ObjectiveTo evaluate the sensitivity and specificity of reverse transcriptase polymerase chain reaction (RT PCR), immunohistochemistry (IHC) and micropartical enzyme immunoassay (MEIA) in detecting metastasis of human colonic carcinoma (HCC) in a nude rat xenograft model. Methods A metastatic model of HCC in nude rats was established by injecting HT 29 cells, a HCC cell line, into the mesenteric vein of the rats. Hepatic and pulmonary metastasis were detected by RT PCR for cytokeratin 20 (CK20) expression, APAAP immunostaining with monoclonal antibody (McAb) KL 1 (anti cytokeratin) for human CK expression, routine pathology (RP) and MEIA for tissue carcinoembryonic antigen (CEA) determination, respectively. Results The detection rate of HCC metastasis by RT PCR, IHC, RP or MEIA was 73 8%, 64 3%, 50% and 33%, respectively. The detection rate of RT PCR was higher than that of RP (? 2 =5 048, P 0 05). The detection rate of IHC was higher than that of MEIA (? 2 =8 052, P 0 05). RT PCR, IHC and MEIA were all highly specific in detecting HCC cells in nude rats. ConclusionsRT PCR for CK20 expression and APAAP immunostaining with the McAb KL 1 for human CK expression were highly sensitive and specific diagnostic techniques in detecing HCC metastasis. MEIA for tissue CEA determination was highly specific, but with a low sensitivity.

OBJECTIVE: To analyze the relationship between metastatic potential and related facters of colorectal tumor cell lines. METHODS: The variants HT-29c and HT-29d cell lines derived from the selection of HT-29 cells were injected into nude rats and the metastatic potential of the two tumor cell variants was analyzed. Expression of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) were measured with ELISA in vitro in colorectal carcinoma cell lines WiDr, HT-29 and HT-29d. Expression of carcinoembryonic antigen (CEA) and phosphoinositide 3-kinase (PI3-Kinase) were determined with immunohistochemistry, (IHC) in vitro and in vivo in WiDr, HT-29 and HT-29d cell lines. In addition, CEA expression was demonstrated with fluorescence activated cell sorter (FACS) in vitro. RESULTS: The liver metastasis rate of the variant HT-29d (with 4 cycles of selection), increased as compared with that of parental HT-29 cells and that of variant HT-29b cells (with 2 cycle of selection). The uPA concentration of variant HT-29d cell line was significantly higher than that of the non metastatic WiDr and the low metastatic HT-29 cells (P<0.05). The variant HT-29d cells produced stronger PI3-kinase expression as compared with the non-anetastatic WiDr cells and the low metastatic HT-29 cells in vivo. CONCLUSION: The selected variant cell lines can exhibit an enhanced metastatic potential. The level of uPA and PAI-1 are positively correlated with the metastatic capacity of tumor cells. The expression of PI3 kinasecorrelates with tumor development and metastasis.

OBJECTIVETo evaluate the gene transfer and expression of enhanced green fluorescent protein (EGFP) in retrovirally transducted variant HT-29c cells in vitro and in vivo.

METHODSThe retroviral vector prkat EGFP/neo was constructed and was transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (in vivo selected HT-29 cells) were transduced by a retroviral vector encoding the EGFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transfected with the EGFP gene bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransfected and transfected in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo.

RESULTSAfter being transduced, HT-29c cells with the EGFP gene displayed a stable and long-term EGFP expression under the nonselective conditions in vitro. After culturing cells successively to passage 50 in vitro, EGFP expression level was still high. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransfected parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed.

CONCLUSIONSAn EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo. These cells are a valuable tool for in vivo analysis of metastatic spread.

Animals ; Colorectal Neoplasms ; genetics ; pathology ; Disease Models, Animal ; Gene Expression ; Gene Transfer Techniques ; Green Fluorescent Proteins ; HT29 Cells ; Humans ; Luminescent Proteins ; genetics ; Neoplasm Metastasis ; Neoplasm Transplantation ; Rats ; Rats, Nude ; Transduction, Genetic ; Transfection