OBJECTIVEThis study was designed to clarify the significance of DNA methylation in the expression of runt-related transcription factor 3 (Runx3) gene.

METHODSReverse transcription-PCR (RT-PCR) was used to measure the expression level of Runx3 mRNA in paired samples of primary gastric cancer and corresponding non-cancerous gastric mucosa, taken from surgical specimens of 70 gastric cancer patients. Western blot was used to detect the protein expression level of Runx3 gene. The promoter methylation status of Runx3 gene was detected by methylation specific PCR (MSP). Furthermore, RT-PCR was used to mesure the expression of DNA methyltransferase 1 (Dnmtl) mRNA . The correlation of Runx3 expression and methylation with Dnmt1 mRNA expression was analyzed.

RESULTSThe mRNA expression level of Runx3 gene was significantly lower in gastric cancer than that in the matched normal gastric mucosa (0.5740 +/- 0.3580 vs. 1.7250 +/- 0.4080, P < 0.05), and the Runx3 protein expression level in gastric cancer was also significantly lower than that in the matched normal gastric mucosa (P < 0.05). Promoter hypermethylation of Runx3 gene was detected in 50.0% (28/56) of the gastric cancer samples, which resulted in a reduced expression of Runx3 mRNA. It was found that the mRNA expression level of Dnmt1 gene was significantly higher in the gastric cancer tissues with methylated Runx3 gene than that in the ones without. There was a significant correlation of Runx3 gene methylation with increased expression of Dnmtl mRNA (r = 0.64, P < 0.05).

CONCLUSIONThe promoter hypermethylation may be one of the predominant inactivation mechanisms of the runt-related transcription factor 3 gene, and may be associated with carcinogenesis of human gastric cancer. Reduced Runx3 expression in gastric cancer may be partially correlated with a high level of DNA methyltransferase 1.

Adenocarcinoma ; genetics ; metabolism ; Adult ; Aged ; Core Binding Factor Alpha 3 Subunit ; genetics ; metabolism ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; biosynthesis ; genetics ; DNA Methylation ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; Young Adult

Vascular endothelial growth factor (VEGF), a central mediator of angiogenesis, not only plays an important role in the pathogenesis of leukemia, but also is an independent prognostic factor in patients with hematologic malignancies, like those in solid tumors. However, the importance of VEGF during differentiation or apoptosis of leukemia cells remains to be elucidated. In order to assess the alternation of VEGF gene expression in the process of all-trans retinoic acid (ATRA)-induced differentiation of NB4 acute promyelocytic leukemia cell line, and a competitor DNA fragment, VEGF gene competative template (T-VEGFDelta) was constructed by using gene recombinant technologies, and a competitive quantitative reverse transcriptase-polymerase chain reaction (cQRT-PCR) method was developed. A standard curve was obtained by co-amplification of serial dilutions of the target nulecules with constant amount of competitive template and this curve was used to detect molecular number of target gene in measuring sample. The surface expression of CD11b antigen and nitroblue tetrazolium (NBT) reduction rate of NB4 cells were also assayed at different time points. The results showed that cQRT-PCR was a sensitive, reliable tool for analysis of VEGF gene expression with a detectable range from 1 x 10(4) to 2 x 10(5) molecules. The number of VEGF gene transcripts detected by means of cQRT-PCR assay was 42.3 x 10(5), 12.6 x 10(5), 3.6 x 10(5), and less than 1.0 x 10(5)/microg total RNA at 0, 12, 24 and 48 hours after ATRA treatment, respectively. This rapid down-regulation of VEGF gene expression, during ATRA-induced NB4 cell differentiation, was accompanied by the up-regulation of CD11b expression and an increased NBT reduction rate. In conclusion, cQRT-PCR method was successtully constructed, confirming that ATRA efficiently repressed VEGF, at the same time, the ATRA might exert an antileukemic effect, other than induction of differentiation via inhibition of angiogenesis.
Antineoplastic Agents ; pharmacology ; CD11b Antigen ; genetics ; Cell Differentiation ; drug effects ; genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; pathology ; Molecular Sequence Data ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Tretinoin ; pharmacology ; Vascular Endothelial Growth Factor A ; genetics

Objective To analyzed the treatment of intracranial aneurysms by mierosurgical clipping, endovascular embolization and embolization combined clipping therapy.In order to explore the ideal and effec- tive treatment plan of intracranial ruptured aneurysm.Methods The clipped group of 30 aneurysms.The embolized group of 34 aneurysms.The combined group of 15 aneurysms.Results Clipped group:All of aneurysms was clipped well,no recurrence,mortality 6%(2/30).Embolized Group:complete embolization rate 70.6%(24/34),recurrence rate 17.6%(6/34),mortality 11.8%(4/34).Combined group:no recur- rence,mortality 6.7%(1/15).All patients of three groups were evaluated by Glasgow Outcome Scale one month later and the rate of recovery well was 80.0%,79.4%and 80.0%.Following up for six months the data were 90.0%,88.2%and 86.7%.Conclusion Microsurgical clipping aneurysm(?)neck is still an ef- fective therapy.Meanwhile it has an absolute advantage of high completely cure rate and low recurrence rate, furthermore it is an available remediation method for those cases that failure of embolization,and for those re- currence aneurysms that have been embolized,microsurgical clipping should be,taken as soon as possible in case of aneurysms re-ruptured.For the patients the aneurysms are narrow shape,irregular shape,small(≤3 nun)or with cerebral hematomas microsurgical clipping is a fitting choice.

OBJECTIVETo investigate the level and significance of interleukin-8 (IL-8), soluble intercellular adhesion molecule (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in patients peripheral blood (PB) during mobilization for peripheral blood stem cells harvesting.

METHODSThe levels of IL-8, sICAM-1 and sVCAM-1 in patients were dynamically assayed by ELISA during the mobilization procedure and the number of CD(34)(+) cell, white blood cell (WBC) and platelet (BPC) by flow cytometric analysis and hematometry respectively. Colony formation was assayed by using semisolid methycellulose culture.

RESULTSThere was a significant increase in plasma levels of IL-8 and both adhesion molecules [IL-8 (247.4 +/- 84.2) microg/L (P < 0.01); sICAM-1 (530.3 +/- 286.1) microg/L (P = 0.002 7); sVCAM-1 (575.3 +/- 350.4) microg/L (P = 0.001 3)] during the mobilization process; furthermore, IL-8 and sVCAM-1 concentration in the patient's plasma was paralleled to the numbers of CD(34)(+) cell, CFU-GM, WBC and BPC (P < 0.001).

CONCLUSIONThe levels of IL-8, sICAM-1 and sVCAM-1 in the patient's plasma were correlated to the PB number of CD(34)(+) cells, CFU-GM, WBC and BPC during the mobilization process. It suggested that analysis of IL-8, and sVCAM-1 dynamic changes may serve as markers for CD(34)(+) cells.

Adolescent ; Adult ; Enzyme-Linked Immunosorbent Assay ; Female ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Intercellular Adhesion Molecule-1 ; blood ; Interleukin-8 ; blood ; Male ; Middle Aged ; Treatment Outcome ; Vascular Cell Adhesion Molecule-1 ; blood

A patient with skin rash, skin denudation, anuria, general dropsy and dyspnea for unknown etiology underwent continuous renal replacement therapy (CRRT) for 3 consecutive days. The biochemical indexes were monitored during the therapy and biopsy was performed on the right thigh. Pathological examination of the biopsy sample established the diagnosis of polymyositis(PM) and dermatomyositis(DM). After the start of CRRT, the patient's heart, liver, kidney and lung injuries showed obvious improvement, and the urine volume (UV) increased and serum creatinine (Cr), urea, total bilirubin (TBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) levels all decreased promptly. The patient showed progressive improvement of the physiological condition even after CRRT, and was discharged 10 days later. This case suggests the efficacy of CRRT in the management of severe PM/DM and its value as a good option for treatment of severe autoimmune disease, especially systemic inflammatory response syndrome.
Adult ; Dermatomyositis ; therapy ; Humans ; Male ; Polymyositis ; therapy ; Renal Replacement Therapy ; Treatment Outcome

OBJECTIVETo study the expression of annexin II (AnnII) and the fibrinolytic activity in NB4 cells and their alterations in the presence of arsenic trioxide (ATO) and daunorubicin (DNR).

METHODSLeukemia cell line NB4 was treated with ATO or DNR for 24 ∼ 72 h. Cell surface expression of AnnII and its mRNA were analysed by flow cytometry and real time PCR, respectively, the fibrinolytic activity by chromogenic assay.

RESULTSCompared with other acute leukemia cell lines, the expression of AnnII on untreated NB4 cells was relatively higher. The AnnII positive cell rates on NB4, HL-60, K562, and A3 cells were (94.5 ± 1.6)%, (40.1 ± 2.1)%, (36.3 ± 1.5)% and (11.8 ± 2.5)%, respectively. The fibrinolytic activity of NB4 cells was the greatest with a A value of 0.68 ± 0.02. The fibrinolytic activity of NB4 cells was obviously decreased by ATO, DNR or monoclonal antibody against AnnII, being decreased by 60.4%, 35.8% and 26.0% of the pretreatment level, respectively. The expressions of AnnII and its mRNA in NB4 cells were decreased dramatically after ATO and DNR treated for 48 h. Annexin II positive cells rate were (55.46 ± 4.72)% and (27.00 ± 6.18)%, respectively.

CONCLUSIONNB4 cells have strong ability to enhance the catalytic efficiency of the t-PA-dependent plasminogen activation and AnnII on the cell membrane contributes to this activity. Its high fibrinolytic activity can be corrected by ATO and DNR through down-regulating AnnII.

Annexin A2 ; Apoptosis ; Daunorubicin ; HL-60 Cells ; Humans ; Leukemia ; metabolism

Multiple myeloma (MM) is an incurable heterogeneous disease derived from malignant clonal expansion of plasma cells. The evaluation of prognosis, detection of minimal residual disease (MRD) and treatment of MM are unclear for decades. Recently, Velcade and autotransplantation have been broadly applied to MM patients and achieved better outcomes, but there is yet no effective and universal marker for MRD detection in MM. Both genetic and epigene-tic aberrations play important roles in the pathogenesis and development of cancer. Our preliminary data showed that aberrant promoter methylation of zo-1 and id4 genes was correlated with their gene silencing in several types of hematological malignancies. Therefore, this study was aimed to identify the promoter methylation status of zo-1 and id4 genes in MM and their relationship with the prognosis, MRD and treatment of MM. The methylation status of zo-1 and id4 genes of MM cell lines U266, H929 and IM9 was tested by using MS-PCR; the methylation status of zo-1, id4 gene promoters in bone marrow samples of 20 MM patients and 6 healthy persons was detected by MS-PCR. The results showed that the zo-1, id4 gene in MM cell lines all were methylation positive (complete or partial methylation), the zo-1, id4 gene in samples of 5 healthy persons all were completely unmethylated. The methylation positive rate of zo-1 and id4 genes were 50% and 85% respectively, which were significantly higher than that in normal bone marrow (0%). The coverage rate of zo-1 and id4 gene methylation was 95%. There were no significant differences in the methylation status of both genes among the patients with different heavy chains, different light chains and symptoms. It is concluded that the change of zo-1, id4 genes methylation status occurs in MM patients and has specificity, which may be a new gene marker of MM, methylation analysis of zo-1 and id4 genes may be important for MRD monitoring in patients with MM.
Adult ; Aged ; Bone Marrow ; metabolism ; Cell Line, Tumor ; DNA Methylation ; Female ; Humans ; Inhibitor of Differentiation Proteins ; genetics ; metabolism ; Male ; Membrane Proteins ; genetics ; metabolism ; Middle Aged ; Multiple Myeloma ; genetics ; metabolism ; Neoplasm, Residual ; genetics ; Phosphoproteins ; genetics ; metabolism ; Zonula Occludens-1 Protein

It is hard to discriminate myelodysplastic syndrome(MDS) from many benign hematological diseases. To identify the methylation status of zo-1 gene in MDS, the methylation specific PCR (MS-PCR) and reverse transcription-PCR (RT-PCR) were applied to detect the MDS cell line MUTZ-1, bone marrow of a healthy donor and an aplastic anemia patient. MS-PCR was also employed to detect the bone marrow of 72 patients with benign hematological diseases, 35 MDS-RA patients, and 20 MDS-like patients. The results showed that MDS cell line MUTZ-1 displayed complete methylation of zo-1 promoter without mRNA expression. Inversely, a patient with benign hematological disease and a donor with normal bone marrow showed complete unmethylation of this gene with unaffected mRNA expression. No zo-1 promoter methylation was detected in patients with benign hematological diseases, while aberrant hypermethylation of zo-1 gene promoter were found in 48.6% (18/37) of MDS-RA patients. The positive rate of zo-1 methylation in MDS-RA patients was higher than that in patients with benign hematological diseases (p < 0.05). Seven suspected MDS patients manifested hypermethylation status of zo-1 gene (7/20), 2 were followed up for 1 year and transformed into MDS. It is concluded that relatively high hypermethylation rate of zo-1 promoter is observed in MDS-RA, and no methylation in patients with benign hematological diseases. Therefore, zo-1 gene hypermethylation may be served as a useful epigenetic marker in the differential diagnosis for MDS.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; DNA Methylation ; Diagnosis, Differential ; Female ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; diagnosis ; genetics ; Young Adult ; Zonula Occludens-1 Protein ; genetics ; metabolism

OBJECTIVETo study the relationship between pretransplantation host thymic recent output function and prognosis in HLA-matched sibling bone marrow transplantation (MSD-BMT) and determine whether pretransplantation host thymic recent output function can act as a marker for predication of prognosis after HSCT.

METHODST-cell receptor excision circle (TREC) in DNA of pretransplantation peripheral blood mononuclear cells from 64 patients underwent MSD-BMT was detected by real-time quantitative PCR. The content of TREC in 70 normal donors was detected as well. All clinical data of patients after HSCT were collected and studied. Survival rates of patients after HSCT were estimated with Log-rank test. Univariate and multivariate analysis of prognostic factors were carried out by COX's proportional hazard regression model.

RESULTSThe mean value of TREC in normal donors was (3351 +/- 3711) copies/10(5) cells. There was an inverse correlation between TREC and age in the donor groups. Before transplantation, all patients were detected TREC, with a mean TREC number of (180 +/-332) copies/10(5) cells being significantly lower than that of normal donors. The results of univariate analysis showed that the counts of pre-HSCT TREC were closely, correlated with long term survival and chronic graft versus host disease (cGVHD) (P < 0.05) and with CMV infection (P = 0.084) but not with acute graft versus host disease (aGVHD). The results of multivariate analysis showed the same thing as that of univariate analysis.

CONCLUSIONPretransplantation host thymic recent output function is closely correlated with prognosis in MSD-BMT and can be a factor for predicting the outcome of HSCT.

Adolescent ; Adult ; Child ; Female ; Hematopoietic Stem Cell Transplantation ; methods ; Humans ; Male ; Middle Aged ; Prognosis ; Receptors, Antigen, T-Cell ; genetics ; Retrospective Studies ; Siblings ; Thymus Gland ; immunology ; Transplantation, Homologous ; immunology ; methods

OBJECTIVETo analyze the relationship between BK viruria and the late onset hemorrhagic cystitis (LOHC) after hematopoietic stem cell transplantation (HSCT), and investigate the role of BK virus load in the development of LOHC.

METHODSFrom August 2006 to April 2008, urine samples were collected weekly from 113 patients undergoing HSCT. Virus DNA were extracted from the urine samples and amplified by qualitative PCR. Real-time quantitative PCR was used to quantify BKV DNA in the urine samples of all BK viruria patients.

RESULTSLOHC occurred in 22 patients (19.5%), including grade 1 in 7, grade 2 in 11, grade 3 in 3, and grade 4 in one. The median onset time was 44 (13 - 114) days after transplantation. Twenty-one of which (95.5%) were BK virus positive, being significantly higher than that in non-LOHC patients (31.9%) (P = 0.000). No BK virus was detected in the healthy control group at the same time. Quantitative PCR detection showed that the mean BK virus DNA copies in LOHC patients at a week before occurring HC was higher than that at the first positive samples (10(5) copies/microl versus 10(4) copies/microl, P = 0.025), and it was no significant change at the onset and a week after HC. Meanwhile, there was no statistical difference in the mean level of BK virus DNA copies among the LOHC patients with different grades. The mean level of BK virus DNA copies in non-HC patients was 10(3) to 10(4) copies/microl, being lower than that in LOHC patients.

CONCLUSIONSBK viruria is an important pathogenic cause of the LOHC after HSCT. The occurrence of BKV viruria in HSCT patients, together with the increasing of BK virus DNA copies in urine, over the level of 10(5) copies/microl may indicate a possible development of LOHC.

Adolescent ; Adult ; BK Virus ; isolation & purification ; Child ; Cystitis ; etiology ; virology ; DNA, Viral ; urine ; Female ; Hematopoietic Stem Cell Transplantation ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; Postoperative Complications ; etiology ; virology ; Transplantation, Homologous ; Viral Load ; Young Adult