Neural stem cells (NSCs) have the ability to proliferate and differentiate into various types of cells that compose the nervous system. To study functions of genes in stem cell biology, genes or siRNAs need to be transfected. However, it is difficult to transfect ectopic genes into NSCs. Thus to identify the suitable method to achieve high transfection efficiency, we compared lipid transfection, electroporation, nucleofection and retroviral transduction. Among the methods that we tested, we found that nucleofection and retroviral transduction showed significantly increased transfection efficiency. In addition, with retroviral transduction of Ngn2 that is known to induce neurogenesis in various types of cells, we observed facilitated final cell division in rat NSCs. These data suggest that nucleofection and retroviral transduction provide high efficiency of gene delivery system to study functions of genes in rat NSCs.
Electroporation; Lipid-Mediated transfection; Neural stem cells; Nucleofection; Retrovirus
Pulmonary tumor thrombotic microangiopathy (PTTM) is a rare, malignancy-related complication that causes marked pulmonary hypertension, right heart failure, and death. We report on a patient with locally advanced breast cancer whose course was complicated by fatal PTTM based on clinical and laboratory findings.
Breast neoplasms; Pulmonary hypertension; Pulmonary tumor thrombotic microangiopathy
We investigate cytotoxicity and mechanism of action of AS703026, a novel, selective, orally bioavailable MEK1/2 inhibitor, in human multiple myeloma (MM). AS703026, more potently (9-10 fold) than AZD6244, inhibited growth and survival of MM cells and cytokine-induced osteoclast differentiation. Inhibition of proliferation induced by AS703026 was mediated by G0-G1 cell cycle arrest and was accompanied by reduction of c-maf oncogene expression. AS703026 further induced apoptosis via caspase 3 and PARP cleavage in MM cells, both in the presence or absence of bone marrow stromal cells (BMSCs). Importantly, AS703026 sensitized MM cells to a broad spectrum of conventional (dexamethasone, melphalan), novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti-MM therapies. Significant tumor growth reduction in AS703026- vs. vehicle-treated mice bearing H929 MM xenograft tumors correlated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nM) was cytotoxic against the majority of tumor cells tested from patients with relapsed and refractory MM (84%), regardless of mutational status of RAS and BRAF genes. Importantly, BMSC-induced viability of MM patient cells was similarly blocked within the same dose range. Our results therefore support clinical evaluation of AS703026, alone or in combination with other anti-MM agents, to improve patient outcome.
multiple myeloma (MM); MEK1/2 inhibitor; bone marrow stromal cells (BMSCs); novel kinase inhibitor therapy
Pathologic changes in the growth plate remain unknown in Legg-Calvé-Perthes (LCP) disease. Spontaneously hypertensive rats have proven to be a good model for studying LCP disease. This study investigated the histopathologic changes and the expression of vascular endothelial growth factor in the growth plate of spontaneously hypertensive rats (SHR).
Materials and Methods
Sixty SHR rats were divided into two groups: those showing osteonecrosis (SHR+n group: 32), and those showing normal ossification (SHR-n group: 28). Thirty Wister Kyoto rats served as a control. For histomorphological measurement, the length of each zone of the growth plate was measured. Cell kinetics was measured by 5-bromo-2'-deoxyuridin (BrdU) immunohistochemistry and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assays. Vascular endothelial growth factor (VEGF) immunohistochemistry was used to identify of expression of VEGF.
The lengths of growth plates of the SHR+n group were significantly shorter in the initial growth period than those of the other groups. The lowest proliferative rate and the highest apoptosis rate were observed in the SHR+n group at the initial growth period. The expression of VEGF in the growth plate of the SHR group was lower than the control group, and it was lower in the SHR+n group than in the SHR-n group.
The growth plate of the SHR+n group was found to be affected by disease process of ischemic necrosis of the femoral head, and this might explain the relative overgrowth of the greater trochanter in the later stages of LCP disease.
Spontaneous hypertensive rats; growth plate; avascular necrosis; apoptosis; vascular endothelial growth factor
To examine the effects of change in weight bearing on the growth plate metabolism, a simulated animal model of weightlessness was introduced and the chondrocytes' cellular kinetics was evaluated.
Materials and Methods
Unloading condition on the hind-limb of Sprague-Dawley rats was created by fixing a tail and lifting the hind-limb. Six rats aged 6 weeks old were assigned to each group of unloading, reloading, and control groups of unloading or reloading. Unloading was maintained for three weeks, and then reloading was applied for another one week thereafter. Histomorphometry for the assessment of vertical length of the growth plate, 5-bromo-2'-deoxyuridin immunohistochemistry for cellular kinetics, and biotin nick end labeling transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay for chondrocytes apoptosis in the growth plate were performed.
The vertical length of the growth plate and the proliferative potential of chondrocytes were decreased in the unloading group compared to those of control groups. Inter-group differences were more significant in the proliferative and hypertrophic zones. Reloading increased the length of growth plate and proliferative potential of chondrocytes. However, apoptotic changes in the growth plate were not affected by the alterations of weight bearing.
Alterations in the weight bearing induced changes in the chondrocytic proliferative potential of the growth plate, however, had no effects on the apoptosis. This may explain why non-weight bearing in various clinical situations hampers normal longitudinal bone growth. Further studies on the factors for reversibility of chondrocytic proliferation upon variable mechanical stresses are needed.
Cellular kinetics; growth plate; changes in weight bearing
The epidermal growth factor receptor (EGFR) has been reported to be overexpressed in anaplastic thyroid carcinoma (ATC). In vitro studies have shown that EGFR tyrosine kinase inhibitors (TKIs) greatly inhibit cellular growth and induced apoptosis in the ATC cell lines, while somatic mutations in the tyrosine kinase domain or an increased gene copy number are associated with increased sensitivity to TKIs in non‐small cell lung cancer.
To investigate the prevalence of EGFR overexpression, gene amplification and activating mutation in the tyrosine kinase domain in patients with ATC.
The EGFR gene status and protein expression were investigated by direct DNA sequencing of the hot‐spot regions in exons 18, 19 and 21, fluorescence in situ hybridisation (FISH), and immunohistochemistry in tumour tissues from 23 patients with ATC.
On mutational analysis and FISH, neither mutations in the hot‐spots nor gene amplification was observed. However, high polysomy was identified in 14/23 (60.9%) patients with ATC. All cases with immunohistochemistry (IHC) positivity (n = 6) had high polysomy, whereas 8/17 (47.1%) cases with IHC negativity had high polysomy (p = 0.048). High polysomy was observed in all 10 cases with giant cell subtype, but in only 4/11 (36.3%) with squamoid and 0/2 with spindle cell sarcomatoid subtype. There was no statistically significant correlation between FISH positivity of ATC tumour and presence of well‐differentiated component.
Despite the low incidence of somatic EGFR gene mutation and amplification in the study samples, in view of the fact that high polysomy was often identified by FISH, as well as the current lack of therapeutic options, EGFR TKIs are worth investigating for treating the patients with ATC who have at least giant cell subtype.
anaplastic thyroid carcinoma; EGFR; high polysomy
Biomarkers play a key role in risk assessment, assessing treatment response, and detecting recurrence and the investigation of multiple biomarkers may also prove useful in accurate prediction and prognosis of cancers. Immunohistochemistry (IHC) has been a major diagnostic tool to identify therapeutic biomarkers and to subclassify breast cancer patients. However, there is no suitable IHC platform for multiplex assay toward personalized cancer therapy. Here, we report a microfluidics-based multiplexed IHC (MMIHC) platform that significantly improves IHC performance in reduction of time and tissue consumption, quantification, consistency, sensitivity, specificity and cost-effectiveness.
By creating a simple and robust interface between the device and human breast tissue samples, we not only applied conventional thin-section tissues into on-chip without any additional modification process, but also attained perfect fluid control for various solutions, without any leakage, bubble formation, or cross-contamination. Four biomarkers, estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR) and Ki-67, were examined simultaneously on breast cancer cells and human breast cancer tissues. The MMIHC method improved immunoreaction, reducing time and reagent consumption. Moreover, it showed the availability of semi-quantitative analysis by comparing Western blot. Concordance study proved strong consensus between conventional whole-section analysis and MMIHC (n = 105, lowest Kendall's coefficient of concordance, 0.90). To demonstrate the suitability of MMIHC for scarce samples, it was also applied successfully to tissues from needle biopsies.
The microfluidic system, for the first time, was successfully applied to human clinical tissue samples and histopathological diagnosis was realized for breast cancers. Our results showing substantial agreement indicate that several cancer-related proteins can be simultaneously investigated on a single tumor section, giving clear advantages and technical advances over standard immunohistochemical method. This novel concept will enable histopathological diagnosis using numerous specific biomarkers at a time even for small-sized specimens, thus facilitating the individualization of cancer therapy.
We describe a girl with Diamond-Blackfan anemia with accompanying red cell enolase deficiency. At the age of 9 yr old, the patient received allogeneic bone marrow transplantation from her HLA-identical sister who had normal red cell enolase activity. While the post transplant DNA analysis with short tandem repeat has continuously demonstrated a stable mixed chimerism on follow-up, the patient remains transfusion independent and continues to show a steady increase in red cell enolase activity for over two and a half years following bone marrow transplantation.
Anemia, Diamond-Blackfan; Erythrocyte Enzyme Deficiency; Red Cell Enolase Deficiency; Bone Marrow Transplantation
Non-Hodgkin lymphoma (NHL) is a hematologic malignancy for which good diagnostic markers are lacking. Despite continued improvement in our understanding of NHL, efforts to identify diagnostic markers have yielded dismal results. Here, we translated low-mass-ion information in urine samples from patients with NHL into a diagnostic marker.
To minimize experimental error, we tested variable parameters before MALDI-TOF analysis of low-mass ions in urine. Urine from 30 controls and 30 NHL patients was analyzed as a training set for NHL prediction. All individual peak areas were normalized to total area up to 1000 m/z. The training set analysis was repeated four times. Low-mass peaks that were not affected by changes in experimental conditions were collected using MarkerView™ software. Human Metabolome Database (HMDB) searches and ESI LC-MS/MS analyses were used to identify low-mass ions that exhibited differential patterns in control and NHL urines. Identified low-mass ions were validated in a blinded fashion in 95 controls and 66 NHL urines to determine their ability to discriminate NHL patients from controls.
The 30 highest-ranking low-mass-ion peaks were selected from the 60-urine training set, and three low-mass-ion peaks with high intensity were selected for identification. Of these, a 137.08-m/z ion showed lower mass-peak intensity in urines of NHL patients, a result that was validated in a 161-urine blind validation set (95 controls and 66 NHL urines). The 130.08-m/z ion was identified from HMDB searches and ESI LC-MS/MS analyses as hypoxanthine (HX). The HX concentration in urines of NHL patients was significantly decreased (P < 0.001) and was correlated with the mass-peak area of the 137.08-m/z ion. At an HX concentration cutoff of 17.4 μM, sensitivity and specificity were 79.2% and 78.4%, respectively.
The present study represents a good example of low-mass-ion profiling in the setting of disease screening using urine. This technique can be a powerful non-invasive diagnostic tool with high sensitivity and specificity for NHL screening. Furthermore, HX identified in the study may be a useful single urine marker for NHL screening.
The aim of the current study is to test whether immortalized B-lymphocyte cell line via Ebstein-Barr virus (EBV) transformation is feasible and can be an unlimited source of genome wide study.
We obtained peripheral whole blood from 5 ovarian cancer patients and immortalized the B-cell lines using EBV transformation. The success rate was analyzed and the bio-identity of the genome was performed using human leukocyte antigen (HLA) identity test.
EBV transformation was successful in all 5 cases (95% confidence interval, 46.3% to 100%). After cryopreservation of EBV-transformed B-cell lines and subsequent thawing, we observed that all cell lines were viable and proliferative. To check bio-identity, HLA-A, B, and DR were tested between the genome of the original samples and the transformed samples. The HLA typing revealed that all observed HLA-A, B, and DR type was identical in 5 cases before and after EBV-transformation.
The current results suggest that EBV-transformation of peripheral blood is an efficient tool in genome banking. The EBV-transformed B-cell lines may be a valuable resource of genome in multi-center translational research by the Korean Gynecologic Oncology Group.
Genomics; Epidemiology; DNA storage; EBV transformation; Cryopreservation
Exposure to environmental tobacco smoke (ETS) is a major risk to human health, and the home is the greatest single source of ETS for children. The authors investigated fetal exposure to paternal smoking at home during pregnancy. Korean families were included as trios of fathers, mothers, and neonates identified in 2005–2007. Sixty-three trios were finally enrolled in this study after exclusion of those in which the mother was a smoker or was regularly exposed to ETS at places other than the home. Nicotine and cotinine concentrations in hair were measured by using liquid chromatography–tandem mass spectrometry to determine long-term exposure to ETS. The difference between neonatal nicotine concentrations in the smoker and nonsmoker groups was not statistically significant. However, in the indoor-smoker group, neonatal nicotine concentrations were significantly higher than in the outdoor and nonsmoker groups (P < 0.05). Furthermore, neonatal nicotine concentrations in the outdoor-smoker group were not different from those in the nonsmoker group. These findings indicate that paternal smoking inside the home leads to significant fetal and maternal exposure to ETS and may subsequently affect fetal health. Conversely, findings show that paternal smoking outside the home prevents the mother and her fetus from being exposed to ETS.
fetus; hair; nicotine; smoking; tobacco smoke pollution
To analyze the effect of allogeneic blood transfusion on clinical outcome in 119 patients with stage IIB cervical cancer who were treated with radiotherapy ± chemotherapy.
Patients and Methods
Medical records were examined for hemoglobin levels before and during radiotherapy, history of allogeneic blood transfusions and the time point when transfusions were given. These factors were retrospectively analyzed along with other clinical risk factors for influences on the patients' clinical outcomes.
Thirty-two patients (26.9%) received packed red blood cell transfusion (mean, 3.4 units; range, 1 - 12 units) before or during radiotherapy. Median follow-up period was 39.3 months (range, 7.6 - 58.4 months). Patients with history of transfusion showed poorer metastasis-free survival and a trend toward poorer overall survival than non-transfused patients. When patients who received transfusions were sub-divided by the time of transfusion, those who received transfusions before radiotherapy had significantly poorer clinical outcome than those who received transfusions during radiotherapy. In a multivariable analysis, patients with pretreatment transfusion showed a higher risk of distant metastasis (HR = 3.75, 95% CI: 1.28 - 12.15, p = 0.017) and decreased overall survival rates (HR = 4.62, 95% CI: 1.15-18.54, p = 0.031) compared with those of other patients.
Our results suggest that allogeneic blood transfusions given before radiotherapy may be associated with higher incidence of distant metastases and decreased survival in patients with stage IIB cervical cancer.
Anemia; blood transfusion; cervical cancer; metastasis; radiotherapy
Elucidating the molecular mechanisms by which tumors become resistant to Herceptin is critical for the treatment of Her2-overexpressed metastatic breast cancer.
To further understand Herceptin resistance mechanisms at the molecular level, we used comparative proteome approaches to analyze two human breast cancer cell lines; Her2-positive SK-BR-3 cells and its Herceptin-resistant SK-BR-3 (SK-BR-3 HR) cells.
Heat-shock protein 27 (HSP27) expression was shown to be upregulated in SK-BR-3 HR cells. Suppression of HSP27 by specific siRNA transfection increased the susceptibility of SK-BR-3 HR cells to Herceptin. In the presence of Herceptin, Her2 was downregulated in both cell lines. However, Her2 expression was reduced by a greater amount in SK-BR-3 parent cells than in SK-BR-3 HR cells. Interestingly, co-immunoprecipitation analysis showed that HSP27 can bind to Her2. In the absence of Herceptin, HSP27 expression is suppressed and Her2 expression is reduced, indicating that downregulation of Her2 by Herceptin can be obstructed by the formation of a Her2-HSP27 complex.
Our present study demonstrates that upregulated HSP27 in human breast cancer cells can reduce Herceptin susceptibility by increasing Her2 protein stability.
The aim of study was to evaluate the usefulness of serum HER2 as a therapeutic response indicator in patients with HER2 positive metastatic breast cancer (MBC).
Materials and Methods
The levels of serum HER2 and CA15.3 were assayed in 148 serial serum samples from 50 HER2 positive MBC patients at both the baseline and follow-ups. The changes in the levels of serum HER2 and CA15.3 in relation to the tumor responses to the various chemotherapy regimens were monitored.
The levels of serum HER2 and CA15.3 were elevated in 82% and 62% of tissue HER2 positive patients, respectively, prior to therapies, with the changes in both tumor markers showing statistical significance in relation to the tumor responses (p<0.01) in patients with elevated baseline serum markers.
The level of serum HER2 could be a valuable response indicator, not only for trastuzumab containing therapy, but also for other common MBC chemotherapeutic agents. Also, as it is more frequently elevated, the serum level of HER2 may also be a more useful tumor marker than CA15.3 in HER2 positive MBC.
Serum HER2; CA15.3; Metastatic breast cancer; Response; Trastuzumab; Monitoring
In CD5 positive (CD5+) mature B-cell lymphomas, newly recognized CD5+ diffuse large B-cell lymphoma (DLBCL) has been characterized by aggressive features. We studied twenty-five cases with CD5+ lymphomas involving bone marrow. Eleven cases were diagnosed as chronic lymphocytic leukemia, six cases were diagnosed as mantle cell lymphoma (MCL), and three cases with morphologic characteristics of MCL and without both the cyclin D1 expression and IGH/CCND1 rearrangement were unclassifiable. The remaining five cases, showing large to medium-sized lym-phoid cells with prominent nucleoli and a moderate amount of cytoplasm, were diagnosed as DLBCL. Five DLBCL cases were positive for CD5, CD20, surface immuno-globulin, but negative for CD23. Patients with CD5+ DLBCL showed a high age of onset (median, 68 yr) and two patients expired one month after the diagnosis. Since CD5+ DLBCL forms a distinct subgroup of DLBCL, a study of CD5 expression in DLBCL would be helpful to predict prognosis and to determine future therapeutic strategy. To the best of our knowledge, this is the first report on de novo CD5+ DLBCL in Koreans.
Antigens, CD5; Leukemia, Lymphocytic, Chronic; Lymphoma, Mantle-Cell; Lymphoma, Large-Cell, Diffuse
The identification of marker chromosomes is important for genetic counseling. However, the origin or composition can rarely be defined with conventional cytogenetic technique alone. In this study, we investigated the incidences and types of marker chromosomes in Korean patients and attempted to establish a cost-effective diagnostic approach for marker chromosomes. We reviewed the karyotypes of 2,984 patients that were requested for the cytogenetic analysis between 1997 and 2003 at the Samsung Medical Center. Ten marker chromosomes were found and identified using fluorescent in situ hybridization (FISH). Among the ten marker chromosomes, six were supernumerary marker chromosomes (SMCs) and the rest were marker chromosomes in Turner syndrome (TS). The incidence of SMCs was 2.01/1,000, slightly higher than that previously reported. Five of six SMCs were satellited marker chromosomes. Three bisatellited marker chromosomes originated from chromosome 15 and two from chromosome 22. The origin of one SMC could not be identified. All marker chromosomes in TS originated from X- or Y chromosome. The application of FISH is indispensable to identify marker chromosomes, and the appropriate selection of probes is necessary for cost-effective analysis. For analyzing satellited marker chromosomes, application of probes for chromosome 15 followed by those for chromosome 22 is recommended and in cases of TS, probes for sex chromosomes should take precedence.