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Background and purpose:Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is one of the ASPP family. It binds to p53 to inhibit the transcriptional activity of p53-target genes and cell apoptosis, which is asso-ciated with tumor formation. Previously, we found a new subtype of iASPP, iASPP splice variant (iASPP-SV), which is a nuclear protein containing 407 amino acid residues and can bind to p53, inhibiting p53 transcriptional activity. However, the relationship of iASPP-SV and breast cancer is still obscure. Therefore, the purpose of this research was to study the role of iASPP-SV on breast cancer tumorigenesis and progression.Methods:5’-rapid ampliifcation of cDNA ends (RACE) was used to identify the 5’-end of iASPP-SV mRNA in MCF-7 cells. HEK 293 cells were transfected with pFLAG-iASPP-SV and pFLAG-iASPP (828). Then Western blot was used to identify whether endogenous iASPP-SV was expressed in HEK 293 cells and 8 types of human tumor cell lines. This study established the stable clones of NIH 3T3 expressing FLAG-iASPP-SV and FLAG-iASPP (828). Cell proliferation assay, colony formation and soft agar colony formation assay were used to identify whether iASPP-SV and iASPP (828) can promote cell proliferation and iASPP-SV is an oncogene. Real-time lfuorescent quantitative polymerase chain reactive (RTFQ-PCR) was used to de-tect the levels of iASPP-SV and iASPP (828) mRNA in primary breast cancers. Luciferase assays were used to identify the relationships between iASPP-SV, iASPP (828), p53 and NF-κB p65.Results:The study identiifed that iASPP-SV was encoded by previously reported NF-κB p65 subunit (RelA)-associated inhibitor (RAI), and endogenously expressed in many human cancer cell lines. Analysis of cell proliferation, colony formation assay and soft agar assay for colony formation identiifed that similarly to iASPP (828), iASPP-SV promoted tumor cell proliferation and acted as an onco-gene. RTFQ-PCR result showed that the median values of iASPP-SV and iASPP (828) in breast cancers with wild-type p53 were more signiifcantly over-expressed than those of mutant p53. Luciferase assays showed that iASPP-SV and iASPP (828) could suppress NF-κB p65 transcriptional activity. Thus iASPP family may participate in the regulation of p53 and NF-κB activity, which imply that iASPP perhaps shows pro- or anti-survival activities when it interacts with different proteins.Conclusion:These ifndings indicate that iASPP-SV may be a potential target for breast cancer thera-py.
ABSTRACT
Objective To investigate the expressions of apoptosis stimulating protein of P53 (ASPP)family in colorectal carcinoma tissue and to explore their relationship with the clinicopathological characteristics of colorectal carcinoma,and to clarify the effect of ASPP family in the development of colorectal carcinoma.Methods 45 cases of colorectal carcinoma tissue and 20 cases of healthy controls were selected. Among 45 cases of colorectal carcinoma tissue, there were 1 1 cases of well differentiated colorectal carcinoma, 2 1 cases of moderately differentiated colorectal carcinoma,and 13 cases of poorly differentiated colorectal carcinoma;7 cases of T1 stage colorectal carcinoma,8 cases of T2 stage colorectal carcinoma,25 cases of T3 stage colorectal carcinoma,and 5 cases of T4 stage colorectal carcinoma;19 cases of N1 stage with lymph node metastasis,26 cases of N0 stage without lymph node metatasis.The expressions of ASPP1,ASPP2,and iASPP in 45 cases of colorectal carcinoma tissue and 20 cases of normal colon tissue were detected by immunohitochemistry SP method,and the correlations between the expressions of ASPP family and the pathologic typing,infiltrative depth,and lymph node metastasis of colorectal carcinoma were analyzed. Results ①The immunohitochemical staining results showed that the ASPP family members expressed in colorectal carcinoma tissue and normal colon tissue, and there were no significant differences in ASPP1 and ASPP2 positive rates between colorectal carcinoma tissue and normal colon tissue (P>0.05);the positive expression rate of iASPP in colon cancer tissue was higher than that in normal colon tissue (P0.05);the expression of ASPP2 positive rate was decreased when the differentiation degree of tumor cells reduced,they had positive correlation (rs=0.454,P=0.002);the expression of iASPP in colon cancer tissue had no correlation with the differentiation degree of tumor cells (rs=-0.171,P>0.05).③ The expression of ASPP1 in colon caner tissue had no correlation with the infiltrative depth of tumor (rs=-0.268,P>0.05);the expression of ASPP2 positive rate was decreased when the tumor infiltrative depth increased,they had negative correlation (rs=-0.348,P0.05).④The expressions of ASPP1,ASPP2, and iASPP in colon caner tissue had no correlation with lymph node metastasis (rs=0.089,rs=0.044,rs=0.210, P>0.05).Conclusion The expression levels of iASPP in colon cancer and normal colon tissues are different,it may be useful for the diagnosis, differential diagnosis and evaluation in benign and malignant colorectal diseases. The expression of iASPP is negatively correlated with the pathologic typing and neoplasm staging of colorectal carcinoma,it indicates that iASPP can be used as a indicator in judging the prognosis of colorectal carcinoma.
ABSTRACT
Objective Construct the eukaryotic expression vector of inhibitory member of the ASPP family (iASPP) and trans-fect it into colon carcinoma cell lines SW480 and Lovo by liposome .Then observe the expression of iASPP and detect the cell apop-tosis by flow cytometry .Methods The amplified PCR product was digested and inserted into pMD19-T simple vector and sub-cloned into eukaryotic expression vector pcDNA3 .1(+ ) .The recombinant eukaryotic expression plasmid pcDNA3 .1(+ )-iASPP was transfected into colon carcinoma cell lines SW480 and Lovo by liposome ,the iASPP expression was analyzed by RT-PCR .The cell apoptosis was detected by FCM .Results The eukaryotic expression plasmid pcDNA3 .1(+ )-iASPP was constructed success-fully ,the gene squence of iASPP was consistent with that reported (gi 60457962) in GenBank .The mRNA expression levels of iASPP gene of SW480 and Lovo cell lines which transfect the positive plasmid were increased ,and the cell apoptosis rates were de-creased .Conclusion We successfully constructed the recombinant expression plasmid pcDNA 3 .1(+ )-iASPP ,and the plasmid were successfully expressed in colon carcinoma cell lines SW 480 and Lovo ,the cell apoptosis rates of those cell lines were decreased .These facts indicated that reducing the high expression of iASPP may be a new strategy to renew the abilities of P 53 tumor suppressor .
ABSTRACT
Objective To investigate the expression and regulation of ASPP family mRNA in NSCLC cell lines.and study the diagnostic and therapeutic significances of apoptosis stimulating of ps3 expression in NSCLC Patients.Methods Real-time fluorescence quantitative PCR wag established and used to measure the expression Ievels of ASPP mRNA in NSCLC cell lines A549(wild-type p53)and NCI-H157 (mutant-type p53)with p53 different genetic background,and ASPP mRNA was also detected in NSCLC tissue,peripheral blood of 37 NSCLC patients.Western Bloting was used to examine the levels of ASPP1 and ASPP2 proteins of NSCLC cell Iines.Chemosensitivity of the cell lines to CDDP was analyzed by MTT assay,and detect the change of ASPP1 and ASPP2.Compared with the ASPPs mRNA expression between NSCLC patients and healthy controls.and monitoring ASPPs mRNA expression in patients with NSCLC chemotherapy.Results The slope rotes of standard curves were-3.249,-3.358 respectively.and correlation coefficients were more than 0.98.Amplification efficiency of standard curves were 1.156.1.028 respectively.The IC_(50) values of NCI-H157 and A549 cells were 3.70.10.48 μg/ml respectively.Mean ASPP1 ASPP2 mRNA levels were a statistically significant 2.66 folds and 6.98 folds higher in NCI-H157 cell compared to A549 cell The levels of ASPP1 mRNA in tumor tissues and peripheral blood were (4.27±0.57)×10~3,(2.49±0.32)×10~3 in patients with NSCLC respectively,and the levels of ASPP2 mRNA in tumor tissues and peripheral blood were(2.34±0.75)×10~3,(7.00±1.17)×10~3 in Patients with NSCLC respectively.The levels of ASPP1 and ASPP2 mRNA in tissues and peripheral blood were(1.32±0.21)×10~4,(1.46±0.31)×10~4 and(1.38±0.19)×10~4,(1.28±0.18)×10~4 in control groups respectively.The ASPP1,ASPP2 mRNA levels in patients with NSCLC were significantly lower than those of control groups(t=2.58,3.94,3.62,3.76,P＜0.05).Before chemotherapy the levels of ASPP1 and ASPP2 mRNA in patients with NSCLc were(2.34±0.56)×10~3 and(6.64±0.72)×10~3,and after 2 periods of postoperative chemotherapy the levels were(3.66±0.64)×10~3 and(9.42±0.44)×10~3,the expression was higher than those of ehemotherapy before(t=3.02,4.50,P＜0.05).Conclusions The expression of ASPP1.ASPP2 mRNA in p53 mutant-type cell lines is higher than that in p53 wild-type cell lines.The expression of ASPP maybe has close relationship with chemotherapeutic sensitivity of NSCLC.In NSCLC patient chemotherapy process,the change of ASPP1 and ASPP2 mRNA expression may be related with chemotherapy medicine function.Enhance the expression of ASPP1 and ASPP2 will be beneficial to the tumot treatment.
ABSTRACT
Objective To establish a reverse transcription-PCR(RT-PCR) assay for detecting ASPP1 mRNA expression levels in normal blood monocyte and leukemia jurkat,HL-60 and K-562 cell lines.And to analyze the differences in relative expression levels of ASPP1 in respective cell line by calculating the value of ASPP1/?-actin for evaluating the effects of ASPP1 on inhibiting the malignant transformation of normal cells.Methods The monocyte isolation reagent was used to isolate normal blood monocyte,and the RPMI 1640 medium was used to culture jurkat,HL-60 and K562 cell lines.The cell RNA was extracted with TripureTM reagent and amplified by RT-PCR.The amplified products were E.B.stained and 10?l of the products were used for agarose gel electrophoresis.The expression levels of ASPP1 mRNA and ?-actin in respective cell line were measured and then compared.Result The ASPP1 mRNA was expressed both in normal blood monocyte and all the leukemic cells.The value of ASPP1/?-actin was 0.545?0.019 in normal blood monocyte,and it was 0.155?0.081,0.199?0.016 and 0.191?0.015,respectively,in the leukemic cells jurkat,HL-60,and K-562.The expression level of ASPP1 mRNA was higher in normal cells than that in the three leukemic cell lines.Conclusion On the process of tumor development,an ASPP1-p53 compound is formed by collaborated effects of ASPP1 and p53,which may act on the arch-apoptosis gene promoter,specifically enhance the ability of p53 binding DNA,and reinforce the function of p53 in enhancing apoptosis.The declined expression and/or deprived function of ASPP1 might lead to tumorigenesis,implying that the function of ASPP1 in enhancing p53 apoptosis may play an important role on inhibiting the malignant transformation of normal cells.