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Abstract
Purpose:
Our aim of research is to find novel genes that overexpressed in various samples such as cell tines and tissues that infinitely proliferate; so-called immortalized cells, cancer cells and tissues. In this study, we obtained a gene from immortalized cell line (WI-38 VA13) then identified it from various cell lines and human lung tissues.
Materials and Methods:
Using suppressive subtractive hybridization (SSH) method, we obtained many genes and selected a novel gene of them. And then the novel gene fragment was amplified by PCR and ligated in Τ easy vector for sequencing. And finally we found a differentially expressed gene in cell lines and tissues when it was performed by reverse transcriptase-PCR (RT-PCR),
Results:
As the result of transformation of genes that we gained using SSH, we obtained about 150 clones. And then we certificated several genes by DNA prep and confirmed it by sequencing. We examined whether the gene sequences are novel or known genes by genome homology search and we confirmed the gene expressions by RT-PCR. As a result, we identified a differentially overexpressed gene (named “clone 58”) in immortalized cells, cancer cell lines and lung squamous cell carcinomas.
Conclusion:
The “clone 58” mRNA was significantly up-regulated in various human cell lines and also human lung cancer tissues compared to the normal. We suppose that this gene can carry out a specific role related to the induction of cancer and/or the mechanism of the changeover of a normal cell to an immortalized cell. In short, the discovery of our gene has an importance in the point that they are thought to have a connection with immortalization and carcinogenesis of human cells and tissues, (J Lung Cancer 2006;5(2):96 — 101)
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Fig. 1.
Reduction of G3PDH abundance by PCR-select subtraction. Normal cDNA was prepared from WI38 (human lung fibroblast cell) total RNA and immortalization cDNA was prepared from WI38 VA13 (transformed WI38 by SV40), PCR was performed on subtracted (lanes 1 〜4) or unsubtracted (lanes 5-8) secondary PCR product with the G3PDH 5’ and 3’ primers. Lanes 1, 5- 18 cycleså lanes 2ô6: 23 cycles; lanes 3, 7’ 28 cycles, lanes 4, 8: 33 cycles.
Fig. 3.
Expression of clone 58 in Wᅵ38ôWᅵ38 VA13 (A) and various cell lines (B), (A) Lane 1æ WI38 (lung fibroblast normal cell), lane 2æ WI38 VA13 (SV-40 transfected cell), lane 3æ WI26VA4 (SV-40 transfected cell). (B) Lane 1- skin normal cell, lane 2’ skin tumor cell, lane 3: colon normal cell, lane 4: colon tumor cell, lane 5- prostate normal cell, lane 6: prostate tumor cell, lane 7: SK (SK- N-SH): brain neuroblastoma, metastasis to bone marrow, lane 8: A172: brain, glioblastoma, lane 9: HeLa, lane 10æ Lung cancer. 18 s rRNA was used as the reference gene.
Fig. 4.
Clone 58 expression in non-small cell lung carcinoma tissues. (A) Næ normal lung tissues J: squamous cell carcinoma. (B) Næ normal lung tissues, T: adenocarcinoma 18s rRNA was used as the reference gene.
Fig. 5.
Serum siimulatioa Clone 58 expressions have no significant on time dependent manner. Lung fibroblast cell line (WI38) was used Lane 1: arrest stage, lane 2æ 0.5 hr, lane 3: 1 hr, lane 4: 2 hr, lane 5: 4 hr, lane 6: 8 hr, lane 7æ 12 hr, lane 8: 24 hr after serum stimulation. 18 s rRNA was used as the reference gene.
Table 1.
Identification of Known Tumor Specific Genes from SSH Libraries
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