Effect of different cryo-devices on in vitro maturation and development of vitrified-warmed immature buffalo oocytes

The aim of the study was to identify a cryo-device that would be best suited for the vitrification of buffalo immature cumulus-oocyte complexes (COCs) as judged by viability and meiotic competence of the vitrified-warmed oocytes and their development ability following in vitro fertilization (IVF). The expression of oocyte secreting factors and their receptors (GDF9, BMP15, BMPR2, TGFBR1) and apoptosis related genes (BCL2, BAX, P53, C-MYC) were compared in vitrified-warmed oocytes after in vitro maturation. COCs from the ovaries of slaughtered buffaloes were vitrified in a combination of dimethyl sulfoxide, ethylene glycol, and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). The fresh COCs were exposed to vitrification and warming solutions as in other vitrification methods without plunging in to liquid nitrogen (EC). The viability of vitrified-warmed COCs, 2 h post warming in HS and CT was similar to fresh and EC groups but significantly higher than CS and OPS methods. The proportions of oocytes with first polar body after 24 h in vitro maturation were significantly higher in HS and CT methods than in CS, OPS and CL methods. The development ability of these vitrified-warmed oocytes to blastocyst stage following IVF in all vitrified groups was significantly lower than control and EC groups. Among the vitrified groups, the blastocyst rate in HS, CT and CL groups was significantly higher than in OPS and CS groups. It was also observed that the expression levels of GDF9, BMP15, BMPR2, TGFBR1, BCL2, BAX, P53 and C-MYC genes in vitrified-warmed COCs in CT, HS and CL groups were similar to control. The results indicated that HS, CT and CL are more suitable cryo-devices for vitrification of buffalo immature oocytes.     

Apoptotic and antiproliferative effects of silk protein sericin conjugated-AgNO3 nanoparticles in human breast cancer cells

Bombyx mori silk sericin is a globular-like protein that is used as an antioxidant, antibacterial, and antitumor agent. In this current research, we isolated sericin by degumming process and formation of sericin-AgNO₃ NPs confirmed by UV–vis spectra, SEM, EDX, FTIR, and XRD patterns. The sericin and sericin-AgNO₃ NPs mediated changes in human breast cancer cells were determined. The antiproliferative activity of sericin-AgNO₃ NPs was analyzed by MTT dye reduction assay. Alterations at molecular levels were investigated by qRT-PCR, while apoptotic effects were studied by nuclear DNA staining. After 72 h treatment, sericin and sericin-AgNO₃ NPs showed significant antiproliferative effects in MDA-MB-231 (26 %) and MCF-7 (41 %) cells. Expression modification showed prominent stimulation of cell cycle arrest and stress related genes such as cyclin-dependent kinase inhibitors (CDKN1A, CDKN1B), and GADD family genes. RT-PCR results of the GADD family include GADD45A, B, G, 34, 153 and cyclin-dependent kinase inhibitors (CDKN1A, 1B) showed pronounced induction of 3.1 to 19.8-folds in MCF-7 cell line while induction in MDA-MB-231 cell line was 2.5 to 34.3-folds. Nuclear DAPI staining showed significant induction of apoptosis and nuclear fragmentation in MDA-MB-231 cells at a concentration of 1 mg/mL for both sericin and sericin-AgNO₃ NPs. Meanwhile, in case of MCF-7 cells, after treatment with sericin and sericin-AgNO₃ NPs (1 mg/mL), the cells changed into a round shape and lost their original spindle outlook in dose-dependent manners. We concluded that sericin-AgNO₃ NPs have significant antiproliferative, apoptosis, and genetic profiling effects in both breast cancer cell lines at the highest concentration.     

Induction of HLA expression in Daudi cells after cell fusion

The Daudi cell line, established from a Burkitt lymphoma, has recently been found to be HLA- andΒ ₂-microglobulin-negative, although it expresses B lymphocyte alloantigens. This report is concerned with the reexpression of HLA-A10, B38, and B17 on the Daudi cell, after cell fusion with another human cell line (Raji) or with mouse fibroblasts. In the latter fusion, the same HLA specificities are re-expressed, but not humanΒ ₂-microglobulin while mouseΒ ₂-microglobulin andH-2 could be detected. No such reexpression was observed when Daudi was fused with the F9 mouse teratocarcinoma, which lacks mouseΒ ₂-m andH-2. No HLA activity (alloantigenic and xenogenic activity) was detected in the membrane or cytoplasm of Daudi, using salt extraction and sonication. Therefore we postulate thatΒ ₂-microglobulin could be necessary for the expression and possible synthesis of the HLA antigen.     

p16INK4A Sensitizes Human Leukemia Cells to FAS- and Glucocorticoid-induced Apoptosis via Induction of BBC3/Puma and Repression of MCL1 and BCL2

Loss of CDKN2A/p16^INK4A in hematopoietic stem cells is associated with enhanced self-renewal capacity and might facilitate progression of damaged stem cells into pre-cancerous cells that give rise to leukemia. This is also reflected by the frequent loss of the INK4A locus in acute lymphoblastic T-cell leukemia. T-cell acute lymphoblastic leukemia cells designed to conditionally express p16^INK4A arrest in the G₀/G₁ phase of the cell cycle and show increased sensitivity to glucocorticoid- and tumor necrosis factor receptor superfamily 6-induced apoptosis. To investigate the underlying molecular mechanism for increased death sensitivity, we interfered with specific steps of apoptosis signaling by expression of anti-apoptotic proteins. We found that alterations in cell death susceptibility resulted from changes in the composition of pro- and anti-apoptotic BCL2 proteins, i.e. repression of MCL1, BCL2, and PMAIP1/Noxa and the induction of pro-apoptotic BBC3/Puma. Interference with Puma induction by short hairpin RNA technology or retroviral expression of MCL1 or BCL2 significantly reduced both glucocorticoid- and FAS-induced cell death in p16^INK4A-reconstituted leukemia cells. These results suggest that Puma, in concert with MCL1 and BCL2 repression, critically mediates p16^INK4A-induced death sensitization and that in human T-cell leukemia the deletion of p16^INK4A confers apoptosis resistance by shifting the balance of pro- and anti-apoptotic BCL2 proteins toward apoptosis protection.     

Middle Infrared Radiation Induces G2/M Cell Cycle Arrest in A549 Lung Cancer Cells

There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3–5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G₂/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker γ-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G₂/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression.     

Copy number aberrations of BCL2 and CDKN2A/B identified by array-CGH in thymic epithelial tumors

The molecular pathology of thymic epithelial tumors (TETs) is largely unknown. Using array comparative genomic hybridization (CGH), we evaluated 59 TETs and identified recurrent patterns of copy number (CN) aberrations in different histotypes. GISTIC algorithm revealed the presence of 126 significant peaks of CN aberration, which included 13 cancer-related genes. Among these peaks, CN gain of BCL2 and CN loss of CDKN2A/B were the only genes in the respective regions of CN aberration and were associated with poor outcome. TET cell lines were sensitive to siRNA knockdown of the anti-apoptotic molecules BCL2 and MCL1. Gx15-070, a pan-BCL2 inhibitor, induced autophagy-dependent necroptosis in TET cells via a mechanism involving mTOR pathways, and inhibited TET xenograft growth. ABT263, an inhibitor of BCL2/BCL-XL/BCL-W, reduced proliferation in TET cells when administered in combination with sorafenib, a tyrosine kinase inhibitor able to downregulate MCL1. Immunohistochemistry on 132 TETs demonstrated that CN loss of CDKN2A correlated with lack of expression of its related protein p16^INK4 and identified tumors with poor prognosis. The molecular markers BCL2 and CDKN2A may be of potential value in diagnosis and prognosis of TETs. Our study provides the first preclinical evidence that deregulated anti-apoptotic BCL2 family proteins may represent suitable targets for TET treatment.     

Inducement of mitosis delay by cucurbitacin E,a novel tetracyclic triterpene from climbing stem of Cucumis melo L., through GADD45γ in human brain malignant glioma (GBM) 8401 cells

Cucurbitacin E (CuE) is a natural compound previously shown to have anti-feedant, antioxidant and antitumor activities as well as a potent chemo-preventive action against cancer. The present study investigates its anti-proliferative property using MTT assay; CuE demonstrated cytotoxic activity against malignant glioma GBM 8401 cells and induced cell cycle G₂/M arrest in these cells. CuE-treated cells accumulated in metaphase (CuE 2.5–10 μM) as determined using MPM-2 by flow cytometry. We attempted to characterize the molecular pathways responsible for cytotoxic effects of CuE in GBM 8401 cells. We studied the genome-wide gene expression profile on microarrays and molecular networks by using pathway analysis tools of bioinformatics. The CuE reduced the expression of 558 genes and elevated the levels of 1354 genes, suggesting an existence of the common pathways involved in induction of G₂/M arrest. We identified the RB (GADD45β and GADD45γ) and the p53 (GADD45α) signaling pathways as the common pathways, serving as key molecules that regulate cell cycle. Results indicate that CuE produced G₂/M arrest as well as the upregulation of GADD45 γ and binding with CDC2. Both effects increased proportionally with the dose of CuE, suggesting that the CuE-induced mitosis delay is regulated by GADD45γ overexpression. Our findings suggest that, in addition to the known effects on cancer prevention, CuE may have antitumor activity in glioma therapy.     

Association between variations in cell cycle genes and idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating and progressive lung disease. Its aetiology is thought to involve damage to the epithelium and abnormal repair. Alveolar epithelial cells near areas of remodelling show an increased expression of proapoptotic molecules. Therefore, we investigated the role of genes involved in cell cycle control in IPF. Genotypes for five single nucleotide polymorphisms (SNPs) in the tumour protein 53 (TP53) gene and four SNPs in cyclin-dependent kinase inhibitor 1A (CDKN1A), the gene encoding p21, were determined in 77 IPF patients and 353 controls. In peripheral blood mononuclear cells (PBMC) from 16 healthy controls mRNA expression of TP53 and CDKN1A was determined.Rs12951053 and rs12602273, in TP53, were significantly associated with survival in IPF patients. Carriers of a minor allele had a 4-year survival of 22% versus 57% in the non-carrier group (p = 0.006). Rs2395655 and rs733590, in CDKN1A, were associated with an increased risk of developing IPF. In addition, the rs2395655 G allele correlated with progression of the disease as it increased the risk of a rapid decline in lung function. Functional experiments showed that rs733590 correlated significantly with CDKN1A mRNA expression levels in healthy controls.This is the first study to show that genetic variations in the cell cycle genes encoding p53 and p21 are associated with IPF disease development and progression. These findings support the idea that cell cycle control plays a role in the pathology of IPF. Variations in TP53 and CDKN1A can impair the response to cell damage and increase the loss of alveolar epithelial cells.     

Primary mediastinal B-cell lymphoma: detection of BCL2 gene rearrangements by PCR analysis and FISH

Primary mediastinal large B-cell lymphoma (PMBCL) has a characteristic clinical presentation, morphology, and immunophenotype, representing a clinically favorable subgroup of diffuse large B-cell lymphoma (DLBCL). By gene expression profiling (GEP), PMBCL shares features with classical Hodgkin lymphoma (cHL). Of further interest, BCL6 gene mutations and BCL6 and/or MUM1 expression in a number of PMBCLs have supported an activated B-cell (ABC) origin. Several studies, including GEP, have failed to detect BCL2 gene rearrangements (GRs) in PMBCL. An index case of t(14; 18)+ PMBCL prompted our study of the incidence of BCL2 GRs in PMBCL by polymerase chain reaction (PCR)/fluorescence in situ hybridization (FISH) analyses and its possible clinical impact. Twenty-five retrospectively identified, well-defined PMBCLs (five with cytogenetics) from three institutions were analyzed for a BCL2 GR by PCR/FISH analyses. The formalin-fixed, paraffin-embedded tissue blocks of 24 available cases were also analyzed by BCL2 immunohistochemistry (IHC). Of the five with cytogenetics, two had a t(14; 18) (q32; q21). Of the 25 analyzed by PCR, 2 had no amplifiable DNA (aDNA), including 1 t(14; 18)+ case. Of those with aDNA, two showed a BCL2 GR; by FISH analysis, three demonstrated a BCL2 GR. BCL2 protein expression by IHC analysis was variably detected in 21 out of 24 (strongly, uniformly expressed: 6, including all with a t(14; 18) or a BCL2 gene rearrangement; moderately weakly expressed in a subset of the malignant cells: 15). Available clinical follow-up of this BCL2+ subset showed a similar course to the other PMBCL cases. Our results imply that a subset of PMBCL [(4 out of 24 analyzed) in our series] may be of GC origin. A larger study is necessary to determine any clinical significance.     

Differential expression of genes linked to the leukemia inhibitor factor signaling pathway during the estrus cycle and early pregnancy in the porcine endometrium

The objective of this study was to determine the expression profiles of leukemia inhibitory factor (LIF) and its receptor (LIFR), interleukin 6 receptor (IL6R), tumor protein p53 (TP53) and B-cell CLL/lymphoma 2 (BCL2) in the porcine endometrium on selected days of the estrous cycle and pregnancy. Time- and reproductive status (estrous cycle/pregnancy)-specific patterns of expression were identified for all investigated genes. The most pronounced changes were seen on Days 12 and 14 of pregnancy when maternal recognition of pregnancy and implantation, respectively, occurs in pigs.