Effect of p27Kip1 on the ability of invasion and metastasis of an oral cancer cell line

p27Kip1 is a cyclin-dependent kinase inhibitor which regulates progression of cells from G1 into S phase in a cell cycle. p27Kip1 has been reported to be an important prognostic factor in various malignancies. As the ability of invasion and metastasis has been thought to be the most important factor for patient's prognosis, we examined whether up-regulation or down-regulation of p27Kip1 can affect the ability of invasion and metastasis of an oral cancer cell (B88t cell) in vitro and in vivo. We constructed an expression vector containing sense- or antisense-oriented human p27Kip1 cDNA with pcDNA3.1. We transfected B88t cells with the empty vector, the sense- or antisense-oriented expression vector to regulate the expression of p27Kip1 gene. Up-regulation of p27Kip1 protein markedly inhibited the migration of cancer cells in a Boyden chamber. Down-regulation of p27Kip1 protein markedly enhanced the migration of cancer cells in a Boyden chamber, and the tumor invasion in nude mice. Moreover, we detected up-regulation of E-cadherin and down-regulation of Tiam-1 (tumor invasive and metastasis-1) in the sense transfectants, up-regulation of Tiam-1 and down-regulation of E-cadherin in the antisense transfectants by Western blotting. These results suggest that p27Kip1 may play an important role in the invasion and metastasis of an oral cancer cell involved in regulation of E-cadherin and Tiam-1.     

Characteristics of antitumor activity of mutant type p27Kip1 gene in an oral cancer cell line

It is well known that loss of the cyclin-dependent kinase inhibitor p27Kip1 protein correlates with the poor prognosis of various cancers including oral squamous cell carcinoma (SCC). Posttranslational degradation of p27Kip1 protein is mediated by phosphorylation of Thr-187 of p27Kip1 protein, which follows ubiquitination. In this study, we constructed an expression vector expressing mutant type p27Kip1 gene (pcDNA3.1-p27Kip1 mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), which is not influenced by ubiquitin-mediated degradation. We transfected mutant and wild type p27Kip1 genes into an oral SCC cell line, B88 to up-regulate the expression of mutant or wild p27Kip1 gene in each transfectant. B88-p27Kip1 mt showed significant growth inhibition than B88-p27Kip1 wt or B88-neo in vitro (p < 0.01). Also, both types of B88-p27Kip1 showed G1 arrest and apoptosis, however, B88-p27Kip1 mt showed remarkable G1 arrest. In addition, B88-p27Kip1 mt and B88-p27Kip1 wt showed markedly inhibition of the migration and out-growth of cancer cells than B88-p27Kip1 wt or B88-neo. Moreover, B88-p27Kip1 mt also showed remarkable suppression of tumor growth and cervical lymph metastasis than B88-p27Kip1 wt or B88-neo in vivo (p < 0.01). In short, the mutant type p27Kip1 gene could show more potent antitumor effects than wild type p27Kip1 gene in B88 cells. These findings suggest that mutant type p27Kip1 gene has the potential to become a novel and powerful gene therapy tool for patients with oral cancers.     

Overexpression of p27(Kip1) induces growth arrest and apoptosis in an oral cancer cell line

p27(Kip1) is a cyclin-dependent kinase inhibitor which regulates progression of cells from G1 into S phase in a cell cycle. Loss of p27(Kip1) has been associated with disease progression and an unfavorable outcome in several malignancies. In the present study, we conducted to examine whether up-regulation or down-regulation of p27(KiP1) can affect the growth of oral cancer cell (B88 cell) in vitro and in vivo. We constructed an expression vector containing sense- or antisense-oriented human p27(Kip1) cDNA with pcDNA3.1(Invitrogen). We transfected B88 cells with the sense or antisense expression vector to regulate the expression of p27(Kip1) gene in each transfectant. The expression of p27(Kip1) protein was up-regulated in the sense transfectants and down-regulated in the antisense transfectants. Moreover, up-regulation of p27(Kip1) protein exerted the growth inhibitory effect, and down-regulation of p27(Kip1) protein enhanced the growth of B88 cells in vitro and in vivo. Furthermore, we detected the G1 arrest and sub-G1 peak in the sense transfectants by flow cytometry analysis. These results suggest that up-regulation of p27(Kip1) protein may exert the growth inhibitory effects through induction of G1 arrest and apoptosis on oral cancer cell line.     

Suppression of murine mammary carcinoma growth and metastasis by HSVtk/GCV gene therapy using in vivo electroporation

The effectiveness of electroporation as a means of gene transfection, both in vitro and in vivo, was tested using the herpes simplex virus 1 thymidine kinase (HSVtk) gene in combination with ganciclovir (GCV) administration as therapy against murine mammary cancer. Approximately 80% of BJMC3879 metastatic mammary carcinoma cells, derived from MMTV-infected BALB/c mice, died as a result of HSVtk/GCV treatment 72 hours after the transfection; decreased DNA synthesis was also seen. Mammary tumors induced by inoculation of syngeneic mice with BJMC3879 cells were subsequently treated by direct injection of vector containing HSVtk (pHSVtk) alone, empty vector or saline alone twice a week. After each injection, the tumors were subjected to in vivo electroporation. Mice treated with pHSVtk or saline were intraperitoneally injected with GCV at 40 mg/kg five times a week. Significantly reduced tumor volumes were observed for the pHSVtk+GCV group in experimental week 2 and thereafter throughout the 2-month study. DNA synthesis was significantly decreased as well in the pHSVtk+GCV group compared with all other groups. Furthermore, metastasis to lymph nodes and lungs was significantly suppressed by HSVtk/GCV treatment. Expression of HSVtk in the tumors was confirmed by RT-PCR. Macrophage accumulations were frequently observed in the peripheries of necrotic regions in HSVtk/GCV-treated tumors, where levels of apoptosis were significantly higher than those observed in other groups. We therefore conclude that in vivo electroporation can result in efficient gene transfer and that the HSVtk/GCV prodrug system strongly suppresses tumor growth and metastases in this model.     

Transfection of p27(Kip1) threonine residue 187 mutant type gene,which is not influenced by ubiquitin-mediated degradation,induces cell cycle arrest in oral squamous cell carcinoma cells

OBJECTIVE: It is well known that reduction of the cyclin-dependent kinase inhibitor p27(Kip1) protein correlates with the malignant behavior of various cancers including oral squamous cell carcinoma (OSCC). The loss of p27(Kip1) protein is suggested to be due to the enhancement of its posttranslational degradation. In the present study, to evaluate the effects of p27(Kip1) transfection on the cell cycle, we transfected OSCC cell lines with a high activity of p27(Kip1) degradation with p27(Kip1) threonine 187-to-alanine (T187A) mutant gene, which is not influenced by ubiquitin-mediated degradation, as well as with wild type gene. METHODS: We transfected p27(Kip1) T187A mutant and wild type gene into OSCC cell lines (HSC2 and HSC3) by using an ecdysone-inducible gene expression system. RESULTS: After treatment with ponasterone A, we could find an induction of both p27(Kip1) wild type and T187A mutant protein. Both wild type and T187A mutant protein induced by 5 microM ponasterone A inhibited cell growth and increased cell number at the G1 phase. After treatment with 1 microM ponasterone A, ectopic p27(Kip1) protein was degraded in wild type clones, but not in T187A mutant clones. Moreover, transfection of the T187A mutant gene was more effective in inhibiting cell growth even by induction of a small amount of protein. CONCLUSION: We suggest that the transfection of the p27(Kip1) T187A mutant gene can be a modality of cancer gene therapy for OSCC.     

Gene delivery using a receptor-mediated gene transfer system targeted to hepatocellular carcinoma cells.

For gene therapy to be effective in cancers, it is necessary to deliver therapeutic genes into cells with high specificity and efficiency. In this study, we examined the in vitro and in vivo gene delivery efficiency of a new, growth receptor-mediated gene transfer system in hepatocellular carcinoma (HCC). The effects of transfection of wild-type p53 using this system were also studied. The system consisted of a ligand oligopeptide for epidermal growth factor receptor (EGFR) recognition, a polypeptide for DNA binding, and an endosome-releasing oligopeptide for endosomolysis. Two human HCC cell lines and a normal liver cell line were used, and pCMV-beta-galactosidase (beta-gal) was used as a reporter gene. Both HCC cell lines had strong expression of EGFR and the in vitro transfer efficiency peaked at day 5 at about 50%. This finding was in contrast to the normal liver cell line, which had weak EGFR expression and less than 1% transfer efficiency throughout. For in vivo gene transfer in tumors produced by inoculating HCC cells in nude mice and with the vector-beta-gal gene complex injected peritumorally, beta-gal expression was detected within the tumors at 12 hr, peaked at day 5 involving about 50% of the tumor cells and persisted at 2 weeks. Using this vector system, transfection of wild-type p53 into Huh-7 cells that had mutated p53 resulted in significant growth inhibition of cancer cells accompanied by a decreased G2/M phase and increased p53 protein. In conclusion, this receptor-mediated gene transfer system appears to work specifically in HCC cells with high efficiency, and may be promising in delivering apoptotic and other genes into HCC cells.     

Combinatory anti-tumor effects of electroporation-mediated chemotherapy and wild-type p53 gene transfer to human esophageal cancer cells

Delivery of electric pulses to an established solid tumor augments the permeability of cell membrane and increases the susceptibility of tumors to an anti-cancer agent that is administered in the vicinity of tumors. Forced expression of the wild-type p53 gene in tumor cells that have non-functional p53 gene(s) can also enhance their sensitivity to a DNA-damaging agent. To investigate the feasibility of electroporation-mediated therapy for cancer, electric pulses were delivered to human esophageal tumors developed in nude mice after they received an anti-cancer agent and/or plasmid DNA containing the wild-type p53 gene. The growth of esophageal tumors was suppressed with electroporation-mediated chemotherapy compared with the treatment with an anti-cancer agent or electroporation alone. Intratumoral injection of the wild-type p53 gene into p53-mutated esophageal tumors followed by electroporation also inhibited tumor growth. When mice were administered with the wild-type p53 gene and an anti-cancer agent, subsequent electroporation produced a synergistic therapeutic effect. Combinatory transfer of plasmid DNA and a pharmacological agent by electroporation is thereby a possible therapeutic strategy for the treatment of solid tumors.     

Progress of gene therapy for esophageal cancer in Japan

Retrovirally expressed interleukin-2 gene, granulocyte macrophage-colony stimulating factor gene, herpes simplex virus-thymidine kinase gene and p53 gene in human esophageal cancer cells showed antitumor effects in a nude mice xenotransplant model. We established a clinical protocol of gene therapy for advanced esophageal cancer using the wild type p53 gene with an adenovirus vector. In December of 2000, we began the first tumor suppressor gene therapy trial. Now, this trial, which has 9 patients. There have been no serious adverse event excluding fever and local pain. The feasibility of this treatment appears fairly good in these 9 cases. Furthermore, we developed a new method for transducing genes without a virus vector since a virus vector has several potentially unwanted properties. In vivo electroporation is a useful strategy for cancer gene therapy. Moreover, electric pulse to established solid tumors increases intracellular concentrations of chemotherapeutic agents. Transduction of the wild-type p53 gene by electroporation decreased the amount of nedaplatin required for tumor suppression. Electrochemo-gene therapy is a relatively simple method and can produce a better therapeutic effect.     

Overexpression of tuberous sclerosis complex 2 exerts antitumor effect on oral cancer cell lines

Tuberous sclerosis is caused by mutations in tuberous sclerosis complex (TSC) 2 on chromosome 16p13.3, encoding tuberin which is thought to be essential for p27^Kip1 to regulate the cell cycle. In this study, we conducted to examine whether overexpression of TSC2 can affect the growth of oral cancer cells which have different expression level of p27^Kip1 protein. We constructed an expression vector containing sense-oriented rat TSC2 cDNA with pcDNA3.1. We transfected oral cancer cells, B88t (high expression of p27^Kip1 protein) and HI (low expression of p27^Kip1 protein) with the sense expression vector to up-regulate the expression of TSC2 gene. Overexpression of TSC2 exerted the growth inhibitory effect of B88t and HI in vitro and in vivo. These findings suggest that overexpression of TSC2 may exert the antitumor effect on oral cancer cells whether they have high expression of p27^Kip1 protein or not.     

p27Kip1 repression of ErbB2-induced mammary tumor growth in transgenic mice involves Skp2 and Wnt/beta-catenin signaling

Expression of the cyclin-dependent kinase (Cdk) inhibitor (p27(Kip1)) is frequently reduced in human tumors, often correlating with poor prognosis. p27(Kip1) functions as a haploinsufficient tumor suppressor; however, the mechanism by which one allele of p27(Kip1) regulates oncogenic signaling in vivo is not well understood. We therefore investigated the mechanisms by which p27(Kip1) inhibits mammary tumor onset. Using the common background strain of FVB, p27(Kip1) heterozygosity (p27(+/-)) accelerated ErbB2-induced mammary tumorigenesis. We conducted microarray analyses of mammary tumors developing in mice with genetic haploinsufficiency for p27(Kip1) expressing a mammary-targeted ErbB2 oncogene. Global gene expression profiling and Western blot analysis of ErbB2/p27(+/-) tumors showed that the loss of p27(Kip1) induced genes promoting lymphangiogenesis, cellular proliferation, and collaborative oncogenic signaling (Wnt/beta-catenin/Tcf, Cdc25a, Smad7, and Skp2). Skp2 expression was induced by ErbB2 and repressed by p27(Kip1). Degradation of p27(Kip1) involves an SCF-type E3 ubiquitin ligase, including Skp2. The Skp2 component of the SCF(SKP2) complex that degrades p27(Kip1) was increased in ErbB2 tumors correlating with earlier tumor onset. In both murine and human ErbB2-overexpressing breast cancers, p27(Kip1) levels correlated inversely with Skp2. p27(Kip1) haploinsufficiency activated Wnt/beta-catenin/hedgehog signaling. Reintroduction of p27(Kip1) inhibited beta-catenin induction of Tcf-responsive genes (Siamosis, c-Myc, and Smad7). p27(Kip1) is haploinsufficient for ErbB2 mammary tumor suppression in vivo and functions to repress collaborative oncogenic signals including Skp2 and Wnt/beta-catenin signaling.     

Reduced expression and altered subcellular localization of the cyclin-dependent kinase inhibitor p27(Kip1) in human colon cancer

The p27(Kip1) protein is a negative regulator of the cell cycle and a potential tumor suppressor gene. Reduced expression of the p27(Kip1) protein has been reported in several human tumors and has been associated with higher tumor grade and increased mortality in breast, lung, colon, prostate, bladder, and gastric cancers. On the other hand, increased expression of the p27(Kip1) protein, in the absence of gene mutation, has been observed in primary colon and breast cancers. It was recently suggested that sequestration in the cytoplasm might be an alternative way to inactivate p27(Kip1)-associated inhibitory activity. This study was undertaken to further evaluate p27(Kip1) expression in primary colon tumors and to verify whether differences exist between normal and cancer tissues in terms of subcellular localization of this protein. Both normal and neoplastic tissues expressed variable amounts of the p27(Kip1) protein, as assessed by western blot analyses. Although the mean values were not different between tumor and normal mucosa samples, the expression of total p27(Kip1) was reduced in a subset of tumors. Decreased levels of total p27(Kip1) were associated with high tumor grade (P=0.03) and stage (P=0.04). Moreover, while there was no significant difference in nuclear p27(Kip1), the amount of p27(Kip1) in the cytoplasmic fraction was significantly higher in the tumor samples than in the normal mucosa samples (P=0.0001). These results suggest that p27(Kip1) expression is lost in a subset of colorectal tumors and that alterations in the subcellular localization of this protein might play a role in colon carcinogenesis.     

Reduced expression of p27(Kip1) correlates with an early stage of cancer invasion in oral squamous cell carcinoma

Down-regulation of p27(Kip1) has been reported to correlate with poor survival of various carcinoma patients including oral squamous cell carcinomas (OSCCs). It is still unclear, however, at what stage of oral carcinogenesis the down-regulation of this protein occurs. In this study, therefore, we evaluated immunoexpression of p27(Kip1) protein in 17 cases of oral epithelial dysplasia and succeeding invasive OSCC in the same patient. We reported here that 88% cases showed high p27(Kip1) expression in dysplastic lesions, whereas 82% cases of succeeding invasive OSCC exhibited reduced expression. The reduction of p27(Kip1) expression was also observed in 16 of 19 (84%) early invasive lesions and well correlated with Ki-67 expression which is good indicator of cell proliferation. We also investigated immunoexpression of p53 protein of which abnormality has been known to occur during the early stage of OSCC development. Overexpression of p53 protein was demonstrated in 29% of dysplastic lesions, 42% of early invasive and 71% of invasive OSCCs. These findings suggest that abnormalities of both p53 and p27(Kip1) are involved in the carcinogenesis of OSCC, but they seem to play their role at different stages of oral cancer development, respectively. Reduced expression of p27(Kip1) may concern the cancer invasion directly or indirectly as well as abnormal proliferation.     

Prognostic role of p27(Kip1) expression in oral squamous cell carcinoma in Taiwan

The cyclin-dependent kinase inhibitor p27(Kip1) can inhibit the G1 to S transition of the cell cycle and is a putative tumor suppressor. Decreased expression of p27(Kip1) protein has been correlated with poor prognosis in a variety of human tumors. We examined the expression of p27(Kip1) in oral squamous cell carcinoma (SCC), epithelial dysplasia (ED) and normal oral mucosa (NOM) using antibodies to p27(Kip1). Positive p27(Kip1) nuclear staining was detected in all the specimems from ED and NOM, whereas positive p27(Kip1) staining was observed in 16 of the 63 (25%) cases of oral SCC. The labeling index for p27(Kip1) protein was significantly reduced from NOM through ED to oral SCCs, indicating that changes of p27(Kip1) protein expression may be an early event in oral carcinogenesis in Taiwan. The Kaplan-Meier analysis showed patients with p27(Kip1)-positive tumors had significantly higher overall survival than those with p27(Kip1)-negative tumors in a total of 63 patients (P=0.015) and 47 patients with areca quid chewing habit (P=0.026). Multivariate analysis showed decreased p27(Kip1) protein expression was an independent significant predictor of poor overall survival in the patients with oral SCCs. These results indicate that p27(Kip1) protein expression may serve as a putative new adjuvant prognostic marker for routine assessment of oral SCC patients.     

Usefulness of repeated direct intratumoral gene transfer using hemagglutinating virus of Japan-liposome method for cytosine deaminase suicide gene therapy

To investigate the feasibility of repeated gene transfection in suicide gene therapy against human solid tumors by a combination of 5- fluorocytosine (5-FC) and its converting enzyme, cytosine deaminase (CD), we repeatedly transfected the yeast CD gene into the human pancreatic cancer cell line BXPC3 using the hemagglutinating virus of Japan-liposome in a new gene transfer method. The in vivo growth of the s.c. transplanted BXPC3 tumor in nude mice given CD-gene transfection was significantly suppressed by i.p. injection of 5-FC when compared with tumors treated with the control vector. Furthermore, the tumor transfected with the CD gene during a 7-day interval was suppressed much more than that of a single transfection. These results suggest that repeated transfection of the suicide gene together with the combination of 5-FC and the yeast CD gene using the hemagglutinating virus of Japan-liposome gene transfer method may be useful for the treatment of human solid tumors, including pancreatic cancer.     

p53 dependence and apoptosis in response to FP treatment with p53-transfected colon cancer cell lines by use of thin layer collagen gel

The combination of 5-fluorouracil (5-FU) plus Cisplatin (CDDP) (FP treatment) possesses synergistic cytotoxicity against colon cancer. The molecular mechanisms by which chemotherapeutic agents induce apoptosis have been clarified by identifying apoptosis-related genes such p53 and bcl-2. We previously established a new experimental technique in which cancer cells are distributed in thin collagen gel as 1 or 2 cell layers. additionally, we evaluated the efficacy and toxicity of FP treatment in the gastric and colon cancer cell lines, and examined the relationship between the response to FP treatment and apoptosis. In these results we reported transfection of normal p53 gene into p53 mutant and analyzed the impact of the p53 gene in a sensitivity test. In this study, we examined induced apoptosis in colon cancer cell lines and the status of p53 expression in response to treatment of HCT116, COLO320, SW480 and DLD1 with 5-FU alone, CDDP alone and FP treatment under flow cytometric analysis. Transfection of SW480 and DLD1 cells was performed to compare the chemosensitivity of naturally occurring mutant-type p53 SW480 and DLD1 cells with neo-transfected SW480 and DLD1 cells and transfected SW480 and DLD1 cells. Appreciable apoptosis was induced in HCT116 and COLO320 (p53 wild-type) but not in SW480 and DLD1 cells (p53 mutant-type). Transfected SW480 and DLD1 cells underwent significantly more apoptosis (p相似文献   

Caspase-induced proteolysis of the cyclin-dependent kinase inhibitor p27Kip1 mediates its anti-apoptotic activity.

The caspase-mediated cleavage of a limited number of cellular proteins is a common feature of apoptotic cell death. This cleavage usually inhibits the function of the target protein or generates peptides that actively contribute to the death process. In the present study, we demonstrate that the cyclin-dependent kinase inhibitor p27Kip1 is cleaved by caspases in human leukemic cells exposed to apoptotic stimuli. We have shown recently that p27Kip1 overexpression delayed leukemic cell death in response to cytotoxic drugs. In transient transfection experiments, the p23 and the p15 N-terminal peptides generated by p27Kip1 proteolysis demonstrate an anti-apoptotic effect similar to that induced by the wild-type protein, whereas cleavage-resistant mutants have lost their protective effect. Moreover, stable transfection of a cleavage-resistant mutant of p27Kip1 sensitizes leukemic cells to drug-induced cell death. Altogether, these results indicate that proteolysis of p27Kip1 triggered by caspases mediates the anti-apoptotic activity of the protein.     

Electrogene transfer of an Epstein-Barr virus-based plasmid replicon vector containing the diphtheria toxin A gene suppresses mammary carcinoma growth in SCID mice

Experimental mammary cancer therapy in mice was conducted using electrogene transfer of a non-viral EBV-based plasmid vector (reduced size of the oriP gene), containing the DT-A gene. Because the EBV-based plasmid vector exhibits high transfer efficiency and strong persistent transgene expression due to autonomous replication in human cells, it is particularly suitable as a tool for cancer gene therapy. In vitro, 79% of MDA-MB231 human mammary carcinoma cells died as a result of the EBV-based vector containing DT-A (pEB-DTA) by 48 h after transfection. DNA synthesis was also significantly decreased as compared to levels with a control vector. In vivo, mammary tumors induced by inoculation of SCID mice with MDA-MB231 cells were subsequently treated by direct injection of pEB-DTA vector or pEB-GFP vector as a control once a week for 5 weeks. After each injection, the tumors were subjected to in vivo electrogene transfer. Significantly reduced tumor volumes were observed for the pEB-DTA group in experimental week 1 and thereafter throughout the study. At necropsy, strong and extent expression of GFP was still observed in tumors receiving pEB-GFP 6 days after the last electrogene transfer. The ratio of histological necrotic area to viable area was significantly increased in the pEB-DTA-treated tumors, where levels of apoptosis were significantly higher than those observed in the pEB-GFP group. DNA synthesis showed a tendency to decrease in the pEB-DTA group but this was not significant. The incidence and multiplicity of lung metastasis were similar between the groups. There was also no difference in the density of microvessels between groups. We therefore conclude that the EBV-based plasmid vector system combined with in vivo electrogene transfer can result in efficient gene transfection and that the non-viral replicon vector containing DT-A suppresses tumor growth due to apoptotic cell death in this model.