Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested with Xho I, and less than 400 bp cDNA fragment was removed by Sephacryl-S400 spin column, the remaining were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E. coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the NB4 cell line cDNA library consisting of 1.65 x 10(6) recombinant bacteriophages was constructed with the recombinant ratio of 99.6%. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.
Bacteriophages/genetics ; DNA, Complementary/*biosynthesis ; *DNA, Neoplasm/biosynthesis ; DNA, Recombinant/biosynthesis ; *Gene Library ; Genetic Vectors ; Leukemia, Promyelocytic, Acute/*genetics ; Leukemia, Promyelocytic, Acute/metabolism ; Leukemia, Promyelocytic, Acute/pathology ; RNA-Directed DNA Polymerase/metabolism ; Transcription, Genetic/genetics

Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested with Xho I, and less than 400 bp cDNA fragment was removed by Sephacryl-S400 spin column, the remaining were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E. coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the NB4 cell line cDNA library consisting of 1.65 x 10(6) recombinant bacteriophages was constructed with the recombinant ratio of 99.6%. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.
Bacteriophages ; genetics ; DNA, Complementary ; biosynthesis ; DNA, Neoplasm ; biosynthesis ; DNA, Recombinant ; biosynthesis ; Gene Library ; Genetic Vectors ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; metabolism ; pathology ; RNA-Directed DNA Polymerase ; metabolism ; Transcription, Genetic ; genetics

OBJECTIVETo explore the correlation of chemotherapy efficacy in tongue squamous cell carcinoma(SCC) with expression level of P-glycoprotein(Pgp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathiones-tranferase (GST-pi), DNA topo-isomerase II alpha (Topo II alpha).

METHODSThe expression patterns of Pgp, MRP, LRP, GST-pi and Topo II alpha in 40 patients (pre and post-chemotherapy, respectively) with tongue SCC were examined by immunohistochemically labelled streptavidin bioein method (LsAB).

RESULTSThe expression ratios of Pgp, MRP, LRP, GST-pi and Topo II alpha in pre-chemotherapy cases were 47.5%, 50%, 35%, 45%, 82.5%, respectively. No relations between expression of Pgp, MRP, LRP, GST-pi, Topo II alpha and clinic indexes were established (P > 0.05). Expression ratios of Pgp, MRP in post-chemotherapy cases were higher than that in pre-chemotherapy cases (P < 0.05). Expression of Pgp and MRP showed relevance with drug resistance (P < 0.05). The co-expression was common, the ratios of co-expression of Pgp, MRP, GST-pi and MRP, GST-pi in chemotherapy non-responders were 40% and 50%, respectively, but 0 in responders.

CONCLUSIONThe intrinsic multidrug resistance of tongue SCC is relevant to the effects of Pgp, MRP, GST-pi.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Adult ; Antigens, Neoplasm ; Carcinoma, Squamous Cell ; metabolism ; DNA Topoisomerases, Type II ; biosynthesis ; genetics ; DNA-Binding Proteins ; Female ; Glutathione S-Transferase pi ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Isoenzymes ; biosynthesis ; genetics ; Male ; Middle Aged ; Multidrug Resistance-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; Random Allocation ; Tongue Neoplasms ; metabolism ; Vault Ribonucleoprotein Particles ; biosynthesis ; genetics

To explore the change of sensitivity to chemotherapy of antisense RNA targeting survivin on hepatocarcinoma carcinoma cells in vitro. Survivin mRNA structure region was amplified by RT-PCR and inserted inversely into eukaryotic expression vector pcDNA3. The antisense expression plasmid pcDNA3/survivin was transfected into HepG2 with lipofectAMINE 2000 (LF2000), with low concentration of 5-fluorouracil (5-Fu) added. Survivin protein was detected by Western-blot, the growth activity was measured by MTT, and apoptosis was detected by Flow Cytometry 12 h, 24 h, 48 h after transfection. The activity of caspase-3 was found by quantitative assay 48 h after transfection. The construction of antisense RNA vector pcDNA3/survivin was verified by restricted endonuclease digestion and nucleotide sequencing. Compared with normal group, 5-Fu and antisense survivin group, the cells growth inhibition, apoptosis index, and caspase-3 activity were increased in antisense survivin transfected + 5-Fu group. The threshold of apoptosis was decreased after survivin was silenced, and the sensitivity to chemotherapy was increased. These findings suggest the existence of a potential new target for gene therapy.
Antigens, Neoplasm ; biosynthesis ; genetics ; Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; DNA, Antisense ; Fluorouracil ; pharmacology ; Humans ; Inhibitor of Apoptosis Proteins ; Liver Neoplasms ; metabolism ; pathology ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; Plasmids ; RNA, Messenger ; biosynthesis ; genetics ; Transfection

AIMTo characterize the coexpression of survivin, an inhibitor of apoptosis (IAF), and human telomerase reverse transcriptase (hTERT) in human testes with varying spermatogenic function.

METHODSTranscript levels of survivin mRNA and hTERT mRNA were determined in normal testes (n=11) and testes with defective spermatogenesis (n=28) using real-time reverse-transcription polymerase chain reaction (RT-PCR). The histological work-up was performed according to a modified Johnsen score.

RESULTSExpressions of both survivin and hTERT were highest at median levels of 96.8 and 709 in normal spermatogenesis and dropped to 53.3 and 534 in testes with postmeiotic spermatogenic arrest (n=10). In severe spermatogenic failure (n=18), survivin expression was lacking in most specimens (n=16), whereas at least low levels of testicular hTERT expression were largely detectable with a normalized expression of 73 in premeiotic spermatogenic arrest (n=7) and 45 in patients with Sertoli cell-only syndrome (SCOS) (n=3). Both survivin and hTERT expressions increased with a progressing Johnsen score (P for trend=0.001).

CONCLUSIONAlthough both survivin and hTERT are correlated with spermatogenic function, they show different expression patterns in testes of infertile patients. These findings substantiate results from studies in the rodent testis suggesting a predominant expression of survivin in meiotically dividing germ cells.

Biopsy ; DNA-Binding Proteins ; biosynthesis ; Gene Expression ; physiology ; Humans ; Infertility, Male ; metabolism ; Inhibitor of Apoptosis Proteins ; Male ; Microtubule-Associated Proteins ; biosynthesis ; Neoplasm Proteins ; biosynthesis ; Spermatogenesis ; physiology ; Telomerase ; biosynthesis ; Testis ; metabolism

OBJECTIVETo study the expression and clinical significance of P-gp, GST-pi and Topo II alpha in gastric and colorectal cancers.

METHODSThe expression of P-gp, GST-pi and Topo II alpha in 83 cases with gastric or colorectal cancer were examined by immunohistochemistry S-P.

RESULTSThe positive expression rates of P-gp, GST-pi, Topo II alpha in normal tissue and gastric and colorectal cancers were 69.9%, 65.1%, 50.6% and 83.1%, 85.5%, 45.8%, respectively. The positive rates of P-gp and GST-pi in gastric and colorectal cancer were significantly higher than those in normal gastric and colorectal tissue (P < 0.05). The expression of Topo II alpha in poorly differentiated cancers was significantly higher than that in well-and moderately differentiated cancers. There was no correlation between other items and clinicopathological parameters (P > 0.05).

CONCLUSIONP-gp, GST-pi and Topo II alpha play important role in multidrug resistance. Their mechanisms of drug resistance were different. The detection of expression of P-gp, GST-pi and Topo II alpha has an important guiding significance in chemotherapy for gastric and colorectal cancers.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Adenocarcinoma ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Antigens, Neoplasm ; biosynthesis ; genetics ; Colorectal Neoplasms ; metabolism ; DNA Topoisomerases, Type II ; biosynthesis ; genetics ; DNA-Binding Proteins ; biosynthesis ; genetics ; Drug Resistance, Neoplasm ; Female ; Gastrointestinal Neoplasms ; metabolism ; Glutathione S-Transferase pi ; biosynthesis ; genetics ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Stomach Neoplasms ; metabolism

To investigate the antitumor immune responses induced by MAGE-3 DNA vaccine, the recombinant mammalian expression plasmid pcDNA3.1/MAGE-3 was constructed by ligating MAGE-3 gene, which was amplified by RT-PCR, and the pcD-NA3.1 + vector. The recombinant plasmids were transfected into B16 cells by liposome, the expression of MAGE-3 was checked by RT-PCR, immunocytochemistry and Western blot. Then, 100 ug recombinant plasmids were injected intramuscularly per C57BL/6 mouse on 0, 10 and 20 days, with pcDNA3.1 + plasmid and PBS as controls. Splenocytes CTLs, the level of antibodies against MAGE-3 the changes of the T lymphocyte subsets and the levels of cytokines were checked after 3 times immunization. As a result, the mice immunized with pcDNA3.1/MAGE-3 plasmid can produce MAGE-3 specific immune response. The CTLs kill activities against B16/MAGE-3 cells was 51.08 +/- 7.41%, and had significant difference (P < 0.01) compared with that of pcDNA3.1 + group (8.44 +/- 1.89%) and PBS group (5.76 +/- 1.75%). The titre of antibody against MAGE-3 was 1:15, while controls were negtive. The number of CD4 + CD8 + and the levels of IFN-gamma IL-2 increased significantly after immunization with pcDNA3.1/MAGE-3 plasmid as compared with those of control groups (P < 0.01). It is concluded that the pcDNA3.1-MAGE-3 DNA vaccine are able to induce both cellular and humoral immune responses in vivo.
Animals ; Antibodies, Neoplasm ; blood ; Antigens, Neoplasm ; biosynthesis ; genetics ; immunology ; Cancer Vaccines ; biosynthesis ; immunology ; Female ; Melanoma, Experimental ; prevention & control ; Mice ; Mice, Inbred C57BL ; Neoplasm Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; T-Lymphocyte Subsets ; immunology ; Vaccination ; Vaccines, DNA ; biosynthesis ; genetics ; immunology

OBJECTIVETo investigate trichorhinophalangeal syndrome 1 gene (TRPS-1) expression patterns in different subtypes of breast cancer and its correlations with other genes and survival using microarray data sets.

METHODSThe transcripts of TRPS-1 and its role in survival in breast cancer were analyzed using published microarray data sets#x02014;Netherlands Cancer Institute (NKI) cohort and Wang cohort.

RESULTSTRPS-1 expression was lower in basal-like breast cancer. The mRNA levels of TRPS-1 negatively correlated with Slug (Pearson correlation coefficient=-0.1366, P=0.0189 in NKI data set and Pearson correlation coefficient=-0.1571, P=0.0078 in Wang data set), FOXC1 (Pearson correlation coefficient=-0.1211, P=0.0376 in NKI data set and Pearson correlation coefficient=-0.1709, P=0.0037 in Wang data set), and CXCL1 (Pearson correlation coefficient=-0.1197, P=0.0399 in NKI data set and Pearson correlation coefficient=-0.3436, P<0.0001 in Wang data set), but positively correlated with BRCA1 (Pearson correlation coefficient=0.1728, P=0.0029 in NKI data set and Pearson correlation coefficient=0.1805, P=0.0022 in Wang data set). Low TRPS-1 expression associated with poor overall survival (hazard ratio 1.79, 95% CI of ratio 0.9894 to 3.238, P=0.054) and relapse-free survival (hazard ratio 1.913, 95% CI of ratio 1.159 to 3.156, P<0.05). The low TRPS-1 mRNA levels predicted poor outcome in breast cancer patients by the 70-gene signature.

CONCLUSIONThe strong expression of TRPS-1 may serve as a good prognostic marker in breast cancer.

Adult ; Biomarkers, Tumor ; biosynthesis ; Breast Neoplasms ; metabolism ; mortality ; pathology ; Cell Line, Tumor ; Cohort Studies ; DNA-Binding Proteins ; biosynthesis ; Disease-Free Survival ; Epithelial-Mesenchymal Transition ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Middle Aged ; Neoplasm Proteins ; biosynthesis ; RNA, Messenger ; biosynthesis ; RNA, Neoplasm ; biosynthesis ; Survival Rate ; Transcription Factors ; biosynthesis

OBJECTIVETo study glycyrrhizin's anticancer effect and its mechanism.

METHODS3-methylcholanthrene were injected into mice's submandibular glands to induce tumor, then transplanted the tumor pieces (1 mm3) to mice. The transplanted tumors were measured, and flow cytometry analysis and cytomorphology observation were conducted.

RESULTSGlycyrrhizin (GL) inhibited the transplanted mandibular gland fibro-sarcoma of mice and the suitable GL dose for inhibiting fibrosarcoma of mice was 1.61 mg per 20 g weight. The GL dose below 3.22 mg per 20 g weight didn't produce remarkable toxicity and side effects. GL induced cytomorphological changes of tumor cells and enhanced immunosuppression of macrophage on fibrosarcoma. The result of flow cytometry showed that tumor cell counts of GL1 and GL2 groups increased remarkably in DNA synthetic prophase, and decreased in DNA synthetic phase.

CONCLUSIONGL can inhibit transplanted mandibular gland fibro-sarcoma of mice. The anticancer mechanism of GL may be acting on related enzymes with phagocytosis. The result of flow cytometry showed that the shift of fibrosarcoma cells from G1 phase to S phase was blocked. This suggests that the anticancer action of GL is related to its inhibition of ribonucleotide reductase, a rate-limiting enzyme in DNA synthesis.

Animals ; Antineoplastic Agents ; pharmacology ; DNA, Neoplasm ; biosynthesis ; Fibrosarcoma ; chemically induced ; pathology ; Glycyrrhizic Acid ; pharmacology ; Macrophages ; physiology ; Male ; Methylcholanthrene ; Neoplasm Transplantation ; Phagocytosis

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antigens, Neoplasm ; biosynthesis ; genetics ; Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; drug effects ; Colorimetry ; DNA Topoisomerases, Type II ; biosynthesis ; genetics ; DNA-Binding Proteins ; biosynthesis ; genetics ; Drug Resistance, Neoplasm ; Epirubicin ; pharmacology ; Female ; Fluorouracil ; pharmacology ; Humans ; Immunohistochemistry ; Liver Neoplasms ; drug therapy ; metabolism ; pathology ; Male ; Middle Aged ; Predictive Value of Tests ; RNA, Messenger ; biosynthesis ; genetics