The Study Of The Level Of P27/Kip1 And Apoptosis In HL60 Cells After Treatment With TGFβ1 And (or) Arsenic Trioxide

The Study of the Level of p27/Kipl and Apoptosis inHL60 cells after Treatment withTGFβl and (or) Arsenic TrioxideAbstract InstructionThe cell division cycle includes G1 phase, S phase, G2 phase and M phase. The major molecules joining in the cell cycle include cyclin, CDKs (cy-clin dependent kinases) and CDKI(cyclin dependent kinase inhibitors). Cyclin and CDK conform cell cycle regulating complex( cyclin - CDK) , activating CDK and making cyclin - CDK alive, which catalyzes corresponding substrates phos-phorylation and accelerates cell cycle progression. CDKI combined with CDK or Cyclin — CDK compounds restrains the activation of CDK, and then leads to cell cycle blocking. In the interphases of cell cycle Gl phase is the most important, because the major regulating events that induce cell mitosis and differentiation of mammal happen in Gl phase, and much evidence indicates that apoptosis commonly happens in Gl phase. The cell cycle blocking of late Gl phase accelerates apoptosis.P27/Kipl is a kind of heat — stable protein found by Polyak when he studied contact restrain of cells and TGFβ - induced Gl phase blocking of cell growth. The molecular weight of P27/Kipl is 27KD. It subjects to KIP family of CDKI and is a negative factor of Gl phase. It is regarded as a kind of suppress cancer gene .This paper studied the effects of TGFβ1 and arsenic trioxide ( As₂ O₃) on cell apoptosis and cell cycle blocking, and the protein expressions of p27 , Bcl -2, CyclinE, indogenesis TGFpl in HL60 cells.Methods1. HL60 cells dealed with different concentration of As2O3 and TGFfJl. HL60 cells were cultured in RPMI 1640 medium supplemented with 10%FCS and humidified atmosphere containing 5% CO2 at 37 T!. The cell concentration was regulated to 5 x 105/ml during log - phase growth. Then the cells were dealed with As2O3(l, 5, lOjimol/L) and (or) TGF(3l(5ng/ml).2. Trypan -blue exclusion to examine cell growth restrain rateThe cells of treated and control groups were harvest after 16, 24, 32, 40 hours of incubation, stained, counted for the percentage of dead cells ( trypan blue stained cells ) .3. Wright — Giemsa staining to observe morphologic change of apoptosis cellsThe HL60 cells treated with 5jxmol/L of As2O3 after 48 hours were harvest and made into cell smears by cytospin. The apoptosis bodies were observed in the microscope after the cells were stained and air - dry.4. Flow cytometry to examine the interphase of Gl phase and apoptosis The cells of all groups were collected to centrifuge tubes, washed withphosphate - buffered saline ( PBS) twice, resuspended in 1 mL of PBS at a concentration of 2 x 10 /mL and fixed by 2mL of ethanol overnight at A°C. Then the cells were washed and incubated with 200|xl of RnaseA( lmg/ml) at 37Tl for 30 minutes. Subsequently, the cells were added into 800jjd of propidium iodide(0. lmg/ml) for 30 minutes in the dark at 4T1. The cell cycle distribution and DNA content were determined using Flow cytometer.5. Immunohistochemistry SP method to examine the expressions of p27, Bel - 2, CyclinE, and TGFfU proteinsThe cells of all groups were harvested, washed, and made into cell smears by cytospin. The cells were fixed with pure acetone for 10 minutes at 4T! , washed, blocked by 3% H2O2 for 10 minutes at room temperature, followed by incubation with goat blood serum for 20 minutes at 37°C ,then the goat blood serum was threw off. Subsequently, the cells were incubated with anti - humanp27, Bel -2, CyclinE, TGFfJl proteins, overnight at4Ti in wet box. The cells were washed with PBS prior to 30 - min incubation with second antibody at 371! for 30min. The cells were washed with PBS prior to 1 - h incubation with strept avidin - peroxidase at 37^1 and subsequent washed. The cells were incubated with diamino - benzidine (DAB) at room temperature for 7 minutes. The cells were further washed, and then dyed blue with hematoxylin. After being washed with distilled water, the cells were enclosed and observed under light microscopy. The background as well as the blank control is negative ( - ). The positive stain is the cells that have yellow conglomeration or granules in the cytoplasm and nucleus. 400 of cells were counted, then the positive cell percentage was calculated.6. Statistical analysisAll results were expressed as mean ± SD data obtained from three or more independent experiments. The statistical significance of differences between groups was determined using the analysis of varianceResult1. Effects of TGFpl and As2O3 on HL60 cell proliferationAs the time prolonged, the percentage of living cells in TGF|31 and (or) As2O3 treated groups fell compared with the control group. The effect of TGFpl treated group is most distinct at 24 hour.2. The apoptosis of HL60 cells treated with As2O3in microscopeThe apoptosis bodies formed by integrated cell membrane wraping the remains of apoptosis cell were observed in microscope.3. The cell cycle and apoptosis of HL60 cells treated with TGF@1 and As2O3The statistics analysis showed the effect of 5jxmol/L of As2O3 was the most strong among the different concentrations of As2O3, and the effect on apoptosis at 48 hour was more strong than 24 hour( P <0. 05 ) . The cell interphase of Gl phase in As2 O3 treated groups did not block. The TGFpl (5ng/ml) treated groups induced cell cycle blocking(P <0.05) in Gl phase conmpared with As203 treated and combined treated groups, and had no difference with control group.4. The expressions of p27, Bel -2, CyclinE and TGFpi proteins in HL60 cells treated with TGFfU and As2O3The statistics analysis showed p27 was up regulated (P <0. 05) , CyclinE and Bel -2 was down regulated(P < 0. 05) in TGFfU - treated group. Bel - 2 was down regulated, endogenesis TGFpl was up regulated(P <0.05) , and the level of p27 and CyclinE did not change in As2O3 - treated group (P >0. 05) . The effect of TGFpi combined with As2O3 on down -regulating Bel -2 protein was more strong than single factor treated group ( P <0. 05) .DiscussionTGFp is the most strong in the haematogenous control factors that inhibit the proliferation of blood cells. It induces apoptosis via cell cycle blocking. TGFpi is found earliest and has the most strong biological activity in TGFp ancestry. TGFjB induces cell cycle blocking via a series of mechanisms. In the epithelium , TGF|3 inhibits Cyclin - CDK of Gl phase, and p27 is the key effector during TGF(3 induces cell cycle blocking. At present, the studies about the relationship of p27 and solid tumors were reported much. Barata' s study showed that in the presence of IL - 7, T - ALL cells not only up — regulated bcl - 2 expression and escaped apoptosis but also progressed in the cell cycle, resulting in sequential induction of cyclin D2 and cyclin A. Down - regulation of p27/kipl was mandatory for IL - 7 - mediated cell cycle progression and temporally coincided with activation of cyclin - dependent kinase ( cdk) 4 and cdk2 and hyper-phosphorylation of Rb. Strikingly, forced expression of p27/kipl in T - ALL cells not only prevented cell cycle progression but also reversed IL - 7 - mediated up - regulation of bcl - 2 and promotion of viability. But Eymin' s study showed up - regulation of p27 by drugs prevented cell from apoptosis. Muthu induced granulocytic leukemia cell line Ml apoptosis with TGFpi in 1994. Lotem also reported TGFpi induced leukemia cell apoptosis. In this study, 5ng/ml of TGFpi induced cell cycle blocking in Gl phase and cell apoptosis, contempora-ry p27 was up regulated and Bel -2 was down regulated.In 70 years, our homeland scholar first applied arsenics to treat acute pro-myelocytic leukemia (APL). Arsenics induce cell apoptosis via up regulating the expression of Bel - 2 gene and digesting the fusing protein of PML - RARa. As2 O3 induces cell apoptosis of NB4 cell line and primary APL cells quickly and effectively. Pilots study showed the effective concentration(lfjunol/L) that induced NB4 cells apoptosis did not affect the expressions of c - MycNp53^BAX and bcl - x genes, but it quickly down regulated Bel - 2 expression in protein and mRNA levels. The reduction of Bcl - 2 protein reduced the ratio of Bcl - 2/ BAX, then promoted apoptosis. This maybe one of the effects of As2O3 on cells. This study showed As2O3(l 10u,mol/L) induced HL60 cells apoptosis, contemporary Bcl—2 protein was down regulated, endogenesis TGF(31 protein was up regulated, and the level of p27 protein did not change. This indicated p27 was independent of As2O3 - induced apoptosis in HL60 cells. The HL60 cells do not contain PML - RARa fusing protein, that indicates digesting PML - RARa fusing protein and down - regulation of Bcl - 2 expression in protein and mRNA levels in the program of As2 O3 - induced apoptosis are two independent mechanisms.The effect of TGFfU combined with As2O3 on HL60 cells apoptosis as well as the degree of down - regulation of Bcl - 2 protein was more strong than single factor - treated groups. The Bcl - 2 protein was down regulated in TGFfJl and As2O3 —treated groups, which indicated the passageways intercross with each other. However endogenesis TGFfSl was up regulated in As2O3 -treated group, but the level of CyclinE protein and the cell cycle did not change, which maybe related with disturbance of CyclinE protein in tumor cells. In As2 O3 - treated groups, the level of p27 protein did not change, but in TGFpi -treated groups p27 protein was up regulated and cell cycle was blocked, that further proved p27 was the key effector during TGF(3 induced cell cycle arrest.Conclusionl.TGFpi induces cell cycle arrest and apoptosis in HL60 cells. In thisprogress p27 protein is up regulated. p27 protein is the key effector of TGF(3 -induced cell cycle arrest.2. Bel -2 is down regulated and the level of p27 protein does not change during As2O3 induces apoptosis in HL60 cells, which indicates p27 protein is independent of As2O3 -induced apoptosis.3. The effect of TGFfil combined with As2O3 on apoptosis as well as the down - regulation of Bel - 2 protein in HL60 cells is more strong than single factor — treated groups, which indicates the passageways intercross with each other.4. Digesting PML - RARa fusing protein and down - regulation of Bel -2 expression in protein and mRNA levels in the program of As2O3 - induced apoptosis are two independent mechanisms.