Ki-67 Promoter Research Articles

TS-2021, a third-generation oncolytic adenovirus that carried Ki67 promoter, TGF-β2 5'UTR, and IL-15 against experimental glioblastoma.

Oncolytic virotherapy is a promising therapeutic approach for glioblastoma (GBM) treatment, although the outcomes are partially satisfactory. Hence, more effective strategies are needed urgently to modify therapeutic viruses to enhance their efficiency and safety in killing tumor cells and improve the survival rate of GBM patients. This study generated a new-generation oncolytic adenovirus Ad5 KT-E1A-IL-15 (TS-2021) and evaluated its antitumor efficacy. Ex vivo analyses revealed Ki67 and TGF-β2 co-localized in GBM cells. In addition, TS-2021 selectively replicated in GBM cells, which was dependent on the expression of Ki67 and TGF-β2. The immunocompetent mice model of GBM demonstrated the in vivo efficacy of TS-2021 by inhibiting tumor growth and improving survival proficiently. Notably, TS-2021 effectively reduced MMP3 expression by inactivating the MKK4/JNK pathway, thereby reducing tumor invasiveness. Altogether, the findings of the present study highlight that TS-2021 can effectively target GBM cells expressing high levels of Ki67 and TGF-β2, exerting potent antitumor effects. Additionally, it can improve efficacy and suppress tumor invasiveness by inhibiting the MKK4/JNK/MMP3 pathway. Thus our study demonstrates the efficiency of the novel TS-2021 in the mouse model and provides a potential therapeutic option for patients with GBM.

Recombinant oncolytic adenovirus armed with CCL5, IL-12, and IFN-γ promotes CAR-T infiltration and proliferation in vivo to eradicate local and distal tumors

The efficacy of chimeric antigen receptor T (CAR-T) cells for solid tumors remains unsatisfactory due to the limited tumor infiltration and immunosuppressive microenvironment. To overcome these limitations, the genetically engineered recombinant oncolytic adenoviruses (OAVs) that conditionally replicate in tumor cells were developed to modify the tumor microenvironment (TME) to facilitate CAR-T-mediated tumor eradication. Here in the present study, a novel recombinant OAV carrying CCL5, IL12, and IFN-γ controlled by Ki67 promoter was constructed (named AdKi67-C3). The antitumor activity of AdKi67-C3 was tested in vitro and in vivo by using mono administration or combing with CAR-T cells targeting B7H3. It proved that CCL5 expressed by AdKi67-C3 indeed induced more CAR-T migration in vitro and CAR-T infiltration in tumor mass in vivo. Meanwhile, cytokines of IFN-γ and IL12 secreted by AdKi67-C3-infected tumor cells significantly promoted proliferation and persistence of CAR-T cells in vitro and in vivo. In tumor-bearing xenograft mouse models of kidney, prostate or pancreatic cancer, local pretreatment with AdKi67-C3 dramatically enhanced CAR-T cell efficacy and eliminated local and distant tumors. More importantly, mice achieving complete tumor regression resisted to re-challenge with the same tumor cells, suggesting establishment of long-term antitumor immune response. Therefore, OAVs armored with cytokines could be developed as a bioenhancer to defeat the immunosuppressive microenvironment and improve therapeutic efficacy of CAR-T in solid tumors.

A highly sensitive strategy for monitoring real-time proliferation of targeted cell types in vivo

Cell proliferation processes play pivotal roles in timely adaptation to many biological situations. Herein, we establish a highly sensitive and simple strategy by which time-series showing the proliferation of a targeted cell type can be quantitatively monitored in vivo in the same individuals. We generate mice expressing a secreted type of luciferase only in cells producing Cre under the control of the Ki67 promoter. Crossing these with tissue-specific Cre-expressing mice allows us to monitor the proliferation time course of pancreatic β-cells, which are few in number and weakly proliferative, by measuring plasma luciferase activity. Physiological time courses, during obesity development, pregnancy and juvenile growth, as well as diurnal variation, of β-cell proliferation, are clearly detected. Moreover, this strategy can be utilized for highly sensitive ex vivo screening for proliferative factors for targeted cells. Thus, these technologies may contribute to advancements in broad areas of biological and medical research.

Mesenchymal stem cells loaded with Ad5-Ki67/IL-15 enhance oncolytic adenovirotherapy in experimental glioblastoma

The conventional treatment strategy for glioblastoma multiforme (GBM) is surgical resection followed by radiotherapy and chemotherapy. Oncolytic adenovirotherapy is a promising alternative to conventional treatment. It provides a strategic combination of direct tumor-specific cell lysis and antitumor immune promotion. Despite advances in oncolytic adenovirotherapy, limitations remain, including the host's antiviral immune response and insufficient viral infiltration into the tumor. Mesenchymal stem cells (MSCs) have emerged as innovative vehicles due to their ability to home to tumors and protect oncolytic adenovirus (oAd) from the host antiviral immune system. We developed an Ad5-Ki67/IL-15 driven by the Ki67 promoter and armed with IL-15. Using this construction, viral replication is related to Ki67 expression in GBM cells. Thus, MSCs with background Ki67 expression can help deliver higher levels of oncolytic viruses and can strike a balance between viral load and cell viability. Using in vitro assay, MSCs loaded with Ad5-Ki67/IL-15 (MSC-Ad5) were shown to exert anti-glioblastoma efficacy. Compared to previous attempts at direct intratumoral injection of high doses of viruses, MSCs loaded with lower doses of viruses exerted stronger therapeutic effects and promoted macrophage/microglia infiltration in a Vivo model. Collectively, our results suggest that the use of MSCs as vehicles of oAd is a promising strategy and deserves further investigation for the treatment of GBM.

Optimization of Thymidine Kinase-Based Safety Switch for Neural Cell Therapy.

Cell therapies based on pluripotent stem cells (PSC), have opened new therapeutic strategies for neurodegenerative diseases. However, insufficiently differentiated PSC can lead to tumor formation. Ideally, safety switch therapies should selectively kill proliferative transplant cells while preserving post-mitotic neurons. In this study, we evaluated the potential of nucleoside analogs and thymidine kinase-based suicide genes. Among tested thymidine kinase variants, the humanized SR39 (SR39h) variant rendered cells most sensitive to suicide induction. Unexpectedly, post-mitotic neurons with ubiquitous SR39h expression were killed by ganciclovir, but were spared when SR39h was expressed under the control of the cell cycle-dependent Ki67 promoter. The efficacy of six different nucleoside analogs to induce cell death was then evaluated. Penciclovir (PCV) showed the most interesting properties with an efficiency comparable to ganciclovir (GCV), but low toxicity. We tested three nucleoside analogs in vivo: at concentrations of 40 mg/kg/day, PCV and GCV prevented tumor formation, while acyclovir (ACV) did not. In summary, SR39h under the control of a cell cycle-dependent promoter appears most efficient and selective as safety switch for neural transplants. In this setting, PCV and GCV are efficient inducers of cell death. Because of its low toxicity, PCV might become a preferred alternative to GCV.

Diagnostic roles of proliferative markers in pathological Grade of T1 Urothelial Bladder Cancer.

The stage T1 urothelial bladder cancer (T1 UBC) tumor grade classification is important for prognosis and clinical management. However, the reproducibility of this two-grade classification system is limited in regards to pathological diagnosis, and there is lack of ideal, objective and easily detected markers for pathological diagnosis. In our study, bladder urothelial lesions from a total of 124 patients diagnosed pathologically after transurethral resection of the bladder tumor (TURBT) were collected, including non-cancerous lesions from 33 patients and lesions from 91 T1 UBC patients. A series of previous studies have suggested some common and valuable factors in the diagnosis and prognosis of UBC, but there are still some controversial factors, such as the mitotic figure (MF) of tumor cell, cell proliferation index Ki-67, graded differentiation marker CK20, P53, P504S and carcinogenesis associated telomerase reverse transcriptase (TERT) promoter mutations. The purpose of this study was to evaluate the value of these factors in the pathological grading diagnosis of T1 UBC. The results showed that gender, lesion size, mitotic index (MI), CK20, P53, Ki-67, P504S and TERT promoter hot spot mutations (C228T and C250T) were correlated with T1 UBC diagnosis (P<0.05). The MI, Ki-67 and P504S were correlated with the pathological grade of T1 UBC (P<0.05). Logistic regression analysis showed that the MI and Ki-67 were independent risk factors for high-grade (HG) of T1 UBC (P<0.05). The combined detection of the MI, Ki-67 and P504S in a multivariate diagnostic model improved the diagnostic accuracy of assigning the T1 UBC pathological grade (AUC=0.904, 95%CI: 0.824~0.956, P<0.05). In conclusion, MI and Ki-67, as important markers of histopathology and cell proliferation, can be easily measured and have good reproducibility. These markers may be meaningful parameters for assigning the pathological grade of UBC.

Efficacy of a novel double-controlled oncolytic adenovirus driven by the Ki67 core promoter and armed with IL-15 against glioblastoma cells

BackgroundGlioblastoma (GBM) is an immunosuppressive, highly vascular and devastating malignant brain tumor. Even with progressive combination treatment that includes surgery, radiotherapy, and chemotherapy, the prognosis for GBM patients is still extremely poor. Oncolytic adenovirus (OAd) can specifically replicate in GBM cells, permitting the rapid copy of the therapeutic genes it carries. Moreover, E1A is an essential gene in adenoviral replication and is the first gene expressed upon viral infection. E1A expression can be regulated by the Ki67 promoter, while the CMV promoter drives therapeutic gene expression. However, the efficacy of a double-controlled OAd driven by the Ki67 core promoter and armed with IL-15 against GBM cells has not been investigated.MethodsFluorescence microscopy was performed to evaluate infection ability in the viruses. Cell viability was detected by CCK-8 assay. Levels of cytokines in different supernatants were determined by ELISA, and IL-15 gene expression was measured by RT-PCR. Angiogenic capacity was analyzed by tube formation assay.ResultsWe successfully constructed a double-controlled oncolytic adenovirus driven by the Ki67 core promoter and armed with IL-15 that selectively infected and killed GBM cells while sparing normal cells. The adenoviruses prime IL-15 gene expression to significantly enhance anti-GBM efficacy through effective activation of microglial cells. Moreover, OAd not only directly inhibits angiogenesis but exhibits potent antiangiogenic capacity mediated by the reduction of VEGF secretion.ConclusionsThese results provide new insight into the effects of a novel double-controlled OAd driven by the Ki67 core promoter and armed with IL-15 in glioblastoma treatment, which may help in the development of novel therapies in solid tumors.

Antitumor effect of hypoxia response element chimeric tumor specific promoter regulated oncolytic adenovirus on renal cell carcinoma

Objective To investigate the effect of chimeric promoter composed of hypoxia response element (HRE) and tumor specific cell proliferation-associated nuclear antigen (Ki-67) promoter on the replication ability of oncolytic adenovirus and its cytotoxic activity under hypoxic conditions. Methods The HRE sequence, as an enhancer, was inserted upstream of the Ki-67 promoter and, at the same time carried the reporter gene enhanced green fluorescent protein (EGFP) to construct oncolytic adenovirus Ad-HRE-Ki-67-EGFP, and the titer of adenoviruses was measured by the tissue culture infective dose (TCID50). Normal renal epithelial cells HK-2 and renal cancer cells OSRC-2 and ACHN were infected with two kinds of viruses under normoxic (21% O2) or hypoxic (1% O2) conditions respectively for 24 h, and the virus tumor-targeting was detected by fluorescence microscopy and flow cytometry. Western blotting was performed to detect the expression of hypoxia-related protein hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF) and the expression of E1A protein. Cell counting kit-8 (CCK-8) assay was used to detect the cytotoxity of virus on cells. Results The recombinant oncolytic virus Ad-HRE-Ki67-EGFP was successfully constructed. Under normoxic conditions, the efficiency of ACHN and OSRC-2 infection by chimeric HRE oncolytic adenovirus in renal cancer cells was (47.5±2.6)% and (35.2±3.4)%, while that in hypoxic conditions was (86.4±3.4)% and (73.5±2.3)%, indicating that the infection efficiency was higher under hypoxic conditions (ACHN: t=15.760; OSRC-2: t=16.190; P<0.01); Normal renal tubular epithelial cells HK-2 were infected with Ad-Ki-67-EGFP or Ad-HRE-Ki-67-EGFP under normoxic conditions. The infection efficiency was (22.4±3.1)% and (22.9±2.2)% respectively, compared with that of ACHN and OSRC-2 cells [ACHN: (68.4±3.3)% and (72.1±2.9)%; OSRC-2: (56.1±3.1)% and (61.4±2.3)%], the difference was statistically significant (Ad-Ki-67-EGFP: t=17.650, 13.390; Ad-HRE-Ki-67-EGFP: t=23.680, 21.430, P<0.01), indicating that the two adenoviruses have a targeting effect on renal cancer cells. Western blotting data showed that the expression of HIF-1α, VEGF and p53 protein were increased under hypoxic conditions and was time-dependent, indicating that hypoxia can regulate the transcription and translation of HIF-1α, VEGF and p53 proteins. Under hypoxic conditions, the expression level of E1A protein in OSRC-2 and ACHN infected with Ad-HRE-Ki67-EGFP was significantly higher than that after Ad-Ki67-EGFP infection. It indicated that HRE can enhance the replication ability of virus under hypoxic conditions. CCK-8 results showed that when the multiple infection (MOI) values were 20, 100, the survival rate of the Ad-HRE-Ki-67-EGFP-normoxia virus group in ACHN and OSRC-2 cells was (73.4±2.0)%, (56.4±1.5)% and (79.9±1.8)%, (61.3±2.7)%, the Ad-HRE-Ki-67-EGFP-hypoxia virus group was (63.1±2.0)%, (31.6±2.1)% and (68.1±2.6)%, (35.8±3.1)%, the Ad-HRE-Ki-67-EGFP-hypoxia virus group showed a stronger proliferation inhibitory effect [MOI (20): t=6.328, 6.441; MOI (100): t=16.410, 10.790, P<0.01]. Conclusion Under hypoxic conditions, chimeric HRE sequence recombinant oncolytic adenovirus has stronger replication ability and cell killing effect in renal carcinoma cells. Key words: Renal cell carcinoma; Oncolytic adenovirus; Hypoxia response element; Hypoxia

Reciprocal Role Of DNA Methylation And Sp1 Binding In Ki-67 Gene Transcription.

PurposeDNA methylation plays major regulatory roles in gene transcription. Our previous studies confirmed that Ki-67 promoter is hypomethylated and Sp1 is a transcriptional activator of Ki-67 gene in cancer cells. However, whether Sp1-mediated transcriptional activation of Ki-67 is related to its methylation has not been studied yet.Materials and methodsIn this study, we confirmed that methylated CpG binding protein 2 (MBD2) binding to methylated DNA hindered the binding of Sp1 to Ki-67 promoter and then repressed Ki-67 transcription through chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qRT-PCR). Co-immunoprecipitation (Co-IP), ChIP, methylation-specific PCR (MS-PCR) and Western blot were utilized to analyze the effects of Sp1 binding to Ki-67 promoter on its methylation status.ResultsLess DNA methyltransferase 1 (DNMT1) bound to the Ki-67 promoter in MKN45 cells than in HK-2 cells. Histone acetyltransferase p300 that was recruited by Sp1 to Ki-67 promoter could attenuate the methylation level of Ki-67 promoter. Furthermore, higher expression of Sp1 and Ki-67 was related to the overall survival (OS), first progression (FP) and post-progression survival (PPS) in gastric cancer by scrutinizing bioinformatics datasets.ConclusionTaken together, our findings suggested that hypomethylation of Ki-67 promoter enhanced the binding of Sp1, which in turn maintained hypomethylation of promoter, leading to increase Ki-67 expression in cancer cells. Sp1 and Ki-67 could act promising prognostic biomarkers for clinical diagnosis and treatment of cancer.

Ki67 targeted strategies for cancer therapy.

Ki67 is a well-known proliferation marker for the evaluation of cell proliferation. Numerous studies have indicated that Ki67 index independently predicts cancer progression. Moreover, because Ki67 is highly expressed in malignant cells but almost could not be detected in normal cells, it has become a promising target for cancer therapy. In this review, we summarize recent advances in Ki67 targeted cancer therapy. In particular, we highlight recent development on the exploitation of Ki67 promoter to drive the expression of siRNAs or therapeutic genes in cancer cells specifically. The use of Ki67 as an attractive target opens a new avenue for cancer therapy.

Novel oncolytic adenovirus sensitizes renal cell carcinoma cells to radiotherapy via mitochondrial apoptotic cell death.

Renal cell carcinoma is the most frequent kidney malignancy and patients with metastatic disease have a poor prognosis. Suppressed apoptosis and marked invasiveness are distinctive features of renal cell carcinoma. In the present study, a dual‑regulated oncolytic adenovirus expressing the interluekin (IL)‑24 gene (Ki67‑ZD55‑IL‑24) was constructed utilizing the Ki67 promoter to replace the native viral promoter of the E1A gene. Whether the combination of Ki67‑ZD55‑IL‑24‑mediated gene virotherapy and radiotherapy produced increased cytotoxicity in renal cell carcinoma cells via mitochondrial apoptotic cell death was investigated. The data indicated that this novel strategy has the potential to be further developed into an effective approach to treat renal cell carcinoma. The results showed that the combination of Ki67‑ZD55‑IL‑24 and radiotherapy significantly enhanced anti‑tumour activity via increasing the induction of apoptosis in melanoma cells compared with the other agents.

Tyrosine phosphorylation of β-catenin affects its subcellular localization and transcriptional activity of β-catenin in Hela and Bcap-37 cells

In order to investigate the relationship between tyrosine phosphorylation of β-catenin and transcriptional activity of β-catenin in Hela and Bcap-37 cells, genistein (a tyrosine kinase inhibitor) was used to inhibit tyrosine phosphorylation in cells. Our results showed the total β-catenin protein levels were mainly equal in Hela, Bcap-37 and HK-2 cells, β-catenin was mainly present in nucleus in Hela and Bcap-37cells, while in HK-2 cell β-catenin was mainly located in cytoplasm. Genistein could inhibit tyrosine phosphorylation of β-catenin and downregulate nuclear β-catenin expression in Hela and Bcap-37 cells. In addition, genistein suppressed Ki-67 promoter activity and Ki-67 protein level, thus promoted cell apoptosis. Furthermore, β-catenin could increase the Ki-67 promoter activity in Hela and Bcap-37 cells. From these findings we conclude that tyrosine phosphorylation of β-catenin can regulate the cellular distribution of β-catenin and affect the transcriptional activity of β-catenin.

A live‐cell reporter that differentiates between quiescent and cycling cells (1148.14)

The transition of a proliferating cell into a state of cell quiescence is a key step in organogenesis and tissue homeostasis. It is currently difficult to distinguish between quiescent cells and those that have entered the cell cycle. This study’s objective was to generate a fluorescent‐based genetically encoded reporter capable of differentiating between arrested and actively cycling cells. The Ki67 promoter (Ki67p) was previously shown in our lab to selectively label cells that have entered the cell cycle. However, using Ki67p to drive GFP as a reporter gene results in fluorescent daughter cells due to GFP’s long half‐life (~26h). We hypothesized using the Ki67p to drive expression of the Fluorescent Ubiquitination‐based Cell Cycle Indicator (FUCCI) reporters would enable indirect detection of quiescent (G0) cells and cells in G1/S and S/G2/M. The existing FUCCI reporters employ a constitutively active promoter, thereby preventing discrimination between cells in G0 and cells in G1. To overcome both reporters’ limitations we generated two lentiviral vector constructs with Ki67p upstream of mcherry‐hCdt1(30‐120) and mAG‐hGem(1‐110). We then generated stable double transgenic cell lines (HT1080 and COS cells) that express mCherry‐hCdt1(30‐120) and mAG‐hGem(1‐110) under control of Ki67p. Fluorescence time‐lapse imaging of Ki67p‐FUCCI cells indicates that low‐density plating induces G0 cell cycle withdrawal and concurrent attenuation of Ki67‐FUCCI reporter activity. Low density compared to high density plating increased the number of Ki67p‐FUCCI− HT1080 cells (44% vs. 5%, P&lt;0.05) indicating G0 entry. Moreover, serum starvation induced G1 arrest (91% Ki67p‐mCherry+ without serum vs. 47% with serum, P&lt;0.01). These reporters will enable studies designed to segregate between quiescent and actively proliferating cells in each of the major phases of the cell cycle in living tissue and in real‐time, facilitating studies in stem cell and cancer biology.Grant Funding Source: NIGMS 2‐R25‐GM‐083275, AHA 10SDG2630130

A dual-regulated oncolytic adenovirus expressing interleukin-24 sensitizes melanoma cells to temozolomide via the induction of apoptosis

Malignant melanoma is one of the most lethal and aggressive human malignancies. Suppressed apoptosis and extraordinary invasiveness are the distinctive features that contribute to malignant melanoma. The alkylating agent temozolomide (TMZ) is one of the most effective single chemotherapeutic agents for patients with malignant melanoma, but resistance develops quickly and with high frequency. We constructed a dual-regulated oncolytic adenovirus expressing interleukin 24 (IL-24) gene (Ki67-ZD55-IL-24) by utilizing the Ki67 promoter to replace the native viral promoter of E1A gene. We investigated whether a combination of Ki67-ZD55-IL-24-mediated gene virotherapy and chemotherapy using TMZ produces increased cytotoxicity against human melanoma cells via the induction of apoptosis. Our data indicate that this novel strategy thus holds promising potentials for further developing an effective approach to treat malignant melanoma.

Construction of conditionally replicative adenovirus expressing proliferation cell nuclear antigen siRNA, human telomerase reverse transcriptase siRNA with proliferation cell nuclear antigen promoter regulation

Objective To construct conditionally replicative adenovirus (CRAds) expressing proliferation cell nuclear antigen (Ki-67) siRNA and human telomerase reverse transcriptase (hTERT) siRNA with Ki-67 promoter regulation.Methods (1) The hTERT siRNA sequence was synthesized and cloned into pSilencer3.1,resulting in the plasmid pShTERT siRNA.(2) Both pZKi-67 and pZD55-Ki-67 siRNA plasmid were digested with Xba I/EcoRV,the fragment containing Ki-67 promoter and the fragment containing Ki-67siRNA were connected,resulting in the plasmid pZKi-67-ZD-Ki-67siRNA.(3) The hTERTsiRNA was amplified from pShTERT siRNA and cloned into pZKi-67-ZD-Ki-67siRNA,resulting in the plasmid pZKi-67-ZD-Ki-67siRNA-hTERT siRNA.(4) pZKi-67-ZD-Ki-67 siRNA-hTERT siRNA was mixed with adenovirus packaging plasmid pBHGE3 and then co-transfected into HEK293 cells.The viral plaques appeared 9-12 days after infection.To verify the sequences of ZKi-67-ZD-Ki-67siRNA-hTERTsiRNA,DNA was extracted from purified viruses and analysed by polymerase chain reaction (PCR).Results Confirmed by PCR assay and sequence assay,a dual-RNA interferenced oncolytic adenovirus,which carried the Ki-67-siRNA and hTERT-siRNA and synchronously was regulated by both Ki-67 promoter was constructed.Conclusion We successfully constructed a dual-RNA interferenced oncolytic adenovirus and provide a new platform for cancer gene therapy. Key words: Conditionally replicative adenovirus; Proliferation cell nuclear antigen promoter; Proliferation cell nuclear antigen; Human telomerase reverse transcriptase; RNA interference

IRF1 suppresses Ki-67 promoter activity through interfering with Sp1 activation

Interferon regulatory factor 1 (IRF1) shows tumor-suppressor activity by suppressing proliferation of cancer cells. To exert its anti-proliferative effects, this factor must ultimately control transcription of several key genes that regulate cell cycle progression. Here, we showed that Ki-67 gene is a novel proliferation-related downstream target of IRF1. IRF1 repressed Ki-67 gene transcription in a dose-dependent manner in human Ketr-3 and 786-O renal carcinoma cells. We previously cloned the Ki-67 core promoter which contained two functional Sp1 binding sites. Mutation of the two Sp1 binding sites abrogated Sp1-dependent enhancement of Ki-67 promoter activity. Forced elevation of IRF1 decreased endogenous Sp1 protein level. However, there was no effect on Sp1 mRNA level after transfected with IRF1. Our findings establish a casual series of events that connect anti-proliferative effects of IRF1 with the Ki-67 gene, which encodes a key regulator of the G1/S phase transition. It suggests that the inhibitory effect on Ki-67 gene expression mediated by decreasing level of Sp1 protein might be a novel function of the anti-tumor activity of IRF1.

Identification of the effect of DNA methylation on Ki-67 promoter activity

Objective To investigate the effect of DNA methylation on Ki-67 promoter activity.Methods Ki-67 promoter fragments were in vitro methylated using M. Hpall, M. HhaI or M. SssI methylases respectively in the presence of S-adenosyl methionine. Methylated promoter fragments were then cloned into the firefly luciferase reporter vector pGL3-Basic, these methylated promoter luciferase reporter constructs and unmethylated Ki-67 promoter constructs were used to transfect normal cell lines ( HL-7702,HUVEC) and cancer cell lines ( A549,EJ, Hela, Kert-3,SGC-7901 ). Promoter activities were determined by Dual Luciferase Reporter Assay System. Results Transient transfection of cultured cell lines with these methylated promoter reporter plasmids showed that dense methylation with M. SssI methylase abolished the promoter activity whereas methylation with HpaII and HhaI methylase resulted in partial reduction of the basal activity of the promoter compared to the unmethylated controls. The rank according to promoter activity reduction from high to low was M. SssI group,M. HhaI group and M. HpaII group respectively. Conclusion Hypermethylation of Ki-67 promoter exerted an inhibitive effect on its transcriptional activity, which was dependent on the density and loci of DNA methylation. Key words: DNA methylation; Ki-67 gene; Promoter; Transcriptional activity

P53 regulates Ki-67 promoter activity through p53- and Sp1-dependent manner in HeLa cells

The expression of the human Ki-67 protein, which is strictly associated with cell proliferation, is regulated by a variety of cellular mediators. In this study, we studied the effects of p53 on Ki-67 promoter in HeLa cells using luciferase reporter assay. The results showed that: (1) p53 inhibited Ki-67 promoter activity in a dose-dependent manner, (2) the p53-binding motifs mediated part of the transcriptional repression of Ki-67 promoter through a sequence-specific interaction with p53, (3) p53 was able to repress the Sp1-stimulated Ki-67 promoter activity, and (4) the Sp1-binding sites were responsible for the p53-mediated transcriptional repression of Ki-67 promoter. In conclusion, p53 inhibited Ki-67 promoter activity via p53- and Sp1-dependent pathways, and the interaction between p53 and Sp1 might be involved in the transcriptional regulatory mechanisms.

The effect of methylated oligonucleotide targeting Ki-67 gene in human 786-0 renal carcinoma cells

To investigate the effect of methylated oligonucleotide (MON) targeting Ki-67 promoter on the expression of Ki-67 gene and the proliferation and apoptosis of the human 786-0 renal carcinoma cells, human 786-0 cells were transfected with MON. The activity of Ki-67 promoter was detected by dual-luciferase reporter assay system. Among the five methylated oligonucleotides (MON(1)-MON(5)), MON(4) is the best excellent one in the inhibition of the Ki-67 promoter activity. The activity of Ki-67 promoter is decreased to 77.88% in 40-nM group, 50.07% in 80-nM group, 35.63% in 120-nM group, 26.09% in 160-nM group, and 16.98% in 200-nM group compared with 0-nM group. The activity of Ki-67 promoter in MON group is decreased to 61.96% at 8 h, 48.93% at 12 h, 15.97% at 24 h, 26.00% at 36 h, 35.01% at 48 h, 46.08% at 72 h, and 66.12% at 96 h compared with pGLBK235 group. These results show that the effect of MON is time- and dose-dependent. The activity of Ki-67 in MON group is decreased to 16.73% compared with pGLBK235 group, while the control groups have no significant difference. The expression of Ki-67 gene in 786-0 cells was detected by RT-PCR and immunohistochemistry, respectively. The expression of Ki-67 mRNA is decreased to 61.04% and that of Ki-67 protein is decreased to 32.07% in MON group compared with the blank group. The proliferation of 786-0 cells was determined by WST-8. The cell proliferation in MON group is decreased to 61.02% at 24 h, 73.78% at 48 h, 79.72% at 72 h, and 91.53% at 96 h compared with the blank group. The cell apoptosis was measured by annexin V and propidium iodide. The number of apoptosis cells in MON group is 2.42 times of that in the blank group at earlier period and 2.57 times at mid-anaphase. We detected the effect of MON on the expression of bax and p53 by Western blot. Compared with the blank group, the expression of bax protein in MON group is increased by 66.12%, while the expression of p53 is decreased to 67.31%. Our study demonstrates that the methylated oligonucleotide targeting Ki-67 promoter has a remarkable effect on the inhibition of Ki-67 expression and the proliferation of the human 786-0 renal carcinoma cells and can induce apoptosis of the 786-0 cells.

Sp1-siRNA and mithramycin effectively inhibit the Ki-67 gene transcription

Objective To investigate the regulation of Ki-67 gene transcription by the inhibition of Sp1 protein with Sp1 small interfering RNAs (Sp1-siRNA) and Sp1 binding sites competitor mithramycin.Methods Sp1 -siRNA and mithramycin ( a drug known to block activity of Sp1 family members by binding to GC-rich regions) were used to down-regulate Ki-67 gene transcription in Hela cells, OS-RC-2 cells and A549 cells. Immunoblotting assay was performed to detect the change of Sp1 protein in Hela cells. Immunohistochemistry was used to detect the change of Ki-67 protein in Hela cells, OS-RC-2 cells and A549cells. The dual-luciferase reporter assay system was used to identify the difference of transcriptional activity between Ki-67 promoter plasmid pGLBK23 terated by Sp1-siRNA or mithramycin and plasmid pGLBK23.Results After treated by Sp1-siRNA, Sp1 protein in Hela cells was increased significantly. Sp1-siRNA and mithramycin significantly decreased the Ki-67 protein in Hela cells, OS-RC-2 cells and A549 cells.The transcription activities of Ki-67 promoter were reduced to 45.9%, 64. 5% and 46.0% by Sp1-siRNA in Hela cells, OS-RC-2 cells and A549 cells, respectively. The transcriptional activities of Ki-67 promoter were affected by mithramycin in a dose-dependent manner. Mithramycin( 500 nmol/L) inhibited the Ki-67gene transcription to 34. 7%, 44. 2% and 29. 8% in Hela cells, OS-RC-2 cells and A549 cells, respectively. Conclusion Inhibition of Sp1 protein by Sp1-siRNA and mithramycin obviously decrease Ki-67gene transcription. Key words: Ki-67 promoter; Transcription factors Sp1; siRNA; Mithramycin