Adenovirus expressing mutant p27kip1 enhanced apoptosis against cholangiocarcinoma than adenovirus-p27kip1 wild type

BACKGROUND/AIMS: The prognosis of cholangiocarcinoma is extremely poor despite the aggressive multidisciplinary cancer therapies that have been used clinically. Thus, it is imperative to develop new and effective treatments, such as gene therapy in order to treat this disease. p53 is the most common target for cancer gene therapy treatment. However, cholangiocarcinoma has a low frequency of p53 mutation, which makes this protein a poor candidate for gene therapy in this disease and another suitable gene therapy target must be found. p27kip1 is a universal cyclin-dependent kinase (CDK) inhibitor that blocks cell cycle progression and inhibits proliferation. Our previous reports have demonstrated the role of p27kip1 in the induction of apoptosis using a recombinant adenoviral vector expressing p27kip1 (Adp27) in several different human cancer cells. p27kip1 is regulated by two mechanisms composed of a ubiquitin-proteasome system and proteolytic processing system that requires the phosphorylation of p27kip1 on Thr-187 by the cyclinE/Cdk2 complex followed by proteolytic degradation. METHODOLOGY: In this study, we focused our aim on the degradation of p27kip1 protein mediated by a recombinant adenoviral expressing a mutant p27kip1 (Adp27-mt), which has a mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC). RESULTS: We observed that the mutated p27kip1 markedly inhibited ubiquitination and the subsequent degradation of p27kip1 when compared to wild type p27kip1. Consequently, we found that mutated p27kip1 induced a stronger induction of apoptosis and cell growth inhibition than wild type p27kip1 in cholangiocarcinoma cell lines TFK-1 and HuCCT-1. Furthermore, we demonstrated that Adp27-mt mainly caused G2/M arrest in the cell cycle progression and a decreased cyclinB1 and Cdc2 protein where as Adp27-wt mediated a G1/S arrest at 48 hours after infection. CONCLUSIONS: In this study, we showed that adenoviral vector expression of mutant p27kip1 protein inhibited degradation by the ubiquitin-proteasome system and strongly induced apoptosis and cell growth inhibition compared to wild type p27kip1. Thus recombinant adenovirus expressing mutant p27kip1 may be potentially useful for gene therapy against cholangiocarcinoma.     

Effect of p27(KIP1) on cell cycle and apoptosis in gastric cancer cells

AIM: To elucidate the effect of p27KIP1 on cell cycle and apoptosis regulation in gastric carcinoma cells. METHODS: The whole length of p27KIP1 cDNA was transfected into human gastric cancer cell line SCG7901 by lipofectamine. Expression of p27KIP1 protein or mRNA was analyzed by Western blot and RNA dot blotting, respectively. Effect of p27KIP1 on cell growth was observed by MTT assay and anchorage-independent growth in soft agar. Tumorigenicity in nude mice was used to assess the in vivo biological effect of p27KIP1. Flow cytometry, TUNEL, and electron microscopy were used to assess the effect of p27KIP1 on cell cycle and apoptosis. RESULTS: Expression of p27KIP1 protein or mRNA increased evidently in SCG7901 cells transfected with p27KIP1. The cell growth was reduced by 31% at 48 h after induction with zinc determined by cell viability assay. The alteration of cell malignant phenotype was evidently indicated by the loss of anchorage-independent growth ability in soft agar. The tumorigenicity in nude mice was reduced evidently (0.55±0.14 cm vs 1.36±0.13 cm, P<0.01). p27KIP1 overexpression caused cell arrest with 36% increase (from 33.7% to 69.3%, P<0.01) in G1 population. Prolonged p27KIP1 expression induced apoptotic cell death reflected by pre-G1 peak in the histogram of FACS, which was also confirmed by TUNEL assay and electron microscopy. CONCLUSION: p27KIP1 can prolong cell cycle in G1 phase and lead to apoptosis. p27KIP1 may be a good candidate for cancer gene therapy.     

Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27KIP1

It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G(1)/S checkpoint and maintain high levels of p27(KIP1) mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27(KIP1) is down-regulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin alpha(2)beta(1), which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals.     

Adenovirus expressing p27kip1 suppresses growth of established esophageal carcinoma xenograffs

AIM: To investigate the growth suppression of adenovirus expressing p27kip1 on established esophageal tumors in nude mice.METHODS: Esophageal carcinoma xenografts in nude mice were established by tumor tissue mass transplantation. The successfully constructed recombinant adenoviral vectors carrying p27kip1 gene (Adp27kip1) were directly injected into the esophageal tumors in nude mice. Compared to control group, the growth curve of tumor was drawn and the growth inhibition rate of tumor was calculated. The histology of tumors was examined by hematoxylin and eosin (H&E) staining. The expression of p27kip1 and survivin was detected in tumors by immunohistochemical technique.RESULTS: The growth of tumors in gene therapy group with Ad-p27kip1 was obviously suppressed compared to control group (0.42±0.08 g vs 1.17±0.30 g, t=6.39,P＜0.01), the inhibition rate of tumor growth reached 64.1%. Pathological detection showed that the tumors in nude mice were poorly differentiated esophageal squamous carcinoma. In addition, the expression of p27kip1 was increased, while the expression of survivin was decreased in tumors after being transfected with Ad-p27kip1.CONCLUSION: p27kip1 gene therapy mediated by adenovirus vector has a significant inhibitory effect on esophageal carcinoma in vivo. Up-regulated p27kip1expression and down-regulated survivin expression may be its important mechanisms.     

Adenovirus expressing p27kip1 suppresses growth of established esophageal carcinoma xenografts

AIM: To investigate the growth suppression of ade novirus expressing p27kip1 on established esophageal tumors in nude mice. METHODS: Esophageal carcinoma xenografts in nude mice were established by tumor tissue mass transplantation. The successfully constructed reco mbinant adenoviral vectors carrying p27kip1 gene (Ad p27kip1) were directly injected into the esophageal tumors in nude mice. Compared to control group, the growth curve of tumor was drawn and the growth inhibition rate of tumor was calculated. The histology of tumors was examined by hematoxylin and eosin (H&E) staining. The expression of p27kip1 and survivin was detected in tumors by immunohistochemical technique. RESULTS: The growth of tumors in gene therapy group with Ad-p27kip1 was obviously suppressed compared to control group (0.42±0.08 g vs 1.17±0.30 g, t=6.39, P<0.01), the inhibition rate of tumor growth reached 64.1%. Pathological detection showed that the tumors in nude mice were poorly differentiated esophageal squamous carcinoma. In addition, the expression of P27kip1 was increased, while the expression of survivin was decreased in tumors after being transfected with Ad-p27kip1. CONCLUSION: p27kip1 gene therapy mediated by adenovirus vector has a significant inhibitory effect on esophageal carcinoma in vivo. Up-regulated p27kip1 expression and down-regulated survivin expression may be its important mechanisms.     

死亡受体通路在曲霉菌素诱导肝星状细胞凋亡中的作用

目的 验证曲霉菌素诱导肝星状细胞HSC-T6凋亡的作用,探讨死亡受体通路在该过程中的意义.方法 将HSC-T6细胞分为曲霉菌素干预组和空白对照组,用流式细胞术检测HSC-T6的凋亡,免疫组化SABC法检测Fas蛋白的表达,分光光度法检测caspase-8的活性.结果 流式细胞术显示,曲霉菌素浓度分别为0.4、0.8、1.2、1.6 mmol/L,作用于干预组HSC-T6细胞1 h后,HSC凋亡指数与对照组相比显著增高,差异有统计学意义(P＜0.05),且随着曲霉菌素浓度增加,凋亡率也相应增高;曲霉菌素以最低有效浓度0.4 mmol/L干预,Fas的表达及caspase-8活性与空白对照组比较均显著增高,差异有统计学意义(P＜0.05).结论 曲霉菌素诱导HSC-T6细胞凋亡具有剂量依赖性,死亡受体通路在该过程中有重要意义.     

Soluble Fas ligand activates the sphingomyelin pathway and induces apoptosis in luteal steroidogenic cells independently of stress-activated p38(MAPK)

Fas ligand (FasL) is implicated as a mediator of luteolysis. However, a gap exists in our understanding of the Fas-mediated signaling mechanisms that are involved in either the loss of progesterone production or the structural regression of the corpus luteum. In the present study we investigated the acute and chronic effects of FasL with respect to activation of cytokine/stress-induced signaling pathways and apoptosis in bovine steroidogenic cells. More specifically, we investigated soluble FasL (sFasL)-activated production of ceramide, a second messenger of the sphingomyelin pathway, and activation of p38(MAPK), a member of the MAPK family. sFasL activated the sphingomyelin pathway, as evidenced by a 2-fold increase (P < 0.05) in the production of ceramide. Pretreatment with imipramine (50 micro M), an inhibitor of acid sphingomyelinase activity, attenuated (75%; P < 0.05) sFasL-induced ceramide production, suggesting that the increase in ceramide was partially the result of acid sphingomyelinase-mediated hydrolysis of sphingomyelin. Treatment of luteal cells with sFasL or a cell-permeable ceramide analog (C8) for 24-48 h resulted in a significant increase (P < 0.05) in apoptosis. Western blot analysis revealed that sFasL had little effect on the activation of p38(MAPK) in primary bovine luteal steroidogenic cells. Furthermore, pretreatment with the p38(MAPK) inhibitor SB203580 failed (P > 0.05) to inhibit sFasL- or C8-induced death. Although sFasL did not alter basal progesterone levels detected in the culture medium, C8 caused a significant increase (P < 0.05) in progesterone concentrations within the medium. Collectively, these data suggest that the role of FasL in luteolysis may be to activate the stress-induced sphingomyelin pathway that, in turn, serves as a mediator of apoptosis.     

Involvement of the Fas/Fas ligand system in p53-mediated granulosa cell apoptosis during follicular development and atresia

In the present study we have examined the presence of Fas, Fas ligand (FasL), and p53 in rat granulosa cells during follicular development and atresia, especially in relation to the granulosa cell cycle progression and the onset of granulosa cell apoptosis. Fas, FasL, and p53 proteins were immunolocalized, and their contents were determined by Western blotting. Granulosa cell apoptosis was assessed by DNA fragmentation analyses (DNA ladder) and in situ terminal deoxynucleotidyl transferase mediated deoxy-UTP-biotin nick end labeling (TUNEL) as well as by flow cytometry. Ovaries not exposed to gonadotropins (control) consisted predominantly of preantral and early (small) antral follicles, the latter of which were mostly atretic and demonstrated intense TUNEL staining in granulosa cells exhibiting positive immunoreactivities for FasL and Fas. Granulosa cells isolated from these follicles were apoptotic, as evident by clear ladder pattern of DNA fragmentation upon electrophoretic analysis and the high percentage (>10%) of the cell population in the A0 phase of the cell cycle. After gonadotropin treatment, these features completely disappeared during each of the 3 days of follicular growth to the medium to large antral stages. Cell cycle analysis showed significantly higher proportion of the cells in S and G2/M phases compared with controls, which was accompanied by marked decrease in immunoreactivities for Fas, FasL, and p53. By days 4 and 5, widespread atresia and extensive granulosa cell apoptosis were noted in large antral and preovulatory follicles and were coincidental to increased expression of p53 and Fas, but not of FasL, as well as an apparent arrest of granulosa cell G1/S progression, as evident by an increased cell population in G0/G1 and a decrease in the S and G2/M. Granulosa cells from equine CG-primed ovaries exhibited marked increases in p53 and Fas protein contents and apoptosis after adenoviral p53-sense complementary DNA infection in vitro and were more responsive to Fas activation by an agonistic Fas monoclonal antibody challenge. Taken together, these findings are consistent with the well accepted concept that gonadotropin plays a central role as a survival factor in the regulation of granulosa cell Fas/FasL and p53 expression during ovarian follicular development. In addition, the control of granulosa cell apoptosis may involve two consecutive cellular/molecular events: cell cycle arrest at G1/S and exit from G0 into A0 phase, via regulation of the p53 and Fas/FasL death pathways.     

Epstein-Barr virus induces Fas (CD95) in T cells and Fas ligand in B cells leading to T-cell apoptosis.

Epstein-Barr virus (EBV) acute infectious mononucleosis (AIM) is characterized by transient immunosuppression in vivo and increased T-cell apoptosis after ex vivo culture of AIM peripheral blood mononuclear cells. We undertook experiments to test whether EBV or purified virion envelope glycoprotein gp350 could contribute to Fas-mediated T-cell apoptosis. Our in vitro results indicate that EBV increased Fas expression in CD4(+) T cells and Fas ligand (FasL) expression in B cells and macrophages. Purified gp350 was also shown to significantly increase CD95 expression in CD4(+) T cells. When T-cell CD95 was cross-linked, EBV-stimulated T cells underwent apoptosis. The induction of T-cell CD95 by EBV followed by CD95 cross-linking with anti-CD95 monoclonal antibody resulted in a loss in the number of T cells responding to the T-cell mitogens, anti-CD3 antibody, and interleukin-2. These results indicate that, in addition to serving as a principal ligand for the attachment of virus to target cells, gp350 may also act as an immunomodulatory molecule that promotes T-cell apoptosis.     

Rapamycin specifically interferes with GM-CSF signaling in human dendritic cells,leading to apoptosis via increased p27KIP1 expression

The longevity of dendritic cells (DCs) is a critical regulatory factor influencing the outcome of immune responses. Recently, we demonstrated that the immunosuppressive drug rapamycin (Rapa) specifically induces apoptosis in DCs but not in other myeloid cell types. The present study unraveled the mechanism used by Rapa to induce apoptosis in human monocyte-derived DCs. Our data demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF) preserves DC survival specifically via the phosphatidylinositol-3 lipid kinase/mammalian target of rapamycin (PI3K/mTOR) signaling pathway, which is abrogated by Rapa at the level of mTOR. Disruption of this GM-CSF signaling pathway induced loss of mitochondrial membrane potential, phosphatidyl-serine exposure, and nuclear changes. Apoptosis of these nonproliferating DCs was preceded by an up-regulation of the cell cycle inhibitor p27(KIP1). Overexpression of p27(KIP1) in DCs using adenoviral gene transduction revealed that apoptosis is directly regulated by p27(KIP1). Furthermore, both overexpression of p27(KIP1) and disruption of the GM-CSF/PI3K/mTOR signaling pathway decreased the expression of the antiapoptotic protein mcl-1. This mTOR/p27(KIP1)/mcl-1 survival seems unique for DCs and may provide novel opportunities to influence immune responses by specific interference with the life span of these cells.     

Resveratrol induces Fas signalling-independent apoptosis in THP-1 human monocytic leukaemia cells

Resveratrol, a natural product present in wine, has recently been shown to inhibit the growth of a number of cancer cell lines in vitro. In the current study, we have demonstrated that resveratrol inhibits the growth of THP-1 human monocytic leukaemia cells in a dose-dependent manner with a median effective dose of 12 microM. It did not induce differentiation of THP-1 cells and had no toxic effect on THP-1 cells that had been induced to differentiate into monocytes/macrophages by phorbol myristate acetate. A significant fraction of resveratrol-treated cells underwent apoptosis as judged by flow cytometric analysis of DNA content, DNA fragmentation and caspase-specific cleavage of poly(ADP-ribosyl) polymerase. Resveratrol treatment had no effect on the expression of Fas receptor or Fas ligand (FasL) in THP-1 cells, nor did it induce clustering of Fas receptors. In addition, THP-1 cells were resistant to activating anti-Fas antibody, and neutralizing anti-Fas and/or anti-FasL antibodies had no protective effect against resveratrol-induced inhibition of THP-1 cell growth. The effect of resveratrol on THP-1 cells was reversible after its removal from the culture medium. These results suggest that (1) resveratrol inhibits the growth of THP-1 cells, at least in part, by inducing apoptosis, (2) resveratrol-induced apoptosis of THP-1 cells is independent of the Fas/FasL signalling pathway and (3) resveratrol does not induce differentation of THP-1 cells and has no toxic effect on differentiated THP-1 cells. Thus, resveratrol may be a potential chemotherapeutic agent for the control of acute monocytic leukaemia.     

Estradiol increases apoptosis in human coronary artery endothelial cells by up-regulating Fas and Fas ligand expression

CONTEXT: In animal models, estrogen inhibits atherogenesis by inhibiting many of the early steps of atherosclerotic plaque formation. However, the lack of cardioprotective effect by postmenopausal hormone replacement therapy and possible increase in cardiovascular events observed during the first year after the initiation of hormone replacement therapy may suggest that once the plaque is formed, estrogen may have additional effects that may counteract its beneficial outcomes. Indeed, the effect of estrogen on plaque stability has not been identified. OBJECTIVE: We hypothesized that 17beta-estradiol (E2) may cause increased apoptosis in human coronary artery endothelial cells (HCAECs). This effect would explain an adverse effect on plaque stability in vivo. INTERVENTION(S) AND MAIN OUTCOME MEASURE(S): The effect of E2 on apoptosis, cell proliferation, and expression of proapoptotic molecules Fas and Fas ligand (FasL) in cultured HCAECs was evaluated. RESULTS: HCAECs in culture treated with E2 showed an increase in DNA strand breaks and nuclear fragmentation indicative of apoptosis. E2 treatment also induced a significant concentration-dependent increase in Fas mRNA and protein expressions in HCAECs. Moreover, the expression of FasL mRNA and secretion of FasL protein by HCAECs were enhanced in response to E2 treatments. CONCLUSIONS: E2 increases the apoptosis in cultured HCAECs. Enhanced Fas and FasL expressions in response to E2 suggest that activation of the Fas/FasL pathway may be a mediator of the proapoptotic effects of E2 in these cells.     

Overexpression of p27(KIP1) induced cell cycle arrest in G(1) phase and subsequent apoptosis in HCC-9204 cell line

AIM We have previously reported that inducible over-expresaion of Bak may prolong cell cycle in G1 phase and lead to apoptosis in HCC-9204 cells. This study is to investigate whether p27KIP1 plays an important role in this process. MEHODS In order to elucidate the exact function of p27KIP1 in this process, a zinc inducible p27KIP1 stable transfectant and transient p27KIP1- GFP fusion transfectant were constructed. The effects of inducible p27KIP1 on cell growth, cell cycle arrest and apoptosis were examined in the mock, control pMD vector, and pMD-KIP1 transfected HCC-9204 cells. RESULTS This p27KIP1-GFP transfectant may transiently express the fusion gene. The cell growth was reduced by 35% at 48 h of p27KIP1 induction with zinc treatment as determined by trypan blue exclusion assay. These differences remained the same after 72 h of p27KIP1 expression, p27KIP1 caused cell cycle arrest after 24 h of induction, with 40% increase in G1 population. Prolonged p27KIP1 expression in this cell line induced apoptotic cell death reflected by TUNEL assay. Fourty-eight h and 72 h of p27KIP1 expression showed a characteristic DNA ladder on agarose gel electrophoresis.     

NS-398 enhances the efficacy of gemcitabine against lung adenocarcinoma through up-regulation of p21WAF1 and p27KIP1 protein

Gemcitabine is a chemotherapeutic drug widely used in the treatment of non-small cell lung carcinoma, especially in advanced lung adenocarcinoma. However, many patients with advanced lung adenocarcinoma show a resistance to gemcitabine. Overexpression of COX-2 has been found in human non-small cell lung cancer tissues and itA s cell lines. Evidences show that COX-2 is involved in drug resistance of tumor. However, It is unknown whether COX-2 inhibitor can augment the efficacy of gemcitabine against lung adenocarcinoma. In this study, A549 cells were treated with gemcitabine and/or NS-398. The cell viability was examined by MTT assay. The cell cycle distribution and apoptotic ratio were tested by flow cytometry. The levels of p21WAF1, p27KIP1, p16INK4a and p15INK4b expression were detected by western blotting. After the cells were treated with gemcitabine along with NS-398, more cells were arrested in G1 phase and went to apoptosis. The levels of p21WAF1 and p27KIP1 protein were elevated, while the levels of p16INK4a and p15INK4b protein were not changed. It can be concluded that NS-398 enhances the efficacy of gemcitabine against lung adenocarcinoma and the efficacy is associated with up-regulation of p21WAF1 and p27KIP1protein.     

Alterations of the p27KIP1 gene in non-Hodgkin's lymphomas and adult T- cell leukemia/lymphoma

The protein p27KIP1 belongs to a recently identified family of proteins termed cyclin-dependent kinase inhibitors (CDKIs). These proteins play an important role in regulating cell-cycle progression and loss of their function has been implicated in tumorigenesis. Transforming growth factor beta (TGF-beta) may induce cell growth arrest through p27 activation. TGF-beta often loses its ability to arrest growth of transformed cells; this could potentially occur through a defect in p27. To determine the role of p27 in tumorigenesis, we examined its mutational status in 74 non-Hodgkin's lymphomas (NHLs) (52 of B-cell phenotype, 22 of T-cell phenotype), 5 lymphoma cell lines, and 42 adult T-cell leukemias/lymphomas (ATLs) using polymerase chain reaction- single strand conformational polymorphism (PCR-SSCP) and Southern blot analyses. A nonsense mutation (stop codon) that could result in expression of a truncated nonfunctional p27 protein was detected at codon 76 in one case of acute lymphomatous ATL, but not in matched normal tissues. Previously undescribed polymorphisms were also identified at codon 109 in the lymphomas and at codon 55 in the ATLs. Two homozygous deletions of the p27 gene were detected in one case of B- immunoblastic NHL and in one case of acute ATL by Southern blot hybridization. These results indicate that p27 gene alterations are rare events in NHLs and ATLs, but may play an important role in the pathogenesis of some hematologic malignancies.     

Bak基因诱导p27KIP1基因的过表达使HCC-9204细胞在G1期停滞

目的Ｂａｋ基因的过表达能够使ＨＣＣ－９２０４细胞的Ｇ１期延长并导致细胞凋亡,探讨ｐ２７ＫＩＰ１基因是否在这一过程中起重要作用。方法建立Ｂａｋ诱导ＨＣＣ—９２０４细胞凋亡和细胞周期停滞的模型。从Ｂａｋ诱导的凋亡模型中获得Ｐ２７ＫＩＰ１基因并测序。构建ＰＭＤ－ＫＩＰ１可诱导型表达系统并转染 ＨＣＣ－９２０４细胞,获得稳定转染子。 进一步研究ｐ２７ＫＩＰ１对细胞生长和细胞周期的调控作用。Ｗｅｓｔｅｒｎ ｂｌｏｔ证实在这一过程中该基因的表达水平上调。结果在诱导 ４８ ｈ后 ｐ２７ＫＩＰ１基因稳定转染细胞的活力降低 ３５％。在诱导后 ２４ｈ时 Ｐ２７ＫＩＰ１能够使 ＨＣＣ－９２０４细胞在Ｇ１期停滞,累积的细胞增加 ４０％。结论 Ｂａｋ使ＨＣＣ－９２０４细胞的 Ｇ１期延长是通过上调 ｐ２７ＫＩＰ１的表达;可诱导型 Ｐ２７ＫＩＰ１基因的过表达能够诱导 Ｇ１期停滞。     

Effect of mutant p27kip1 gene on human cholangiocarcinoma cell line, QBC939

AIM:To investigate the effects of exogenously mutated p27kip1 (p27) on proliferation and apoptosis of human cholangiocarcinoma cell line,QBC939 in vivo.METHODS:Adenviral vectors were used to transfect mutated p27 cDNA into human QBC939 cell line.Expression of p27 was detected by RT-PCR.Western blot.Cell growth,morphological change,cell cycle,apoptosis and cloning formation were determined by MTT assay and flow cytometry.RESULTS:The expression of p27 protein and mRNA was increased significantly in QBC939 cell line transfected with Ad-p27mt.The transfer of Adp27mt could significantly inhibit the growth of QBC939cells,decrease the cloning formation rate and induce apoptosis,p27 over expression caused cell cycle arrest at G0/G1 phase 72 h after infection with Adp27mt.CONCLUSION:p27 may cause cell cycle arrest at G0/G1 phase and subsequently lead to apoptosis.Recombinant adenovirus expressing mutant p27 may be potentially useful in gene therapy for cholangiocarcinoma.     

腺病毒介导MDA-7/IL-24选择性促进肝癌细胞的凋亡和增殖阻滞

目的 观察MDA-7／IL-24基因对不同p53状态人肝癌细胞HepG2、MHCC97L以及Hep3B和正常的肝细胞L02的选择性杀伤作用，为肝癌的基因治疗提供理论基础。方法 将携带人MDA-7／IL-24基因的腺病毒Ad．mda-7感染人正常肝细胞L02和不同p53状态的肝癌细胞HepG2，MHCC97L、Hep3B。通过逆转录聚合酶链反应方法观察MDA7／IL24基因的表达，酶联免疫吸附法检测细胞培养上清液中MDA-7／IL-24蛋白的浓度，通过四甲基偶氮唑盐染色法及Hoechst染色观察MDA-7／IL-24对肝癌细胞的生长抑制和杀伤作用，Annexin-V和碘化丙啶双染后流式细胞仪检测细胞的凋亡，应用流式细胞仪检测细胞周期。结果 复制缺陷型腺病毒能介导外源基因MDA-7／IL-24在肝癌细胞株HepG2，MHCC97L和Hep3B以及正常细胞L02中高效表达。细胞培养上清液中有MDA-7／IL-24蛋白表达。MDA-7／IL-24能明显抑制各种肝癌细胞的生长，Hoechst染色提示MDA-7／IL-24促进肝癌细胞的凋亡，流式细胞仪提示MDA-7能选择性杀伤肝癌细胞而对正常的肝细胞无影响，细胞周期分析提示MDA-7／IL-24阻滞肝癌细胞在G2／M期，同时对正常的肝细胞没有促凋亡作用和增殖阻滞作用。结论 复制缺陷型重组腺病毒载体Ad．mda-7能介导MDA-7／IL-24基因在人肝癌细胞中高效表达，选择性地杀伤肝癌细胞HepG2、MHCC97L和Hep3B，促进细胞增殖阻滞及诱导肿瘤细胞凋亡而与肿瘤细胞的P53基因的状态无关，同时对正常的肝细胞L02无任何毒性作用。