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The aim of this study was to investigate the effect of microRNA-193b (miR-193b) on C-KIT protein expression and biological behaviors in K562 cells. The FAM-labeled miR-193b mimic and negative control were respectively transfected into K562 cells using HiPerFect transfection reagent. The percentage of FAM-positive cells was monitored by flow cytometry. The levels of C-KIT and phosphorylated C-KIT protein were detected by Western blot. The cell growth was measured by CCK-8 reagent. The apoptosis of cells were analyzed by flow cytometry with Annexin V staining. The differentiation of cells were analyzed by flow cytometry with anti-CD11b or anti-CD15 staining. The results demonstrated that the percentage of FAM-positive cells was about 80% in miR-193b or negative control-transfected K562 cells. Compared with the negative control group, overexpression of miR-193b in K562 cells significantly inhibited the levels of C-KIT and phosphorylated C-KIT protein. Meanwhile, the cell growth was inhibited and the percentages of apoptotic cells, CD11b- or CD15-positive cells increased. It is concluded that miR-193b can reduce C-KIT expression and inhibit cell growth in K562 cells. The growth-inhibitory activity of miR-193b is associated with apoptosis and granulocytic differentiation. This study contributed to further investigate the role of miR-193b in leukemogenesis.

The aim of the research was to find the up-regulated microRNA (miRNA) in K562 cells treated by 5-aza, to detect the miRNA expression in healthy people, CML patients and leukemia cell lines and to investigate the influence of miRNA on K562 cell proliferation. Up-regulated miRNA in K562 cells after 5-aza treatment was screened by microarray. The up-regulated miR-638, miR-663 and miR-92b with CpG islands in upstream region were screened by microarray in combination with bioinformatics. The miRNA mentioned above was further ascertained by SYBR-green real-time PCR. Up-regulated miR-663 was confirmed by real-time PCR. Expression level of miR-663 was detected in K562, U937 and Kasumi cell lines, and white blood cells from bone marrow of normal donor and from peripheral blood of newly diagnosed CML patient. Methylation-specific PCR (MSP) was applied to analyze the methylated status of miR-663 CpG island in K562 cells. Proliferation of K562 cells was observed after miR-663 was over expressed by transient transfection. The results showed that the expression level of miR-663 in K562 cells was up-regulated after 5-aza treatment, and the expressions of miR-663 were lower in K562, U937, Kasumi cell lines and newly diagnosed patients, compared with healthy people. The CpG island of miR-663 was methylated in K562 cell line according to detection result of MSP. The proliferation of K562 cell could be suppressed by over-expression of miR-663 in vitro. It is concluded that miR-663 CpG island is methylated in K562 cell line. The miR-663 is down-regulated in K562, U937, Kasumi cell lines and CML patients, compared with healthy people. miRNA-663 in K562 cells is up-regulated after 5-aza treatment. Over-expression of miR-663 can suppress the proliferation of K562 cells, which suggests that miR-663 may possesses suppressive effect for leukaemia.

AIM: To evaluate the role of leptin in the internal disorders during hepatic ischemia/reperfusion injury. METHODS: A rat model of 70% hepatic ischemia/reperfusion injury was established, with groups of sham-operation (Sham), 60 min ischemia/60 min reperfusion (I60’R60’), I60’R150’, I60’R240’ and I60’R360’. Serum leptin was detected by a self-produced radioimmunoassay; serum glucose, total anti-oxidation capacity, myeloperoxidase, alanine transaminase and diamine oxidase were determined by relevant kits, while histological alterations and protein levels of leptin in the lung, liver and duodenum were examined by hematoxylin-eosin staining and immunohistochemistry. Spearman’s rank correlation between leptin and other variables or grading of tissue impairment were analyzed simultaneously. RESULTS: Serum leptin in I60’R360’ was significantly higher than in Sham and I60’R240’ groups (both P < 0.05), serum glucose in I60’R360’ was higher than in Sham and I60’R150’ (both P < 0.05), and serum total anti-oxidation capacity in I60’R240’ and I60’R360’ were higher than in Sham (both P < 0.05) and I60’R150’groups (both P < 0.01). Serum myeloperoxidase in groups of I60’R240’ and I60’R360’ were lower than in I60’R150’group (both P < 0.05), serum alanine transaminase in the four reperfusion groups were higher than in the Sham group (all P < 0.05), while serum DAO in I60’R360’ was lower than in I60’R60’ (P < 0.05). Histological impairment in the lung, liver and duodenum at the early phase of this injury was more serious, but the impairment at the later phase was lessened gradually. Protein levels of leptin in the lung in the four reperfusion groups were significantly lower than in the Sham group (all P < 0.01), decreasing in the order of I60’R150’, I60’R60’, I60’R360’ and I60’R240’; the levels in the liver in I60’R60’ and I60’R240’ were higher than in the Sham group (both P < 0.01), while the levels in I60’R240’ and I60’R360’ were lower than in I60’R60’ (both P < 0.01); the levels in duodenum in I60’R240’ and I60’R360’ were higher than in Sham, I60’R60’ and I60’R150’ (all P < 0.01), while the level in I60’R150’ was lower than in I60’R60’ (P < 0.05). There was a significantly positive correlation between serum leptin and alanine transaminase (ρ = 0.344, P = 0.021), a significantly negative correlation between the protein level of leptin in the lung and its damage scores (ρ = -0.313, P = 0.036), and a significantly positive correlation between the protein level of leptin in the liver and its damage scores (ρ = 0.297, P = 0.047). CONCLUSION: Endogenous leptin fluctuates in hepatic ischemia/reperfusion injury, exerts a potency to rehabilitate the internal disorders and represents a potential target for supportive therapy.

Mutations and/or overexpression of c-Kit proto-oncogene frequently occur in subsets of acute myeloid leukemia (AML) and contribute to abnormal cell proliferation and poor outcomes. We showed that c-Kit expression was subject to post-transcriptional regulation by microRNA (miRNA)-193b. Notably, miR-193b was significantly down-regulated in the examined AML cells and its levels were inversely correlated with c-Kit levels. Restoration of miR-193b expression in AML cells resulted in distinctly reduced c-Kit expression and inhibited cell growth. These data reveal a role for miR-193b dysregulation in myeloid leukemogenesis and the therapeutic promise of regulating miR-193b expression for c-Kit-positive AML.

This study was purposed to investigate the difference of zo-1 gene promoter methylation between healthy individuals and acute leukemia patients. BS-PCR method was used to detect the status of zo-1 gene methylation in healthy individuals, acute leukemia patients and leukemic cell line NB4 cells. The results showed that zo-1 gene was hypomethylated in bone marrow samples from healthy individuals (1.9%). In newly diagnosed AL and relapsed patients, the rate of zo-1 gene methylation was 93.2% and 66.9% respectively, while it was 16.4% in AL patients in complete remission, which was much higher than that in healthy individuals. There was significant difference between them. It is concluded that as compared with healthy individuals, zo-1 gene in acute leukemia patients is hypermethylated and with different degrees in various phases of leukemia. Analysis of zo-1 gene methylation status may be useful to monitor the development of acute leukemia.

MYCN amplification is highly associated with malignancy and correlates with poor prognosis in patients with neuroblastoma. We developed a novel liposome-MYCN siRNA-folic acid complex, and the transfection efficacy was measured in LA-N-5 cells by cy-3 fluorescence density in each microgram of protein from the transfected cell lysate. MYCN expression and cell growth were studied with quantitative RT-PCR and MTT assays, and the expression of MYCN protein was studied with Western blot, respectively. An SCID mouse model with subcutaneous LA-N-5 xenografted tumor was established. The animals were divided into four groups (n = 5) and they were peritoneally injected with liposome-encapsulated MYCN siRNA (siRNA 125 μg/kg/day), lipid-encapsulated control siRNA, MYCN siRNA, or liposome only, respectively, for 5 consecutive days. The animals were killed 24 h after the last injection, and the expression of MYCN mRNA in tumor tissue was detected by RT-PCR. Our results are as follows: the transfect efficacy reached 1808.5 ± 140.2 pg siRNA/μg protein in LA-N-5 lysates after treatment with 100 nmol/L MYCN siRNA encapsulated with lipid, and fluorescence could be visualized in 92% of LA-N-5 cells after transfection. At 72 h post-transfection, MYCN mRNA expression in LA-N-5 cells was downregulated by 79.2%, MYCN protein was downregulated by 71.3% and cell growth was inhibited by 66.2%, as measured by MTT assay. In the in vivo study, MYCN mRNA expression was knocked down 53.1% in tumor tissues with injection of liposome-encapsulated MYCN siRNA as compared to control siRNA. These results suggest that targeted delivery of MYCN siRNA by folate receptor-targeted lipid vesicles into LA-N-5 cells is efficacious and capable of suppressing MYCN mRNA expression both in vitro and in vivo.

To analyze the possible microRNA (miRNA) target sites in the 3' untranslated region (3'-UTR) of FMS-like tyrosine kinase 3 (FLT3) gene, a FLT3 3'-UTR-luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in 293T cell line. The 3'-UTR fragment of FLT3 gene was amplified by PCR from genomic DNA of HepG2 cells. PCR products were cloned into PstI/EcoRI-modified pGL3-control reporter vector (pGL3-control-m). The miRNA targeting FLT3 3'-UTR was predicted by Target Scan 5.1 software. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into 293T cells using FuGENE HD transfection reagent. The Dual-Luciferase Reporter Assay System was used to quantitate the reporter activity. The results showed that a 804-bp 3'-UTR fragment of FLT3 gene was successfully cloned into the pGL3-control-m reporter vector, which authenticated by PstI/EcoRI digestion and DNA sequencing. The predicted miRNA targeting FLT3 3'-UTR included miRNA-15a, miRNA-15b, miRNA-16, miRNA-195, miRNA-424 and miRNA-497. The luciferase activity of reporter construct treated with miRNA-15a, miRNA-15b or miRNA-195 was decreased respectively about 20% compared with the control group. It is concluded that the FLT3 3'-UTR-luciferase reporter vector has been successfully constructed. The luciferase activity of the reporter can be suppressed by miRNA-15a, miRNA-15b or miRNA-195.

To test the hypothesis that methylation of a CpG island is associated with regulation of microRNA expression, we investigated CpG islands in the upstream sequences of microRNA precursors (pre-miRNAs) through bioinformatic analysis and determined whether the CpG islands were methylated by methylation-specific PCR in the k-562 cell line. We used 5-azacytidine for DNA demethylation, and changes in microRNA expression were detected by microarray assay, RT-PCR, and real-time PCR after 5-azacytidine induction. We showed that the CpG islands in the upstream regions of 18 pre-miRNAs were methylated, including miR-663, miR-369, miR-615, and miR-410, and promoter activity was detected in the upstream region of pre-miR-663. We found that a decrease in methylation of a CpG island could up-regulate the expression of miR-663, suggesting that miR-663 could be regulated by DNA methylation. Expression levels of miR-369, miR-615, and miR-410 were not regulated by DNA methylation in this cell line.

In order to investigate the effect of demethylating treatment on the expression of inhibitory KIR and the cytolytic activity of NK-92MI cells, and to study the possible relationship between the demethylation of inhibitory kir gene and the function of NK cells. NK-92MI cells were treated with 5-azacytidine to induce DNA demethylation. The expression of KIR3DL1, KIR2DL2/KIR2DL3, KIR2DL1 and the viability of NK-92MI cells were detected by flow cytometry. The KIR3DL1 positive and the KIR3DL1 negative NK-92MI cells were also sorted by flow cytometry. The cytotoxicity of NK-92MI against K562 cells was detected by the LDH release assay. The results demonstrated that the expressions of KIR3DL1, KIR2DL2/KIR2DL3 and KIR2DL1 in NK-92MI cells all increased after treating with 1.0, 2.5 and 5 micromol/L of 5-azacytidine for 72 hours. And the cytotoxicity of NK-92MI against K562 cells decreased. In these dose range, 5-azacytidine did not influence the viability of NK-92MI cells. Additionally, the cytotoxicity of KIR3DL1 positive NK-92MI cells was lower than that of the KIR3DL1 negative cells. It is concluded that the demethylating treatment suppresses the cytotoxicity of NK-92MI cells through increasing the expression of inhibitory KIR.

The aim of this study was to analyze the promoter methylation patterns of inhibitory killer cell immunoglobulin-like receptor (KIR) which gene expression and the effect of demethylation treatment were studied, and to explore the possible regulation mechanism of inhibitory kir gene expression. The promoter methylation levels of kir2DL1 and kir2DL2/kir2DL3 in NK-92MI cell line were detected by bisulfite sequencing technique. Then NK-92MI cells were treated with 5-azacytidine to induce the demethylation of CpG islands. The levels of gene expression of kir were determined by RT-PCR. The results demonstrated that the methylation frequencies of CpG dinucleotides surrounding the promoter regions of kir2DL1 and kir2DL2/kir2DL3 genes were 25% to 88% and 5% to 80% respectively. DNA-demethylating treatment with 5-azacytidine resulted in re-expression of kir2DL1 gene and increased expressions of kir2DL1, kir2DL2 and kir2DL3 genes in NK-92MI cells. In conclusion, the promoter DNA methylation participates in the regulation of kir gene expression in NK-92MI cells.

To study the role of transcription factor E2F1 in the transcription of KIR3DL1 promoter, and to identify its molecular mechanism of regulation of KIR3DL1 gene expression.The mutant promoter fragment of KIR3DL1 gene was amplified from genomic DNA of K562 cells by PCR. The PCR product was cloned into pGL3-basic reporter vector to construct KIR3DL1 promoter-luciferase reporter plasmid (PLRP). The binding of E2F1 to KIR3DL1 promoter was detected by chromatin immunoprecipitation (CHIP) assays. The KIR3DL1 PLRP construction was transfected into K562 cells using cationic liposome SuperFect. The binding of E2F1 to the construction was detected by CHIP assays and reporter activity was quantitated by the dual-luciferase reporter assay system. The mammalian expression vector containing E2F1 cDNA was co-transfected into K562 cells with wild-type KIR3DL1 PLR construction and reporter activity was quantitated.The mutant KIR3DL1 PLR recombinant was constructed successfully and a naturally point mutation (TTTGGCGC--TTCGGCGC) within a putative E2F1 binding site in the KIR3DL1 promoter region was authenticated by DNA sequencing. E2F1 absolutely could not bind to the mutant KIR3DL1 promoter in K562 cells, but could bind to the wild-type one in NK-92MI cells. The binding of E2F1 to the mutant KIR3DL1 PLR construction was partially reserved, however, its relative luciferase activity was decreased by 50% than that of wild-type. On the other hand, when co-transfected with E2F1 mammalian expression vector, the relative luciferase activity of wild-type construction was increased, over 2-fold higher than that of control group.E2F1 participates in the regulation of the transcriptional activation of KIR3DL1 gene. The number of CpG dinucleotide and methylation pattern within the E2F1 binding site probably influence the binding of E2F1 to target sequence.

To investigate the methylation pattern of KIR3DL1 promoter region in NK cell line NK-92MI and the effect of demethylation and histone acetylation on gene expression, and to study the possible regulation mechanism of KIR3DL1 expression.The methylation status of KIR3DL1 promoter in NK-92MI cells was detected by bisulfite sequencing technique. Then NK-92MI cells were treated with the DNA-demethylating compound 5-azacytidine and (or) the inhibitor of histone deacetylase trichostatin A, and the expression of KIR3DL1 gene was observed.The CpG dinucleotides surrounding promoter region of KIR3DL1 gene in NK-92MI cells were consistently methylated with a frequency of 70%-100%. After 72 h treatment with 2.5 micromol/L and 5 micromol/L of 5-azacytidine, the mRNA expression of KIR3DL1 gene in NK-92MI cells increased 66.6% and 114.6%, respectively. However, after 72 h treatment with 50 nmol/L of trichostatin A, the mRNA expression of KIR3DL1 gene in NK-92MI cells did not increase. Additionally, combined treatment with 5-azacytidine and trichostatin A did not lead to a synergistic effect compared with 5-azacytidine alone.KIR3DL1 expression in NK-92MI cells is regulated by promoter methylation.

To explore the distribution of leptin expression and the effect of sepsis on leptin protein and mRNA levels.Vital organ samples including hypothalamus, lung, liver, spleen, stomach, duodenum, kidney, epididymal fat pad and testis of normal rats were collected. The mRNA expressions of leptin in those samples were determined by RT-PCR. The sepsis rat model induce by cecal ligation and perforation (CLP) was established, setting groups of sham-operation, CLP model, CLP + intralipid injection, CLP + estradiol injection and CLP + insulin injection, as the latter three groups were set to intervene energy metabolism and neuroendocrine function. Radioimmunoassay was applied to measure serum leptin concentrations in each group at 12 h after injury, while RT-PCR was also used to detect Leptin mRNA expressions in hypothalamus, fat and lung after injury.Leptin mRNA expressions were confirmed in all the above nine vital organs, with the highest in kidney but the lowest in testis. The serum leptin level showed no significant difference between sham operation group and other four groups. Compared with sham operation group, the Leptin mRNA level in CLP group decreased significantly in hypothalamus, fat and lung, while that in the other three groups showed different changes. The effect of intralipid on Leptin mRNA expression was found to be a dual-direction pattern, with central stimulation but peripheral inhibition.Leptin is widely expressed in multiple vital organs, and it may be a protective factor to promote recovery of sepsis-induced internal disorders.

To analyze the methylation pattern of kir3dl1 promoter and its relationship with gene expression, and to study the possible regulation mechanism of kir3dl1 gene expression, the methylation status of kir3dl1 promoter in K562 cell line was detected by bisulfite sequencing technique. Then K562 cells were treated with 5-azacytidine so as to induce de-methylation of CpG island, and the relationship of CpG island methylation with kir3dl1 gene expression was investigated. The results demonstrated that CpG dinucleotides surrounding core promoter region of kir3dl1 gene was methylated at a frequency of 20% to 100% in K562 cell line. Comparison of promoter sequence with GenBank database revealed a base substitution within a putative binding site for the transcription factor E2F in K562 cell line. This mutation forms a new methylation site in kir3dl1 promoter. DNA-demethylating treatment resulted in de novo expression of kir3dl1 gene in formerly non-expressed K562 cell line. It is concluded that kir3dl1 expression in K562 cells is regulated by DNA methylation. Deeply to study E2F function contributes to profound understanding of its role in kir3dl1 gene expression regulation.

To analyze the function of kir3dl1 core promoter and study the possible regulation mechanism of kir3dl1 gene expression, a kir3dl1 promoter-luciferase reporter vector was constructed and the promoter activity was evaluated in the K562 cell line. A core promoter fragment of the kir3dl1 5'-untranslated region was amplified by PCR. PCR products were cloned into BglII/NcoI-digested pGL3-basic reporter vector; the polycationic compound SuperFect-reporter vector complexes were transferred into K562 cells. The Dual-Luciferase Reporter Assay System was used to quantitate the reporter vector luciferase activity. MTT method was used to measure the influence of SuperFect-DNA complexes on the survival rate of K562 cells. The results indicated that a 254-bp promoter fragment of kir3dl1 gene was successfully constructed and cloned into the pGL3-basic reporter vector, which was authenticated by BglII/NcoI digestion and DNA sequencing. The luciferase activity of the minimal promoter construct was significantly higher than that of the pGL3-Basic promoter in K562 cells. Transiently transfected cells presented continuously optimal luciferase activity and relative luciferase activity up to 3 days. The cell activity was between 76% and 92%. It is concluded that a kir3dl1 promoter-luciferase reporter vector is successfully constructed, the transfection system used in this study can effectively transfer gene in K562 cells. The kir3dl1 core promoter possesses higher activity in K562 cells, and can promote significantly expression of luciferase reporter gene in K562 cells.

The KIR3DL1 gene is a member of killer immunoglobulin-like receptors family, which exhibits a variegated expression pattern in NK cells and subsets of CD4+ and CD8+ T cells. The E2F family of transcription factors plays a crucial role in the regulation of gene expression. The present study reports a naturally occurring point mutation (TT T GGCGC → TT C GGCGC) within a putative E2F binding site in the KIR3DL1 promoter in K562 cells. Interestingly, this mutation introduces a new methylation site. This study shows for the first time that E2F1 binds to the KIR3DL1 promoter in vivo. This point mutation and concomitantly altered methylation pattern within the E2F1 binding site abolishes their binding and reduces the promoter activity, while elevated expression of E2F1 correlates with increased promoter activity. Therefore, E2F1 contributes to the transcriptional activation of the KIR3DL1 gene.

To investigate the clinical features, treatment modalities and the prognosis of advanced neuroblastoma in children.The medical records of 63 children with stage III or IV neuroblastoma from January 1996 to December 2005 were retrospectively reviewed. Sixty patients were treated by tumor resection and (or) chemotherapy and (or) radiation. Fourteen out of the 60 patients received another autologous peripheral blood stem cell transplantation.Of the 63 patients with advanced neuroblastoma, the male/female ratio was 2.7:1 and the median age at diagnosis was 4 years old. Most of the initial symptoms included pyrexia, abdominal pain, abdominal mass, and leg or articular pain. Primary tumor sites were adrenal (38%), retroperitoneal (35%), mediastinal (17%), pelvic (6%) and cervical (2%). The sites of metastasis at diagnosis included local (41%) and (or) distant (37%) lymph nodes, bone marrow (60%), bone (46%) and liver (16%). The median survival time of the 63 patients was 32.7 months. The 2-year survival rate was 44.3%. Statistical analysis demonstrated that unfavorable survival prognostic factors were the following: age1 year at diagnosis (P0.05); serum neuro-specific enolase100 mg/L (P0.05); serum lactic dehydrogenase1500 U/L (P0.01); serum ferritin150 mg/L (P0.05). The overall survival period of the patients was prolonged through total resection of the primary tumor (P0.05). Intensive chemotherapy in combination with autologous peripheral blood stem cell transplantation could also result in a prolonged overall survival period (P0.01).Neuroblastoma with advanced stages often presents with various clinical manifestations and has a poor prognosis. It is beneficial to improve the prognosis of neuroblastoma through an early diagnosis and a comprehensive therapy including total resection of the primary tumor, autologous peripheral blood stem cell transplantation and intensive chemotherapy.

The purpose of this study was to evaluate the safety of cryopreserved and thawed peripheral blood stem cell (PBSC) fractionated return infusions in children. 35 children patients with malignant tumors (13 acute leukaemias, 15 neuroblastomas and 7 malignant lymphomas) received fractionated return infusions of cryopreserved stem cells after undergoing high-dose chemotherapy without or with total body irradiation. The toxicities of 70 return infusions were evaluated. All patients were mobilized by chemotherapy plus recombination human granulocyte colony-stimulating factor (rhG-CSF), and then PBSCs were collected by a separator CS-3000 plus or COBE spectra-4. The grafts were cryopreserved in 10% dimethyl sulfoxide (DMSD) and stored in liquid nitrogen. There were totally 70 PBSC transfusions. The total volume of PBSCs transfused: 190 - 420 ml (265 +/- 73 ml or 13.7 +/- 4.2 ml/kg) with a mean of (4.43 +/- 1.91) x 10(8)/kg of PBSCs, and 0.94 +/- 0.18 g/kg of DMSO. The single dose: 90 - 300 ml (132 +/- 37 ml or 6.6 +/- 5.2 ml/kg) with a mean of 0.68 +/- 0.12 g/kg of DMSO. Symptoms occurring during the infusions were recorded. All patients were monitored for 24 hours after infusion. Pulse, blood pressure, body temperature, and respiratory rate were recorded every 15 minutes. At four hours before and 8 hours after infusion, urinalysis was performed. Serum potassium, sodium, creatinine, total bilirubin, aspartate amino transferase (AST), and alanine amino transferase (ALT) levels were examined within 24 hours before and after the first infusion. The results showed that the toxicities observed included hemoglobinuria in 54 return infusions (77.1%), headache in 28 (40.0%), nausea in 24 (34.3%), vomiting in 17 (24.3%), and abdominal pain in 8 (11.4%). Patients who received a graft200 ml tended to have a higher frequency of hemoglobinuria, headache, nausea, vomiting, or abdominal pain (P0.01), and they disappeared quickly, too. Total bilirubin increased after the first return infusion (P0.01), and there was a significant correlation between the volume of infusion and the degree of total bilirubin increase (r=0.8977, P0.01). No renal failure or shock occurred. It is concluded that transient hemoglobinuria, headache, nausea, vomiting, and abdominal pain are common toxicities associated with PBSC autograft, and these toxicities are related with a single volume of PBSCs transfused. Total bilirubin increase is correlated with the volume of infusion. In a word, the toxicity is less frequent and lower severe in children with fractionated infusions of cryopreserved peripheral blood stem cell.

To examine the feasibility and practicability of quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with SYBR GREEN I fluorescence for detecting the MYCN mRNA expression in neuroblastoma cell line LA-N-5.MYCN mRNA expression in LA-N-5 cells was measured using real time RT-PCR with SYBR GREEN I. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as internal control. The level of the MYCN mRNA was calculated as MYCN copies/GAPDH copies.Standard curves were linear and showed high correlations (R20.99). The ratio of MYCN mRNA copies to GAPDH mRNA copies was calculated based on specific PCR products. The MYCN mRNA level in LA-N-5 cells was obtained (17.4 +/- 1.2).Quantitative RT-PCR with SYBR GREEN I fluorescence may be a sensitive and reliable method for detecting the MYCN mRNA expression. It may also be potential applicable for detecting the MYCN mRNA expression in the small amount neuroblastoma tissues.

To explore the clinical and pathological characteristics of hepatosplenic gammadelta T-cell lymphoma and its relationship with Epstein-Barr virus infection, the clinical features of a 9-year-old girl with hepatosplenic gammadelta T-cell lymphoma were investigated, the smears of bone marrow was stained with Wright' s stain, biopsies of bone marrow and liver specimen were embedded in plastic and sliced about 4 microm in thickness and routinely stained with HE staining, the immunohistochemical staining was used to mark the tumor cells, and EBER probes were used to detect Epstein-Barr virus RNA. The results showed that the girl presented with prolonged fever, anemia, thrombocytopenia, hepatosplenomegaly, chronic active Epstein-Barr virus infection, and elevated levels of serum ferritin and lactate dehydrogenase. Bone marrow aspirate revealed the infiltration of atypical lymphocytes in the bone marrow stroma. The liver biopsy specimen revealed the infiltration of lymphocytes in the sinusoids, which was positive for the T-cell associated marker CD3 and activated cytotoxicity-associated marker granzyme B. In-situ hybridization analysis with EBER probes revealed that the above-mentioned characteristics were negative in neoplastic cells. It is concluded that hepatosplenic gammadelta T-cell lymphoma is a disease with distinctive clinical, histopathologic, and phenotypic characteristics. Hepatic and/or splenic and/or bone marrow biopsy with combined phenotype is beneficial to diagnosis. Epstein-Barr virus infection is late event involving an already transformed gammadelta T-cell clone.

The reason that a certain subgroup of acute myeloid leukemia (AML) patients with t(8;21) translocation (generating the AML1/ETO fusion gene) displays a poor survival remains elusive. The proto-oncogene c-kit is expressed in approximately 80% of AML cases. The kinase domain mutation of the c-kit gene, one of the most common gain-of-function mutations associated with t(8;21) AML, predicts higher relapse risk and poor prognosis. However, the role of c-kit high expression in t(8;21) AML remains poorly understood. Here we evaluated the prognostic significance of c-kit expression levels in AML patients. The mRNA expression of c-kit was determined by real-time quantitative reverse transcription PCR in 132 adult AML patients. Patients were grouped into quartiles according to c-kit expression levels (Q1-Q4, each quartile containing 25% of patients) and divided into c-kit high (Q4; n = 33) and c-kit low (Q1-Q3; n = 99). High c-kit expression was associated with AML1/ETO-positive and with c-kit mutation. Of note, 35.8% of the AML1/ETO-positive AML patients carrying wild-type c-kit expressed high levels of c-kit, suggesting that other factors are involved in c-kit overexpression. High c-kit expression was associated with inferior overall and event-free survival in AML1/ETO-positive patients and was independently predictive for overall and event-free survival in multivariate analyses in a c-kit mutation-independent manner. Thus, high c-kit expression serves as a reliable molecular marker for poor prognosis, supporting a pathogenetic role of c-kit signaling in AML1/ETO-positive AML. AML1/ETO-positive patients with high c-kit expression might benefit from early treatment modifications and molecular target therapies.

A series of receptors expressed in the surface of tumor cells, which are able to mediate internalizing effect by specially connecting with corresponding ligands. These receptors are potential targets for drugs combined with conjugates. So the drug-conjugate compounds can be targeted delivery to tumor cells. The folate receptor is a promising target because of its marrow tissue specificity, its overexpression in malignant tissues, especially in myeloid leukemic cells, and its ability to bind and internalize folic acid conjugates. It is a promising potential method to apply folate receptor in the receptor-mediated targeting therapy of leukemic cells. In this review, the biological features of folate receptor, its chromosome location and its interaction with ligands, the distribution characteristics of folate receptor in normal and tumor tissues, especially in myeloid leukemic cells, and progress of research on folate receptor mediated targeting tumor cells, especially leukemic cells were summarized.