Transfection Of Wild-type P53 Gene Research Articles

Low frequency ultrasound combined with microbubbles to promote the transfection of wild-type P53 gene in human prostate cancer cells mediated by liposome

Objective To study low frequency ultrasound combined with microbubbles to promote the transfection of P53 gene in human prostate cancer cells mediated by liposome. Methods Ultrasound equipment was used with a frequency of 21 kHz and intensity was 46 mW/cm2 and the working time was controlled at 20% (i.e., 2 sontime and 8 sofftime) lasting 5 minutes. The human prostate cancer cell line PC-3 suspension was prepared, the cell concentration was adjusted to 1×105 cell/ ml, and cells were divided into 8 groups: control group, single microbubbles group, single ultrasound group, ultrasound combined with microbubbles group, single liposome group, liposome combined with microbubbles group, liposome combined with ultrasound group, liposome combined with ultrasound and microbubbles group. Each microbubbles group was added SonoVue 200 μl and the wild type P53 plasmid, plasmid∶liposome is 1∶2. At 24 hours after irradiation, Western-blot and RT-PCR were used to detect the gene transfection efficiency, CCK-8 was used to detect cell proliferation, then cell survival rate was calculated, cell apoptosis was detected by flow cytometry. Results After transfection, compared with single liposome group and control group, liposomes combined with ultrasound and microbubbles group can significantly improve expression of the human wild type p53 gene and protein (P<0.001). After transfection, the apoptosis rate of human prostate cancer PC-3 cells in liposomes combined with ultrasound and microbubbles group was significantly higher than that of the liposome group and control group (P<0.001). And after transfection, cell survival rate of liposome combined with ultrasound and microbubble group decreased significantly than those of single liposome group and control group (P<0.001). Conclusions Low frequency and low energy ultrasound combined with microbubbles can promote the transfection of human wild-type P53 gene mediated by liposome. Key words: Sonication; Microbubbles; Liposomes; Prostatic neoplasms

Apoptosis induced by the transfection of wild-type p53 gene into DU145 cells through 20 kHz ultrasound combined with microbubbles and iiposome

Objective To investigate whether the transfection of wild-type p53 gene into DU145 cells through 20 kHz ultrasound combined with microbubbles and liposome can induce cell apoptosis, and to explore the probable mechanism. Methods Ultrasound with a frequency of 20 kHz and power of 80 mW in continuous wave mode was used to transfect wild-type p53 gene into DU145 cells, and microbubble and liposome were added. DU145 cells were divided into four groups： control group（A）, ultrasound combined with microbubbles group（B）, liposome group（C）, and ultrasound combined with microbubbles and liposome group（D）. Twenty-four hours after treatment, the early apoptosis was detected by flow cytometry, and western blotting was used to evaluate the expression of the apoptosis-related proteins, p53 and MDM2, after treatment for thirty-six hours. Results The early apoptosis rate of group D was much higher than that of group A,B and C（ P ~ 0. 01 ）. Western blotting showed that the treatment of group D increase the expression of p53 protein and decreased the expression of MDM2 protein,which has a significance difference when compared with group A,B and C（ P d0. 01 ）. Conclusions The treatment of transfection wild-type p53 gene into DU145 cells through 20 kHz ultrasound combined with microbubbles and liposome induce cell apoptosis may through up-regulation of p53 and down-regulation MDM2. Key words: Sonication; Microbubbles; Liposomes; Apoptosis

Effect of p53 gene transfection on proliferation, apoptosis, and radiosensitivity of cervical cancer cells.

e22024 Background: To investigate the effects of p53 gene transfection on proliferation, apoptosis and radio-sensitivity of cervical cancer cells. Methods: Two cervical cell lines: Siha with wild-type p53 gene and C33a with mutant p53 gene, were transfected with human wild-type p53 gene. Un-transfected cells and transfected cells were exposed to 6 Gy of radiation. Cell proliferation, apoptosis, and protein expressions of p53, MDM2, p21, and bax in the un-treated, p53- transfected, and irradiated cells were analyzed. Results: The protein expression of p53, MDM2, p21, and bax in the untreated Siha cells is very low; High level p53 and MDM2 proteins and low level of p21 and bax proteins were found in the untreated C33a cells. When transfected with recombinant adenoviral human wild-type p53 gene (rAd-p53) the protein levels of p53, MDM2, p21, and bax in both Siha and C33a increased. Highest levels of these proteins were detected at 48 hours after transfection. The increased protein expressions were accompanied with inhibition of cell proliferation. At 72 hours after transfection, 12.3% and 5.5% of Siha and C33a cells became apoptotic, respectively. When exposed to 6 Gy of radiation after 48 hours of the transfection, 46.2% and 38.3% apoptotic Siha and C33a cells were detected, respectively; but only 5.6% and 6.3% of apoptotic cells of their un-transfected counterparts detected at 24 hours after the radiation treatment. Conclusions: Wild-type p53 gene transfection can inhibit cell proliferation, promote apoptosis, and sensitize to radiation of both Siha and C33a cells. The effects on Siha cells are stronger.

Feasibility and effect of ultrasound microbubble-mediated wild-type p53 gene transfection of HeLa cells

Gene therapy holds great promise for the treatment of diseases. The key problem of gene therapy is the choice of an effective vector. Ultrasound-mediated microbubble technique (UMMT) has already shown promising applications in numerous types of tumors apart from cervical carcinoma. In the present study, according to the results of an MTT assay, we initially chose an ultrasound intensity of 0.5 W/cm(2), an ultrasound exposure time of 30 sec and a microbubble concentration of 10% as the optimum experimental condition for wtp53 plasmid transfection into HeLa cells. To further investigate the transfection efficiency of ultrasound combined with microbubbles, RT-PCR analysis was used to examine the mRNA level of p53. The transfection efficiency in the plasmid plus microbubbles and ultrasound group was significantly higher than that of the other groups. Following transfection of the wtp53 gene, flow cytometric analysis showed that the cell cycle of HeLa cells was arrested in the G1 phase. The results of the present study suggest that UMMT, a new gene delivery system, increases the transfection efficiency of the wtp53 gene. Moreover, the growth of HeLa cells was arrested by introducing wtp53. This study may afford a new trend for the gene therapy of cervical carcinoma.

The experimental study of recombinant adenovirus-mediated human wild type p53 gene transfection into neuroblastoma cell line

Objective To observe the effect of transduetion of human wild type gene(wt-p53) mediated by recombinant adenovirus on cell growth, cell cycle and apoptosis of neuroblastoma (NB) cell line SH-SY5Y. Methods The NB cell line SH-SY5Y were transfected by recombinant adenovirus with green fluorescent protein(Ad-GFP). The suitable muhiplieity of infection (MOI) was determined after trans-fer efficiency was tested by flow cytometry (FCM). Both recombinant adenovirus(Ad) and recombinant adenovirus mediating wt-p53 gene (Ad-p53) were transfected into SH-SY5Y cells. SH-SY5Y cells without any transfection were used as blank group. The p53 protein expres-sion was validated by Western blot. Cell number was counted and growth curve was drawn. Both cell cycle and apoptesis were detected by FCM. Results The results indicated that the transfer efficiency of recombinant adenovirus to SH-SY5Y was satisfied, and 100 MOI was suitable. After transfection with Ad-p53, p53 protein was high expressed. Cell growth were inhibited, apoptosis and cell cycle arrest were in-duced in p53 gene transfeetion group. Conclusions Our results indicate that clinical application of the in vitro p53 gone modified tumor cell vaccine might be feasible and effective in clinical treatment of NB. Key words: Genes, P53 ; Neuroblastoma; Transfection ; Cell proliferation; Cell cycle; Apoptosis

Effect of recombinant adenovirus-p53 on growth and chemosensitivity of human lung adenocarcinoma cell lines

p53 gene is the most commonly mutated gene in lung cancer. p53 mutation results in insensitivity of cells when exposed to chemotherapy. It has been reported that adenovirus-mediated wild-type p53 gene transfection into lung cancer cells can enhance the cytotoxic effect of anti-cancer drugs. The aim of this study is to evaluate the effects of domestic recombinant adenovirus-p53 (Ad-p53, Gendicine) on growth and chemosensitivity of human lung adenocarcinoma cell lines. Human lung adenocarcinoma cell lines GLC-82 (including mutant p53) and A549 (including wild-type p53) were treated with Ad-p53, cisplatin (DDP) or Ad-p53+DDP respectively. p53 expression was detected by Western blot. The cell growth inhibition was assessed by MTT, and cell cycle and apoptosis were detected by flow cytometry. High-level p53 expression was detected in Ad-p53 infected GLC-82 and A549 cells by Western blot. There was a dose-dependent and time-dependent inhibition of cell proliferation by Ad-p53. After combined treatment with Ad-p53 (100MOI) and DDP (0.5mg/L) for 72h, the growth inhibition rate of A549 cells was 43.13%±0.72%, which was significantly higher than that in Ad-p53 group ( 23.44%±0.54%, P < 0.001) and DDP group (14.17%±1.39%, P < 0.001); and the growth inhibition rate of GLC-82 cells was 63.73%±0.92%, which was significantly higher than that in Ad-p53 group ( 41.51%±0.59%, P < 0.001) and DDP group (56.11%±1.12%, P < 0.001). Combined administration of Ad-p53 and DDP remarkably arrested A549 and GLC-82 cells in G0-G1, and cells in S phase significantly decreased. Meanwhile the apoptotic rate of A549 cells was 28.99%±1.07% in Ad-p53+DDP group, which was significantly higher than that in Ad-p53 group (15.35%±1.31%, P < 0.001) and DDP group (1.74%±0.77%, P < 0.001). The apoptotic rate of GLC-82 cells was 62.98%±2.43% in Ad-p53+DDP group, which was significantly higher than that in Ad-p53 group (20.88%±0.71%, P < 0.001) and DDP group (6.91%±1.52%, P < 0.001). Ad-p53 (Gendicine) can inhibit the growth of human lung adenocarcinoma cell lines irrespective of the status of endogenous p53 gene. Its combination with DDP may significantly enhance the chemosensitivity of human lung adenocarcinoma cells to DDP.

Effect of wild-type p53 gene transfection on the growth and radiotherapeutic sensitivity of human glioma cells

To evaluate the effect of wild-type p53 gene on the growth and radiotherapeutic sensitivity of human glioma cells, plasmid PC53-SN3 carrying wild-type p53 gene was transfected into U251 cells. p53 gene expression in transfected cells was detected by RT-PCR, and the cell growth inhibition and apoptosis in the absence or presence of irradiation were assessed by MTT and flow cytometry. The transfection of p53 gene into U251 cells was confirmed by RT-PCR. MTT showed that p53 gene alone induced strong inhibitory effect on the growth of U251 cells (inhibition rate (IR), (79.60 +/- 5.69)%). The killing effect of irradiation alone on U251 cells was not strong (IR: (17.06 +/- 4.35)% (17.39 +/- 1.67)% (18.73 +/- 4.68)%) and increased with the irradiation doses (3, 6, 9 Gy). When combined treatment of wild-type p53 gene transfection and irradiation was used, the effect was significantly increased (IR:(80.60 +/- 5.35)%. (90.30 +/- 1.67)%, (91.30 +/- 2.01)%). The apoptosis rate of U251 cells induced by p53 gene transfection was 17.38%. The rate induced by irradiation increased (4.61%, 4.84%, 5.40%) with the irradiation doses (3, 6, 9 Gy). The apoptosis rate was also significantly increased (17.80%, 20.03%, 22.34%) after combined treatment of p53 and irradiation with different doses (3, 6, 9 Gy). It is concluded that wild-type p53 gene and irradiation could result in synergistic inhibitory effect on the growth of human glioma cells.

The effects of wild-type p53 gene transfection on the growth and chemotherapeutic sensitivity of human glioma cells.

To evaluate the effects of wild-type p53 gene on the growth and chemotherapeutic sensitivity of human glioma cells, plasmid PC53-SN3 carrying wild-type p53 gene was transfected into U251 cells. p53 gene expression in transfected cells was detected by RT-PCR, the cell growth inhibition and apoptosis in either the absence or the presence of cisplatin was assessed by MTT and flow cytometry. The transfection of p53 gene into U251 cells was confirmed by RT-PCR. MTT showed that p53 gene by itself induced strong inhibition effect on the growth of U251 cells [inhibition rate, IR (79.60 +/- 5.69)%]. The killing effects of cisplatin by itself on U251 cells was not strong [IR (19.40 +/- 6.69)%, (24.41 +/- 2.68)%, (51.84 +/- 13.38)%, (66.22 +/- 5.02)%] and increased with the increase of cisplatin concentration (1, 2, 4, 8 micrograms/ml). When combined treatment of wild-type p53 gene transfection and cisplatin was used, that was significantly increased [IR (91.64 +/- 1.00)%, (94.98 +/- 1.67)%, (95.32 +/- 2.01)%, (95.65 +/- 1.00)%]. The apoptosis rate of U251 cells induced by p53 gene transfection was 17.38%. That induced by cisplatin increased (5.71%, 5.93%, 6.27%, and 6.81%) with the increase of cisplatin concentration (1, 2, 4, 8 micrograms/ml). The apoptosis rate was also significantly increased (23.50%, 23.54%, 23.89%, and 28.88%) after combined treatment of p53 and cisplatin with different concentration (1, 2, 4, 8 micrograms/ml). It is concluded that wild-type p53 gene and cisplatin could result in synergistic inhibition effects on the growth of human glioma cells.

Wild-type p53 gene transfection in human cultured sarcomas: effect of CDDP.

We examined the susceptibility of five human bone and soft tissue sarcoma cell lines to transfection with recombinant p53 adenovirus vector (AxCA-p53). Transfection efficiency was more than 90% at 72 h with AxCA-lacZ at a multiplicity of infection (MOI) of 50 in all the cell lines, except for MG-63 (p53 gene mutated) cells. Western blot analysis showed overexpression of both P21/Waf1 and Bax protein in all the cell lines, implying sufficient and successful p53 gene transfection. AxCA-p53 transfection at MOI of 50 resulted in a significant decline of viable cells at 72 h, due to apoptosis, in NY (mutated) and Saos-2 (deletion), but not in the other three lines. The two apoptosis-induced cell lines showed a gradual increase in Bax expression up to 72 h and non-detectable expression of Bcl-XL from 48 h, suggesting the involvement of an apoptosis-inducing mechanism. Pre-treatment with cis-diamminedichloroplatinum (II) (CDDP) at 0.1 microg/ml significantly suppressed tumor cell viability in NY and HuO-3N1 (mutated), but not in the other three lines including HT-1080 carrying the wild-type p53 gene, implying the existence of different mechanisms for the tumor suppressive effect of p53 gene transfection and CDDP. These results indicate that wild-type p53 gene transfection with CDDP is a promising therapy for some, but not all, non-resectable bone-and soft tissue sarcomas, regardless of intrinsic p53 gene status.

Heterogeneous induction of apoptosis in colon cancer cells by wild-type p53 gene transfection.

To examine the effects of wild-type (wt)-p53 gene transfer on cancer cell growth and apoptosis induction, we transduced human wt-p53 cDNA into three colon cancer cell lines either with or without a mutation of the p53 gene using the HVJ-cationic liposome method. Wt-p53 gene transfer, thus, induced an apparent growth arrest in all cell lines, but its enhancement of the apoptotic rate varied (from about 4 to 70 times). The simultaneous doxorubicin treatment was able to enhance growth arrest and the apoptosis induction rate. These findings suggest that wt-p53 gene transfer using HVJ-cationic liposomes seems to be a potentially effective therapeutic strategy, however wt-p53 gene transfer still appears to be more effective in combination with other cytotoxic treatments.

Anti-Fas antibody-induced apoptosis in human colorectal carcinoma cell lines: role of the p53 gene.

The cell surface Fas antigen transducts an apoptotic signal by its crosslinking with Fas ligand or anti-Fas antibody in a variety of human cultured cells. In this study, we examined the expression of Fas antigen and its mediation of apoptosis in six human colorectal carcinoma cell lines. A flow cytometric analysis revealed that LoVo, DLD-1, WiDr and SW837 cell lines showed higher expression levels of Fas antigen, in contrast to lower expression in COLO201 and COLO320DM. Interferon-gamma enhanced the expression of Fas antigen in all of the cell lines examined. Both Fas ligand and Fas-associated phosphatase-1 (FAP-1) were expressed only in COLO320DM. Anti-Fas antibody induced apoptosis in LoVo carrying wild-type p53 gene, but not in the other five cell lines carrying mutated p53 gene. The transfection of wild-type p53 gene using an adenovirus vector upregulated P53 protein in WiDr and SW837 cells, both of which showed, however, no increase in apoptotic cells by anti-Fas antibody treatment. These results indicated that (1) Fas antigen was variably expressed, regardless of the p53 gene status and (2) the susceptibility to anti-Fas antibody-mediated apoptosis did not correlate to Fas, Fas ligand or FAP-1 expression levels. Therefore, we conclude that wild-type P53 expression might not necessarily be essential for Fas-mediated apoptosis in human colorectal carcinoma cell lines.

2009 Adenoviral mediated transfection of wild-type p53 gene increases the radiosensitivity of rat glioblastoma tumors treated with radiosurgery

2009 Adenoviral mediated transfection of wild-type p53 gene increases the radiosensitivity of rat glioblastoma tumors treated with radiosurgery