Reciprocal activation between PLK1 and Stat3 contributes to survival and proliferation of esophageal cancer cells.

Background & Aims: Aberrant activation of the signal transducer and activator of transcription (Stat)3 and overexpression of polo-like kinase (PLK)1 each have been associated with cancer pathogenesis. The mechanisms and significance of dysregulation of Stat3 and PLK1 in carcinogenesis and cancer progression are unclear. We investigated the relationship between Stat3 and PLK1 and the effects of their dysregulation in esophageal squamous cell carcinoma (ESCC) cells.

Changes in cropland topsoil organic carbon with different fertilizations under long-term agro-ecosystem experiments across mainland China.

Topsoil soil organic carbon (SOC) data were collected from long-term Chinese agro-ecosystem experiments presented in 76 reports with measurements over 1977 and 2006. The data set comprised 481 observations (135 rice paddies and 346 dry croplands) of SOC under different fertilization schemes at 70 experimental sites (28 rice paddies and 42 dry croplands). The data set covered 16 dominant soil types found in croplands across 23 provinces of mainland China.

Survivin stable knockdown by siRNA inhibits tumor cell growth and angiogenesis in breast and cervical cancers.

Increased resistance to apoptosis is a hallmark of many tumor cells. Survivin, a member of IAP family protein, is expressed in many human cancers and plays an important role in protecting cells from apoptosis. Here we show that vector-based small interfering RNAs (siRNA) stably knockdown survivin expression in several cancer cell lines, leading to increased apoptotic rate in response to different proapoptotic stimuli, such as doxorubicin or TNF-alpha.

Stable knockdown of estrogen receptor alpha by vector-based RNA interference suppresses proliferation and enhances apoptosis in breast cancer cells.

Breast cancer, the most common malignancy in women, has a known association with the steroid hormone estrogen. Estrogen receptor alpha (ERalpha) plays an important role in the clinical care of breast cancer patients, both as a prognostic factor and as a therapeutic target. Here, we show that a small interfering RNA (siRNA) against ERalpha downregulates ERalpha expression in human MCF-7 and Bcap-37 breast cancer cells, causing a significant decrease in breast cancer cell proliferation.

[Study on apoptosis of human cervical carcinoma HeLa cells with RNA interference targeting hTERT].

Aim: To study the induction of tumor cell apoptosis by RNA interference-mediated inhibition of the expression of telomerase in cancer cells.

Methods: HeLa cells were transfected with the successfully established siRNA(small interfering RNA) expression vectors targeting hTERT (human telomerase reverse transcriptase). By electronic microscopy, Western blot and FCM (flow cytometry), the apoptosis of HeLa cells was tested.

[Growth inhibitory effects of recombinant granzyme B containing different N-terminal translocating peptides].

Translocating protein and translocating peptides have therapeutic potential against tumors by exposing the cytotoxic domains of toxic proteins to the cell cytosol. The aim of this study is to investigate the effect of N-terminally fused PE translocating peptides on granzyme B (GrBa) activity. PE II-GrBa fusion protein genes were constructed by replacing N-terminal signal and acidic dipeptide sequence of human granzyme B gene with two truncated translocating sequences of Pseudomonas exotoxin A (PE II aa 280-364/358) by recombinant PCR, and then cloned into pIND inducible expression vector.

[Pro-apoptotic efficiencies of three reconstructed human caspase-8 on cervical cancer cell line HeLa].

Background & Objective: Apoptosis is a kind of evolutional high conservative cell death. Transferring high active pro-apoptotic molecules into cancer cells to induce apoptosis is a potential strategy for cancer gene therapy. Based on our previous generation of reconstructed human caspase-8, which can continuously induce apoptosis of cervical cancer cell line HeLa, by reversing its large and small subunits, this study was designed to investigate the pro-apoptotic efficiencies of 3 reconstructed human caspase-8 (Casp8CD, Rev8, and Rev8L) on HeLa cells, and to explore the feasibility of reconstructed human caspase-8 as potential apoptosis-inducing candidates.

[Cloning and subcellular localization of apr-1--a new gene of tumor specific antigen family].

Background & Objective: apr-1 was cloned by improved polymerase chain reaction (PCR)-based subtractive hybridization from all-trans retinoic acid (ATRA)-induced apoptotic leukemia HL-60 cells in 1999. Preliminary results showed that apr-1 might be an apoptosis-related gene (GenBank ID: NM_014061). This study was to explore the background of apr-1 through gene cloning, bioinformatic analysis, and subcellular locating.

[Establishment of Hela cell line inducibly expressing human active granzyme B].

Aim: To construct inducible expression vector for human granzyme B gene and express it in Hela cell line.

Methods: Human active granzyme B gene was obtained by PCR and cloned into the inducible expression vector pIND. The resulting expression vector, together with a helper plasmid pVgRXR, was stably transfected into Hela cells using Lipofectamine 2000.

A caspase-6 and anti-human epidermal growth factor receptor-2 (HER2) antibody chimeric molecule suppresses the growth of HER2-overexpressing tumors.

Clinical studies have suggested that human epidermal growth factor receptor-2 (HER2) provide a useful target for antitumor therapy. We previously described the generation of a chimeric HER2-targeted immunocasp-3 protein. In this study, we extend the repertoire of chimeric proapoptotic proteins with immunocasp-6, a construct that comprises a HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A, and an active caspase-6, which can directly cleave lamin A leading to nucleus damage and inducing programmed cell death.

[Construction, expression and cell growth inhibition of translocating peptide/granzyme B fusion protein gene].

Aim: To investigate the inhibitory effect of translocating peptide/granzyme B fusion protein on cell growth.

Methods: The translocating peptide gene of Pseudomonas exotoxin A (PE) was fused with active granzyme B gene by recombinant PCR to construct PE II-GrBa fusion protein gene. PE II-mGrBa with a mutation of serine to cystein at active center of GrB was used as negative control.

[Construction and identification of recombinant adenovirus vector containing Tet-on adjustable NT3 and BDNF genes].

Aim: To construct Tetracycline(tet) inducible recombinant adenovirus vectors containing human neurotrophin 3(NT3) and brain-derived neurotrophic factor(BDNF) genes, respectively and perform PCR and restrictive enzyme digestion analysis.

Methods: Full length NT3 and BDNF cDNAs were subcloned into pIND vector, followed by being cloned into pTRE-shuttle2 vector. The NT3 and BDNF gene fragments resulted from the pTRE-shuttle2-NT3 and pTRE-shuttle2-BDNF digested with I-Ceu I and PI-Sce I were linked to the linear adeno-X virus DNA.

[Tumor suppression of a constitutively active caspase-3 in the SKBr-3 breast carcinoma xenograft mice model].

Aim: To explore the apoptosis-inducing effect of a constitutively active caspase-3 molecule on human breast carcinoma SKBr-3 cells.

Methods: A revcaspase-3 gene was generated by arranging the coding sequence of the small subunit preceding that of the large one, i.e.

[The cloning and expression of the truncated human bid gene and its pro-apoptotic effect on Hela cells].

Aim: To explore the expression of the truncated bid gene and its pro-apoptotic effect on Hela cells.

Methods: A full-length human bid gene was cloned by RT-PCR and confirmed by sequence analysis. By deleting the 60 amino acids of N-terminal the truncated bid (tbid) gene was obtained and then inserted into the eukaryotic expression vector pIRES2-EGFP.

[Prokaryotic expression and bioactivity identification of Fab against human gamma-seminoprotein].

Aim: To construct the expression vector containing cDNA encoding Fab against human gamma-seminoprotein and express it in E. coli.

Methods: The genes encoding K chain and Fd against gamma-seminoprotein were acquired from pUC19-K and pBluescript KS( M13-)-Fd by restrictive enzyme digestion and then cloned into the expression vector pComb3 to construct recombinant expression vector pComb3-Fab.

[Construction and expression of recombinant antibody/granzyme B containing truncated translocating peptide].

Aim: To construct an eukaryotic expression vector for recombinant antibody/granzyme B gene containing truncated translocating peptide and express it in HER-2(+) SKBR-3 cells and HER-2(-) Hela cells.

Methods: PCR amplication was used to obtain recombinant DNA encoding antibody/granzyme B containing truncated translocating peptide DNA, and then the DNA fragment was cloned into eukaryotic expression vector pCMV-e23sFv-PE40. After being transfected into SKBR-3 cells and Hela cells, the expression of target gene and its effect on cellular morphology were detected by immunocytochemical staining.

[Inducible expression of reconstructed human caspase-6 gene in Hela cells].

Aim: To observe the pro-apoptotic effect of RCasp-6 gene in Hela cells.

Methods: RCasp-6 gene was amplified by PCR and cloned into the pIND vector. Hela cells were transfected with pIND-RCasp-6 and then inducced with ecdyson analogue.

[Construction of single-chain Fv Gene of Anti-HBsAg and its expression in COS-7 cells].

Aim: To subclone the V(L) gene and V(H) genes of anti-HBsAg and construct the single-chain Fv gene and to analyse the expression of the constructed gene in COS-7 cells.

Methods: A set of oligonucleotide primers were designed and used to amplify the V(H) and V(L) genes from Fab antibodies screened from phage antibody library. The products were cloned into pUC19 vector and their sequences were analysed.

[Expression of a secreted fusion protein consisting of anti-HER2 antibody and reversed caspase-3 and its suppression on growth of SKOV3 cells].

Aim: To investigate the killing effect on SKOV3 cells by fusion protein HER2-specific antibody-reversed caspase-3 in the secreted form.

Methods: The reversed human caspase-3 gene was subcloned into pCMV-e23scFv-PEII-PEIII to construct recombinant eukaryotic expression vector pCMV-e23scFv-PEII-revcasp-3 and transfect Jurkat cells. The secreted expression of the in culture supernatant of transfected Jurkat cells was detected by ELISA.

[Effect of the truncated human apoptosis-inducing factor expression on apoptosis of HeLa cells].

Aim: To observe the expression of the truncated human apoptosis-inducing factor (AIF) gene and its apoptosis-inducing effect on HeLa cells.

Methods: Full-length human AIF gene was cloned by RT-PCR, then the truncated AIF gene was constructed by deleting the N-terminal mitochondrial location sequence (MLS), and inserted into the EGFP co-expression vector pIRES2-EGFP. After being transfected into HeLa cells with Lipofectamin, the expression of the truncated AIF gene and its effect on HeLa cells morphology and growth condition were detected by fluorescence microscope, immunohistochemical staining, indirect fluoroimmunoassay and electron microscope analysis.

Secreted antibody/granzyme B fusion protein stimulates selective killing of HER2-overexpressing tumor cells.

Targeted cell killing is required for effective treatment of cancers. We previously described the generation of a chimeric immunocasp-3 protein and its potent selective antitumor activity (Jia, L. T.

[Targeted tumor suppression by a secreted fusion protein consisting of anti- erbB2 antibody and reversed caspase-3 to SKBr3 cells].

Objective: To investigate the targeted killing effect to SKBr3 cells due to the expression of a secreted fusion protein consisting of anti-erbB2 antibody and reversed caspase-3.

Methods: A recombinant plasmid pCMV-e23scFv-PEII-revcasp 3 was constructed by subcloning reversed caspase-3 gene to the downstream of anti-erbB2 antibody and transfected into Jurkat cells. The cell lines which secreted expressing fusion protein stably were selected.

Specific tumoricidal activity of a secreted proapoptotic protein consisting of HER2 antibody and constitutively active caspase-3.

In this study, a novel approach to antitumor therapy was devised by generating a chimeric tumor-targeted killer protein, referred to as immunocasp-3, that comprises a single-chain anti-erbB2/HER2 antibody with a NH(2)-terminal signal sequence, a Pseudomonas exotoxin A translocation domain, and a constitutively active caspase-3 molecule. In principle, cells transfected with the immunocasp-3 gene would express and secrete the chimeric protein, which then binds to HER2-overexpressing tumor cells. Subsequent cleavage of the constitutively active capase-3 domain from the immunocasp-3 molecule and its release from internalized vesicles would lead to apoptotic tumor cell death.

Inducible Expression of bcl-2 Antisense RNA Promotes Apoptosis of Human Gastric Cancer Cell Line.

A lot of molecular biological techniques was used to observe the effect of bcl-2 antisence RNA on human gastric cancer cell line SGC7901 and to detect the inducibility and promotion of MT-II promoter. The results showed that MT-II promoter could be activated by the induction with 160 &mgr;mol/L Zn(2+) and the expression of Bcl-2 plays an important role in apoptosis of SGC7901 cells.

Upregulation of bax Gene Expression Promotes Paclitaxel-induced Apoptosis in Esophageal Carcinoma Cells.

An inducible mammalian expression vector of bax gene was constructed and the control ability of metallothionein II promoter in esophageal carcinoma cell line was systematically identified with luciferase report gene. After the transfection of it into human esophageal carcinoma cell line Eca109, Bax protein expression was analyzed by immunolcytochemical method. Paclitaxel-induced apoptosis was determined by TUNEL assay, DNA ladder assay and flow cytometry.