BACKGROUND: Ikaros family zinc finger 1 (IKZF1) is a transcription factor with an important role in controlling hematopoietic proliferation and function, particularly lymphoid cell differentiation. It was previously shown that various mechanisms and expression patterns of Ikaros are linked to a variety of cancers. We hypothesized that aberrant methylation (hypomethylation) of the IKZF1 promoter region might be one of the causes of B-cell acute lymphoblastic leukemia (B-ALL). In B-ALL patients, an increased expression of this gene is a potential cause of B-cell differentiation arrest and proliferation induction. Therefore, as more than 90% of patients with ALL are <15 years old, we investigated the methylation pattern of the IKZF1 promoter in childhood B-ALL. METHODS: Twenty-five newly diagnosed B-ALL cases were included (all younger than 15 yr). In addition, we selected 25 healthy age- and sex-matched children as the control group. We collected the blood samples in EDTA-containing tubes and isolated lymphocytes from whole blood using Ficoll 1.077 Lymphosep. Next, we extracted genomic DNA with the phenol/chloroform method. Two microgram of DNA per sample was treated with sodium bisulfite using the EpiTect Bisulfite Kit, followed by an assessment of DNA methylation by polymerase chain reaction (PCR) analysis of the bisulfite-modified genomic DNA. RESULTS: Our data highlighted a hypomethylated status of the IKZF1 promoter in the ALL cases (96% of the cases were unmethylated). In contrast, the control group samples were partially methylated (68%). CONCLUSION: This study demonstrated a hypomethylated pattern of the IKZF1 promoter region in childhood B-ALL, which might underlie the aberrant Ikaros expression patterns that were previously linked to this malignancy.
B-Lymphocytes ; Child ; DNA ; DNA Methylation ; Ficoll ; Hematologic Neoplasms ; Humans ; Leukemia ; Lymphocytes ; Methods ; Methylation ; Polymerase Chain Reaction ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Promoter Regions, Genetic ; Sodium ; Transcription Factors ; Zinc Fingers

OBJECTIVE: The favored method of preserving fertility in young female cancer survivors is cryopreservation and autotransplantation of ovarian tissue. Reducing hypoxia until angiogenesis takes place is essential for the survival of transplanted ovarian tissue. The aim of this study was to investigate the role of angiopoietin-1 (Angpt-1), angiopoietin-2 (Angpt-2), and vascular endothelial growth factor (VEGF) in ovarian tissue grafts that were cryopreserved using two methods. METHODS: Ovarian tissues harvested from ICR mice were divided into three groups: group I (control), no cryopreservation; group II, vitrification in EFS (ethylene-glycol, ficoll, and sucrose solution)-40; and group III, slow freezing in dimethyl sulfoxide. We extracted mRNA for VEGF, Angpt-1, and Angpt-2 from ovarian tissue 1 week following cryopreservation and again 2 weeks after autotransplantation. We used reverse transcriptase-polymerase chain reaction to quantify the levels of VEGF, Angpt-1, and Angpt-2 in the tissue. RESULTS: Angpt-1 and Angpt-2 expression decreased after cryopreservation in groups II and III. After autotransplantation, Angpt-1 and Angpt-2 expression in ovarian tissue showed different trends. Angpt-1 expression in groups II and III was lower than in group I, but Angpt-2 in groups II and III showed no significant difference from group I. The vitrified ovarian tissues had higher expression of VEGF and Angpt-2 than the slowfrozen ovarian tissues, but the difference was not statistically significant. CONCLUSION: Our results indicate that Angpt-2 may play an important role in ovarian tissue transplantation after cryopreservation although further studies are needed to understand its exact function.
Angiopoietin-1* ; Angiopoietin-2 ; Animals ; Anoxia ; Autografts ; Cryopreservation* ; Dimethyl Sulfoxide ; Female ; Fertility ; Fertility Preservation ; Ficoll ; Freezing ; Humans ; Methods* ; Mice ; Mice, Inbred ICR ; Ovary ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger ; Sucrose ; Survivors ; Tissue Transplantation ; Transplantation, Autologous ; Transplants* ; Vascular Endothelial Growth Factor A* ; Vascular Endothelial Growth Factors ; Vitrification

BACKGROUND/OBJECTIVES: The objectives of this study were to investigate the effects of lycopene on the migration, adhesion, tube formation capacity, and p38 mitogen-activated protein kinase (p38 MAPK) activity of endothelial progenitor cells (EPCs) cultivated with high glucose (HG) and as well as explore the mechanism behind the protective effects of lycopene on peripheral blood EPCs. MATERIALS/METHODS: Mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation. EPCs were identified after induction of cellular differentiation. Third generation EPCs were incubated with HG (33 mmol/L) or 10, 30, and 50 microg/mL of lycopene plus HG. MTT assay and flow cytometry were performed to assess proliferation and apoptosis of EPCs. EPC migration was assessed by MTT assay with a modified boyden chamber. Adhesion assay was performed by replating EPCs on fibronectin-coated dishes, after which adherent cells were counted. In vitro vasculogenesis activity was assayed by Madrigal network formation assay. Western blotting was performed to analyze protein expression of both phosphorylated and non-phosphorylated p38 MAPK. RESULTS: The proliferation, migration, adhesion, and in vitro vasculogenesis capacity of EPCs treated with 10, 30, and 50 microg/mL of lycopene plus HG were all significantly higher comapred to the HG group (P < 0.05). Rates of apoptosis were also significantly lower than that of the HG group. Moreover, lycopene blocked phosphorylation of p38 MAPK in EPCs (P < 0.05). To confirm the causal relationship between MAPK inhibition and the protective effects of lycopene against HG-induced cellular injury, we treated cells with SB203580, a phosphorylation inhibitor. The inhibitor significantly inhibited HG-induced EPC injury. CONCLUSIONS: Lycopene promotes proliferation, migration, adhesion, and in vitro vasculogenesis capacity as well as reduces apoptosis of EPCs. Further, the underlying molecular mechanism of the protective effects of lycopene against HG-induced EPC injury may involve the p38 MAPK signal transduction pathway. Specifically, lycopene was shown to inhibit HG-induced EPC injury by inhibiting p38 MAPKs.
Apoptosis ; Blotting, Western ; Centrifugation, Density Gradient ; Ficoll ; Flow Cytometry ; Glucose* ; Humans ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Protein Kinases ; Signal Transduction ; Stem Cells*

BACKGROUND/OBJECTIVES: The objectives of this study were to investigate the effects of lycopene on the migration, adhesion, tube formation capacity, and p38 mitogen-activated protein kinase (p38 MAPK) activity of endothelial progenitor cells (EPCs) cultivated with high glucose (HG) and as well as explore the mechanism behind the protective effects of lycopene on peripheral blood EPCs. MATERIALS/METHODS: Mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation. EPCs were identified after induction of cellular differentiation. Third generation EPCs were incubated with HG (33 mmol/L) or 10, 30, and 50 microg/mL of lycopene plus HG. MTT assay and flow cytometry were performed to assess proliferation and apoptosis of EPCs. EPC migration was assessed by MTT assay with a modified boyden chamber. Adhesion assay was performed by replating EPCs on fibronectin-coated dishes, after which adherent cells were counted. In vitro vasculogenesis activity was assayed by Madrigal network formation assay. Western blotting was performed to analyze protein expression of both phosphorylated and non-phosphorylated p38 MAPK. RESULTS: The proliferation, migration, adhesion, and in vitro vasculogenesis capacity of EPCs treated with 10, 30, and 50 microg/mL of lycopene plus HG were all significantly higher comapred to the HG group (P < 0.05). Rates of apoptosis were also significantly lower than that of the HG group. Moreover, lycopene blocked phosphorylation of p38 MAPK in EPCs (P < 0.05). To confirm the causal relationship between MAPK inhibition and the protective effects of lycopene against HG-induced cellular injury, we treated cells with SB203580, a phosphorylation inhibitor. The inhibitor significantly inhibited HG-induced EPC injury. CONCLUSIONS: Lycopene promotes proliferation, migration, adhesion, and in vitro vasculogenesis capacity as well as reduces apoptosis of EPCs. Further, the underlying molecular mechanism of the protective effects of lycopene against HG-induced EPC injury may involve the p38 MAPK signal transduction pathway. Specifically, lycopene was shown to inhibit HG-induced EPC injury by inhibiting p38 MAPKs.
Apoptosis ; Blotting, Western ; Centrifugation, Density Gradient ; Ficoll ; Flow Cytometry ; Glucose* ; Humans ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Protein Kinases ; Signal Transduction ; Stem Cells*

OBJECTIVE: The aim of this study was to compare vitrification optimization of mouse embryos using electron microscopy (EM) grid, cryotop, and thin plastic strip (TPS) containers by evaluating developmental competence and apoptosis rates. METHODS: Mouse embryos were obtained from superovulated mice. Mouse cleavage-stage, expanded, hatching-stage, and hatched-stage embryos were cryopreserved in EM grid, cryotop, and TPS containers by vitrification in 15% ethylene glycol, 15% dimethylsulfoxide, 10 microg/mL Ficoll, and 0.65 M sucrose, and 20% serum substitute supplement (SSS) with basal medium, respectively. For the three groups in which the embryos were thawed in the EM grid, cryotop, and TPS containers, the thawing solution consisted of 0.25 M sucrose, 0.125 M sucrose, and 20% SSS with basal medium, respectively. Rates of survival, re-expansion, reaching the hatched stage, and apoptosis after thawing were compared among the three groups. RESULTS: Developmental competence after thawing of vitrified expanded and hatching-stage blastocysts using cryotop and TPS methods were significantly higher than survival using the EM grid (p<0.05). Also, apoptosis positive nuclei rates after thawing of vitrified expanded blastocysts using cryotop and TPS were significantly lower than when using the EM grid (p<0.05). CONCLUSION: The TPS vitrification method has the advantages of achieving a high developmental ability and effective preservation.
Animals ; Apoptosis ; Blastocyst ; Dimethyl Sulfoxide ; Embryonic Structures ; Ethylene Glycol ; Ethylenes ; Ficoll ; Mental Competency ; Mice ; Microscopy, Electron ; Plastics ; Sucrose ; Vitrification

PURPOSE: Considering the complications of nonspecific immunosuppression, as well as the availability of insulin, heavy immunosuppressive treatment to pancreatic islet transplantation patients is not justified. Antigen administration via the portal vein has been demonstrated to induce immunosuppression, and may present a possible mechanism for the induction of tolerance. Using a mouse model, without any immunosuppressive treatment, the islet allograft survivals were compared between portal venous transfusion and portal venous saline injection groups. METHODS: Six C57BL/6J mice were used as pancreatic islet donors per Balb/c recipient mouse. Islets were harvested by digestion of the pancreata with collagenase, with subsequent Ficoll density gradient separation. Recipient mice were divided into two groups: seven mice received a portal venous injection of 0.1 cc saline (PVS) and eight a portal venous transfusion of 0.1 cc donor blood (PVT). Islets were transplanted into the subcapsular space of the left kidney. Transplantation failure was determined if the transplanted mouse failed to show a blood glucose level less than 200 mg/dl at 24 hours after the transplantation; these mice were not included in the statistics. Rejection was determined when the normalized blood glucose level (<200 mg/dl) returned to above 300 mg/dl. RESULTS: The mean islet equivalent numbers (IEQ) of the seven PVS and eight PVT mice were 893+/-262 and 911+/-288, respectively. The islet allograft survival of the PVS group ranged between 1 to 9 days; whereas, that of the PVT group ranged between 6 to 16 days. The PVT group showed significantly higher islet allograft survival than the PVS group (P=0.0443). CONCLUSION: A portal venous transfusion prolonged the islet allograft survival.
Allografts* ; Animals ; Blood Glucose ; Collagenases ; Digestion ; Ficoll ; Humans ; Immunosuppression ; Insulin ; Islets of Langerhans* ; Kidney ; Mice* ; Portal Vein* ; Tissue Donors ; Transplantation

PURPOSE: A high yield of viable pancreatic islets is an essential prerequisite for the study of pancreatic islet transplantation. The purpose of this study is to compare the yield between intra-gall bladder (intra-GB) and intra-common bile duct (intra-CBD) injection of collagenase solution for isolation of mouse pancreatic islets. METHODS: The mice were divided into two groups, the intra-GB and intra-CBD groups, and each group included twelve mice, respectively. Collagenase solution was injected via the gallbladder in the intra-GB group mice, while this was done via the common bile duct in the intra-CBD group. After removal and digestion of the mouse pancreases, the pancreatic islets were isolated by Ficoll density gradient centrifugation and hand picking. RESULTS: The intra-GB group yielded 121.67+/-39.86 IEQs, and the intra-CBD group reveled 168.17+/-29.23 IEQs. There was a statistically significant difference in islet yield between the two groups (P=0.005, Mann-Whitney Test). The purities of the isolated islets were 86.42+/-3.99% for the intra-GB group and 87.17+/-4.47% for the intra-CBD group, and there was no difference between the two groups (P=0.755, Mann- Whitney Test). CONCLUSION: Both the intra-GB and intra-CBD groups yielded an average of >120 IEQs. However, the intra-CBD group revealed a higher yield than the intra-GB group for isolating mouse pancreatic islets.
Animals ; Bile Ducts* ; Bile* ; Centrifugation, Density Gradient ; Collagenases* ; Common Bile Duct ; Digestion ; Ficoll ; Gallbladder ; Hand ; Islets of Langerhans* ; Mice* ; Pancreas ; Urinary Bladder*

OBJECTIVE: This study was conducted to find an optimal condition for the vitrification of mouse morulae and expanded blastocysts. MATERIALS AND METHODS: Mouse embryos were obtained at 2-cell stage and cultured to morula and expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% Serum Substitute Supplement (SSS). The vitrification solutions used were EFS30, EFS35 and EFS40 that contains 30%, 35% and 40% ethylene glycol, respectively, with 18% ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% SSS. The vitrification procedure was performed in EFS solution with three steps, followed by thawing in 6 steps with 0.5 M sucrose, and then survival and hatching-hatched rate per embryos recovered were compared among six groups. RESULTS: After 24 h culture in different vitrification and thawing solution, the survival rate of morula embryos was 94.1%, 85.4% and 59.7% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of morula embryos after 72 h culture was 30.6%, 25% and 11.3% for EFS30, EFS35 and EFS40 group, respectively. The survival rate of expanded blastocyst embryos after 24 h culture was 90.4%, 98.5% and 100% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of expanded blastocyst embryos after 48 h culture was 46.2%, 57.6% and 64.3% for EFS30, EFS35 and EFS40 group, respectively. CONCLUSION: The EFS30 solution was the best for vitrification of mouse morulae. The EFS40 solution was the best for vitrification of mouse expanded blastocysts. The mouse expanded blastocyst was better than mouse morula for vitrification of mouse embryos.
Animals ; Blastocyst ; Embryonic Structures* ; Ethylene Glycol ; Ficoll ; Humans ; Mice* ; Morula ; Sucrose ; Survival Rate ; Vitrification*

PURPOSE: A chemosensitivity test can reflect the differences in responses of individual cancer patients to chemotherapeutic agents. The adenosine triphosphate-based chemotherapy response assay (ATP-CRA)is an accurate method, which does not require a large amount of tissue specimen. So far, no studies have evaluated the utility of the ATP-CRA in Korea. Therefore, we investigated the clinical usefulness of the ATP-CRA in 53 patients with lung cancer. MATERIALS AND METHODS: Tumor tissues were obtained from bronchoscopic biopsies or surgical resections. The validity of ATP-CRA was assessed focusing on the success rate, experimental error level (intraassay mean coefficient of variation [CV]) and reproducibility. RESULTS: The overall success rate of ATP-CRA was 90.6% (48/53). Normal cells were effectively eliminated from the tumor tissues with the use of ficoll gradient centrifugation and immunomagnetic separation, which was confirmed using loss of heterozygosity analysis of the 3p deletion. The mean CV of ATP assays was 10.5+/-4.6%. The reproducibility of ATP assays was 94+/-3.8%. The results of the ATP assays were reported to physicians within 7 days of specimen collection. More than 6 anticancer drugs were tested on the tumor specimens obtained from bronchoscopic biopsies. CONCLUSION: The ATP-CRA is a stable, accurate and potentially practical chemosensitivity test in patients with lung cancer.
Adenosine Triphosphate ; Adenosine* ; Biopsy ; Centrifugation ; Drug Therapy* ; Feasibility Studies* ; Ficoll ; Humans ; Immunomagnetic Separation ; Korea ; Loss of Heterozygosity ; Lung Neoplasms* ; Lung* ; Specimen Handling

OBJECTIVE: The aim of this study was to compare the survival and developmental rate of two vitrification solutions for the vitrification of mouse expanded blastocysts. METHODS: Mouse embryos were obtained at 2 cell stage and cultured to expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% serum substitute supplement (SSS). The vitrification solutions used were EFS40 and VS. EFS40 consisted of 40% ethylene glycol, 18% ficoll, and 0.5 M sucrose while VS consisted of 20% ethylene glycol, 20% DMSO, and 10% 1,3-butanediol diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% calf serum (CS). Toxicity was tested by exposing expanded blastocysts to vitrification solution. The vitrification procedure used for EFS40 was performed in three steps, after which they were warmed in 5 steps with 0.5 M sucrose. VS was performed in two steps, after which they were warmed with 1.0 M trehalose. Recovery, survival and hatching rate per expanded blastocysts recovered were compared between two groups. RESULTS: In toxicity test, survival and hatching rate of EFS40 group were 95% and 100%, respectively. In contrast, survival and hatching rate of VS group were 100% and 87.5%, respectively. After vitrification and warming in solution, recovery rate for EFS40 group was 73.7% whereas recovery rate for VS group was 66.5%. After 24 h culture, survival and hatching rate were 80.5% and 20.7% for EFS40 and 66% and 0% for VS group, respectively. After 48 h culture, survival and hatching rate were 69% and 33.3% for EFS40 and 58.3% and 1.9% for VS group, respectively. Survival and hatching rate in EFS40 group were significantly higher than those found in VS group. CONCLUSION: The EFS40 solution was better than VS solution for vitrification of mouse expanded blastocysts.
Animals ; Blastocyst* ; Dimethyl Sulfoxide ; Embryonic Structures ; Ethylene Glycol ; Ficoll ; Humans ; Mice* ; Sucrose ; Toxicity Tests ; Trehalose ; Vitrification*