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Volume 11,Issue 4,2004 Table of Contents
2004, 11(4):239-243.
DOI: 10.3872/j.issn.1007-385X.2004.4.002
Abstract:
Objective: To prepare anti-CEA monoclonal antibody-β-glucosidase conjugate(Anti-CEA McAb-β-glucosidase conjugate)， evaluate its immunoreactivity and enzymatic activity, and study its killing effect against LoVo cells in combination with amygdalin in vitro.Methods: Anti-CEA monoclonal antibody-β-glucosidase conjugate was prepared to use heterobifunctional crosslinking reagent 3-maleimidobenzoic acid N-succinimidyl ester (MBS) conjugating β-glucosidase to a anti-CEA monoclonal antibody, the immunoreactivity and enzymatic activity of the conjugate was tested by ELISA method. The cytotoxicity to targeted cells and untargeted cells following specific activation of amygdalin by the conjugate was evaluated by MTT assy in vitro.Results: Anti-CEA McAb-β-glucosidase conjugate kept both immunoreactivity and enzymatic activty. Anti-CEA McAb-β-glucosidase conjugate could bird LoVo cells specifically and activate amygdalin to kill Love cells in a dose-dependent manner, but it had not the same effect against MCF-7 cells.Conclusion: Anti-CEA McAb-β-glucosidase conjugate has a good character for binding LoVo cells in vitro and can activate amygdalin prodrug to kill targeted cells specifically.This way might become a hopeful model of colorectal carcinoma immuonotherapy.
Objective: To prepare anti-CEA monoclonal antibody-β-glucosidase conjugate(Anti-CEA McAb-β-glucosidase conjugate)， evaluate its immunoreactivity and enzymatic activity, and study its killing effect against LoVo cells in combination with amygdalin in vitro.Methods: Anti-CEA monoclonal antibody-β-glucosidase conjugate was prepared to use heterobifunctional crosslinking reagent 3-maleimidobenzoic acid N-succinimidyl ester (MBS) conjugating β-glucosidase to a anti-CEA monoclonal antibody, the immunoreactivity and enzymatic activity of the conjugate was tested by ELISA method. The cytotoxicity to targeted cells and untargeted cells following specific activation of amygdalin by the conjugate was evaluated by MTT assy in vitro.Results: Anti-CEA McAb-β-glucosidase conjugate kept both immunoreactivity and enzymatic activty. Anti-CEA McAb-β-glucosidase conjugate could bird LoVo cells specifically and activate amygdalin to kill Love cells in a dose-dependent manner, but it had not the same effect against MCF-7 cells.Conclusion: Anti-CEA McAb-β-glucosidase conjugate has a good character for binding LoVo cells in vitro and can activate amygdalin prodrug to kill targeted cells specifically.This way might become a hopeful model of colorectal carcinoma immuonotherapy.
2 Expression，Purification of Fusion Protein TGFα-PE40 and the Cytotoxicity of TGFα-PE40 on Tumor Cells
2004, 11(4):244-247.
DOI: 10.3872/j.issn.1007-385X.2004.4.003
Abstract:
Objective：To express and purify transforming growth factor α (TGFα)-pseudomonas exotoxin 40 and investigate its cytotoxic effect on cancer cells overexpressing epidermal growth factor (EGF) receptor. Methods：Recombinant plasmid pV28 was constructed by inserting the gene coding TGFα-PE40 into the vector pET28a Expression of fusion protein was conducted using the host BL21. Production of the recombinant protein was induced by IPTG, following extraction and purification of inclusion bodies with His-tag purification system. Cell viability assay (by MTT) was performed to determine the cytotoxic effect of TGFα-PE40 on cancer cells (A431 and SK-OV3).Results：Recombinant plasmid pV28, which expresses TGFα-PE40, was constructed successfully. Purity of TGFα-PE40 was about 98% after a purification procedure using His-tag column. Cytotoxic experiment showed that at a concentration of 0.86±0.07 μg/ml, TGFα-PE40 could reduce 50% viability of A431, which has high expression of EGFR. Whereas the IC50 for ovarian cancer cell SK-OV3, which expresses less EGFR, was 6.37±2.18 μg/ml.There was a significant difference between these two groups (P<0.05). Conclusion：The cytotoxicity ability of the fusion protein on tumor cells depends on the number of the EGFR presented on tumor cells.
Objective：To express and purify transforming growth factor α (TGFα)-pseudomonas exotoxin 40 and investigate its cytotoxic effect on cancer cells overexpressing epidermal growth factor (EGF) receptor. Methods：Recombinant plasmid pV28 was constructed by inserting the gene coding TGFα-PE40 into the vector pET28a Expression of fusion protein was conducted using the host BL21. Production of the recombinant protein was induced by IPTG, following extraction and purification of inclusion bodies with His-tag purification system. Cell viability assay (by MTT) was performed to determine the cytotoxic effect of TGFα-PE40 on cancer cells (A431 and SK-OV3).Results：Recombinant plasmid pV28, which expresses TGFα-PE40, was constructed successfully. Purity of TGFα-PE40 was about 98% after a purification procedure using His-tag column. Cytotoxic experiment showed that at a concentration of 0.86±0.07 μg/ml, TGFα-PE40 could reduce 50% viability of A431, which has high expression of EGFR. Whereas the IC50 for ovarian cancer cell SK-OV3, which expresses less EGFR, was 6.37±2.18 μg/ml.There was a significant difference between these two groups (P<0.05). Conclusion：The cytotoxicity ability of the fusion protein on tumor cells depends on the number of the EGFR presented on tumor cells.
2004, 11(4):248-251.
DOI: 10.3872/j.issn.1007-385X.2004.4.004
Abstract:
Objective:To investigate the effect of survivin and bcl-2 antisense oligodeoxynucleotides (AsODN) combined transfection on the human gallbladder carcinoma cell line GBC-SD in vitro.Methods: Survivin and Bcl-2 protein expressions were detected by immunohistochemical method; Cultured cells were divided into 4 groups: Nomal control group, survivin antisense observed group, bcl-2 antisense observed group and combined group. After transfected for 24 h, the expression of survivin mRNA was detected by reverse transcription polymerase chain reaction(RT-PCR). Cell morphological changes were observed under electron microscopy. Apoptosis index (AI) was examined by flow cytometry; Inhibitory rate (IR ) was determined by the colorimetri MTT cell viability and proliferation assay.Results:Survivin and Bcl-2 protein were highly expressed in gall bladder carcinoma cells; The expression of survivin mRNA was decreased 47.8%. Abnormal morphological changes of cells were observed in the three AsODN transfection groups; The AI in survivin antisense observed group,bcl-2 antisense observed group,and combined group was 11.38％±3.91％, 9.26％±4.15％, 28.45％±6.34％ respectively and significantly higher than the nomal control group (P<0.01), and that in the combined group was significantly higher than the other two observed groups(P<0.05). The IR was 54.3％,47.6％,76.5％ respectively in the three AsODN transfection groups. Conclusion:Survivin and bcl-2 protein were highly expressed in gallbladder carcinoma cells. Survivin AsODN and bcl-2 AsODN can induce GBC-SD cell apoptosis and the effect was more markedly by combining the two AsODNs.
Objective:To investigate the effect of survivin and bcl-2 antisense oligodeoxynucleotides (AsODN) combined transfection on the human gallbladder carcinoma cell line GBC-SD in vitro.Methods: Survivin and Bcl-2 protein expressions were detected by immunohistochemical method; Cultured cells were divided into 4 groups: Nomal control group, survivin antisense observed group, bcl-2 antisense observed group and combined group. After transfected for 24 h, the expression of survivin mRNA was detected by reverse transcription polymerase chain reaction(RT-PCR). Cell morphological changes were observed under electron microscopy. Apoptosis index (AI) was examined by flow cytometry; Inhibitory rate (IR ) was determined by the colorimetri MTT cell viability and proliferation assay.Results:Survivin and Bcl-2 protein were highly expressed in gall bladder carcinoma cells; The expression of survivin mRNA was decreased 47.8%. Abnormal morphological changes of cells were observed in the three AsODN transfection groups; The AI in survivin antisense observed group,bcl-2 antisense observed group,and combined group was 11.38％±3.91％, 9.26％±4.15％, 28.45％±6.34％ respectively and significantly higher than the nomal control group (P<0.01), and that in the combined group was significantly higher than the other two observed groups(P<0.05). The IR was 54.3％,47.6％,76.5％ respectively in the three AsODN transfection groups. Conclusion:Survivin and bcl-2 protein were highly expressed in gallbladder carcinoma cells. Survivin AsODN and bcl-2 AsODN can induce GBC-SD cell apoptosis and the effect was more markedly by combining the two AsODNs.
2004, 11(4):252-256.
DOI: 10.3872/j.issn.1007-385X.2004.4.005
Abstract:
Objective: To investigate the cell cycle change of K562 cells and bone marrow mononucleate cells(BMMNC) of leukemic patient induced by DDP and the role of antisense oligonucleotide targeting Chk1/2.Methods: The change of cell cycle was assayed by means of flow cytometer after different interval in which the K562 cells and BMMNC of leukemia are treated by DDP. K562 cells or BMMNC are transfected with the antisense oligonucleotide complementary to Chk1/2 by lipofection. The apoptosis of K562 cells and BMMNC induced by DDP was investigated by flow cytometer after transfection. Results: K562 cells and BMMNC of leukemia arrested at S phase at 10μmol/L of DDP. The antisense oligonucleotide targeting Chk1 or Chk2 could increase the apoptosis of K562 cells treated with DDP. The antisense oligonucleotide targeting Chk1 could increase the apoptosis of leukemic BMMNC treated with DDP, but the antisense oligonucleotide targeting Chk2 could not increase the apoptosis of BMMNC of leukemia induced by DDP.Conclusion: Chk1 may take part in apoptosis regulation of leukemia cells and be used as target of leukemia therapy.
Objective: To investigate the cell cycle change of K562 cells and bone marrow mononucleate cells(BMMNC) of leukemic patient induced by DDP and the role of antisense oligonucleotide targeting Chk1/2.Methods: The change of cell cycle was assayed by means of flow cytometer after different interval in which the K562 cells and BMMNC of leukemia are treated by DDP. K562 cells or BMMNC are transfected with the antisense oligonucleotide complementary to Chk1/2 by lipofection. The apoptosis of K562 cells and BMMNC induced by DDP was investigated by flow cytometer after transfection. Results: K562 cells and BMMNC of leukemia arrested at S phase at 10μmol/L of DDP. The antisense oligonucleotide targeting Chk1 or Chk2 could increase the apoptosis of K562 cells treated with DDP. The antisense oligonucleotide targeting Chk1 could increase the apoptosis of leukemic BMMNC treated with DDP, but the antisense oligonucleotide targeting Chk2 could not increase the apoptosis of BMMNC of leukemia induced by DDP.Conclusion: Chk1 may take part in apoptosis regulation of leukemia cells and be used as target of leukemia therapy.
LI Yu-ying
,
QIAN Gui-sheng
,
HUANG Gui-jun
,
YU Shi-cang
,
WANG Xing-you
,
CHEN Wei-zhong
,
LI Shu-ping
2004, 11(4):257-262.
DOI: 10.3872/j.issn.1007-385X.2004.4.006
Abstract:
Objective:To construct a mammal expression system of human canstatin and study its anti-tumor effects on lung cancer. Methods:Canstatin cDNA was acquired by RT-PCR, and cloned into a mammal exprssion vector named pCMV-Script. Canstatin expression was detected by Real-time PCR. The proliferation, apotosis of the cells transfected with recombinant canstatin vector were measured by trypan blue exclusive assay, 3H-thymidine incorporation and TUNEL method respectively. Then the recombinant vector encoding canstatin cDNAs was transferred into tumors of cancer-bearing nude mice with electroporator in vivo, and micro-vessel count was proceeded of each tumor by anti-CD31 antibody immunohistochemical staining.Results: The recombinant vector pCMV-Script-Cans was successfully constructed , and the canstatin mRNA was detected in both of the transformed HUVE and A549 cells. The 3H-TdR intake rate in pCMV-Script-Cans transformed HUVE cells is significant lower than that of the naked plasmid transformed cells (P<0.001) , while the apotosis rate of them is significant higher than that of the control cells(P<0.001). The micro-vessels in the recombinant vector transformed tumors were significant lower than that of the control group. Conclusions: Canstatin only inhibit cell proliferation and induce apotosis in endothelial cell, and it also has a good anti-tumor effect in vivo.
Objective:To construct a mammal expression system of human canstatin and study its anti-tumor effects on lung cancer. Methods:Canstatin cDNA was acquired by RT-PCR, and cloned into a mammal exprssion vector named pCMV-Script. Canstatin expression was detected by Real-time PCR. The proliferation, apotosis of the cells transfected with recombinant canstatin vector were measured by trypan blue exclusive assay, 3H-thymidine incorporation and TUNEL method respectively. Then the recombinant vector encoding canstatin cDNAs was transferred into tumors of cancer-bearing nude mice with electroporator in vivo, and micro-vessel count was proceeded of each tumor by anti-CD31 antibody immunohistochemical staining.Results: The recombinant vector pCMV-Script-Cans was successfully constructed , and the canstatin mRNA was detected in both of the transformed HUVE and A549 cells. The 3H-TdR intake rate in pCMV-Script-Cans transformed HUVE cells is significant lower than that of the naked plasmid transformed cells (P<0.001) , while the apotosis rate of them is significant higher than that of the control cells(P<0.001). The micro-vessels in the recombinant vector transformed tumors were significant lower than that of the control group. Conclusions: Canstatin only inhibit cell proliferation and induce apotosis in endothelial cell, and it also has a good anti-tumor effect in vivo.
2004, 11(4):263-266.
DOI: 10.3872/j.issn.1007-385X.2004.4.007
Abstract:
Objective : To investigate the specific anti-flk1 immunity and the anti-angiogenesis effect of attenuated Salmonella typhimurium strain carrying flk1 expression vector encoding flk1 cDNA. Methods：Plasmid pcDNA3.1-flk1 was constructed and transformed into live attenuated Salmonella typhimurium strain SL7207. The recombinant bacteria were used to infect murine macrophage in vitro and the expressed products were detected by Western blot. C57BL/6J mice were immunized with recombinant bacteria encoding flk1, and CTL activity of the splenocytes derived from the immunized mice was measured by MTT assay. Alginate bead assay was designed to measure in vivo angiogenesis induced by tumor cells. Result: Murine macrophage infected with recombinant S. typhimurium transformed with plasmid pcDNA3.1-flk1 could express flk1 protein. A strong CTL activity against flk1 was found in pcDNA3.1-flk1 immunized animals. The recombinant S. typhimurium vaccination inhibited tumor-induced angiogenesis in vivo. Conclusion: Attenuated Salmonella typhimurium strain encoding flk1 cDNA can elicit specific cellular immune response and inhibit tumor-induced angiogenesis in vivo.
Objective : To investigate the specific anti-flk1 immunity and the anti-angiogenesis effect of attenuated Salmonella typhimurium strain carrying flk1 expression vector encoding flk1 cDNA. Methods：Plasmid pcDNA3.1-flk1 was constructed and transformed into live attenuated Salmonella typhimurium strain SL7207. The recombinant bacteria were used to infect murine macrophage in vitro and the expressed products were detected by Western blot. C57BL/6J mice were immunized with recombinant bacteria encoding flk1, and CTL activity of the splenocytes derived from the immunized mice was measured by MTT assay. Alginate bead assay was designed to measure in vivo angiogenesis induced by tumor cells. Result: Murine macrophage infected with recombinant S. typhimurium transformed with plasmid pcDNA3.1-flk1 could express flk1 protein. A strong CTL activity against flk1 was found in pcDNA3.1-flk1 immunized animals. The recombinant S. typhimurium vaccination inhibited tumor-induced angiogenesis in vivo. Conclusion: Attenuated Salmonella typhimurium strain encoding flk1 cDNA can elicit specific cellular immune response and inhibit tumor-induced angiogenesis in vivo.
2004, 11(4):267-271.
DOI: 10.3872/j.issn.1007-385X.2004.4.008
Abstract:
Objective:To investigate the p16 gene expression in lung cancer cell line introduced by adenovirus, and to observe the effect of p16 gene on cell cycle arrest.Methods: The replication-deficient adenovirus Ad5-p16 was homologously recombined in 293 cells and used to infect the lung cancer cell line SPC-A1. The expression of P16 protein was identified by immunohistochemistry. Trypan blue staining was used to count the alive cells and to draw a cellular growth curve. The changes of cell cycle and apoptosis were analyzed by flow cytometry (FCM).Results:After 48 hours since the SPC-A1 cells were infected by the replication-deficient adenovirus Ad5-p16 at MOI=10, the positive rate of the cells for P16 expression was 71%. The cellular growth inhibition and cytopathic effects were present when the cells were infected with Ad5-p16 at MOI=100. The cell cycle arrest and the cell apoptosis were found by FCM.Conclusion:The expression of p16 gene in SPC-A1 lung cancer cells induced by the replication-deficient adenovirus may result in the inhibition of cell growth and the introduction of cell apoptosis. The strategy targeted to the tumor suppressor gene that regulates the cell cycle directly, obtained satisfactory results, and provided a reliable theory for lung cancer gene therapy.
Objective:To investigate the p16 gene expression in lung cancer cell line introduced by adenovirus, and to observe the effect of p16 gene on cell cycle arrest.Methods: The replication-deficient adenovirus Ad5-p16 was homologously recombined in 293 cells and used to infect the lung cancer cell line SPC-A1. The expression of P16 protein was identified by immunohistochemistry. Trypan blue staining was used to count the alive cells and to draw a cellular growth curve. The changes of cell cycle and apoptosis were analyzed by flow cytometry (FCM).Results:After 48 hours since the SPC-A1 cells were infected by the replication-deficient adenovirus Ad5-p16 at MOI=10, the positive rate of the cells for P16 expression was 71%. The cellular growth inhibition and cytopathic effects were present when the cells were infected with Ad5-p16 at MOI=100. The cell cycle arrest and the cell apoptosis were found by FCM.Conclusion:The expression of p16 gene in SPC-A1 lung cancer cells induced by the replication-deficient adenovirus may result in the inhibition of cell growth and the introduction of cell apoptosis. The strategy targeted to the tumor suppressor gene that regulates the cell cycle directly, obtained satisfactory results, and provided a reliable theory for lung cancer gene therapy.
2004, 11(4):272-276.
DOI: 10.3872/j.issn.1007-385X.2004.4.009
Abstract:
Objective: To evaluate the mechanism of the enhanced antitumor immune effect of the locally expressed soluble molecule sPD-1 on mouse H22 hepatoma.Methods: The mRNA expression level of PD-L1 and PD-L2, the ligands of PD-1, were investigated in mouse H22 cells as well as H22 tumor tissues by using semi-quantitative RT-PCR method. The cytotoxicity assay in vitro was used to evaluate the lysis activity of HSP70-peptides complex-stimulated spleen cells on H22 cells when the tumor cells or spleen cells were pretreated with sPD-1. The antitumor effect of sPD-1 on H22 hepatoma was investigated by experiment in vivo after mice were inoculated with H22 tumor cells. Results: PD-L1 but not PD-L2 mRNA was expressed in H22 hepatoma cells. Both PD-L1 and PD-L2 mRNAs were expressed in tumor tissues of tumor-bearing mice and upregulated as compared with muscle tissues in normal mice. Blocking PD-Ls on either tumor cells or spleen cells by sPD-1 mediated enhanced lysis of H22 cells by HSP70-peptides complex-stimulated spleen cells. sPD-1 also mediated strong antitumor immune effects on mouse H22 tumor model in vivo. Conclusion: The results provide a novel antitumor method of expression of soluble receptor of PD-1 in tumor sites by local gene therapy, which could block the action of PD Ls on both immune cells and H22 tumor cells，and then increase the antitumor immune activity.
Objective: To evaluate the mechanism of the enhanced antitumor immune effect of the locally expressed soluble molecule sPD-1 on mouse H22 hepatoma.Methods: The mRNA expression level of PD-L1 and PD-L2, the ligands of PD-1, were investigated in mouse H22 cells as well as H22 tumor tissues by using semi-quantitative RT-PCR method. The cytotoxicity assay in vitro was used to evaluate the lysis activity of HSP70-peptides complex-stimulated spleen cells on H22 cells when the tumor cells or spleen cells were pretreated with sPD-1. The antitumor effect of sPD-1 on H22 hepatoma was investigated by experiment in vivo after mice were inoculated with H22 tumor cells. Results: PD-L1 but not PD-L2 mRNA was expressed in H22 hepatoma cells. Both PD-L1 and PD-L2 mRNAs were expressed in tumor tissues of tumor-bearing mice and upregulated as compared with muscle tissues in normal mice. Blocking PD-Ls on either tumor cells or spleen cells by sPD-1 mediated enhanced lysis of H22 cells by HSP70-peptides complex-stimulated spleen cells. sPD-1 also mediated strong antitumor immune effects on mouse H22 tumor model in vivo. Conclusion: The results provide a novel antitumor method of expression of soluble receptor of PD-1 in tumor sites by local gene therapy, which could block the action of PD Ls on both immune cells and H22 tumor cells，and then increase the antitumor immune activity.
9 The Interference Effect of Vector-based siRNA on Survivin Gene of Human Breast Cancer SKBr-3 Cells
LI Qing-xia
,
LIU Jia-yun
,
HUANG Hong-yan
,
XU Yan-ming
,
JIA Lin-tao
,
ZHAO Jing
,
WANG Cheng-ji
,
YANG An-gang
2004, 11(4):277-280.
DOI: 10.3872/j.issn.1007-385X.2004.4.010
Abstract:
Objective:To construct the eukaryotic expression vector for RNA interferencing human survivin gene and detect its interference effect in human breast cancer cell line(SKBr-3). Methods: Two target gene segments were synthesized and cloned into pSUPER vector respectively to construct two recombinant eukaryotic expression vectors: pSUPER-S1 and pSUPER-S2. The two recombinant vectors were identified by enzyme digestion analysis and DNA sequencing. Then SKBr-3 cells were transfected with pSUPER-S1 or pSUPER-S2, together with pCDNA3 plasmid, and subjected to G418 selection. In G418-resistant cells, the interference effect was detected by RT-PCR, Western blot and immunocytochemical staining. Results: Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into pSUPER vector respectively. The results of RT-PCR, Western blot and immunocytochemical staining indicated that both vectors could knock down the transcription and expression of survivin gene, and that pSUPER-S1 had better interference effect than pSUPER-S2. Conclusion:The transcription and expression of survivin gene were inhibited effectively by the constructed RNAi eukaryotic expression vectors in the breast cancer cells.
Objective:To construct the eukaryotic expression vector for RNA interferencing human survivin gene and detect its interference effect in human breast cancer cell line(SKBr-3). Methods: Two target gene segments were synthesized and cloned into pSUPER vector respectively to construct two recombinant eukaryotic expression vectors: pSUPER-S1 and pSUPER-S2. The two recombinant vectors were identified by enzyme digestion analysis and DNA sequencing. Then SKBr-3 cells were transfected with pSUPER-S1 or pSUPER-S2, together with pCDNA3 plasmid, and subjected to G418 selection. In G418-resistant cells, the interference effect was detected by RT-PCR, Western blot and immunocytochemical staining. Results: Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into pSUPER vector respectively. The results of RT-PCR, Western blot and immunocytochemical staining indicated that both vectors could knock down the transcription and expression of survivin gene, and that pSUPER-S1 had better interference effect than pSUPER-S2. Conclusion:The transcription and expression of survivin gene were inhibited effectively by the constructed RNAi eukaryotic expression vectors in the breast cancer cells.
2004, 11(4):281-284.
DOI: 10.3872/j.issn.1007-385X.2004.4.011
Abstract:
Objective：To study the inhibition of antisense TRP 1 on cell growth of malignant melanoma(MM) and explore a new way for therapy of melanoma.Methods：Antisense TRP-1 recombinant vector was constructed and transfected into MM cells. According to the results of MTT, cell growth curves were drawn and then clonogenic assay was performed in vitro. At last, tumorigenesis assay was undertaken in nude mice in vivo.Results：Cell proliferations of TRP-1 transfected MM cells were inhibited compared with the control cells. The results of clonogenic assay displayed the difference of clonogenic percentage between TRP-1 transfected MM cells (52%, P<0.01) and the control cells (68%, P<0.01). Tumorigenesis assay in vivo 30 days after implantation showed that the growth of TRP-1 antisense vector transfected MM cells was significantly suppressed. Conclusion：Antisense TRP-1could inhibit the proliferation of MM cells both in vivo and in vitro. It could be expected that antisense TRP-1 be used in the therapy of MM in the future.
Objective：To study the inhibition of antisense TRP 1 on cell growth of malignant melanoma(MM) and explore a new way for therapy of melanoma.Methods：Antisense TRP-1 recombinant vector was constructed and transfected into MM cells. According to the results of MTT, cell growth curves were drawn and then clonogenic assay was performed in vitro. At last, tumorigenesis assay was undertaken in nude mice in vivo.Results：Cell proliferations of TRP-1 transfected MM cells were inhibited compared with the control cells. The results of clonogenic assay displayed the difference of clonogenic percentage between TRP-1 transfected MM cells (52%, P<0.01) and the control cells (68%, P<0.01). Tumorigenesis assay in vivo 30 days after implantation showed that the growth of TRP-1 antisense vector transfected MM cells was significantly suppressed. Conclusion：Antisense TRP-1could inhibit the proliferation of MM cells both in vivo and in vitro. It could be expected that antisense TRP-1 be used in the therapy of MM in the future.
2004, 11(4):285-290.
DOI: 10.3872/j.issn.1007-385X.2004.4.012
Abstract:
Objective:To study the repair mechanisms of ICLs in mammalian cells. Methods: A site-specific MMC cross-link was placed between the promoter and the coding region to inactivate the expression of luciferase genes from reporter plasmids. An in vivo reactivation assay was developed to examine the removal of ICLs in cultured cells. Results: MMC cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the NER pathway were examined and found to be highly defective in the recombination independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near the positions of MMC crosslinks. Conclusion: Recombination-independent ICL pathway exists in mammalian cells and NER involve in the repair with an error-prone mechanism.
Objective:To study the repair mechanisms of ICLs in mammalian cells. Methods: A site-specific MMC cross-link was placed between the promoter and the coding region to inactivate the expression of luciferase genes from reporter plasmids. An in vivo reactivation assay was developed to examine the removal of ICLs in cultured cells. Results: MMC cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the NER pathway were examined and found to be highly defective in the recombination independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near the positions of MMC crosslinks. Conclusion: Recombination-independent ICL pathway exists in mammalian cells and NER involve in the repair with an error-prone mechanism.
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
Supported by:Beijing E-Tiller Technology Development Co., Ltd.