|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
After stimulating of mouse peritoneal cells with a cytokine, the supernatant from the culture strongly induced the apoptosis of mast cells and several tumor cell lines such as NS-1, indicating a presence of an apoptotic factor in the culture. By using the deficient mice, neutralizing antibodies, recombinant cytokines, and RT-PCR analyses, we confirmed that this apoptosis-inducing activity is not related to the well-known apoptosis-inducing factors such as TNF-α, TNF-β, TRAIL, Fas/FasL, Perforin etc. The ultimate aim of the project is to identify and clone the unknown apoptotic factor.
Until now, we tried to clone the factor via the following two ways. Firstly, the mRNA from cytokine-stimulated and -non-stimulated peritoneal cells was isolated and converted into 1^<st> strand cDNA. After subtractive hybridization using CLONTECH PCR-Select cDNA Subtraction Kit, the cDNA was PCR amplified and the PCR products were cloned into a pCR 2. 1-TOPO vector of INVITROGEN. The ligated plasmids were
then transformed into TOPO10 competent cells. We analyzed more than 200 clones by sequence analysis of DNA. Although several novel cDNA sequences were identified, all of them were not related to the apoptosis-inducing activity. Secondly, by using ZAP Express cDNA Synthesis Kit and cDNA Gigapack III Gold Cloning Kit of STRATAGENE, the cDNA from the cytokine-stimulated peritoneal cells was ligated into an eukaryotic expression vector (pBK Phagemid Vector). Then the phagemids from each 50 clones were isolated and transformed into COS-7 cells for protein expression. The culture supernatants were collected and analyzed for a possible apoptosis-induction of NS-1 cells. More than 10,000 clones were analyzed ; however, no functional clone was identified.
According to the above results of the two methods, we realized that it is very difficult to clone the apoptosis-inducing factor by the generally used methods. Therefore, we begin to focus on a new strategy, that is, the progressing technique of PROTEOMICS. This strategy is based on the 2-D Electrophoresis and Mass Spectrometry and is mostly suitable for the post-genome researches. The techniques provide a possibility for us to successfully clone the apoptosis-inducing factor. This project is being continued. Less