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Objective To establish a radiation-resistant cell subline from a human small cell lung cancer ( SCLC) cell line NCI-H446, providing a pairing research tool for investigating mechanism of radiation tolerance and the reverse strategy in lung cancer .Methods The NCI-H446 cell was radiated repeatedly by increased dose of radiation gradually (total 7500cGy) and a radiation-resistant cell substrain was induced and selected from the survival of cells .The doubling time and cell cycle distribution of the substrain were detected by ATP kit and flow cytometry Assay respectively ;Radiation biology parameters were calculated and analyzed by cell survival curve fitting from multi -target model, SF=1-(1-e-D/D0) N.Results Comparing with the control , The resistant substrain radiobiology parameter values were D 0 ( 1.9673, 2.2756), N(1.0016,2.6008), Dq (0.6783,1.6860)and SF2(0.3623,0.7134) respectively.Cell morphology is more slender and has more tentacles .The SF2 value of radiation-resistant subline is 1.97 times more than that of wild cell line . The proportion of radiation-resistant cells in G2/M-phase was down to 7.84%, compared with the 18.52%of wild cells. Conclusions A radiation-resistant SCLC subline NCI-H446-R is established and may be a useful tool for studying resistant to radiation of SCLC in the future .
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Objective To predict clinical chemotherapy sensitivity of primary ovarian cancer by jointing adenosine triphosphate(ATP) - tumor chemo-sensitivity assay(TCA) method in vitro and detection of drug resistance genes, provide reference for clinical treatment. Methods Forty-seven primary epithelial ovarian tumor samples were collected from the patients who received cytoreductive surgery. Viable ovarian cancer cells obtained from malignant tissue were tested for their sensitivity to carboplatin (CBP), cisplatin (DDP), paclitaxel(PTX) and CBP + PTX using ATP-TCA method in vitro; at same time, real-time quantitative PCR was used to analysis BRCA1 and ERCC1 mRNA relative expression in forty-six specimens (1 frozen tumor samples mRNA were not detected due to serious degradation). The relationship between ATP-TCA test results, clinical indicators, and the effectiveness of the joint prediction on clinical chemosensitivity by combining these two methods were statistically analyzed using chi-square test. Results (1)The results showns that three programs of DDP,CBP and PTX + CBP were significantly related with clinical results(P<0.05) in vitro, in which the compliance rate in PTX + CBP program was the highest 83%(39/47) ,and the predictive sensitivity, predictive specificity, positive predictive value, negative predictive value and predictive accurate rate were 90%,71%,84% and 80% ,respectively.PTX + CBP combined in vitro test results was also related with residual tumor size and neoadjuvant chemotherapy, which was more prone to drug resistance with residual tumor larger than 2 cm (P = 0. 023) and with neoadjuvant chemotherapy (P = 0.011). (2) BRCA1 mRNA expression levels in the clinical-resistant group and the clinical-sensitive group was 0.673 ± 2.143 and - 1.436 ± 2.594 (P=0.008), ERCC1 mRNA expression levels in the clinical-resistant group and the clinical-sensitive group was -0.529 ± 1.982 and - 3.188 ±2.601 (P =0.001). There were also significant correlation among the expression levels of BRCA1 ,ERCC1 mRNA and clinical efficacy (P<0.01). (3)ATP-TCA and detection of drug resistance genes combined to predict the clinical application of PTX + CBP resistance may occur in 8/9 cases. Conclusions ATP-TCA may be an ideal method of in vitro drug sensitivity testing method, which could effectively predict clinical chemotherapy sensitivity. Combination of the drug-resistant associated genes detection method and the ATP-TCA method can increase the predictive effectiveness of ovarian cancer chemosensitivity and guide individual chemotherapy of ovarian cancer.
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Objective: To develop a therapeutic adjuvantfree protein vaccine against HPV16 which is closely related to cervical cancer of China. Method: First the E6/E7 gene by PCR technology from HPV16z virus strain was isolated in the highrisk cervical cancer area of Shanxi province of China in1990s, and again got the gene segment of Hsp65 from BCG by the same method, mutated the transforming codes in sequences of HPV 16 E6/E7 genes and thus constructed the expression vector pET28aHsp65E6/E7, expressed the Hsp65E6/E7 fusion protein in E.coli BL21(DE3) strain and researched optimal protein purification procedures. Results: The expression vector pET28aHsp65E6/E7 was constructed successfully and E6/E7 gene was mutated correctly. Hsp65E6/E7 fusion protein was renatured and purified on the affinity chromatography column simultaneously. The protein purity achieved 95% after the anionic exchange chromatograph purification. conclusions: This research laid a foundation for further functional study of the therapeutic adjuvantfree protein vaccine——Hsp65E6/E7.
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Objective To investigate the heterogeneity of chemosensitivity in colorectal cancer using an ATP-tumor chemosensitivity assay (ATP-TCA) and the feasibility of individual chemotherapy.Methods An ATP-TCA were used to determine the effect of 16 single or combined cytotoxic drugs in surgical specimens from 50 patients with colorectal cancer.Results There were considerable differences in chemosensitivity between individuals.The most active single drugs in the assay was identified as Navelbine, Hydroxycamptothecin, 5-Fluorouracil and Paclitaxel; 34.1%, 31.6%, 27.6% and 24.3% of specimens showed sensitivity to them, respectively.5-Fluorouracil+Mitomycin+Aytarabine was found to be the most effective combination, for 100% (11/11)specimens were sensitive to this regimen.5-Fluorouracil+Cisplatin+Adriamycin and Gemcitabine+Cisplatin were moderately active regimens.Conclusions There was the heterogeneity of the in vitro chemosensitivity in colorectal cancer.The use of the ATP-TCA provides a method of selecting appropriate anti-cancer drugs in colorectal cancer.
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<p><b>OBJECTIVE</b>To develop and identify the monoclonal antibodies (mAb) against c-erbB-2 p185 intracellular domain for detection of c-erbB-2 protein overexpression in breast tumor cells.</p><p><b>METHODS</b>BALB/C mice were immunized with a synthesized p185 peptide of intracellular domain. The biological characteristics and immunoactivities were identified by different techniques.</p><p><b>RESULTS</b>Three hybridoma cell strains secreting mAbs to c-erbB-2 protein were established and one of the mAbs, No. 035-E61 was tested for c-erbB-2 protein immunostaining on 39 breast carcinoma sections, 30 breast fibroadenoma sections and 16 sections from various normal organs. The results showed that the positive detection frequency of protein expression was 26% (10/39) in breast cancer, none (0/30) in fibroadenoma and 3 sections from normal organs were positively stained. The consistency between the 035-E61 and the DAKO reagent approved by FDA was 74%.</p><p><b>CONCLUSIONS</b>No. 035-E61 mAb can specifically recognize the p185 intracellular domain and may be useful in guiding clinical Herceptin treatment.</p>
Subject(s)
Animals , Female , Humans , Mice , Antibodies, Monoclonal , Allergy and Immunology , Antibody Specificity , Binding Sites , Allergy and Immunology , Breast Neoplasms , Allergy and Immunology , Pathology , Fibroadenoma , Allergy and Immunology , Pathology , Immunoglobulin G , Allergy and Immunology , Immunohistochemistry , Mice, Inbred BALB C , Receptor, ErbB-2 , Allergy and Immunology , Tumor Cells, CulturedABSTRACT
Objective: To study the biological effects of the HPV16Z-Hsp65-E6/E7 fusion protein vaccine on the tumor associated with HPV16 infection. Methods: We tested the cellular immune responsive intensity to the vaccine by the lymphocyte proliferation and CTL response, and studied the therapeutic effect of the vaccine on mouse TC-1 cell transplanted cancer in vivo and the influence on mouse lifetime. Results: The spleen lymphocytes from the C57BL/6 mouse immunized by the Hsp65-E6/E7 vaccine could proliferate obviously in the presence of the protein and TC-1 tumor cell could be lysed specifically by immune activated lymphocytes in vitro. This animal therapeutic experiment in vivo showed that the vaccine suppressed the growth of TC-1 cell transplanted tumor remarkably. Conclusion: The recombined vaccine can induce specific cellular immune response in vitro and suppress HPV16 positive TC-1 tumor cell growth obviously in vivo.