Correlation between a gene polymorphism of tumor necrosis factor and inflammatory bowel disease.

OBJECTIVES: To analyze polymorphism of the tumor necrosis factor (TNF) gene in inflammatory bowel disease (IBD) patients from the Han Chinese ethnic group, and to investigate the role of polymorphism in the pathogenesis of IBD. METHODS: Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques were used to analyze gene polymorphisms in the TNF-alpha and TNF-beta genes in 131 patients with IBD. RESULTS: The genotype frequency and allelic frequency of TNF-alpha-308 in patients with ulcerative colitis (UC) were 15.5% and 8.7%, respectively, significantly higher than the control population (4.1% and 2.0%, respectively; P < 0.001). There was no significant difference between patients with Crohn's disease (CD) and the normal population with regard to the genotype frequency and allelic frequency of TNF-alpha-308, and neither were there any differences with regard to TNF-beta+252 in patients with IBD (UC and CD) and the normal population. The TNF-alpha-308 polymorphism and the TNF-beta+252 loci did not correlate with age, gender, disease activity or lesion site for IBD patients. CONCLUSIONS: The TNF-alpha-308 allele may be related to susceptibility to UC. The TNF-alpha-308 gene polymorphism is not involved in pathogenesis of CD. No correlation was found between the TNF-beta+252 polymorphism and IBD. Polymorphisms of the TNF-alpha-308 and TNF-beta+252 loci do not correlate with age, gender, disease activity or lesion site.

[A study on the regulatory mechanisms of mitogen-activated protein kinase phosphatase-1 in hypoxia inducible factor-1 trans-activation].

OBJECTIVE: To investigate the regulatory mechanisms of mitogen-activated protein kinase phosphatase-1 (MKP-1) in hypoxia inducible factor (HIF)-1 trans-activation. METHODS: (1) Gastric cancer cells of the line SGC7901 were cultured, then continued to be cultured in hypoxic environment, and was lysed. The supernatant was collected. Western blotting was used to detect the content of total extracellular signal-regulated kinase (ERK) and phosphorylated ERK. (2) Another SGC7901 cells were cultured with PD98059, inhibitor of ERK passway, or SB203580, inhibitor of p38 passway, in the same manner as above-mentioned. Dual luciferase reporter (DLR) was used to detect the luciferase activity so as to measure the HIF-1 trans-activation. (3) siRNA vector U6M2 plasmid against MKP-1 mRNA was constructed. In another experiment SGC7901 cells were cultured and U6M2 and blank vector U6 were transfected into the cells respectively. 24 hours later, the cells were cultured in hypoxic environment with added PD98059 of different concentrations for 12 hours. Dual luciferase reporter (DLR) was used to detect the luciferase activity HIF-1 trans-activation. (4) Another SGC7901 cells were co-transfected with U6M2, pGL-3SV40HRE vector containing promoter SV40, and pRL-TK (internal control vector). Then PD98059 was added, the cells were lysed, and the activity of fluorescein was tested. (5) SGC7901 cells were cultured, transfected with UdM2 or U6 respectively, and 24 hours later cultured under hypoxia with PD98059 of different concentrations for 12 hours. ELISA was used to examine the VEGF protein concentration in the culture fluid. RESULTS: (1) The content of phosphorylated ERK in the SGC7901 cells increased along with the time of hypoxia, peaked at the 12th hour, and then decreased. However, there was no difference in total ERK expression. (2) After 12 hours of hypoxia, different concentrations of PD98059 inhibited the luciferase activity, however, SB203580 of different concentrations had no effect. (3) 24 hours after transfection, the expression of phosphorylated form of ERK in the SGC7901cells transfected with siRNA plasmid against MKP-1 mRNA was higher compared with that in cells transfected with blank vectors after 12 hour of exposure to hypoxia. (4) PD98059 inhibited the luciferase activity either in U6 cells or in U6M2 cells. Notably, when the PD98059 concentration was above 50 micromol/L, there was no difference in HIF-1 activity between the U6 and U6M2 cells. (5) PD98059 of different concentrations all inhibited the VEGF expression either in U6 cells or in U6M2 cells, and when the concentration of PD98059 was over 50 micromol/L there was no difference in VEGF expression level between the U6 cells and the U6M2 cells. CONCLUSION: In SGC7901 cells, the function of MKP-1 is involved in regulation of HIF-1 trans-activation via inactivation of the ERK pathway.

[Effect of a hypothetical gene Af116609 on multi-drug resistance of gastric cancer cells].

OBJECTIVE: To investigate the effect of gene Af116609 on gastric cancer multi-drug resistance (MDR) by introducing it into gastric cancer multi-drug resistant (MDR) cell line SGC7901/VCR. METHODS: Gene Af116609 was cloned from SGC7901/VCR by RT-PCR and its differential expression between gastric cancer MDR cells and its parental cells was displayed by Northern blot. The gene was introduced to gastric cancer cells by transfection of recombinant eukaryotic expression vector by electroporation. MTT assay in vitro was applied to investigate its effect on multi-drug resistance phenotype of gastric cancer cells. RESULTS: The full length CDS of gene Af116609, as long as 327 bp, was cloned from gastric cancer MDR cell line SGC7901/VCR and its sequence was coincident with the hypothetical gene Af116609 in GenBank. It was overexpressed in MDR cells than its parental cells at mRNA level. In the MTT assay in vitro, the drug sensitive cells transfected with sense eukaryotic expression vector showed upregulated targeted gene, with increased resistance to vincristine, 5-fliorouracil and arabinoside, and decreased resistance to adriamycin, but no influence on resistance to methotrexate. However, the drug resistant cells transfected with anti-sense eukaryotic expression vector, showed down regulated targeted gene, with less resistance to all the five anticancer drugs to different degrees. CONCLUSION: Gene Af116609 is related to MDR phenotype of gastric cancer cells and may become a candidate molecular target to reverse the MDR of gastric cancer.

Diagnosis of atrophic body gastritis in Chinese patients by measuring serum pepsinogen.

OBJECTIVE: Atrophic body gastritis (ABG) is common in China. Although histology via endoscopy is an efficient and reliable means of diagnosing ABG, it is an invasive procedure. Therefore, in the present study serum pepsinogen (PG) was used as a biomarker to develop a novel noninvasive test as the first option for screening of ABG in certain groups of Chinese. METHODS: The study population consisted of 81 selected dyspeptic patients (mean age, 64.8 +/- 0.7 years; M:F, 43:38) who underwent diagnostic gastroscopy. At least four biopsy specimens were taken from the antrum and corpus of the stomach (two specimens from each site) for histological diagnosis. Blood samples for ELISA assays of serum pepsinogen I (PGI), pepsinogen II (PGII) and IgG antibodies against Helicobacter pylori (Hp IgG) were drawn after endoscopy. Cut-off points were calculated using receiver operating curves (ROC). RESULTS: There was no correlation between serum PG and atrophy in the antral mucosa. The mean serum concentration of PGI was lower (P < 0.05) in patients with ABG (89.9 microg/L) than in those with normal mucosa (NM) and non-ABG (123.7 microg/L and 139.1 microg/L). The mean ratio of PGI:PGII was also lower (P < 0.01) in patients with ABG (6.2) than in those with NM and non-ABG (11.6 and 11.7). There was no difference in serum PGI or the PGI:PGII ratio between patients with and without H. pylori infection. For diagnosing ABG, the area under the ROC of PGI and the PGI:PGII ratio was 0.741 (95% CI: 0.627-0.856) and 0.874 (95% CI: 0.788-0.961), respectively. The maximum of the Youden's index (YI) of PGI and the PGI:PGII ratio was 0.426 and 0.722, respectively. The best cut-off point for PGI was 97.1 microg/L with sensitivity of 67% and specificity of 76%, and for PGI:PGII ratio was 8.1 microg/L, with sensitivity of 89% and specificity of 83%. CONCLUSIONS: The serum PGI:PGII ratio appears to be a sensitive and specific assay for corpus atrophy, thus providing a noninvasive and indicative test for diagnosis of atrophic gastritis.

Characterization of a specific phage-displayed Peptide binding to vasculature of human gastric cancer.

Antivascular therapy provides a promising method for anticancer therapy. But targeting to gastric cancer vessels is nonselective due in part to the lack of specific cell-surface receptors identified on target vascular cells. Herein we used in vivo screening of phage displayed peptide library to identify some peptides that bind selectively to endothelial cells of human gastric cancer rather than nonendothelial cells. After four rounds of selection, one phage was obtained with a cyclic 7-mer peptide CGNSNPKSC homing to human gastric adenocarcinoma . There was a 4.6 approximately 137.26-fold increase in the number of the selected phage in gastric cancer xenograft in comparision with control organs brain, heart, liver, spleen and kidney. Immunohistochemistry in mouse and human tissue showed that this phage peptide only bind to the endothelial cells of human gastric cancer. This peptide was observed only specific binding to HUVEC not to SGC-7901, Eca-109, LoVo and Hep-G2 by ELISA. The competitive and inhibitory result between the synthetic CGNSNPKSC peptide and the phage displaying the peptide CGNSNPKSC on HUVEC and in vivo was also confirmed its specific binding effect. This peptide may be a possible candidate for targeted drug delivery in antivascular therapy.

[Multidrug resistant effect of alternative splicing form of MAD2 gene-MAD2beta on human gastric cancer cell].

OBJECTIVE: To study the effect of alternative splicing form -MAD2beta of mitotic arrest deficient protein 2 (MAD2) on the formation of multidrug resistance in human gastric adenocarcinoma cell SGC7901. METHODS: RNA was extracted from a multidrug resistance cell line SGC7901/ADR. The full-length MAD2beta cDNA was obtained by RT-PCR and cloned into the pUCm-T vector, and then recombined into the eukaryotic expression vector pcDNA3.1 in forward direction. Subsequently, pcDNA3.1/MAD2beta vectors were then transfected into SGC7901 cells by lipofectamine. Sensitivity to drug was detected by MTT assay. Cell cycle alteration and intracellular fluorescence intensity were determined by FACS. RESULTS: A fragment of 0.53 Kb was obtained and confirmed by DNA sequencing which was a new alternative splicing form of MAD2 named as MAD2beta. pcDNA3.1/MAD2beta transfected SGC7901 cells (SGC7901/MAD2beta) were more resistant to ADR, VCR and MMC than the control cells (SGC7901/pcDNA3.1), and also ADR fluorescence intensity of SGC7901/MAD2beta cells was lower (P < 0.05) than that of SGC7901/pcDNA3.1 cells. CONCLUSION: MAD2beta could increase the multidrug resistance of SGC7901 cell line.

[Prokaryotic expression of human calcyclin-binding protein and preparation of mouse polyclonal antibody against hCacyBP].

AIM: To express hCacyBP in E.coli and prepare mouse polyclonal antibody against hCacyBP so as to study tissue distribution of hCacyBP and evaluate its role during formation of multidrug resistance to gastric cancer. METHODS: hCacyBP gene was subcloned into an expression vector pET28a, and then the recombinant vector was transformed into E. coli BL21. The recombinant protein was expressed in BL21 under IPTG induction. Using purified hCacyBP as immunogen, mouse polyclonal antibody against hCacyBP was prepared. hCacyBP was detected by Western blot in 10 kinds of rabbit tissues. Expression and distribution of hCacyBP in SGC7901/VCR and SGC7901 cells were detected by Western blot and immunohistochemical staining. RESULTS: hCacyBP was successfully expressed in E. coli. The Western blot analysis showed that hCacyBP was expressed in all 10 kinds of rabbit tissues, but expression of brain and liver tissues were higher as compared with other tissues. Expression and distribution of hCacyBP in both SGC7901/VCR and SGC7901 cells had no significant difference. CONCLUSION: CacyBP expressed widespreadly in varied tissues. Polyclonal antibody against hCacyBP that we prepared has high specificity, which provides a powerful tool for studying the function of hCacyBP.

[Expression pattern of tumor-associated antigen MG7-Ag in gastric cancer: difference between Tibetans and Hans].

OBJECTIVE: To investigate the difference of the expression pattern of tumor-associated antigen MG(7)-Ag in gastric carcinoma between Hans and Tibetans. METHODS: Avidin-biotin peroxidase complex immunohistochemical methods were adapted to examine the expression pattern of MG(7)-Ag in 200 patients with gastric carcinoma, including 100 Tibetan patients and 100 Hans patients, 10 patients with chronic artrophic gastritis, 18 with gastric mucosal dysphasia and 10 subjects with normal gastric mucosa served as control. RESULTS: The positive rate for MG(7)-Ag was 92.4% and 88.7% in Tibetan and Han gastric carcinoma patients, 55.6% and 38.9% in Tibetan and Han gastric mucosal dysplasia patients, 20.0% and 10.0% in Tibetan and Han chronic atrophic gastritis patients, respectively. There was no positive expression in normal gastric mucosa of both Hans and Tibetans. >From normal gastric mucosa, chronic artrophic gastritis and gastric mucosal dysplasia to gastric carcinoma, expression of MG(7)-Ag showed an ascending tendency in the same group of subjects or patients (P < 0.01). However between Hans and Tibetans there was no significantly different expression of MG(7)-Ag. In gastric carcinoma, the expression pattern of MG(7)-Ag was not related to tumor cell differentiation and metastasis of lymph nodes in Hans and Tibetans (P > 0.05), but in the same group the expression of MG(7)-Ag was related to metastasis of lymph nodes. CONCLUSIONS: The expression pattern of MG(7)-Ag displayed no significant difference between Hans and Tibetans, The tumor associated antigen MG(7)-Ag in gastric carcinoma can be used in Tibetans as a reliable marker to predict early gastric cancer.

[The overexpression and significance of MKP-1 in oxygen-deprived gastric cancer cell line SGC7901].

OBJECTIVE: To investigate the expression status of Mitogen-activated Protein Kinase Phosphatase-1 in oxygen-deprived gastric cancer cell line SGC7901 and its role in HIF-1 regulation. METHODS: The expression of MKP-1 in gastric cell line SGC7901 was detected with Western blot and semiquantity RT-PCR; Eukaryotic sense expression vector was constructed based on DNA recombination technology. Transfections of SGC7901 were performed using liposome; The luciferase activity was determined using Dual Luciferase Reporter System and the levels of VEGF in SGC7901cells under normoxia and hypoxia were measured by ELISA. RESULTS: (1) Semiquantity RT-PCR and Western blot suggested that the expression of MKP-1 was elevated in oxygen-deprived gastric cancer cell line SGC7901; (2) 48 hours after transfection, the phosphorylated form of HIF-1alpha in cell line transfected with recombinant plasmids was lower compared with that in cell line transfected with empty vectors after 12 hours of exposure to hypoxia; (3) There was very low luciferase activity under nomoxia while under hypoxia luciferase activity increased in a time-dependent manner and at all time points there was significant lower luciferase activity in SGC7901 cells than in cells transfected with empty plasmids. (4) At different points of time course, the expression of VEGF in SGC7901 was significantly higher under hypoxia than that in SGC7901 under nomorxia, while under hypoxia, the expression of VEGF in SGC7901 transfected with recombinant plasmids was significantly lower than that in SGC7901 transfected with empty vectors at all time points as indicated. CONCLUSION: The expression of MKP-1 in SGC7901 was elevated under hypoxia, which could downregulate the HIF-1 trans-activition activity thereby repressing the expression of downstream target gene VEGF.

[The overexpression of prion protein in drug resistant gastric cancer cell line SGC7901/ADR and its significance].

OBJECTIVE: To investigate the overexpression of prion protein (PrP) in drug-resistant gastric cancer cell line SGC7901/ADR and its role in multidrug resistance in gastric cancer. METHODS: (1) The expression of PrP in SGC7901/ADR, SGC790/VCR and their parental cell line SGC7901 was detected with Northern and Western blot at the mRNA and protein level. (2) Eukaryotic sense and antisense expression vector were constructed based on DNA recombination technology and (3) introduced into SGC7901 and SGC7901/ADR cell lines through electroporation. (4) The accumulation and retention of ADR in transiently transfected cells were detected by flow cytometry. RESULTS: (1) Northern and western blot suggested significantly higher expression of PrP in SGC7901/ADR and SGC7901/VCR than that in SGC7901. (2) 48 hours after the vectors transfection, the average fluorescence intensity of Adr in transfected cells were detected. The accumulation intensity were 8.9 +/- 0.7 in BS, 6.6 +/- 0.3 in PS and 7.5 +/- 0.6 in PA. The retention intensity were 9.3 +/- 0.6 in SGC7901, 5.9 +/- 0.5 in PS and 7.1 +/- 0.5 in PA. There were significant difference between PS and BS with P < 0.01, as well as RA and BA with P < 0.01. These data suggested that PrP gene could affect the drug accumulation in gastric cancer cells after its transfected into cells. CONCLUSION: PrP was highly expressed in gastric cancer cell lines SGC7901/ADR and SGC7901/VCR. Overexpression of PrP had certain effect on drug accumulation in gastric cancer cells.

[Differential expression of RPL6/Taxreb107 in drug resistant gastric cancer cell line SGC7901/ADR and its correlation with multiple-drug resistance].

OBJECTIVE: To investigate the differential expression of RPL6/Taxreb107 between drug-resistant gastric cancer cell line SGC7901/ADR and gastric cancer cell line SGC7901 as well as its correlation with multiple-drug resistance (MDR) in gastric cancer cells. METHODS: Total RNA was extracted from SGC7901 and SGC77901/ADR, with internal control RT-PCR, Northern blot, gene cloning and expression, construction of eukaryotic expression vector, gene transfection by electroporation. The accumulation and retention of ADR in transiently transfected cell was detected by flow cytometry. RESULTS: The internal control RT-PCR and Northern blot showed high RPL6/Taxreb107 expression in SGC7901/ADR cell line. Sense and antisense eukaryonic expression vectors demonstrated by double enzyme digestion were successfully transfected into gastric cancer cell line SGC7901 and SGC7901/ADR respectively by electroporation. The accumulation and retention of ADR detected 48 hours after transfection showed that RPL6 gene had shown effect on drug resistance in gastric cancer cell. CONCLUSION: The high expression of RPL6/Taxreb107 in drug resistant gastric cancer cell shows its correlation with multiple-drug resistance in gastric cancer.

[In-vivo screening and characterization of peptides specific for vasculature of gastric cancer].

AIM: To screen in vivo from a phage-displayed peptide library polypeptide fragments specific binding to vascular endothelial cells of gastric cancer xenografts, so as to provide for anti-angiogenesis therapy of tumor. METHODS: Immunosupressed mice models for human gastric cancer xeno-grafts were established by subrenal capsular assay (SR-CA). The 12-peptide library was panned through 4 rounds. Phages were recovered and titrated from tumor xenografts and control tissue (brain). The distribution of phage were detected in transplanted tumor tissues by immunohistochemical staining. RESULTS: Phage homing to gastric cancer xenografts were enriched through four rounds of panning,being 3.4-fold of that recovered from brain tissue. Peptide sequences were characterized for randomly picked-upclones and the peptide sequence YESIRIGVAPSQ appeared most frequently. Immunohistochemical staining for the homing phage revealed a specific vascular endothelial cell localization in gastric cancer xenografts 5 min after injection of the enriched phages. When the specific phage individually test-ed, the phage recovered from gastric cancer xenografts were as 4. 2 times as those from control tissue ( brain) , as 4.9 times as those from lung, as 5.4 times as those from heart. CONCLUSION: The tumor-specific homing peptides may provide a effective tool for targeting tumor vasculature in anti-angiogenesis therapy of cancer. The in vivo selection technique in this study was feasible and applicable to screening peptides homing to vascular endothelial cells.

[Screening of bioactive peptide that mimic the epitope of gastric cancer associated antigen].

AIM: To screen bioactive peptides that mimic the epitope of gastric cancer associated antigen. METHODS: Anti-gastric cancer monoclonal antibody (mAb) MG7 was purified by ion chromatography, and then coated on ELISA plate. By using the mAb as selective molecular, a 12-meres phage displaying peptide library was biopanned, and the positive clones were selected. RESULTS: 12 positive phage clones were selected. CONCLUSION: A candidate mimic epitope of gastric cancer associated antigen was identified, which provided the foundation for developing tumor peptide vaccine.

[Preparation of phage-displayed single chain variable fragments of monoclonal antibody MC5 recognizing colorectal carcinoma].

BACKGROUND & OBJECTIVE: MC5 is a murine monoclonal antibody with a good specificity to human colorectal carcinoma and smaller murine antibody can significantly decrease the possibility of developing human antimouse antibody response in vivo study. The aim of this study was to prepare single chain variable fragments (ScFv) of MC5. METHODS: mRNA was isolated from the hybridoma cell line producing MC5, and the DNAs encoding variable domains of heavy and light chains(VH and VL DNAs) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNAs with a linker DNA. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E. coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage antibody ScFvs. After two rounds of panning with cell line SW480 highly expressing MC5-binding antigen, the phage clones displayed ScFv fragments of the antibody were selected by ELISA, and the affinity of the positive phage clones was assayed by competition ELISA. RESULTS: The VH, VL, and ScFv DNAs were about 340 bp, 320 bp, and 750 bp, respectively. Ten phage clones displayed ScFv of MC5 were selected from 25 enriched phage clones, and 3 of the 10 phage clones had higher affinity of binding to the antigen. CONCLUSION: The phage-displayed ScFv fragments of monoclonal antibody MC5 are successfully produced by phage display technique, which may provide a way for broadening the application range of the antibody.

Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3.

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

Immuno-protective effect of tumor cell vaccine on Kunming mice bearing Ehrlich ascites tumor.

AIM:To evaluate the immunity of chemically modified tumor cell vaccine.METHODS:Tumor cell vaccines (TCV) were prepared by incubating the live Ehrlich ascites tumor cells with concanavalin A-mitomycin C (ConA-MMC), mitomycin C (MMC), concanavalin A-glutaraldehyde (ConA-Glu), glutaraldehyde (Glu), or paraformaldehyde (Para), respectively. The whole cell or soluble forms of the vaccines were administered intraperitoneally into Kunming mice once a week for three times prior to the intraperitoneal inoculation of a lethal dose of live tumor cells. A second challenge with live tumor cells was given four weeks later. Survival and antibody production of the mice were analyzed.RESULTS:After the first challenge, the mice, received whole TCV of ConA-MMC, MMC (P < 0.01) and Glu (P < 0.05) promoted survival incidence than the controls. All the treated mice had the survival time prolonged. ConA-MMC vaccine treated mice had longer survival days than that of ConA-Glu ones (P < 0.05). For the soluble TCV immunized mice,those treated with vaccines of Para (P < 0.01), ConA-Para and ConA-Glu (P < 0.05) had longer survival periods compared with that of the controls. Following the second challenge, survival incidence of the mice received vaccines of ConA-MMC, MMC, ConA-Glu or Glu was significantly increased (P < 0.01). Moreover, all the treated mice had the survival time prolonged, and ConA-MMC vaccine treated mice had longer survival days than that of Para treated ones (P < 0.05). Antibodies against Ehrlich ascites tumor cells were found to be positive in sera of the mice treated with whole TCV of ConA-MMC.CONCLUSION:Ehrlich ascites tumor cells are immunogenic when treated with ConA-MMC, MMC, ConA-Glu, Glu or Para, which might act as safe and effective tumor vaccines with safety and effectiveness.

Transduction of Fas gene or Bcl-2 antisense RNA sensitizes cultured drug resistant gastric cancer cells to chemotherapeutic drugs.

AIM:To compare the expression level of Fas gene and Bcl-2 gene in gastric cancer cells SGC7901 and gastric cancer MDR (multidrug resistant) cells SGC7901/VCR,to transduce Fas cDNA and Bcl-2 antisense nucleic acid into SGC7901/VCR cells respectively, and to observe the expression of two genes in transfectants and non-transfectants as well as their drug sensitivity.METHODS:Eukaryotic expression vector pBK-Fas cDNA and pDOR-anti Bcl-2 were constructed and transfected into SGC7901/VCR cells by lipofectamine,respectively.Northern blot and Western blot were used to detect the expression of mRNA and protein in SGC7901/VCR and SGC7901 cells and transfectants, and drug sensitivity of transfectants for VCR, CDDP and 5-FU was analyzed with MTT assay.RESULTS:After gene transfection, 80 for Fas and 120 for antisense Bcl-2 drug-resistant clones were selected from 2 X10(5) cells, transfection rate being 0.04% and 0.06%. Two clones of SGC7901 Fas/VCR cells and SGC7901 anti Bcl-2/VCR cells were randomly selected for further incubation. Hybridization results showed that the expression level of Fas mRNA and protein in SGC7901/VCR cells was much lower,but that of Bcl-2 mRNA and protein was higher than that in SGC7901 cells. The expression of Fas mRNA and protein in SGC7901 Fas/VCR cells was higher,and of Bcl-2 mRNA and protein was lower in SGC7901 anti Bcl-2/VCR cells than that in non-transfectants. MTT assay showed that transfectants were more sensitive to VCR, CDDP, 5-FU than non-transfectants. CONCLUSION:Bcl-2 gene displayed high expression while Fas gene had low expression in drug resistant gastric cancer cells. Expression of Bcl-2 protein was effectively blocked in SGC7901 anti Bcl-2/VCR cells by gene transfection. In contrast, the expression of Fas mRNA and protein in SGC7901 Fas/VCR cells increased. Fas gene and Bcl-2 antisense nucleic acid transfection sensitized drug resistant gastric cancer cells to chemotherapeutic drugs. These results suggest cell apoptosis plays an important role in the mechanism of MDR, and enhancing apoptosis might reverse MDR.