OBJECTIVETo investigate the effect of SHIP gene mutation on the cell cycle and its related gene expression in K562 cells.

METHODSThe recombined green fluorescent protein (GFP) containing FIV-SHIP gene was transfected into K562 cells. The transfection efficiency and cell cycle of K562/SHIP were assessed by flow cytometry (FCM). The proliferation of K562 cells was detected by MTT assay, the mRNA levels of SHIP by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR), and the protein levels of SHIP, Cyclin D1, p21(WAF1/CIPI) and p27(KIP1) by Western blot.

RESULTSWild type SHIP inhibited K562 cell proliferation and caused a G(0)/G(1) arrest \[(34.2 +/- 7.8)% vs (0.7 +/- 8.3)% (P < 0.01)\]; while the point mutation of SHIP gene did not show such effect. Western blot results showed that the Akt phosphorylation and cyclin D1 expression was significantly decreased (P < 0.01), and the expression of p27(KIP1) and p21(WAF1/CIPI) increased. Site-directed mutation of SHIP gene SH2 domain (TTC-->CTC, Phe-->Leu) did not influence the Akt phosphorylation and cyclins (P > 0.05).

CONCLUSION(1) wtSHIP gene can down-regulate Akt phosphorylation and result in inhibition of cyclin D1 expression, up-regulating p27(KIP1) and p21(WAF1/CIPI) expression, finally leading to the reduction of K562 cell proliferation, and inducing G(0)/G(1) phase arrest. (2) SHIP gene suppresses the proliferation of K562, being dependent on its intact structure and function.

Cell Cycle Proteins ; metabolism ; Humans ; Inositol Polyphosphate 5-Phosphatases ; K562 Cells ; Mutation ; Phosphoric Monoester Hydrolases ; genetics ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Transfection

OBJECTIVETo explore the effect of mutation in PxxP domain of SHIP on migration and invasion of leukemia cells and its mechanism.

METHODSThe lentiviral vector mediated wild type SHIP (wtSHIP) and mutant SHIP (muSHIP) plasmids were transfected into K562 cells through gene transfection techniques. Expression of SHIP at mRNA and protein level was detected by real-time PCR and Western blot, respectively. Transwell assay was used to analyze the difference between the migration and invasion ability of the K562/wtSHIP and the K562/muSHIP cells after transfection. Primary migration associated factor FAK, MMP and NF-κB were assayed by Western blot.

RESULTSAfter transfection, the SHIP expression in transfected K562 cells were significantly increased. Compared with the migration ability of K562/wtSHIP\[(15.8 ± 1.4)%\], that of K562/muSHIP cells \[(54.3 ± 2.4)% \] increased greatly and almost at the same level of that of K562/pFIV\[(50.3 ± 3.8)%\] (P < 0.01). The invasion assay also showed that K562/wtSHIP\[(32 ± 6)/HP\] has a lower invasion ability than that of the K562/muSHIP group \[(83 ± 16)/HP\] and K562/pFIV group \[(78 ± 13)/HP\] (P < 0.01). Western blot analysis showed that the expression of p-FAK and NF-κB was up-regulated in K562/muSHIP group compared to that of the K562/wtSHIP group.

CONCLUSIONSThe results confirmed that mutation in PxxP domain of SHIP gene played an important role in negative regulating function of SHIP gene. The mutation affects the cell migration and invasion ability through increase in MMP-9 expression, FAK phosphorylation and NF-κB activation. It suggested that the mutation of PxxP domain in SHIP gene might be pathogenic, and be one of the reasons for SHIP abnormality in leukemia.

Cell Movement ; Genetic Vectors ; Humans ; Inositol Polyphosphate 5-Phosphatases ; K562 Cells ; Leukemia ; pathology ; Mutation ; Phosphoric Monoester Hydrolases ; genetics ; Plasmids

The src homology 2 (SH2)-domain containing inositol-5-phosphatase (SHIP) is another recently identified lipid phosphatase after phosphatase and tensin homology deleted on chromosome ten gene (PTEN). It plays an important role in negatively regulating the proliferation of hematopoietic cells. The relationship between SHIP and the inhibition of tumor proliferation is rarely reported. The purpose of this study is to evaluate the apoptosis induced by SHIP gene in K562 cell line and to explore the involved signaling pathway. The K562 cells were transfected with human SHIP gene by using the lentiviral vector containing SHIP, and the transfection was verified by fluorescent quantitative PCR (FQ-PCR) and Western blot. Then the effects of SHIP protein expression on cell growth and apoptosis were measured. The levels of p-Akt, bcl-2 family, caspase and the activity of NFkappaB were assayed by Western blot and ELISA, respectively. The results are as follows: (1) Human leukemia cell line K562 was SHIP-negative; (2) Transfection with SHIP gene led to the re-expression of SHIP mRNA and protein in K562, as shown by FQ-PCR and Western blot; (3) The expression of SHIP protein inhibited cell growth and significantly increased apoptosis in K562 cells; (4) Compared to that in control group, the expression level of p-Akt-308 and p-Akt-473 in SHIP-expressing cell group decreased significantly (P<0.01); SHIP activated caspase-9, caspase-3, up-regulated protein levels of bad, p27, down-regulated expression of bcl-xL, while it had no effect on the expression of bcl-2 and bax. Furthermore, the inhibition of NF-kappaB was achieved along with the inactivation of Akt. These data suggest that SHIP gene has potential abilities to inhibit K562 leukemic cell proliferation and induce its apoptosis via inactivating PI3K/Akt pathway. The loss of SHIP might be the explanation of aberrant high-level p-Akt in human leukemia. It may be at least one of the mechanisms by which the loss of SHIP expression contributes to leukemia progression.
Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Cell Proliferation ; Down-Regulation ; Genetic Vectors ; Humans ; Inositol Polyphosphate 5-Phosphatases ; K562 Cells ; NF-kappa B ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphoric Monoester Hydrolases ; genetics ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; Transfection

Objective To investigate iodine deficiency disorders(IDD) in Chongqing and Linzhi, and to provide scientific basis for IDD control and prevention. Methods According to the national program developed in 2007, investigation was conducted in Chengkou and Wuxi county in Chongqing municipality, and Linzhi, Bomi,Milin and Langxian county in Linzhi prefecture. Five towns were sampled in Linzhi county, and 3 in other counties.In each town, one township primary school and two village primary schools were selected to inspect thyroid by B ultrasound and palpation, and urinary iodine of children aged 8 to 10 years was tested in these schools. Meanwhile,2 villages were selected in each town for test of salt iodine level and urinary iodine of childbearing age women and search cretin cases. Results Three hundred and forty families in Chongqing and 915 families in Linzhi were investigated. The coverage of iodized salt in Chongqing was 98.82%(336/340), which was significantly higher than that in Linzhi[66.34%(607/905), x2 = 139.56, P ＜ 0.01]. Goiter rate of children in Chongqing was 9.27%(89/960) by palpation and 8.34% (61/731) by B ultrasound, while goiter rate of children in Linzhi was 7.80%(102/1308) by palpation and 5.53% (69/1248) by B ultrasound. The difference of goiter rate by palpation between Chongqing and Linzhi was not statistically significant (x2 = 1.37, P ＞ 0.05 ). But goiter rate of children by B ultrasound in Chongqing was higher than that in Linzhi (x2= 5.51, P ＜ 0.05). In Chongqing, the median urinary iodine was 319.15 μg/L, and 345.75 μg/L in Chengkou county and 281.39 μg/L in Wuxi county. In Linzhi prefecture, the median urinary iodine was 189.81 μg/L, and 207.81 μg/L in Linzhi county, 161.12 μg/L in Bomi county, 131.83 μg/L in Milin county and 334.60 μg/L in Langxian county. The median urinary iodine in childbearing women were 248.42 μg/L in Chongqing and 121.25 μg/L in Linzhi. The median urinary iodine in Chongqing both in children and women were higher than those in Linzhi. No new cretin case was found in these two areas. Conclusions Goiter rate in high risk areas of IDD in Chongqing and Linzhi has decreased to less than 10%.No new cretin case is found in these areas. It can be concluded that the work of control and prevention is effective.There is excess iodine in Chongqing. In Linzhi county and Langxian county, iodine is excess in children and deficient in women. Further investigation should be conducted to find out the reason. Population iodine is excess in Bomi and Milin counties. The concentration of salt iodine should be decreased in Chongqing. In Linzhi prefecture,adding iodine measures should be adjusted based on further investigation.

OBJECTIVETo study the effects of benzo(a)pyrene (BaP) on the cell cycle distribution and activities of mitogen-activated protein kinase (MAPK) signal molecules (ERK1/2, JNK1/2 and p38) in human embryo lung cells (HELF), and to investigate the relationship between alterations of MAPK protein phosphorylation and the cell cycle distributions.

METHODSThe phosphorylation of MAPK were induced by exposing HELF cells to BaP at 0.1, 0.5, 2.5 and 12.5 micromol/L. The phosphorylation and protein expression levels of ERK1/2, JNK1/2 and p38 were determined through western-blotting assay. And the flow cytometry assay was used to measure the cell cycle effects in HELF cells after treatment with 2.5 micromol/L BaP for 24 h.

RESULTSThe phosphorylation levels of ERK1/2, JNK1/2 and p38 were significantly increased through BaP exposure. In addition, the phosphorylation of these three MAPKs has similar alteration pattern. We found that exposure of cells to 2.5 microM of BaP for 24 h resulted in a decrease of G(0) and G(1) population by 11.9% (F = 41.38, P < 0.01) and an increase of S population by 17.2% (F = 68.13, P < 0.01). Three chemical inhibitors of MAPK (AG126, SP600125 and SB203580) could significantly inhibit the cell cycle alteration because of BaP treatment.

CONCLUSIONERK1/2, JNK1/2 and p38 could positively regulate the BaP independently induced cell cycle alterations.

Benzo(a)pyrene ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Fibroblasts ; drug effects ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lung ; cytology ; embryology ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinase 8 ; metabolism ; Mitogen-Activated Protein Kinase 9 ; metabolism ; Signal Transduction ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo study the role of E2F1/4 pathway in vitamin C reversing benzo (a) pyrene [B (a) P]-induced changes of cell cycle in human embryo lung fibroblasts (HELF) and the relationship between E2F1 and cyclin D1/CDK4.

METHODSThe stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established to detect the relationship of signaling pathway. Cells were cultured and pretreated with vitamin C before stimulation with B (a) P for 24 hours. The expression levels of cyclin D1, CDK4, E2F1 and E2F4 were determined by Western blot and the band intensity was analysed as the relative value to control by using the Gel-Pro 3.0 software. Flow Cytometric Analysis was employed to detect the distributions of cell cycle.

RESULTSB (a) P significantly elevated the expression levels of cyclin D1, CDK4, E2F1 and E2F4 in HELF cells. Vitamin C decreased the expression levels of above proteins in B (a) P-stimulated HELF cells. The expression levels of these proteins in B (a) P-treated above transfectants were lower than those in B (a) P-treated HELF cells. The expression levels of above proteins with vitamin C combined with antisense cyclin D1 were decreased as compared to those with antisense cyclin D1 alone. B (a) P increased the percentage of S phase as compared to the controls [(41.1 +/- 0.2)% vs (33.5 +/- 3.2)%, P < 0.05]. Both vitamin C [(33.2 +/- 0.6)% vs (41.1 +/- 0.2)%, P < 0.05] and antisense cyclin D1 [(31.2 +/- 1.3)% vs (41.1 +/- 0.2)%, P < 0.05] suppressed the changes of cell cycle induced by B (a) P. Vitamin C combined with antisense CDK4 markedly suppressed B (a) P-induced changes of cell cycle as compared to those with antisense CDK4 alone.

CONCLUSIONVitamin C might reserve the B (a) P-induced changes of cell cycle via intracellular signaling pathway of cyclin D1-CDK4/E2F-1/4.

Ascorbic Acid ; pharmacology ; Benzo(a)pyrene ; antagonists & inhibitors ; toxicity ; Cell Cycle ; drug effects ; Cyclin D1 ; metabolism ; E2F1 Transcription Factor ; metabolism ; E2F4 Transcription Factor ; metabolism ; Humans ; Lung ; cytology ; embryology ; Signal Transduction

OBJECTIVETo study the role of mitogen activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway in benzo(a)pyrene (B(a)P)-induced changes of cell cycle in human embryo lung fibroblasts (HELF).

METHODSAP-1 luciferase activity was determined by the Luciferase reporter gene assay using a luminometer. The expression levels and activity of extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle. The dominant negative mutant of ERK2, JNK1 and p38 were applied to detect the upstream or downstream relationship of signaling pathways.

RESULTSB(a)P treatment resulted in a marked activation of AP-1 and its upstream MAPK, including ERK, JNK and p38 in human embryo lung fibroblasts (HELF). B(a)P exposure also led to increase the population of cells at S phase compared to control (P < 0.01) with a concomitant decline of cells at G(1) phase. B(a)P-induced cell cycle alternation was markedly impaired by stable expression of a dominant negative mutant of ERK2 or JNK1, but not p38. B(a)P-induced AP-1 transactivation was inhibited by the overexpression of dominant-negative mutant of ERK2 or JNK1, but not p38. Inhibition of the activation of AP-1 by curcumin, a chemical inhibitor of AP-1, significantly inhibited the cell cycle changes in response to B(a)P treatment.

CONCLUSIONERK and JNK, but not p38, mediated benzo(a)pyrene-induced cell cycle changes by AP-1 transactivation in HELF.

Benzo(a)pyrene ; pharmacology ; Blotting, Western ; Cell Cycle ; drug effects ; Cells, Cultured ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Lung ; cytology ; embryology ; Mitogen-Activated Protein Kinase 1 ; metabolism ; physiology ; Mitogen-Activated Protein Kinase 8 ; metabolism ; physiology ; Phosphorylation ; Transcription Factor AP-1 ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo examine the incidence and clinical significance of NPM1 mutations in childhood acute myeloid leukemia (AML) patients.

METHODSNPM1 mutations of 70 newly diagnosed childhood AML were detected by high resolution melting (HRM) analysis on the LightCycler 480. The incidence and clinical significance were analyzed.

RESULTSNPM1 mutations were identified in 32 (45.7%) of the 70 AML children. There was no significant difference in clinical characteristics between patients with or without NPM1 mutation, but patients with NPM1 mutation had a higher platelet count (P = 0.013). There was also no significant difference in NPM1 mutation between normal and abnormal karyotype groups. In AML-ETO or PML-RARα positive groups, the incidence of NPM1 mutations was significant lower (P = 0.048). There was no significant difference in response rates after induction therapy (P = 0.217), but the complete remission (CR) rate was higher in the NPM1-mutated group (81.3%). There was a trend toward higher event-free survival (EFS) and overall survival (OS) rates in the NPM1 mutated patients than that in wild NPM1 patients (EFS = 53.8% vs 41.4%, OS = 52.7% vs 39.2%), but the difference was not statistically significant (P = 0.374 and 0.380).

CONCLUSIONNPM1 mutations were relatively common in our cohort of AML patients. There was no significant difference in clinical characteristics between patients with and without NPM1 mutation. The NPM1 mutation patients group seemed to have better therapy response, but the difference was not statistically significant.

Child ; Disease-Free Survival ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Mutation ; Nuclear Proteins ; genetics ; Prognosis

OBJECTIVETo investigate the role of mitogen activated protein kinases (MAPKs) signaling pathways in the regulation of benzo(a)pyrene (B(a)P)-induced c-Jun activation in human embryo lung fibroblasts (HELFs).

METHODSHELFs were cultured with 2.0 micromol/L B(a)P for various time (0, 3, 6, 12, 24 h) or with various concentration of B(a)P (0.0, 0.5, 1.0, 2.0 micromol/L) for 12 h. Western blot was performed to examine the effect of B(a)P on c-Jun activation. The dominant negative mutants of p38, c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) were applied to establish stable transfectant, and to detect the relationship of MAPK signal molecules and c-Jun activation in B (a) P-treated cells.

RESULTSB(a)P treatment resulted in a marked activation of c-Jun in time-dependent manner with a peak at 12 h (the densitometric ratios of phosphorylated c-Jun Ser63, Ser73 to actin were 20.1, 15.2 times for control respectively) and in dose-dependent manner. However, there was no evident change on total c-Jun expression in B(a)P-treated HELFs. Moreover, B(a)P-induced activation of c-Jun was inhibited by stable expression of dominant negative mutants of JNK or ERK, but not by dominant negative mutant of p38.

CONCLUSIONJNK and ERK signaling pathways, but not p38 pathway regulate B(a)P-induced c-Jun activation in HELFs.

Benzo(a)pyrene ; pharmacology ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Fibroblasts ; drug effects ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lung ; cytology ; metabolism ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-jun ; metabolism ; Signal Transduction ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the methylation of CpG island in the SHP-1 gene promoter and its significance in lymphoma. To evaluate the effects of As2O3 on demethylation of SHP-1 in human lymphoma cell line T2 and on proliferation of T2 cells.

METHODST2 cells were treated with AsO3. Methylation specific PCR was used to detected the status of SHP-1 methylation in newly diagnosed lymphoma tissues and the T2 cells. The mRNA and protein expression of SHP-1 were determined by FQ-PCR and Western blot. The expression of phospha-c-kit was examined by Westren blot. MTT and flow cytometry were used to determine the growth and apoptosis in T2 cells.

RESULTST2 cells contained completely methylated SHP-1. Furthermore, there was constitutive c-kit phosphorylation. The expression of SHP-1 was recoverd when the cells exposed to AsO3, and concomitant with increasing SHP-1, a parallel down-regulation of phosphorylated c-kit occurred, so that by day 3 phosphorylated c-kit was barely detectable. As2O3 inhibited the cell growth, and the effects were dose- and time-dependent. As2O3 also increased apoptosis rate of T2 cells in a dose- and time-dependent manner, too, and on the 1, 2, 3 d treatment with AsO3 (2.5 micromol/L), the apoptosis rates were 6.12%, 26.53%, 50.90%, respectively. The frequency of methylation in SHP-1 gene promoter in lymphoma tissues was 87.5% (28/32). In the control group, however, 12 specimens of benign lymph node proliferation showed no methylation in CpG island of SHP-1 gene promoter.

CONCLUSIONHypermethylation of SHP-1 gene promoter in lymphoma indicates the inactivation of SHP-1 gene and its possible role in the tumorigenesis of lymphoma. As2O3 can effectively cause demethylation and inhibit the growth of tumor by reactivating the SHP-1 gene transcription. SHP-1 methylation leading to epigenetic activation of c-kit may have a tentative role in the pathogenesis of lymphoma. Therefore, As2O3 is potentially useful in the treatment of lymphoma as a demethylating agent.

Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Arsenicals ; administration & dosage ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; CpG Islands ; DNA Methylation ; drug effects ; Dose-Response Relationship, Drug ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphoma ; metabolism ; pathology ; Lymphoma, Non-Hodgkin ; genetics ; metabolism ; pathology ; Oxides ; administration & dosage ; pharmacology ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; metabolism ; Proto-Oncogene Proteins c-kit ; metabolism ; RNA, Messenger ; metabolism ; Transcriptional Activation ; drug effects ; Up-Regulation