Src Family Kinase Inhibitor PP2 Has Different Effects on All-Trans-Retinoic Acid or Arsenic Trioxide-Induced Differentiation of an Acute Promyelocytic Leukemia Cell Line / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Leukemic promyelocytes have the unique ability to undergo differentiation after exposure to retinoic acid and both differentiation and apoptosis after exposure to arsenic trioxide (ATO). Recent studies have shown that inhibition of Src family kinases (SFKs) resulted in enhancement of retinoic acid-induced myeloid differentiation. MATERIALS AND METHODS: In this study, we investigated the question of whether the SFK inhibitor PP2 enhanced the differentiation of NB4 cells when combined with ATO as well as when combined with all-trans-retinoic acid (ATRA). In addition, we attempted to determine the difference in retinoic acid-induced gene expression between cells treated with PP2 in combination with ATRA and in combination with ATO. RESULTS: SFK inhibitor PP2 induced significant enhancement of ATRA- or ATO-induced differentiation of NB4 cells. A significantly stronger synergistic effect was observed when PP2 was combined with ATRA than when combined with ATO. Flow cytometric analysis demonstrated a significant increase in CD11b-positive granulocytes up to 60.73% and 31.58%, respectively. These results were confirmed by nitroblue tetrazolium staining. These effects were not related to apoptosis. Results of Annexin-V-fluorescein staining revealed that PP2 combined with ATRA or PP2 combined with ATO did not induce apoptosis in NB4 cells. Retinoic acid-induced gene expression was different in both groups. Intercellular adhesion molecule-1 expression showed a significant increase in cells treated with PP2 in combination with ATRA, whereas cathepsin D expression showed a significant increase in cells treated with PP2 in combination with ATO. CONCLUSION: Our data showed that SFK inhibitor PP2 enhanced acute promyelocytic leukemia cell differentiation when combined with either ATRA or ATO with difference in activation of retinoic acid-induced genes.

Monitoring Transplanted Human Mesenchymal Stem Cells in the Penile Cavernosal Tissues of Streptozotocin-induced Diabetic Rats Using Molecular Magnetic Resonance Imaging / 대한남성과학회지

PURPOSE: A molecular magnetic resonance (MR) imaging technique using superparamagnetic iron oxide (SPIO) nanocrystals was developed for monitoring stem cells noninvasively. This study was performed to investigate whether the presence of transplanted human mesenchymal stem cells in the penile cavernosum of a diabetic rat model could be evaluated noninvasively using molecular MR imaging. MATERIALS AND METHODS: SPIO nanocrystals (Feridex; AMI, Cambridge, USA) were transferred to human mesenchymal stem cells (hMSCs) using GenePORTER. The SPIO-transferred hMSCs were examined with Prussian blue staining. SPIO-labeled hMSCs were transplanted to the penile cavernosum of a diabetic rat model and MR images were examined in vivo using 1.5 T MR. RESULTS: SPIO was transferred to hMSCs successfully. MR signal intensity at the areas of the SPIO-transferred hMSCs decreased in the penile cavernosum of the diabetic rat model. SPIO particles were confirmed in the transplanted penile cavernosum with Prussian blue staining. CONCLUSIONS: The SPIO-labelled hMSCs in the penile cavernosum of a diabetic rat model can be monitored noninvasively using molecular MR imaging.

Short & Long-term Repopulating Capacity of Cord Blood Cells That Are Stored Overnight before Cryopreservation / 대한수혈학회지

BACKGROUND: Umbilical cord blood (UCB) is generally stored overnight and it undergoes a CD34 positive selection process the next day for reducing the cost and due to the convenience. We intended to determine whether overnight storage of cord blood cells affects the short and long-term repopulating capacity. METHODS: Five individuals' UCB samples were analyzed by colony assay, apoptotic cell counts and long term bone marrow culture. All the samples were divided to four groups, which were the fresh group (immediate use of harvest), the overnight storage group (overnight storage at room temperature after harvest), the immediate cryopreservation group (immediate cryopreservation after harvest) and the overnight cryo group (cryopreservaton after overnight storage at room temperature after harvest). RESULTS: The number of colony forming units-granulocyte macrophage (CFU-GM) was 116.2+/-20.1 in the fresh group and 90.8+/-15.8 in the overnight storage group (P=0.07). The number of CFUs-GM was similar between the immediate and overnight cryo groups (P=0.79). The immediate cryo group showed a significantly lower number of CFUs-GM as compared to that of the fresh group (P=0.03). The apoptotic cells were detected at 21+/-6.8% in the fresh group and this was 24.2+/-2.4% in the overnight storage group (P=0.32), and this was similar between immediate and overnight cryo groups (P=0.80). The fresh group had a significantly lower number of apoptotic cells compared to that of the immediate cryo group (P=0.02). After long term stromal-based culture, the mean production of CFU-GM colonies was similar between all the groups (P>0.05). CONCLUSION: These results support the continue use of overnight storage of UCB before cryopreservation as a convenient, cost reducing measure.

Magnetic resonance evaluation of human mesenchymal stem cells in corpus cavernosa of rats and rabbits / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To investigate whether the biological process of superparamagnetic iron oxide (SPIO)-labeled human mesenchymal stem cells (hMSCs) may be monitored non-invasively by using in vivo magnetic resonance (MR) imaging with conventional 1.5-T system examinations in corpus cavernosa of rats and rabbits.</p><p><b>METHODS</b>The labeling efficiency and viability of SPIO-labeled hMSCs were examined with Prussian blue and Tripan blue, respectively. After SPIO-labeled hMSCs were transplanted to the corpus cavernosa of rats and rabbits, serial T2-weighted MR images were taken and histological examinations were carried out over a 4-week period.</p><p><b>RESULTS</b>hMSCs loaded with SPIO compared to unlabeled cells had a similar viability. For SPIO-labeled hMSCs more than 1 X 10 (5) concentration in vitro, MR images showed a decrease in signal intensity. MR signal intensity at the areas of SPIO-labeled hMSCs in the rat and rabbit corpus cavernosa decreased and was confined locally. After injection of SPIO-labeled hMSCs into the corpus cavernosum, MR imaging demonstrated that hMSCs could be seen for at least 12 weeks after injection. The presence of iron was confirmed with Prussian blue staining in histological sections.</p><p><b>CONCLUSION</b>SPIO-labeled hMSCs in corpus cavernosa of rats and rabbits can be evaluated non-invasively by molecular MR imaging. Our findings suggest that MR imaging has the ability to test the long-term therapeutic potential of hMSCs in animals in the setting of erectile dysfunction.</p>

Human Umbilical Cord Blood-derived Cells Generate Insulin-producing Cells In Vitro / 대한이식학회지

PURPOSE: Here we showed that human umbilical cord blood (hUCB)-derived cells, when cultured under defined conditions, generated insulin-producing cells (IPCs). METHODS: hUCB mononuclear cells (MNCs) were cultured in serum-free low (5.5 mM glucose) DMEM at a cell density of 3x10(6)/cm2 in the presence of 1% DMSO for 3 days followed by high (25 mM glucose) DMEM supplemented with 10% FBS for 7 additional days. They were plated in plastic six well plates on slide coverslips (22x22 mm2) coated with 0.006% type I collagen. RESULTS: These IPCs formed clusters similar to islets of Langerhans. We confirmed these clusters were positive for insulin and C-peptide by immunohistochemistry. CONCLUSION: Our data demonstrated that in vitro hUCB-derived cells generated IPCs, which can be a potential source for the treatment of diabetes via a stem cell therapy approach.

In vitro Differentiation of Human Embryonic Stem Cells into Definitive Endodermal Cells / 대한이식학회지

PURPOSE: Whole liver transplantation has limitation including donor shortage and fatal surgical complications. Hepatocyte transplantation, which is simpler and less expensive than whole liver transplantation, allows the use of living related donors, permits the use of a single donor organ for multiple recipients. However, hepatocytes have limitation in proliferation and lose their property during culture period. To over come these problems, here we performed differentiation of human embryonic stem cells (hESCs) into definitive endoderm in order to differentiate into hepatocytes efficiently. METHODS: Undifferentiated hESCs were maintained on mouse embryo fibroblast feeder (MEF) layer for 5~7 days. For endoderm differentiation, we used modified Kevin A D'Amour's method that added 100 ng/mL Activin A for 5 days. After differentiation, differentiated endodermal cells were collected and RT-PCR and immunostain analysis were performed. RESULTS: After 5 days of differentiation period, hES cells showed endoderm committed-cells and increased expression of endoderm-specific marker genes (Sox17 and Foxa2). Also differentiated endoderm cells were stained with Sox17 and Foxa2 whereas undifferentiated hES cells were not stained with Sox17, Foxa2. CONCLUSION: In vitro differentiotion from hES cells to definitive endoderm was done repetitively by our methods. Further well defined protocol for differentiation of definitive endoderm to hepatocytes should be made.

Molecular MRI Images of the Transplanted Human Mesenchymal Stem Cells in the Liver, Kidney, Bladder and Penile Cavernosum of Rats / 대한비뇨기과학회지

PURPOSE: Molecular magnetic resonance (MR) imaging techniques using superparamagnetic iron oxide nanocrystals (SPIO) have been developed for noninvasively monitoring stem cells. This study was performed to investigate if the presence of transplanted human mesenchymal stem cells in the liver, kidney, bladder and penile cavernosum can be evaluated noninvasively with using molecular MR imaging. MATERIALS AND METHODS: SPIO (Feridex; AMI, Cambridge, USA) were transferred to the human mesenchymal stem cells (hMSCs) using GenePORTER. The labeling viability, efficiency and differentiation of the SPIO transferred hMSCs were examined with Tripan blue, Von Kossa, alkaline phosphatase, toluidine blue, oil red O and Prussian blue staining. The SPIO labelled hMSCs were transplanted to the liver, kidney, bladder and penile cavernosum of rats, and the MR images were examined in vitro or in vivo using 1.5 T MR. RESULTS: The viability and efficiency of the SPIO transferred hMSCs were good. Osteogenic, chondrogenic or adipogenic differentiation from the SPIO transferred hMSCs was observed. A decrease of the MR signal intensity of the SPIO transferred hMSCs with using GenePORTER was found in vitro. A decrease of the MR signal intensity was found at concentrations that were more than 1x10(5) hMSCs in vitro. The MR signal intensity at the areas of the SPIO transferred hMSCs decreased in the liver, kidney, bladder and penile cavernosum. The intracellular SPIOs were confirmed in the SPIO labelled hMSCs that were transplanted in the liver, kidney, bladder and penile cavernosum with Prussian blue staining. CONCLUSIONS: The SPIO labelled hMSCs in the liver, kidney, bladder and penis can be evaluated noninvasively by using molecular MRI.

In vitro Differentiation of Human Mesenchymal Stem Cells into Hepatocytes / 대한이식학회지

PURPOSE: Whole liver transplantation has limitation including donor shortage and fatal surgical complications. Hepatocyte transplantation, which is simpler and less expensive than whole liver transplantation, allows the use of living related donors, permits the use of a single donor organ for multiple recipients, and makes possible the cryopreser-vation of hepatocytes for future use. However, hepatocytes have limitation of proliferation and lose their property during culture period. To over come this problems, here we performed differentiation of bone marrow derived mesenchymal stem cells into hepatocytes. METHODS: Human bone marrow cells were harvested from posterior iliac spine of male and then mononuclear cells were obtained by Ficoll-Paque density-gradient centrifuge and plated in tissue culture flasks. For hepatogenic differentiation, we used modified Kuan-Der Lee's method. After differentiation, hepatocytes were collected and RT-PCR and PAS stain analysis were performed. RESULTS: After 5 weeks of cultivation period, mesenchymal stem cells showed cuboidal morphology and contained abundant granules in the cytoplasm. RT-PCR analysis showed increased expression of hepatocyte-specific marker genes (albumin,CK18, PERCK, CPS). Undifferentiated MSCs were not stained with PAS and differentiated hepatocytes from human MSCs stained with PAS indicating that hepatocytes contained glycogen in the cytoplasm. CONCLUSION: Hepatocyte transplantation could be one of the most effective treatments for chronic liver disease. However, hepatocyte has several disadvantages and problems. For alternative cell therapy sources, human bone marrow derived MSCs are considered as transplantable cells. Human MSCs are able to differentiate into functional hepatocytes in vitro and can be a possible cell transplantation source for chronic liver disease patients. Further studies should be done for differentiating human MSCs to hepatocytes in vivo condition.

Arsenic Trioxide Induces Erythroid Differentiation and Apoptosis of K562 Human Leukemia Cells through the Down-Regulation of Bcl-2 / 대한혈액학회지

BACKGROUND: Arsenic trioxide (As2O3) has been identified as an effective drug for the treatment of acute promyelocytic leukemia (APL). However, the role of As2O3 during the erythroid differentiation of human leukemic cells remains unknown. In this study, we investigated the in vitro effects of As2O3 on the erythroid differentiation of the K562 cell line and also on the expression and regulation of the apoptotic modulators of this process. METHODS: The K562 cells were cultured in the presence of 0.1, 0.5 and 1.0micrometer As2O3, or they were cultured in the presence of 1.0 and 10micrometer all trans retinoic acid (ATRA). The expression of glycophorin A before and after treatment with As2O3 or with ATRA in the K562 cells was assessed by flow cytometry and western blotting. The expressions of Bcl-2 and caspase-3 were determined by western blotting. RESULTS: The viability of the K562 cells was not decreased after treating with 0.1 and 0.5micrometer of As2O3, but the viability was significantly reduced at a dose of 1.0micrometer Caspase 3 activation was not observed at 0.1 and 0.5micrometer of As2O3 until 12 days, but Caspase 3 was activated by 1.0micrometer of As2O3 from day 3. The expression of glycophorin A was increased in dose dependent manner by As2O3 treatment, but this was not changed in the ATRA treated K562 cells. The expression of Bcl-2 was increased by 0.1 and 0.5micrometer of As2O3, but it was abruptly reduced by 1.0micrometer of As2O3. CONCLUSION: These results suggest that As2O3 induces the erythroid differentiation of K562 cells and that 1.0micrometer of As2O3 induces apoptosis through the down-regulation of Bcl-2.

Histologic Monitoring of the Transplanted Superparamagnetic Iron Oxide Labelled Human Mesenchymal Stem Cells in the Rat Bladder

PURPOSE: Molecular magnetic resonance(MR) imaging technique using the superparamagnetic iron oxide nanocrystals(SPIO) are developed for the monitoring the stem cells noninvasively. This study was performed to monitor the transplanted SPIO labelled human mesenchymal stem cells(hMSCs) with the molecular MR imaging and histologically in the rat bladder. MATERIALS AND METHODS: SPIO(Feridex; AMI, Cambridge, MA, USA) were labelled to hMSCs using GenePORTER. The labeling viability, efficiency of the SPIO labelled hMSCs were examined with tripan blue, prussian blue staining. SPIO labelled hMSCs are transplanted to the bladder in rats and MR images of them were examined using 1.5 T MR. Histologic examination of the SPIO labelled hMSCs transplanted bladder was performed with H&E and prussian blue staining. RESULTS: The viability, efficiency of the SPIO labelled hMSCs were good. MR signal intensity at the areas of the SPIO labelled hMSCs in the bladder decreased after transplantation of 1x10(6) SPIO labelled hMSCs and were confined locally. Intracellular SPIO were confirmed in the hMSCs transplanted bladder up to 5 weeks. CONCLUSION: The SPIO labelled hMSCs in the bladder can be monitored using the molecular MR imaging and SPIO labelled hMSCs were found in the transplanted bladder.

Effects of a Network Program for Preventing Obesity of Patients Taking Antipsychotics or Antidepressants / 간호학회지

PURPOSE: This study was designed to investigate the effects of a network program to prevent obesity and improve dietary habits for patients taking antipsychotics or antidepressants. METHOD: Thirty-seven patients in two hospitals were assigned to a control group (21 patients) or an intervention group (16 patients). The intervention group was evaluated to analyze the effect of the network program for six weeks after the program. RESULT: There was a difference in the rate of increased body weight between the control group and the intervention group. Notably, the body weight of both groups before the intervention was significantly increased. However, after the intervention the body weight of the intervention group rarely increased, whereas, the body weight of the control group was significantly increased as expected. There was an observed difference in diet between the control group and the intervention group. After the intervention, caloric intake per day of the intervention group decreased. Also, the duration of the meal of the intervention group after the intervention was longer than before. CONCLUSION: The network program for preventing obesity and improving dietary habits of patients taking antipsychotics or antidepressants was effective. The study shows that a network program can be an important part of a nursing intervention in clinical practice.

Monitoring Transplanted Human Mesenchymal Stem Cells in the Rabbit Bladder Using Molecular MRI / 대한남성과학회지

PURPOSE: Monitoring the biological changes in transplanted stem cells is important. Molecular magnetic resonance (MR) imaging using superparamagnetic iron oxide nanocrystals(SPIO) has been developed for noninvasive monitoring of stem cells. This study was performed to investigate whether the biological status of transplanted human mesenchymal stem cells(hMSCs) in the rabbit penis can be monitored noninvasively using molecular MR imaging. MATERIALS AND METHODS: SPIO(Feridex; AMI, Cambridge, MA, USA) were transferred into hMSCs using GenePORTER. The labeling efficiency, viability, and differentiation of the SPIO-containing hMSCs were examined with Trypan blue, Von Kossa, alkaline phosphatase, toluidine, blueoil red O, and Prussian blue staining. SPIO-labeled hMSCs were transplanted into the rabbit penis, and MR images of them were examined in vitro or in vivo using 1.5 T MR. Histologic examination was performed. RESULTS: The viability and efficiency of the SPIO-transferred hMSCs were good. Osteogenic, chondrogenic and adipogenic differentiation from SPIO-containing hMSCs were found. A decrease in MR signal intensity in the SPIO-containing hMSCs was found in vitro. MR signal intensity in the areas of the SPIO-transferred hMSEc in the rabbit penis decreased and were confined locally. Intracellular SPIO were confirmed in the hMSCs transferred into penis. CONCLUSIONS: SPIO-labeled hMSCs in the rabbit penis can be evaluated noninvasively using molecular MR imaging.

Monitoring Transplanted Human Mesenchymal Stem Cells in the Rabbit Bladder Using Molecular MRI / 대한남성과학회지

PURPOSE: Monitoring the biological changes in transplanted stem cells is important. Molecular magnetic resonance (MR) imaging using superparamagnetic iron oxide nanocrystals(SPIO) has been developed for noninvasive monitoring of stem cells. This study was performed to investigate whether the biological status of transplanted human mesenchymal stem cells(hMSCs) in the rabbit penis can be monitored noninvasively using molecular MR imaging. MATERIALS AND METHODS: SPIO(Feridex; AMI, Cambridge, MA, USA) were transferred into hMSCs using GenePORTER. The labeling efficiency, viability, and differentiation of the SPIO-containing hMSCs were examined with Trypan blue, Von Kossa, alkaline phosphatase, toluidine, blueoil red O, and Prussian blue staining. SPIO-labeled hMSCs were transplanted into the rabbit penis, and MR images of them were examined in vitro or in vivo using 1.5 T MR. Histologic examination was performed. RESULTS: The viability and efficiency of the SPIO-transferred hMSCs were good. Osteogenic, chondrogenic and adipogenic differentiation from SPIO-containing hMSCs were found. A decrease in MR signal intensity in the SPIO-containing hMSCs was found in vitro. MR signal intensity in the areas of the SPIO-transferred hMSEc in the rabbit penis decreased and were confined locally. Intracellular SPIO were confirmed in the hMSCs transferred into penis. CONCLUSIONS: SPIO-labeled hMSCs in the rabbit penis can be evaluated noninvasively using molecular MR imaging.

Clinical and Cytogenetic Analysis of Children with Maternal Chromosomal Balanced Translocation / 소아과

PURPOSE: Parents' genetic information plays an important role in their children's genetic expression. Human chromosome has 23-paternal chromosomes and 23-maternal chromosomes. Parental chromosomal translocation can induce clinical problems in their children because of imbalance in genetic information. We intent to analyze the cytogenentic and clinical features about children with maternal balanced translocation between chromosome 15 and 18. METHODS: We detected by one family's FISH study of chromosome 15. We have evaluated children born to clinically normal parents about peripheral bood analysis, endocrine, metabolic, radiologic study, electroencephalogram and social & intelligence scale. and We analysis their clinical manifestation by hospital records. RESULTS: Patient's father and elder sister are normal clinically and genetically. Her mother's chromosome show balanced translocation, 46, XX, t (15; 18) (p11.2; p11.3). One child has 46, XX, der (18) t (15; 18) (p11.2; p11.3), mental retardation, growth retardation, speech & social developmental delay, recurrent infection and mild mitochondria dysfunction. Her young brother has 46, XY, der (15) t (15; 18) (p11.2; p11.3), mental retardation, aggressive behavior, obesity and speech developmental delay. CONCLUSION: In this study we observed the children with developmental delay, dysmorphic facial features, mental retardation, growth retardation associated with growth hormone deficiency and aggressive behavior due to unbalanced translocation between chromosome 15 and 18.

The Expression of Fas antigen and Bax and Apoptosis in Ex Vivo Expanded Hematopoietic Progenitor Cells / 대한혈액학회지

BACKGROUND: During ex vivo expansion of cord blood (CB) CD34+ cells, differentiation of the expanded cells happened and hematopoietic potential of the progenitor cells decreased. In this study, we evaluate the effect of the expression of Fas antigen, Bcl-2, and Bax on CD34+ or AC133+ hematopoietic progenitor cells during ex vivo expansion. METHODS: CD34+ and AC133+ cells isolated from human CB were cultured in serum free medium supplemented with several cytokines for 7 days. After expansion culture, we re isolated CD34+ and AC133+ cells and compared the numbers of granulocyte-macrophage colony-forming units (CFU-GM) and granulocyte, erythrocyte, monocyte, and macrophage colony-forming units (CFU-GEMM), and expression of Fas antigen, Bcl-2, and Bax with unexpanded cells. RESULTS: CFU-GM was expanded 23.94 fold in CD34+ cells and 15.22 fold in AC133+ cells at day 7 of culture but CFU-GEMM was not expanded. The expression of Fas antigen and Bax was 7.44% and 2.75%, respectively, in fresh isolated CD34+ cells and increased to 19.71 % and 33.67%, respectively, in expanded CD34+ cells at day 7 culture, but Bcl-2 was not changed. In case of AC133+ cells, the expression of Fas antigen and Bax were also increased from 5.87% and 6.19% to 24.85% and 22.83%, respectively, and Bcl-2 was slightly decreased. Apoptosis was not changed in CD34+ cells and AC133+ cells during ex vivo expansion. CONCLUSION: These results indicate that the nature of expansion was similar between CD34+ and AC133+ cells, and expression of Fas antigen and Bax increased on CD34+ and AC133+ cells during ex vivo expansion. Selection of the expanded progenitor cells without apoptosis may be useful for the hematopoietic stem cell transplantation.

Restoration of the TRAIL Resistance with a Low Dose Doxorubicin in Acute Leukemia Cell Lines / 대한혈액학회지

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to induce apoptosis in various tumor cells but not in normal cells, which suggests its potential use as a tumor-specific antineoplastic agent. In the present study, we examined here that the treatment of TRAIL-resistant leukemia cells with a low dose doxorubicin in combination of TRAIL induces a synergistic apoptotic response. METHODS: Cell growth inhibition was assessed by MTT assay, and the induction of apoptosis was examined using flow cytometry after stained with Annexin V. To find out the molecular change of the TRAIL receptors and caspase expression in acute leukemia cell lines and human umbilical cord blood (UCB) mononuclear cells, RT-PCR for TRAIL receptors and western blot analysis for DR4, DR5, and caspase 3, 7, 8, and 9 were performed. RESULTS: The Jurkat cell line was TRAIL sensitive and TRAIL-resistant Molt-4 cell line became sensitive after treatment with TRAIL and a low dose of doxorubicin (0.1micrometer), but UCB mononuclear cells remained resistant. DR4 expression was increased when TRAIL-sensitive Jurkat cells were treated with TRAIL. DR5 expression was increased after exposing TRAIL- resistant Molt-4 cell line to TRAIL plus a low dose of doxorubicin for 24 hours. The expression of DR4 and DR5 in UCB mononuclear cells was unchanged after treatment with TRAIL, a low dose doxorubicin, or TRAIL plus a low dose of doxorubicin. Activated caspase 3 expression was augmented by TRAIL plus a low dose of doxorubicin than TRAIL or a low dose of doxorubicin alone in TRAIL-resistant Molt-4 cells. CONCLUSION: A low dose doxorubicin in combination with TRAIL may effectively promote caspase activation in TRAIL-resistant leukemia cells. Apoptosis synergistically induced by the combination of a low dose doxorubicin and TRAIL might be considered as an effective and safe tool in treating TRAIL-resistant acute leukemia.

Effect of Arsenic Trioxide in TRAIL (Tumor Necrosis Factor-related Apoptosis Inducing Ligand)-Mediated Apoptosis in Multiple Myeloma Cell Lines / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The potential therapeutic application of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in the treatment of multiple myeloma (MM), was recently proposed. However, there have been some problems with the use of TRAIL, due to the appearance of TRAIL-resistant cells in MM. The effect of arsenic trioxide (As2O3) on the rate of apoptosis induced by TRAIL was evaluated in MM cells. MATERIALS AND METHODS: Using TRAIL-sensitive (RPMI- 8226) and TRAIL-resistant (ARH-77 and IM-9) MM cell lines, the cell viability, induction of apoptosis, and change in the caspases were examined after treatment with TRAIL alone, or in combination with various concentrations of As2O3. RESULTS: Incubating the cell lines with As2O3 augmented the TRAIL-induced apoptosis in the MM cell lines, according to the As2O3 concentration. Apoptosis was mediated through caspase activation. When TRAIL was used alone, caspase8 was activated in the RPMI-8226 cell lines, but not in the ARH-77 and IM-9 cell lines. When As2O3 was added to TRAIL, caspase-9 was activated in the ARH-77 and IM-9 cells. CONCLUSION: The use of As2O3, in combination with TRAIL, would help enhance the level of TRAIL-induced apoptosis, and overcome the TRAIL-resistance, in MM cells.

In Vitro Effect of Interleukin-11 (IL-11) on Megakaryopoiesis from Umbilical Cord Blood Cells

BACKGROUND: The megakaryopoiesis and platelet production is regulated by several hematopoietc factors such as thrombopoietin (TPO), interleukin-11 (IL-11) and interleukin-3 (IL-3). IL-11 is a potent stimulator of megakaryopoiesis in vivo, and acts primarily as a megakaryocyte maturation factor in vitro and it can act synergistically with IL-3 and TPO. We performed this study to investigate the effects of recombinant human IL-11 (rhIL-11) with other hematopoietic factors on megakaryocyte colony formation in vitro. METHODS: CD34+ cells were separated from umbilical cord blood and megakaryocyte colonies using MegaCult Assay Kit were cultured with rhIL-11, recombinant human IL-3 (rhIL-3), and recombinant human TPO (rhTPO) for 7 and 14 days. The number and percentage of CD34+ and CD41a+ cells were determined by flowcytometry. RESULTS: The number of CD41a+ cells were 0.54+/-0.05x10(4) (rhIL-11 100 ng/ml), 5.32+.-0.23x10(4) (rhIL-3 100 ng/ml), and 8.76+/-0.15x10(4) (rhTPO 50 ng/ml) of total expanded cells during the culture of the purified CD34+ cells in liquid phase for 7 days. The number of CD41a+ cells were increased to 7.47+/-0.69x10(4) (rhIL-3 rhIL-11), 11.92+/-0.19x10(4) (rhTPO rhIL-11) of total expanded cells, respectively, during the culture of the purified CD34+ cells in liquid phase for 7 days in the presence of rhIL-11 (100 ng/ml). When the purified CD34+ cells were cultured in semisolid media including various concentration of rhIL-11, the megakaryocyte colonies were not formed. When the purified CD34+ cells were cultured with rhIL-11 and rhTPO or with rhIL-11 and rhIL-3, the number of megakaryocyte colonies were increased compared with rhTPO or rhIL-3 alone. CONCLUSION: These results indicate that IL-11 exerts a potent proliferative activity to colony forming unit-megakaryocyte from human umbilical cord blood, and it acts with other hematopoietic factors synergistically

Arsenic Trioxide (As2O3) Induced Apoptosis in NB4 Cell lines / 대한혈액학회지

BACKGROUND: Recently, inorganic arsenic trioxide (As2O3) was reported to induce complete remission in a high proportion of patients with refractory acute promyelocytic leukemia (APL). To illustrate cellular and molecular mechanisms of As2O3 in the treatment of APL, many experimental studies were performed on APL-derived cell lines in vitro. Previous studies showed that As2O3 inhibited proliferation and induced apoptosis in the APL-derived cell lines. This study was done to clarify the in vitro mechanisms of As2O3-induced apoptosis in APL-derived NB4 cell lines. METHODS: To determine the effects of As2O3 in the various concentrations, NB4 cells were cultured with 0.1 to 2micro M/L of As2O3. To assay the apoptosis in NB4 cell lines, DNA fragmentation assay and TUNEL were performed. To find out the molecular change of As2O3- induced apoptotic NB4 cell lines, RT-PCR and Western blot analysis for PML-RARalpha chimeric protein expression and flow cytometry for bcl- 2/bax expression were performed. To clarify the caspase activation pathway, Western blot analysis and flow cytometry for procaspase expression were performed. RESULTS: As2O3 induces apoptosis on NB4 cells in relatively high concentration (0.5 to 2 micro M/L) for 2 days. After 2 days of culture the PML-RARalpha chimeric protein expression decreased rapidly by Western blot and RT-PCR analysis and bcl-2 expression also decreased by flow cytometry. The expression of bax by flow cytometry showed a marked increase in high concentration (2micro M/L) but there was no change in low concentration (0.5micro M/L). In the Western blot analysis, the amount of pro`enzyme of caspase-3 was significantly decreased in the cells with high concentration (2micro M/L) compared with that in the cells with low concentration (0.5micro M/L). As2O3 induces proteolytic processing of pro-caspase 7 but not pro-caspase 9 and 8. CONCLUSION: Apoptosis of APL-derived NB4 cell lines was induced by As2O3 and progressed rapidly in higher concentrations. During apoptosis, activation of caspase-7 pathway and degradation of PML-RARalpha chimeric protein, decrease in bcl-2 and increase in bax were shown.

Increased Expression of Fas Antigen and Apoptosis in Aplastic Anemia Bone Marrow Cells

BACKGROUND: Clinical observations and laboratory studies have supported an immune basis for most acquired aplastic anemias, with the majority of patients responding to immunosuppressive therapy. Fas, a member of the tumor necrosis factor (TNF) receptor superfamily is a critical downregulator of cellular immune responses. Proinflammatory cytokines like interferon gamma (IFN-gamma) and TNF-alpha can induce Fas expression and render hematopoietic progenitor cells susceptible to Fas-induced growth suppression and apoptosis. METHODS: In order to investigate the involvement of apoptosis in the pathogenesis of aplastic anemia (AA), we measured the expression of Fas antigen and caspase-3 on bone marrow (BM) mononuclear cells (MNCs) of AA in the presence or absence of IFN-gamma, TNF-alpha, or macrophage inflammatory protein 1-alpha (MIP-1alpha). RESULTS: We confirmed that AA BM MNCs were more apoptotic and highly expressed Fas antigen than normal donors. Stimulation by IFN-gamma, TNF-alpha, or MIP-1alpha increased Fas antigen and caspase-3 expression in AA BM MNCs than BM MNCs of normal donors. Anti-Fas monoclonal antibody enhanced IFN-gamma, TNF-alpha, or MIP-1alpha mediated caspase-3 expression in BM MNCs of normal donors. Among these three cytokines, IFN-gamma enhanced apoptosis most strongly via Fas-caspase-3 pathway. CONCLUSION: These results suggest that Fas signal pathway may play a role in the pathophysiology of aplastic anemia and negative hematopoietic regulators like IFN-gamma can induce apoptosis of bone marrow progenitors in part by Fas induction.