| Background: Phosphatase and tensin hemology deleted on chromosome ten gene (PTEN),is a novel tumor suppressor gene in phosphatase family. PTEN protein has protein phosphatase and lipid phosphatase dual activity. PTEN is a lipid phosphatase for the second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3). By converting PIP3 into phosphatidylinositol 4,5-bisphosphate (PIP2), PTEN negatively regulates the PI3 kinase-Akt signaling pathway. Because of this function, PTEN can inhibit proliferation, induce apoptosis and regulate cell cycle progression. The protein phosphatase activity of PTEN can regulate cellular processes such as migration and focal adhesion, by targetting mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK) pathway and inhibiting phosphorylation of several proteins. Briefly PTEN can regulate the growth, differentiation, adhesion and cell cycle of normal cells; supress the proliferation, invasion and metastasis of tumor cells; induce apoptosis of tumor cells.Previous studies found that genetic mutation/deletion or lower expression of PTEN gene is so common in many types of human solid cancers, indicating that PTEN is one of the most frequently candidate of tumor suppressors. The above mentioned abnormality of PTEN may decrease its tumor suppression function that is closely corelated with the poor prognosis of human malignant diseases.In the past decade, the function of PTEN gene in the genesis of hematopoietic malignancies such as leukemia has been generally focused. Studies showed that PTEN gene frequently deleted or methylated, expressed at low level but seldom mutated in leukemia. Mice with PTEN-mutant or PTEN- deletion had an increased proliferation of myeloid and T-lymphoid lineages and developed myeloproliferative disorder (MPD), and then transformed to acute myeloid/lymphoid leukaemia in the late stage of MPD. If the hemetopoietic stem cells from these mice were transplanted to SCID mice, the recipient mice also developed leukemia and with extramedullary infiltrations of leukemia cells. Meanwhile, PTEN gene played an important role in distinguishing leukemia stem cell and haemopoietic stem cell. Resent study indicated that the abnormality of PTEN gene probably involved in leukemia pathogenesis, multidrug resistance, angiogenesis and extramedullary infiltrations.In many solid tumors, the ability of PTEN gene in inhibiting tumor cells proliferation and promoting apoptosis was confirmed already by many studies. However, the effect and its mechanism of PTEN gene in the genesis of hematopoietic malignancies such as leukemia were still not clear.K562 cells, a leukemia cell line established from a patient with chronic myelocytic leukemia (CML) in blast crisis of erythroleukemia, have high activity of PI3K/Akt pathway and low-expression levels of PTEN gene and protein. In the present study, we transfected fresh human leukemia cells or K562 cell line cells with wild-type PTEN gene mediated by adenovirus vector. The effects and the machinesim of wild-type PTEN gene on leukemia cells proliferation, apoptosis, invasion, cell cycle progression, multidrug resistance, and angiogenesis were studied. The result of this study may provid an important theoretical basis for leukemia therapy with PTEN gene. This paper consists of the following three parts:Part one: Effects of wild-type PTEN gene on proliferation, apoptosis and cell cycle progression of K562 cells and primary leukemia cellObjective To investigate in vitro the effects of wild-type tumor-suppressing gene PTEN on proliferation, apoptosis, cell cycle of human primary leukemia cells and human chronic myeloid leukemia (CML) cell line K562 cells and to explore its possible machinesims.Method The recombinant adenovirus vector-PTEN (Ad-PTEN) gene containing green fluorescent protein (Ad-PTEN-GFP) or the empty adenovirus vector containing green fluorescent protein (Ad-GFP) was transfected into K562 cells or primary leukemia cells from CML patients in blast crisis. The growth inhibition rate of leukemia cells was observed by MTT assay; the transfection efficiency, the apoptosis rate and cell cycle distribution were assessed by flow cytometry (FCM). We also obsereved apoptosis by morphological characteristics under optical microscope; DNA ladder, Hoechst 33342 fluorescent staining and cell colony fomation methods were also used to detect proliferation and apoptosis of K562 cells. The mRNA expression levels of PTEN, and the members of apoptosis family Bcl-2, Bcl-xL, Bax, Caspase-3, Caspase-7, Caspase-9, Survivin, Xiap, Smac, and cell cycle related molecular CyclinD1, CyclinD2, CDK4, and P27kip1 were measured by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR); The expression levels of PTEN, Bcl-2, Akt and p-Akt protein were detected by western blotting; Caspase-3/7, Caspase-9 protein activity were detected by caspase activity measuring kits.Results1 The transfection efficiency was about 81.2±5.4% in K562 cell line and 7.32±6.41% in five sample of primary leukemia cells after the cells were transfected with Ad-PTEN-GFP or Ad-GFP for 3 days, detected by flow cytometry at the condition of multiple of infection (MOI)=200.2 The expression of PTEN mRNA and protein increased to the highest level at the third day of transfection, with MOI=200. In Ad-PTEN-GFP group, PTEN mRNA expression level was (23.58±4.52) and protein level was (0.912±0.102) that was higher than that in Ad-GPF group (PTEN mRNA was 0.650±0.516 and protein was 0.117±0.028) and that in untransfected group (PTEN mRNA was 0.575±0.226 and protein was 0.086±0.021), P<0.01.3 Compared with empty vector adenovirus (Ad-GFP) group, the proliferation inhibiting rate of five primary leukemia cells transfected with Ad-PTEN-GFP was 33.8±10.4% at the fifth day. The maximum proliferation inhibiting rate was 38.67% in K562 cells transfected with Ad-PTEN-GFP for 5 days, which was higher than the 5.34% in Ad-GPF groups. The apoptosis rate was higher in Ad-PTEN-GFP group (22.4±2.11%) than that in Ad-GFP group (2.8±0.67%) and untransfected group (0.2±0.02%), P<0.05. 4 Cell morphology examination result showed that in the untransfected group and in Ad-GFP group, K562 cells were plump, the cytoplasm was abundant, the chromatin was delicate and loose, but in Ad-PTEN-GFP group the cells shrank and displayed the morphological sign of apoptosis: chromatin condensation, marginalization, nuclear pyknosis and fragmentation, the appearance of apoptotic bodies. Hoechst33342 fluorescent staining showed that the nucleus of the non-apoptosis cells were round with light blue clour, but the nucleus of apoptosis cell were sapphirine clour because of the nuclear pyknosis. The apoptosis rate was 35.6±2.2% in Ad-PTEN-GFP and 3.1%±1.13% in untransfected group, 6.2%±0.87% in Ad-GFP group at the 5th day of transfection。5 After transfection with PTEN gene, 180bp to 200bp DNA bands appeared on agarose gel electrophoresis, among DNA extracted from the transfected K562 cells. The cells in Ad-GFP group and untransfected group did not show the phenomenon of DNA degradation at the 5th day of transfection with MOI=200.6 After transfection with Ad-PTEN-GFP at MOI=200 for seven days, cell cycle distribution showed the ratio of G0/G1 phase cells increased from 54.9±3.61% to 78.5±5.17% (P<0.05), the ratio of G2/M phase decreased form 30.2±2.42% to 13.6±2.01% (P<0.05), and the cell cycle was arresd at G0/G1 phase.7 After transfection with Ad-PTEN-GFP at MOI=200 for three days, western blot results showed the Akt protein expression level had no change but p-Akt expression level (0.021±0.011) decreased, compared with that in Ad-GFP group (0.587±0.078) and that in untransfected group (0.547±0.061), the difference was statisticly significant, P<0.01.8 After transfection with Ad-PTEN-GFP at MOI=200 for 3 days, the activitay of caspase-9 (0.786±0.032), caspase-3/7 (1.362±0.039) was up-regulated compared with the activity of caspase-9 (0.591±0.024) and Caspase-3/7 (1.103±0.024) in Ad-GFP group, and the difference was significant (P<0.05). 9 After K562 cells had been transfected Ad-PTEN-GFP at MOI=200 for the third days, the expression level of survivin mRNA (0.0700±0.0059), xiap mRNA (0.00889±0.0006), smac mRNA (0.0600±0.0039) was lower than the expression level of survivin (0.437±0.079), xiap (0.0661±0.0072), smac (0.158±0.0078) in Ad-GPF group and the expression level of Survivin (0.453±0.081), xiap (0.070±0.0079), smac (0.177±0.0085) in untransfected group. Compared with the expresson level of the apoptosis related genes in Ad-GFP group, the expression levels of survivin, xiap, smac mRNA in the K562 cells transfected with Ad-PTEN-GFP decreased 6.14 fold, 7.44 fold and 2.95 fold, respectively.10 After K562 cells transfected with Ad-PTEN-GFP at MOI=200 for 3 days, the Bcl-2 mRNA expression levels in untransfected group, Ad-GFP group and Ad-PTEN-GFP group were 0.732±0.078, 0.723±0.148, 0.195±0.043 respectively; the Bcl-xL mRNA expression levels were 0.0181±0.0009, 0.0176±0.0011, 0.0047±0.0008; the Bax mRNA expression levels were 0.376±0.031, 0.395±0.036, 0.912±0.117, respectively. This result showed that compared with that in the untransfected group and Ad-GFP group, the expression levels of Bcl-2, Bcl-xL mRNA and Bcl-2 protein were down-regulated, but Bax mRNA was up-regulated in Ad-PTEN-GFP group. The ratio of Bcl-2 to Bax mRNA was 1.83 in Ad-GFP group to 0.214 inAd-PTEN-GFP group, and decreased 8.55 fold.11 After K562 cells transfected with Ad-PTEN-GFP at MOI=200 for 3 days, the expression levels of cell cycle related moleculars in untransfected group, Ad-GFP group and Ad-PTEN-GFP group changed as following: CyclinD1 mRNA (0.711±0.174; 0.698±0.162; 0.243±0.102), CyclinD2 mRNA (0.123±0.0196; 0.106±0.0178; 0.0441±0.0144), CDK4 mRNA (0.859±0.165; 0.876±0.142; 0.272±0.092), P27kip1 mRNA (0.0721±0.0102; 0.0683±0.00883; 0.470±0.172). Compared with the levels in untransfected group and Ad-GFP group, in Ad-PTEN-GFP group the expression levels of CyclinD1 mRNA, CyclinD2 and CDK4 mRNA expression levels decreased, but P27kip1mRNA increased and the difference was statistically significant (P<0.05). ConclusionsThe over-expression of wild-type PTEN gene inhibited proliferation and induced apoptosis of the primary leukemia cells and K562 cell line cells, probably via inhibition PI3K/Akt pathway and regulating the expression levels of many apoptosis related genes, such as Bcl-2 family, Caspase family and IAP family. Cell cycle distribution examination results showed the cell cycle arresd at G0/G1 phase through regulating the cell cycle related gene expression.Part two: Mechanism of PTEN gene on multidrug resistance reversal in K562/ADM cellsObjective To explorer in vitro the effects of wild-type tumor-suppressing gene PTEN on proliferation, apoptosis of Adriamycin resistant human chronic myeloid leukemia (CML) cell line K562/ADM cells and the possible molecular machinesim of multidrug resistance reversal by investigating the synergistic effect of PTEN with different chemotherapeutic drugs.Method The recombinant Ad-PTEN gene containing green fluorescent protein gene (Ad-PTEN-GFP) or the empty vector (Ad-GFP) was transfected into K562/ADM cells, then, the cells were treated with Adriamycin (ADM) or cytarabine (Ara-c) or arsenic trioxide (As2O3) to investigate the synergistic effect of chemotherapeutics with PTEN gene. The growth inhibition rate of K562/ADM cells was measured by MTT assay after the cells were transfected with PTEN gene and combined with or without chemotherapeutic drugs; the drug resistance reversal index was measured by the IC50 of different chemotherapeutic drugs combined with or without PTEN gene transfection; the transfection efficiency and the apoptosis rate were assessed by flow cytometry (FCM). The mRNA expression levels of PTEN, NF-κB, I-Κb, P53, MDR1, MRP1 and apoptosis related gene Bcl-2, Bcl-xL and Bax were detected by FQ-PCR. The protein expression levels of PTEN, Akt, p-Akt and P65 were detected by western blot.Results1 The transfection efficiency was about 82.2±5.8% in K562/ADM cell line cells after the cells transfected with Ad-GFP or Ad-PTEN-GFP on the third day detected by flow cytometry at the condition of multiple of infection (MOI)=200.2 With MOI=200, at the third day of transfection, the expression level of PTEN mRNA and protein increased to the highest level. In Ad-PTEN-GFP group, PTEN mRNA expression level was (25.43±3.77) and protein level was (1.240±0.219), which were higher than that in Ad-GPF group (PTEN mRNA was 0.473±0.117 and protein was 0.105±0.014) and untransfected group (PTEN mRNA was 0.477±0.071 and protein was 0.073±0.011), P<0.01. PTEN mRNA and protein expression levels in K562/ADM cells had no statistical significance difference when compared with that of K562 cells, P>0.05.3 Compared with empty vector adenovirus (Ad-GFP) group, the proliferation inhibiting rate was 32.26% in Ad-PTEN-GFP transfected K562/ADM cells at the fifth day, that was higher than the inhibition rate in Ad-GPF group (3.31%), P<0.01; and was lower than the inhibition rate in parental K562 cells, but the difference had no statistical significance, P>0.05.4 The apoptosis rate was detected by FCM. After transfection with Ad-PTEN-GFP or Ad-GFP at MOI=200, the apoptosis rate in Ad-PTEN-GFP group combined with 10mg/L ADM or 5μmol Ara-c or 1μmol/L As2O3 were 17.8±2.3, 28.6±2.9, 61.7±3.2 respectively which were higher than that in Ad-GFP combined with chemotherapeutic drugs (2.3±0.7, 15.2±1.8, 20.4±2.1 respectively), P<0.01.5 After transfection with Ad-PTEN-GFP and combined with the treatment of different chemotherapeutic drugs such as ADM, Ara-c, As2O3 for 72h, the drug resistance reversal index was 3.8 in ADM, 2.64 in Ara-c, 2.65 in As2O3 respectively.6 After transfection with Ad-PTEN-GFP for 3 days, western blot examination results showed the Akt expression levels had no change but Ser473 p-Akt expression levels (0.022±0.007) decreased when compared with that in Ad-GFP group (0.646±0.066) and untransfected group (0.633±0.065), P<0.01.7 After K562/ADM cells transfected with Ad-PTEN-GFP or Ad-GFP at MOI=200 for 3 days, the FQ-PCR results showed that in untransfected group, Ad-GFP group, and Ad-PTEN-GFP group, the mRNA expression level of NF-κB (P65) was 16.08±2.85, 15.20±2.08, 1.17±0.06, respectively, P<0.05, and P65 protein expression level was down-regulated after transfection with PTEN gene; The I-κB mRNA expression levels in untransfected group, Ad-GFP group and Ad-PTEN-GFP group were 0.21±0.08, 0.18±0.07, 0.23±0.11, the difference had no statistical significance (P>0.05). The P53 mRNA expression levels in untransfected group (2.95±0.32), Ad-GFP group (3.07±0.75) were lower than that in Ad-PTEN-GFP group(5.93±1.51),P<0.05.8 After K562/ADM cells transfected Ad-PTEN-GFP at MOI=200 for 3 days, in untransfected group, Ad-GFP group and Ad-PTEN-GFP group, the MDR1 mRNA expression levels were 0.25±0.02, 0.24±0.03, 0.13±0.01 (P<0.05) respectively; The MRP mRNA expression levels were 0.44±0.07, 0.42±0.07, 0.47±0.09 (P>0.05). The results showed that after transfection with PTEN gene, the MDR1 mRNA was down-regulated and MRP mRNA had no significant change.9 After K562/ADM cells transfected Ad-PTEN-GFP at MOI=200 for 3 days, in untransfected group, Ad-GFP group and Ad-PTEN-GFP group, the Bcl-2 mRNA expression levels were 0.040±0.013, 0.036±0.010, 0.011±0.004, respectively (P<0.05). Bcl-xL mRNA expression levels were 0.0071±0.0003, 0.0076±0.0003, 0.0037±0.0001, respectively (P<0.05). Bax mRNA expression levels were 0.129±0.028, 0.141±0.018, 0.432±0.071 (P<0.05). The results showed the Bcl-2 and Bcl-xL mRNA expression levels were down-regulated and Bax mRNA up-regulated after transfection PTEN gene,ConclusionsWild-type PTEN gene inhibited proliferation and induced apoptosis and enhanced the drug sensitivity or reversed drug resistance via inhibiting PI3K/Akt pathway and its downstream partener of the cell signaling transduction pathway in K562/ADM cells, such as down-regulated NF-κB, MDR1, Bcl-2 and up-regulated p53 and Bax.Part three:Regulatory mechanism of PTEN gene on VEGF in leukemia cells and its clinical significanceObjective To investigate the clinical significance of wild-type PTEN gene with VEGF and VEGFR1 (FLT1) mRNA expression in myeloid leukemia; and explore the anti-angiogenesis effects of wild-type PTEN gene via regulating VEGF and FLT1 expresson.Method (1) The expression of PTEN, VEGF and FLT1 mRNA were detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR) in 50 de novo acute myeloid leukemia (AML) patients, 10 AML patients in complete remission (CR), 10 chronic myelogenous leukemia (CML) patients in chronic phase (CML-CP), 10 CML patients in blast crises (CML-BC) and 10 normal controls.(2)The recombinated adenovirus containing green fluorescent protein (GFP) and PTEN gene(Ad-PTEN-GFP)or empty vector (Ad-GFP)was transfected into K562 cells. Then the VEGF, FLT1 and PTEN mRNA expression levels and the correlation between the expression of these genes were measured. (3) Human umbilical vein endothelial cell (HUVEC) line ECV304 cells were also transfected with or without PTEN gene; the proliferation inhibition rate and apoptosis rate were measured by MTT and flow cytometry (FCM). (4)Chick chorioallantoic membrane (CAM) test was used to testify the effect of PTEN gene on angiogenesis.Results1 The expression levels of PTEN, VEGF, FLT1 mRNA in myeloid leukemia and its relationship.1.1 PTEN mRNA expression levels in the newly diagnosed acute myeloid leukemia (AML) patients (expression level were 0.051±0.059) were higher than that in AML-CR patients (1.015±0.411) and NC group (1.059±0.595), (P<0.05). In CML-BC patients, the PTEN mRNA expression levels (0.022±0.021) were lower than that in CML-CP patients (1.134±1.124) and in NC, (P<0.01).1.2 VEGF mRNA expression levels in the newly diagnosed AML patients (expression levels were 0.442±0.192) were higher than those in AML-CR patients (0.075±0.042) and NC group(0.059±0.039), (P<0.01). In CML-BC patients (0.512±0.322) the VEGF mRNA expression levels were higher than CML-CP patients (0.219±0.131) and NC, (P<0.01).1.3 VEGFR1 (FLT1) mRNA expression levels in the newly diagnosed AML patients (expression levels were 1.569±0.694) were higher than those in AML-CR patients (0.375±0.241) and NC group(0.402±0.238), (P<0.01). In CML-BC patients (1.579±0.623) the VEGF mRNA expression levels were higher than those in CML-CP patients (0.598±0.311) and NC, (P<0.01).1.4 Pearson correlation analysis showed PTEN mRNA expression levels had negative correlation with VEGF and FLT1 mRNA expression levels, and the correlation coefficient were r=-0.631, (P<0.05) and r=-0.522, (P<0.05) respectively.2 The effect of wild-type PTEN gene transfection on invasion activity of K562 cells and the regulation of p-Akt, VEGF and VEGFR1 (FLT1)2.1 The Ad-GFP or Ad-PTEN-GFP transfection efficiency was about 81.2±5.4% in K562 cell line on the third day detected by flow cytometry with multiple of infection (MOI) of 200.2.2When K562 cells were transfected with Ad-GFP or Ad-PTEN-GFP for 72 hours at different MOI (MOI=50, 100, 200 and 400), PTEN mRNA expression level increased while the VEGF and FLT1 mRNA expression levels decreased. The expression levels of PTEN mRNA and VEGF mRNA or FLT1 mRNA showed negative correlation, and the correlation coefficient were r=-0.903, (P<0.05) and r=-0.900, (P<0.05) respectively. VEGF proteins were also decreased. After transfection with Ad-GFP or Ad-PTEN-GFP for 3 days, compared with Ad-GFP group, in Ad-PTEN-GFP group, PTEN mRNA expression levels increased 41.01 fold but VEGF and FLT1 mRNA expression levels decreased 4.80 and 4.68 fold. The expression level of VEGF protein decreased 2.21 fold detected by ELISA method. 2.3 After transfection with Ad-PTEN-GFP at MOI=200 for 3 days, Akt expression levels in K562 cells had no change, but p-Akt expression level (0.021±0.011) decreased compared with that in Ad-GFP group (0.587±0.078) and in untransfected group (0.547±0.061), P<0.01.2.4 After transfection with Ad- GFP or Ad-PTEN-GFP at MOI=200 for 48 hours, transwell invasion test results showed that the number of fluorescent staining cells, that were adhered to the bottom of the upper chamber of transwell, was 50±11 in Ad-GFP group, which was higher than the number of cells in Ad-PTEN-GPF group (24±6). These results indicated that the invasion or migration ability of K562 cells was suppressed when the cells were transfected with Ad-PTEN-GFP.3 The anti-proliferation and apoptosis induction effect of PTEN gene on human umbilical vein endothelial cell (HUVEC) line ECV304 cells.3.1 After ECV304 cells were transfected with Ad-PTEN-GFP at MOI=100, the maximum proliferation inhibition rate was 50.38±4.15% at the fifth day of transfection, which was higher than that in Ad-GFP group (4.28±0.73), P<0.01.3.2 The apoptosis rate in Ad-PTEN-GFP group was 18.56±2.11, which was higher than that in Ad-GFP group (1.34±0.32), P<0.01, after ECV304 cells transfected at MOI=100 for 5 days.4 PTEN gene significantly suppressed blood vessels'formation in chick embryo choriallantoic membrane (CAM). In control group and Ad-GFP group, blood vessels surrounding gelatin sponge grew well, it was tree-like and well-branched. While avascular area surrounding gelatin sponge could be observed in Ad-PTEN-GFP group, its blood vessels were less than control group. In Ad-GFP and Ad-PTEN-GFP group, the blood vessel index was 92.2±4.37% and 47.6±3.37% respectively P<0.05.Conclusions1 PTEN gene expressed at low level in the newly diagnosed AML patients and its expression level was negatively correlated with the expression level of VEGF and FLT1 gene. 2 PTEN gene inhibited the invasion ability of K562 cells and had anti-angiogenesis effect by inhibiting PI3K/Akt signaling transduction pathway and down-regulated the expression of VEGF and FLT1 genes. PTEN gene may be a candidate of effective anti-leukemia gene.
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