Dynamic change of the reticulin fibres and collagen fibres in vitro long-term bone marrow culture of acute myeloid leukemia

AIM and METHOD: The relationship between the evolution of the reticulin fibres(RF) or the collagen fibres(CF) and the growing of hematopoietic cells in long-term bone marrow culture (LTBMC) from 15 paticnts with acute myeloid leukemia (AML) and form 6 normal subjects was observed by inverted microscope, Gomoris staining and Massons staining were used. RESULTS: (1)The amount of RF contents of 8 AMLs, with self- maintained(AMLsm) in the 1~8 weeks-old-culture was significant less than that of normal control and 7 AMLs, without self-maintained(AMLnsm) (P＜0.05). On the contrary, the amount of CF coutents of AMLsm in the 1~6 weeks-old-culture was significant more than that of normal and AMLnsm(P＜0.05). (2) There were many hematopoietic cells in the areas of CF, and there were few in the areas of RF. (3)During the whole culture, the most part of confluence of the adherent layer only observed in 3 AMLsm. CONCLUSION: A proper amounts of fibres in the extracellular matrixes of bone marrow is necessary for hematopoiesis, but the change of the proportion of RF contents to CF contents may play an important role in the development of AML and other malignant blood diseases.     

Serum acute phase reactants sCRP,SAA, IL-6 in acute coronary syndrome and its clinical significance

AIM: To investigate the possible involvement of CRP, SAA, IL-6 and its significance in acute coronary syndrome. METHODS: Serum SAA, IL-6 and sCRP levels in 40 patients with AMI, 40 patients with UAP and 40 controls were measured by means of ELISA. RESULTS: Serum SAA, IL-6 and CRP were significantly higher in ACS group (AMI or UAP) than in control group. In the Spearman correlation analysis , we found a correlation among IL－6, SAA, sCRP, fibrinogen both in the AMI group and UAP group. There was no correlation between CK-MB and serum SAA, IL-6 and sCRP levels either in AMI group or in UAP group. In the AMI group, there was no difference of serum SAA,IL-6 and sCRP levels when divided into ≤6 score group and >6 score group using Wagner's QRS scoring system. CONCLUSIONS: Serum levels of certain inflammatory markers have some diagnostic value for ACS. The high levels of serum SAA, IL-6 and sCRP are not mainly caused by myocardial death, but by the inflammation in the multifocal unstable plague.     

Expression of DNA repair gene ERCC1 and its relationship with PAH-DNA adducts in lung cancer tissues

AIM: To investigate the expression of nucleotide excision repair gene ERCC1 and its relationship with PAH (polycyclic aromatic hydrocarbons)-DNA adducts in lung cancer tissues. METHODS: ERCC1 mRNA expression and the PAH-induced DNA adducts were detected in 150 lung cancer tissues, 120 adjacent lung tissues without cancer cells, 40 benign lung lesions and 40 normal lung tissues. The effects of some exposure factors on the expression of ERCC1 gene and the connection between ERCC1 and PAH-DNA adduct was analyzed. RESULTS: Reduced expression levels of ERCC1 were observed in 46 of 150 (30.7%) lung cancer specimens and 1 of 40 (2.5%) normal lung tissues. Smoking may suppress the expression of ERCC1 gene. The level of PAH-DNA adduct was negatively correlated with the expression of ERCC1 gene, the Spearman coefficient was -0.648, P<0.01. CONCLUSION: ERCC1 is an important nucleotide excision repair gene and may participate in the repair of DNA damage, such as PAH-DNA adduct. Low expression of ERCC1 may play an important role in the development of human lung cancer.     

Expression of CD44s in lung cancer and its significance

AIM: To investigate expression of CD44s in lung cancer and it's clinical significance. METHODS: A total of 117 primary lung cancer from patients were examined for CD44s expression by immunohistochemical staining. RESULTS: CD44s mostly expressed in non-small cell lung cancer (NSCLC) but not in small ecll lung cancer (SCLC), and squamous cell carcinoma(SCC) showed much stronger expression of CD44s than adenocarcinoma(ADC)(P＜0.05). In comparison of the lung cancer with/ without lymph node metastasis, the latter showed stronger expression of CD44s(P＜0.01). According to TNM, there was a distinct statistic difference between early stage and advanced stage(P＜0.05). CONCLUSION: CD44s might be a better indicant in histological classification of lung cancer, lymph node metastasis, clinical stage and prognosis.     

Methylation of p15INK4B gene and its mechanism in patients with myelodysplastic syndrome and leukemia

AIM: To explore the relationship between hypermethylation of p15INK4B gene and the pathogenesis of hematopoietic malignances. METHODS: The expression and methylation of p15INK4B gene and the expression of DNA methyltransferase genes (DNMTs) in bone marrow cells from 54 cases with hematopoietic malignances were detected by RT-PCR, Western blot, and methylation-specific PCR. RESULTS: The p15INK4B gene was methylated more often in high-risk myelodysplastic syndrome (MDS) patients, patients at blast phase of chronic myeloid leukemia (CML-BP) and acute leukemia patients than that in low-risk MDS patients (P<0.01). The expression levels of DNMT_3A and DNMT_3B in acute leukemia patients, high-risk MDS patients, and CML-BP patients were higher than that in low-risk MDS patients (P<0.05). CONCLUSION: The hypermethylation of p15INK4B gene may be one of the most common genetic event in pathogenesis of acute leukemia, high-risk MDS, and blast phase of chronic myeloid leukemia. Furthermore, DNMT_3A and DNMT_3B are substantially over-expressed in the bone marrow cells of these patients.     

Expression of cytokines and their receptors in leukemia cell lines and normal blood cells

AIM: To study the expression of cytokines and their receptors in leukemia cell lines and normal blood cells. METHODS: RT-PCR was used to detect expression of mRNA for cytokines in leukemia cell lines(HL-60,U937,K562,HEL,DAMI,MEG-01,HUT78 and CA) and normal blood cells, including CD34⁺ cells, megakaryocytes,platelets, peripheral mononucleates cells and granulocytes. RESULTS: ①CD34⁺ cells simultaneously expressed mRNA for IL-1(α,β),IL-3, IL-6 , G-CSF, GM-CSF and their receptors and SCFR,MPL as well. The granulocytes only expressed IL-6,IL-6R,G-CSFR,GM-CSF. Megakaryocytes and platelets only expressed IL-3R,IL-6,IL-6R,MPL.Interestingly, TGFβ₁ ,TNFα and their receptors sustained to express in normal cells.②Most leukemia cell lines were found to simultaneously express at least two or more stimulating cytokines and receptors ,while TGFβ 1 , TNFα and their receptors were expressed in all the leukemia cell lines we observed. CONCLUSIONS: ①Multi-autocrine loops exist in leukemia cells;②Imbalance of autocrine loops of positive and negative cytokines may be related to leukemia.     

Expression of telomerase inhibitor Pinx1 in leukemia cells and its correlation with telomerase activity

AIM:To study the expression of telomerase inhibitor Pinx1 in acute leukemia cells and during the differentiation of acute promyelocytic leukemia cells,and to realize its effect on telomerase activity.METHODS:Realtime quantitative PCR with fluorescence probe hybridization was used to measure the expression of Pinx1 and hTERT mRNA in acute leukemia cells and during differentiation of NB4 cells induced by ATRA.The correlations between Pinx1 and hTERT expression were also analyzed.RESULTS:Pinx1 mRNA expression in acute leukemia samples (0.00312,5.42×10-4-0.024) was significantly higher than that in normal bone marrow mononuclear cells (7.89×10-4,0-0.00863,P<0.01).The expression of Pinx1 mRNA had significant positive correlation with hTERT mRNA expression (r=0.296,P<0.05).Pinx1 mRNA expression decreased during differentiation,its expression was positive correlated with hTERT mRNA expression (r=0.900,P<0.05).CONCLUSIONS:As an inhibitor of telomerase,however,Pinx1 also had the same direction of regulation with telomerase activity in acute leukemia cells,suggesting its expression variation may be a subsequent reaction induced by that of hTERT to stabilize telomerase activity.The exact mechanisms remained to be verified.     

Effects of p27kip1 gene on DNA and protein synthesis in esophageal carcinoma cells

AIM: To investigate the effects of p27kip1 gene on DNA replication and protein synthesis in esophageal carcinoma cells. METHODS: Recombinant adenovirus Ad-p27kip1 was constructed and transfected into esophageal carcinoma cell EC-9706, and its effects on DNA replication, protein synthesis and the growth of esophageal carcinoma cells were explored by means of cell growth count, [3H]-TdR, [3H]-Leucine incorporation method and flow cytometry. RESULTS: The growth of esophageal carcinoma cells was inhibited obviously. The radioactive intensity of -TdR and -Leucine in Ad-p27kip1 group decreased significantly than that in control group after EC-9706 transfection (P<0.01), while the multiplication of esophageal carcinoma cell was inhibited obviously. CONCLUSION: Ad-p27kip1 inhibited the multiplication of the esophageal carcinoma cells, which may be related to its suppressive function for DNA replication and protein synthesis.     

Effects of cigarette smoke extract on DNA damage and cell stress in human bronchi smooth muscle cells

AIM: To investigate DNA damage and cell stress (heat shock protein 70 expression) in human bronchi smooth muscle cells by cigarette smoke extract (CSE) in vitro. METHODS: 30 mL smog was dissolved in 1 mL culture medium as stock solution of CSE. Human bronchi smooth muscle cells were cultured 3 hours with 1∶16, 1∶10, 1∶8, 1∶6 and 1∶4 of CSE. The DNA damage and HSP70 expression were determined by single cell gel assay (comet assay) and Western blot, respectively. RESULTS: Associated with rising CSE concentration, DNA damage aggravated. Compared with the untreated group, except 1∶16 of CSE, the level of DNA damage was significantly different (P＜0.05). The level of HSP70 expression rised in 1∶16 and 1∶10 of CSE, but it gradually decreased in others. There was significantly different in the fifth and sixth group with negative control (P＜0.05). CONCLUSION: CSE results in DNA damage and decreasing of HSP70 expression, which may be related with chronic obstructive pulmonary diseases.     

Changes of serum T-PSA,F-PSA and F/T ratio in patients with prostate cancer (PCa) and its clinical significance

AIM: To evaluate the changes of serum total prostate specific antigen (T-PSA), free prostate specific antigen (F-PSA) and the ratio of F-PSA to T-PSA (F/T) in patients with prostate cancer and its clinical significance. METHODS: The concentrations of T-PSA and F-PSA in serum were measured by micropartical enzyme immunoassay (MEIA) using AxSYM System, and the F/T ratio was calculated. RESULTS: Before operation, the concentrations of T-PSA and F-PSA in patients with PCa were much higher and F/T ratio was significantly lower than that in patients with benign prostate hyperplasia (BPH). T-PSA and F-PSA levels decreased, but F/T ratio increased after operation in PCa and BPH. F/T ratio in 83.5% PCa and 6.5% BPH was less than 0.16. To diagnosis PCa, the sensitivity of F/T ratio was 83.5%, and the specificity was 86.7%. CONCLUSION: Serum T-PSA, F-PSA and F/T ratio are important parameters for the early diagnosis of prostate cancer.     

Expression of miRNA-93 in acute lymphocytic leukemia

AIM: To investigate the expression of microRNA (miRNA)-93 in acute lymphocytic leukemia (ALL) and its effect on the proliferation of acute T-cell leukemia Jurkat cells.METHODS: The expression of miRNA-93 in the bone marrow samples of patients with ALL was measured by real-time PCR. After down-regulation of miRNA-93 by transfection with miRNA-93 inhibitor in the Jurkat cells, the cell viability, cell proliferation and cell cycle distribution were detected by CCK-8 assay, EdU assay and flow cytometry, respectively. Furthermore, the protein levels of cell cycle-related molecules such as cyclin D1, cyclin-dependent kinase 4 (CDK4), phosphorylation retinoblastoma (Rb) and P27 were measured by Western blot.RESULTS: miRNA-93 was highly expressed in the patients with ALL, and the expression level was highest in the high risk patients. Down-regulation of miRNA-93 inhibited Jurkat cell viability, arrested cell cycle in G₁/S transition. In addition, the protein levels of cyclin D1, CDK4 and p-Rb were significantly decreased, the protein expression of P27 was increased in Jurkat cells trasfected with miRNA-93 inhibitor.CONCLUSION: miRNA-93 expression is increased in ALL patients. Down-regulation of miRNA-93 restrains cell proliferation in the acute T cell leukemia cell line Jurkat via regulating cell cycle-related molecules.     

Glucosylceramide synthase upregulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway in leukemia multidrug-resis-tant cell line

AIM: To investigate whether glucosylceramide synthase (GCS) regulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway, thus enhancing drug resistance of K562/A02 human leukemia multidrug resistant cell line. METHODS: siRNA targeting GCS was transfected into K562/A02 cells. Bcl-2, p-ERK and total ERK expression at mRNA and protein levels after GCS knockdown were detected by real-time PCR and Western blotting. After exposed to MEK-ERK pathway inhibitor U0126, the expression of Bcl-2 at mRNA and protein levels also was analyzed by real-time PCR and Western blotting, respectively. The viability of the cells was evaluated by CCK-8 assay. RESULTS: The expression of GCS and Bcl-2, as well as MEK/ERK signaling were significantly inhibited in K562/A02 cells by GCS siRNA transfection compared with negative control group. Inactivation of MEK/ERK signaling due to U0126 treatment decreased Bcl-2 mRNA and protein levels in a concentration-dependent manner, and sensitized K562/A02 cells to adriamycin. CONCLUSION: GCS may affect the expression of apoptosis-related gene bcl-2 by MEK/ERK signaling pathway, thus regulating multidrug resistance of human leukemia K562/A02 cells.     

Expression and clinical significance of Bmi-1 in gastric carcinoma

AIM: To investigate the relationship between expression of Bmi-1 (B cell-specific MLV integration site-1) in gastric cancer and its clinicopathologic significance.METHODS: 146 surgical patients with gastric carcinoma were followed up at least 2 years.Expression of Bmi-1 protein was examined by immunohistochemistry in their archival paraffin embedded tissue specimens.RESULTS: The intensive positive rate of Bmi-1 expression in gastric cancer was 67.8% (99/146).Expression of Bmi-1 was highly correlated with tumor size,clinical stage,lymph node metastasis and T classification (P<0.05),but not with sex,age,tumor differentiation,etc.(P>0.05).The survival rate in the patients with Bmi-1 expression was much lower than that in those patients without Bmi-1 expression (P<0.01).Multivariate analysis indicated that Bmi-1 expression,T classification,lymph node metastasis,distant metastasis,tumor size and postoperative chemotherapy were all significantly prognostic factors of gastric carcinoma.CONCLUSION: Overexpression of Bmi-1 in patients with gastric carcinoma enhances the possibility of invasion and metastasis,implying a poor prognosis.Bmi-1 may serve as fairly a good prognostic factor to indicate biologic behavior and prognosis in gastric carcinoma.     

Expression and clinical significance of Bmi-1 in colorectal cancer

AIM:This study was to investigate the expression and significance of Bmi-1 in colorectal carcinoma (CRC), and to explore the effect of Bmi-1 on Ki67 expression in human CRC.METHODS:The samples from sixty CRC, thirty adenomas and twenty normal colorectal mucosal tissues were used in this study.The expression of Bmi-1 protein was detected by immunohistochemistry.The clinicopathological features and survival rate of patients were also analyzed.RESULTS:The overexpression of Bmi-1 was respectively 25.0％, 6.7％and 0% in CRC and adenomas as well as normal colorectal mucosal tissues.The results showed that the expression of Bim-1 was significantly higher in CRC, compared with that in adenomas and normal colorectal mucosal tissues (P<0.05).The overexpression of Bmi-1 protein in CRC was obviously associated with distant metastasis and TNM stage (P<0.05), but not with gender, age, tumor size, tumor site, histological type, differentiation degree and lymph node metastasis (P>0.05).Kaplan-Meier survival analysis showed that the overexpression of Bmi-1 reduced significantly survival of CRC patients (P<0.05).No statistical relation between expression of Bmi-1 and Ki67 in CRC was observed.CONCLUSION:The overexpression of Bmi-1 protein is significantly correlated with tumorigenesis, metastasis and prognosis of CRC.Bmi-1 might be regarded as a parameter in evaluating prognosis of CRC.     

Association of serum soluble E-selectin concentrations with insulin resistance in essential hypertension patients

AIM: To investigate the relationship between serum soluble E-selectin (sE-selectin) and insulin resistance, serum uric acid, serum lipid in essential hypertension patients. METHODS: Fasting serum sE-selectin concentration, plasma glucose, serum insulin, serum uric acid, total cholesterol, triglycerides, high density lipoprotein-cholesterol, low density lipoprotein-cholesterol were determined in 186 patients with essential hypertension (75 males, 111 females). Homeostasis model assessment was applied to assess the status of insulin resistance (HOMA-IR). RESULTS: Based on the HOMA-IR, the essential hypertension patients were divided into insulin-sensitive individuals (IS) and insulin resistant subjects (IR). The serum sE-selectin concentration was significantly higher in male group [(50.1±17.8)g/L]than in female group [(40.6±16.6)g/L](P<0.01). No difference between IR group [(51.6±16.8)g/L]and IS group [(48.5±18.8)g/L] in male, and significantly higher in IR group [(45.1±18.0)g/L]than in IS group [(36.0±13.7)g/L](P<0.01) in female were observed. A stepwise multiple linear regression analysis showed that HOMA-IR was an independent predictor of serum sE-selectin concentrations in female group and not in male group, and both serum uric acid and serum lipid were not independent predictors of serum sE-selectin concentrations. CONCLUSION: Serum sE-selectin concentrations were directly related to insulin resistance in females with essential hypertension and not in males with essential hypertension. Both serum uric acid and serum lipid were not directly related to serum sE-selectin concentrations.     

Establishment of arsenic trioxide-resistant K562/AS2 cell line and the mechanism of multidrug resistance

AIM:To establish a arsenic trioxide (As₂O₃ )-resistant leukemic cell line to explore the mechanism of resistance to As₂O₃, and the relationship between the resistant cell line and the multidrug resistance was also investigated. METHODS:The arsenic trioxide (As₂O₃ )-resistant leukemic cell line was established by exposing the cells to the increasing concentration of As₂O₃. MTT assay was used to detect the cytotoxicity. Cell cycle was detected by PI assay. Flow Cytometry was used to detect the P-glycoprotein on the surface of the cells, the intracellular concentration of DNR, and the immuetype of the cells. RESULTS:The cell doublings time and the cell cycle of the arsenic trioxide (As₂O₃ )-resistant leukemic cell line, K562/AS2, is similar to that of K562. The relative resistant fold of K562/AS2 to As₂O₃, DNR, VP16 and Ara-C was 7.4, 2.9, 3.8 and 1.1, respectively. The relative resistant fold of multidrug resistant cell line, K562/ A02, to As₂O₃, DNR, VP16 and Ara-C was 0.8、94、2.5 and 0.9, respectively. The fluorescence of the P-glycoprotein on the surface or of the DNR inside the cells detected was not significantly different between the K562 and the K562/AS2 cell lines. CONCLUSIONS:A cell line, K562/AS2, resistant to clinical achieving level (2 μmol/L) of As₂O₃ has been established. The relative resistant fold of K562/ AS2 to As₂O₃ is about 7.4 fold to the parent K562 line sensitive to As₂O₃. Partial resistance of K562/AS2 to DNR and VP16 is observed , the mechanism of which is unrelated to the P-gp, the expression product of multidrug resistance gene 1 (mdr1).