and miRNA. Sequencing after bisulfite treatment (BSP) was employed to detect the methylation of OCT3/4 promoter in A549 cells. And ChIP was conducted to detect the expression of H3K9Me3 and H3K9Ace. RESULTS: Both 5-Fu and CDDP result in the apoptosis of A549 and NCI-H226 cells and improve the expressions of has-miR-134 and has-miR-296. However, 5-Fu enhances the expression of OCT3/4 in A549 cells, and the change of methyltransferase genes and BSP results suggested some genetic differences between CDDP and 5-Fu treatment in A549 cells. ChIP assay showed that the expression of H3K9Me3 significantly decreased and H3K9Ace significantly increased in A549 cells. CONCLUSION: The enrichment effect of CDDP on A549 and NCI-H226 carcinoma stem cells is inconsistent with the enrichment effect of 5-Fu. The enrichment of A549 lung cancer stem cells with 5-Fu might be related to the methylation of OCT3/4 promoter and the expression of H3K9Me3 and H3K9Ace.

Correction: Cycling hypoxia affects cell invasion and proliferation through direct regulation of claudin1 / claudin7 expression, and indirect regulation of p18 through claudin7.

[This corrects the article DOI: 10.18632/oncotarget.14397.].

Fbxw7 and Skp2 Regulate Stem Cell Switch between Quiescence and Mitotic Division in Lung Adenocarcinoma.

BACKGROUND/AIMS: The molecular mechanism of dormancy initiation of cancer stem cells (CSCs) is not clear. This study was to explore the molecular mechanism by which CSCs switch from mitotic division to quiescence. METHODS: MTT assays, flow cytometry, Western blotting, qRT-PCR, and immunofluorescence staining were used to test cell viability, cell cycle and expression of F-box and WD repeat domain-containing 7 (Fbxw7), c-myc, S phase kinase associated protein-2 (Skp2), cyclin-dependent kinase inhibitor 1B (p27), octamer-binding transcription factor 3/4 (Oct3/4), and ß catenin gene in 5-fluorouracil (5-FU)-treated A549 cells. Lung adenocarcinoma xenograft models were employed to detect the effects of Fbxw7 on tumor growth. RESULTS: 5-FU inhibited the proliferation of A549 cells, with a median inhibitory concentration (IC50) of 200 µg/ml after 24 h treatment. 5-FU treatment increased the expressions of Oct3/4, Fbxw7, and p27 and increased the number of A549 cells at G0/G1. 5-FU treatment triggered nuclear translocation of ß-catenin, decreased the expression levels of c-myc and Skp2, and decreased the number of A549 cells at S phase. Release from 5-FU decreased the expressions of Oct3/4, Fbxw7 and p27; decreased the percentage of cells in the G0/G1 phase; increased the expressions of Skp2 and c-myc; and increased the proportion of cells in S phase. 5-FU treatment led to high expressions of Oct3/4, c-myc, and p27, with low expressions of Fbxw7 and Skp2. Knockdown of Fbxw7 augmented the expression of c-myc and decreased the proportion of A549 cells in Go/G1 phase. Skp2 siRNA increased the expression of p27 and the percentage of G0/G1 phase cells and reduced the proportion of S phase cells. Fbxw7 overexpression inhibited tumor growth in mouse lung adenocarcinoma xenograft models. When Fbxw7 expression was low, Skp2 expression was higher in lung adenocarcinoma tissues and associated with the differentiation of lung adenocarcinoma. CONCLUSION: 5-FU enriches the CSCs in lung adenocarcinoma cells via increasing Fbxw7 and decreasing Skp2 expression, followed by downregulation of c-myc and upregulation of p27, which switches cells to quiescence.

[Nasal and pharyngeal non-Hodgkin lymphomas and their relationship with Epstein-Barr virus: a report of 158 cases].

OBJECTIVE: To study the clinical features, immunophenotypes and the significance of Epstein-Barr virus infection in primary nasal and pharyngeal non-Hodgkin's lymphomas in Shenyang. METHODS: One hundred and fifty eight cases of primary nasal and pharyngeal non-Hodgkin's lymphomas were included in this study. The samples were stained with haematoxylin and eosin for histological examination. Immunohistochemistry studies were performed using monoclonal antibodies, including CD3 for T-lymphocytes, CD20 for B-lymphocytes, and CD56 and CD57 for NK cells. All cases were reclassified according to the new WHO classification of lymphomas (2001). In situ hybridization detection of EBV-encoded small nuclear RNA (EBER-1) was performed in 99 cases. RESULTS: Overall, 101 (63.9%) of the 158 NHL were extranodal NK/T cell lymphomas (nasal type), 23 (14.6%) were nonspecific peripheral T cell lymphomas and the remaining 34 cases (21.5%) were B cell lymphomas. The primary sites of involvement were the nasal cavity (53.2%, 84/158), the tonsil (24.7%, 39/158) and the pharynx (22.1%, 35/158). Among 99 cases studied by EBER-1 in situ hybridization, a positive detection was seen in 70/71 cases (98.6%) of extranodal NK/T cell lymphoma (nasal type), 8/12 cases (66.7%) of T cell lymphoma, and 7/16 cases (43.8%) of B cell lymphoma. CONCLUSIONS: Among primary nasal and pharyngeal NK lymphomas, extranodal NK/T cell lymphoma (nasal type) is the most common type and is strongly associated with EBV infection. The pathological diagnosis of nasal and pharyngeal lymphomas should take considerations of the anatomic sites and immunophenotypical features.

Expression and Role of Oct3/4 in Injury-Repair Process of Rat Alveolar Epithelium after 5-Fu Treatment.

OBJECTIVE: We aimed to investigate how the embryonic stem cell-related gene Oct3/4 changes during the injury-repair process of distal pulmonary epithelium induced by 5-fluorouracil (5-Fu). METHODS: We have developed the lung injury model induced by 5-Fu and observed the dynamic changes of Oct3/4 by indirect immunofluorescence, Western blot, and quantitative real-time PCR. Immunofluorescence double staining was used to compare the positions of Oct3/4(+) cells and other reported alveolar epithelial stem cells. RESULTS: Oct3/4(+) cells were not found in normal rat lung epithelial cells. However, after treatment with 5-Fu, Oct3/4(+) cells appeared at 12 h, reached the peak at 24 h, then decreased at 48 h, and eventually disappeared at 72 h. Oct3/4 was localized in the nucleus. We found that the sites of Clara cell secretory protein and surfactant protein-C dual positive cells were apparently different from Oct3/4(+) cells. CONCLUSIONS: Our results revealed that, in rat alveolar epithelium, expression of Oct3/4 could be induced after treatment with 5-Fu, then decreased gradually, and was silenced following the alveolar epithelial differentiation. We hold that Oct3/4(+) cells are lung stem cells, which can provide new evidence for identification and isolation of lung epithelial stem cells.

Influence of body mass index on postoperative complications after thymectomy in myasthenia gravis patients.

OBJECTIVES: It is not clear whether being overweight or obese influences postoperative complications in myasthenia gravis (MG) patients. We retrospectively investigated an association between body mass index (BMI) and postoperative complications in MG. MATERIALS AND METHODS: Fifty-nine MG patients who had undergone transsternal thymectomy were classified as low or high BMI based on the criteria for Asian-Pacific populations. An association between BMI and complications was analyzed. RESULTS: MG patients with high BMI had significantly higher rates of major adverse complications (P = 0.033), postoperative respiratory failure (P = 0.045), and longer postoperative hospitalization (P = 0.005). The optimal cutoff value of BMI for postoperative respiratory failure was 23.3 kg/m2, with a sensitivity of 75.0% and a specificity of 64.7% (P = 0.046). CONCLUSIONS: MG patients with a BMI indicating overweight or obesity have a higher risk of postoperative complications after thymectomy. Thus, close monitoring must be performed when surgery is necessary.

Cycling hypoxia affects cell invasion and proliferation through direct regulation of claudin1 / claudin7 expression, and indirect regulation of P18 through claudin7.

Claudins (CLDNs), the major integral membrane proteins at tight junction, play critical roles in apical cell-to-cell adhesion, maintenance of epithelial polarity, and formation of impermeable barriers between epithelial cells.We investigated in this study the expression of CLDNs- Claudin1 (CLDN1) and Claudin7 (CLDN7), and their relation to tumor progression in nasopharyngeal cancer (NPC). CLDN7, rather than CLDN1, showed higher expression in both undifferentiated tumor tissue and the poorly differentiated CNE2 cells, compared with differentiated tissue and the highly differentiated CNE1 cells. Furthermore, knockdown of CLDN7 dramatically inhibited the metastasis and invasion of CNE2 cells suggesting that CLDN7 could act as a biomarker for NPC metastasis.Cycling hypoxia could induce significant changes in CLDN1 and CLDN7 expression in NPC cells. Genetics analysis demonstrated that CLDN1/CLDN7 were not only regulated directly by HIF1a but also affected each other through a feedback mechanism. CLDN7 acted as a bridge to promote HIF1a-induced P18 expression and cell differentiation. Taken together, our results provide evidence that adjusting the oxygenation time and cycles in NPC might be an effective method to prevent / delay the metastasis of poorly differentiated NPC cells.

Changes in the methylation status of the Oct3/4, Nanog, and Sox2 promoters in stem cells during regeneration of rat tracheal epithelium after injury.

We investigated the relationship between promoter methylation and tracheal stem cell activation. We developed a model of rat tracheal epithelium regeneration after 5-fluorouracil (5-FU)-induced injury. Using immunohistochemistry and Western blotting, the expression levels of the stem cell pluripotency regulator Oct3/4 and differentiation marker CK14 were measured after 5-FU treatment. The methylation status of the Oct3/4, Nanog, and Sox2 promoters was investigated using methylation-specific PCR. Additionally, the effects of 5-azacytidine (5-azaC), a demethylating agent, on Oct3/4, Nanog, and Sox2 mRNA and protein expression were evaluated. Finally, we measured the activity of the maintenance and de novo DNA methyltransferases DNMT1, DNMT3a, and DNMT3b. Our data indicate that Oct3/4, Sox2, and Nanog are transiently expressed in response to 5-FU-induced injury, and then they are gradually silenced as the cells differentiate. DNA methylation can result in silencing of gene expression, and it can determine whether tracheal stem cells are in an active or dormant state. Treatment with 5-FU reversed the methylation of the Oct3/4, Nanog, and Sox2 promoters, which corresponded to increases in Oct3/4, Nanog, and Sox2 mRNA and protein. Thus, both maintenance and de novo methyltransferases are involved in regulating tracheal stem cell dormancy and activation.

Dormancy activation mechanism of tracheal stem cells.

Accurate markers and molecular mechanisms of stem cell dormancy and activation are poorly understood. In this study, the anti-cancer drug, 5-fluorouracil, was used to selectively kill proliferating cells of human bronchial epithelial (HBE) cell line. This method can enrich and purify stem cell population. The dormant versus active status of stem cells was determined by phosphorylation of RNAp II Ser2. The surviving stem cells were cultured to form stem cell spheres expressing stem cell markers and transplanted into nude mice to form a teratoma. The results demonstrated the properties of stem cells and potential for multi-directional differentiation. Bisulfite sequencing polymerase chain reaction showed that demethylation of the Sox2 promoter by 5-FU resulted in Sox2 expression in the dormant stem cells. This study shows that the dormancy and activation of HBE stem cells is closely related to epigenetic modification.

[Study of tracheal regeneration after injury induced by 5-fluorouracil in rats].

OBJECTIVE: Localization of tracheal stem cells in rat trachea. METHODS: Extracorporeal tracheal injury (Wistar rats) was induced by 5-FU. The process of regeneration was observed and analyzed by light microscopy, electron microscopy, and immunohistochemistry. RESULTS: Twelve hours after treatment with 5-FU, the tracheal epithelium shed and cells with naked nuclei were seen located sparsely on the basement membrane. Six hours after removal of 5-FU, the tracheal rings were covered with flattened epithelium. These cells were poorly differentiated under electron microscopy. Immunohistochemistry showed few proliferating cell nuclear antigen (PCNA)-negative cells sparsely scattered among PCNA-positive cells on the basement membrane. Nine hours later, electron microscopy found that these cells differentiated into mucous cells and ciliated cells. Forty-eight hours later, the tracheal rings were entirely covered by pseudostratified ciliated columnar epithelium. CONCLUSIONS: A small number of G(0) cells with naked nuclei are located sparsely on the basement membrane of the trachea. Tracheal epithelium regenerates by proliferation and differentiation of these cells. It is likely that some of these G(0) cells on the tracheal basement membrane represent tracheal stem cells.

The recovery process of mice tracheal epithelium injured by 5-FU and its microarray analysis.

Although the research on the localization of trachea stem cells has made a rapid progress, the mechanism of proliferation and differentiation of trachea stem cells remains unclear. The objective of this study is to observe and analyze the recovery process of mice tracheal epithelium injured by 5-FU, and to investigate the mechanism involved in the regulation of tracheal stem cells proliferation and differentiation through morphological, immunofluorescence, and microarray analysis. After treatment with 5-FU, the mature cells were dead and desquamated. Only a few G0 phase cells remained on the basement membrane. When supplied with normal culture media, the cells eventually became flat, cubic, and restored as pseudostratified epithelium. These G0 phase cells were ABCG2 positive. It suggested that these cells could differentiate into cilia cells or Clara cells, and had the multi-differentiation ability of stem cells. We examinated the expression profile of genes involved in the stem cell differentiation in normal tracheal epithelial cells and the regenerated epithelial cells at 24 and 48 h after injured by 5-FU using gene microarray. After 24 h treatment, 8 genes were up-regulated and 31 genes were down-regulated. After 48 h treatment, 5 genes were up-regulated and 42 genes were down-regulated. The differential gene expressions in gene microarray analysis focused on cell cycle regulation, intercellular junction, fibroblast growth factors, bone morphogenetic protein, Notch and Wnt-signaling pathways, which suggested that the differential gene expressions might be closely associated with the proliferation and differentiation of tracheal stem cells.

Enrichment of Oct3/4-positive cells from a human bronchial epithelial cell line.

Most adult stem cells are in the G0 phase of the cell cycle, accounting for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. The anti-tumor drug 5-fluorouracil (5-FU) selectively kills proliferating cells, sparing cells in the G0 phase. Thus, the objective of this study was to determine whether 5-FU can be used to enrich stem cells in a human bronchial epithelial (HBE) cell population in vitro. Side population (SP) cells were isolated from untreated HBE cells or HBE cells treated with 5-FU, and the resulting cells were subjected to colony formation assays, culturing of cell spheres, and tumorigenicity assays. Expression of Oct3/4, Sox2, PCK, and ß-catenin were examined by Western blot analysis and immunofluorescence. Treatment with 5-FU increased the percentage of SP cells from 0.3% to 1.5%, and the clonogenic ability of 5-FU-treated cells was more than twofold higher than that of HBE cells. Cells that survived after 5-FU treatment exhibited a higher capacity for sphere formation. Furthermore, spheres formed from 5-FU-treated cells possessed the capacity to generate differentiated progenies. Cells treated with 5-FU also exhibited tumorigenic potential, based on tumor formation assays in nude mice, and Oct3/4-positive cell aggregates were identified in the resulting tumors. In this study, we have shown that 5-FU treatment enriched the population of cells expressing the putative embryonic markers Oct3/4 and Sox2 and exhibiting nuclear accumulation of ß-catenin. Furthermore, 5-FU-treated cells expressed low levels of the epithelial differentiation marker PCK. Analysis of epigenetic modifications suggested that Oct3/4-positive cells possessed characteristics of stem cells. These results demonstrate that treatment with 5-FU can enrich the stem cell population present in a human bronchial epithelial cell line, and implicate combined treatment with 5-FU and serum-free medium as a new method for isolation of stem-like cells from the HBE cell line.

Immunohistochemistry of programmed cell death in archival human pathology specimens.

Immunohistochemistry (IHC) for detecting key signal molecules involved in programmed cell death (PCD) in archival human pathology specimens is fairly well established. Detection of cleaved caspase-3 in lymphocytes in rheumatoid arthritis (RA) and gastric surface foveolar glandular epithelia but not in synoviocytes in RA, gastric fundic glandular epithelia, or nasal NK/T-cell lymphoma (NKTCL) cells suggests anti-apoptotic mechanisms in cell differentiation and in oncogenesis such as the induction of survivin. Enzymatically pretreated and ultra-super sensitive detection of beclin-1 in synoviocytes in RA and gastric fundic glandular epithelia suggests enhanced autophagy. The deposition of beclin-1 in fibrinoid necrosis in RA and expression of beclin-1 in detached gastric fundic glandular cells suggest that enhanced autophagy undergoes autophagic cell death (ACD). NKTCL exhibited enhanced autophagy through LC3 labeling and showed densely LC3 labeled cell-debris in regions of peculiar necrosis without deposition of beclin-1, indicating massive ACD in NKTCL and the alternative pathway enhancing autophagy following autophagic vesicle nucleation. Autophagy progression was monitored by labeling aggregated mitochondria and cathepsin D. The cell-debris in massive ACD in NKTCL were positive for 8-hydroxydeoxyguanosine, suggesting DNA oxidation occurred in ACD. Immunohistochemical autophagy and PCD analysis in archival human pathology specimens may offer new insights into autophagy in humans.