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Cell migration and invasion are triggered by a number of chemoattractants that stimulate intracellular signaling pathways through regulating reorganization of the actin cytoskeleton. Rac1, an intracellular signal transducer, regulates a variety of cell functions, including the organization of the cytoskeleton, cell migration, and invasion. However, currently, very little is known about the roles of Rac1 in the cytoskeleton formation and invasion of human colorectal cancer cells. In our study, Rac1-shRNA was used to silence the Rac1 to reduce its expression specifically in Lovo cells. Our studies showed that RNA interference-mediated deletion of Rac1 strongly inhibited lamellipodia formation, cell migration, and invasion of Lovo cells in vitro. The deletion of Rac1 can serve as an alterative therapy to inhibit the invasion and metastasis of colorectal cancer cells.

To evaluate the role of p53 gene mutation in colorectal carcinoma and assess the value of peripheral blood p53 single nucleotide polymorphism (SNP) in early diagnosis of colorectal carcinoma.NSP in axons 5-8 of p53 gene was detected using ligase detection reaction-polymerase chain reaction (LDR-PCR) in the peripheral blood of 100 patients with colorectal cancer and 100 healthy subjects.The mutation rate of p53 gene was 24% (24/120) in colorectal carcinoma patients and 0% (0/120) in the healthy subjects (P0.05).p53 plays an important role in the carcinogenesis of colorectal carcinoma, and SNP analysis for p53 gene can be helpful in early diagnosis of colorectal carcinoma.

We investigated the potential role of mitochondrial DNA (mtDNA) in colorectal carcinogenesis by constructing a eukaryotic expression vector of the mitochondrial D-loop gene from colorectal cancer cell SW480 and transfected NIH3T3 cells. The NIH3T3/SW480 cells exhibited a significantly increased growth rate and colony formation rate, and also had a decreased apoptotic rate. Polyploidy and aberrant chromosomes were detected in the NIH3T3/SW480 cells by chromosome karyotype analysis. Our results suggested that mtDNA from colorectal cancer cells promotes the malignant phenotype of NIH3T3 cells. Further study of the biological functions of NIH3T3/SW480 cells might be helpful in understanding the role of mtDNA in colorectal carcinogenesis.

Laterally spreading tumor (LST) behaves close to malignant tumor. This study was to search for the genes and functional proteins correlated to growth and development of colorectal LST (CLST), and to reveal the molecular mechanisms of CLST growth pattern and malignant transformation.cDNA microarray was used to screen differentially expressed genes among CLST cell line and other 2 colorectal cell lines LoVo and SW480. Different mRNA and protein expressions of the genes were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.Compared with LoVo and SW480 cells, 58 genes were up-regulated and 39 genes were down-regulated in CLST cells. mRNA level of ADP-ribosylation factor 1 (ARF1) was significantly higher in CLST cells than in SW480 and LoVo cells (0.93 vs. 0.74 and 0.47, P = 0.04); protein level of ARF1 was significantly higher in CLST cells than in LoVo cells.CLST has specific gene expression profile. ARF1 is overexpressed in CLST, and may mediate specific biological function of CLST.

To screen differentially expressed genes between laterally spreading tumor (LST) cell line and common colon carcinoma cell lines, and identify new targets and strategies for exploring the pathogenesis of colorectal tumor.The total RNA was extracted from the LST, SW480 and LoVo cells, from which purified mRNAs were obtained. The PCR products of 18 816 genes were blotted onto a fibrous membrane to generate the microarray. The mRNAs from the 3 cell lines were reversely transcribed into cDNA probes and labeled with (33)P before hybridization with the cDNA microarray. After thorough washing, the cDNA microarray was scanned and the 3 samples compared.A series of differentially expressed genes were found between the 3 samples, and 58 up-regulated and 39 down-regulated genes were identified among the 97 differentially expressed genes, which suggest different pathogeneses of the laterally spreading tumor. Further analysis of the obtained genes can be helpful in understanding the molecular mechanism of colorectal tumors.

To construct a cDNA phage expression library for human colorectal carcinoma antigens.After the total RNA was extracted from human colorectal cancer tissues, the single-strand and double-strand cDNA were synthesized through reverse transcriptase PCR and long-distance PCR, with the cDNA fragments smaller than 500 bp removed and the remaining cDNA combined with the right and left arms of dephosphorylated lambdaTriplEx2 phage vector. The recombinant phage were then packaged in vitro by MaxPlax Packaging extract, and a small portion of the packaged phage was used to infect E.coli XL1-Blue. Titer measurement was performed so as to determine the capacity of the library. SfiI restriction endonucleases was used to cut the recombined phage DNA in order to identify the size of inserted cDNA.The constructed cDNA phage expression library for human colorectal cancer antigens consisted of 2.39 x 10(6) pfu/ml bacteriophages with a recombination rate of 97.5% and the length of the inserted cDNA fragment ranged from 600 to 4,000 bp with an average of 1,400 bp.The cDNA phage expression library of human colorectal cancer antigens is successfully constructed to meet the currently recognized standards, and can be well applicable in screening cDNA-cloned genes of human colorectal cancer-associated antigens by immunoscreening.

To express the original human Fab antibody phage display library with positive recombined bacterium XL1-blue-Pcomb3 and identify its specific binding activity with colorectal cancer cells in vitro after screening with human colorectal cancer-related antigens.The recombination rate of Fd fragment of the heavy chain and insertion of kappa chain of the antibodies was determined with PCR, and the original Fab library was expressed. The antigens were extracted from 3 sensitized colorectal cancer tissues previously used for construction of the original Fab library and from 13 non-sensitized colorectal cancer tissues, along with the antigens from LoVo, HT-29 and LS-174T cells cultured in vitro. The original Fab antibody library was screened with the 3 groups of mixed antigens derived in preceding procedure and 3 tertiary Fab antibody libraries were obtained, which were then mixed in equal volume for subsequent tests of binding activity with human colorectal cancer tissues and cells in vitro using enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. Specimens of gastric and esophageal carcinomas and normal intestinal mucosa, together with liver cancer cells and gastric cancer cells were utilized as control.The recombination rate of Fd and kappa chain were 40 % and 70 % respectively, and the rate of their simultaneous insertion into Pcomb3 vector was 28%. The capacity of library for Fab fragment genes was 2.1x10(6), and the original antibody libraries screened with the 3 groups of mixed antigens were enriched to varied degrees, which all displayed relatively specific binding activity with human colorectal cancer tissue and cells in vitro.Colorectal cancer-related antibody Fab fragments are obtained through screening phage display library, which show relatively specific binding activity with human colorectal cancer tissues and cells.

To observe the effect of protein kinase C (PKC) on the multidrug resistance of multidrug-resistant colorectal cancer LoVo/Adr cells and explore the mechanism.The changes of PKC activity in LoVo/Adr cells in response to treatment with staurosporine (SP) and phorbol-12-myristate-13-acetate (PMA) were detected by way of 32P incorporation. The effect of PKC on adriamycin uptake in LoVo/Adr cells was detected by flow cytometry. Reverse transcriptase-PCR was utilized to observe the effect of PKC on mdr1 gene expression.PMA evinced bi-directional regulation of PKC activity in LoVo/Adr cells, and SP significantly inhibited membrane and cytosol fraction of PKC activity. Preincubation with PMA for 30 min caused the uptake of adriamycin to decrease significantly, but when the preincubation was prolonged to 24 h, significant increase occurred in adriamycin uptake. Neither PMA nor SP, however, could affect the expression of mdr1 gene.PKC regulates multidrug resistance of the cells through mechanisms other than regulation of mRNA level of mdr1 gene.

To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer.Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer. RT-PCR was used to amplify the heavy chain Fd and light chain kappa and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected.The amplified fragments of Fd and kappa gained by RT-PCR were about 650 bp. Fd and kappa PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 10(6). The libraries were enriched about 120-fold by 3 cycles of adsorption-elution-multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5 clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer.The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.