ABSTRACT. We have cloned the arginine deiminase (ADI) gene from Mycoplasma hominis PG21 genomic DNA by polymerase chain reaction, and changed four TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The recombinant ADI (rADI) was purified to apparent homogeneity by Ni-affinity chromatography after extraction from inclusion bodies followed by refolding. The rADI expressed in E. coli was estimated to be 50 kDa. Dimeric forms of rADI exerted enzymatic activity. We found that high concentration of potassium dihydrogenphosphate (PDP) and L-arginine addition in refolding reaction increases the enzyme activity. The specific activity of rADl was calculated as 0.618 U/mg. In addition, the enzyme activity of purified rADI remained for at least one month in 100 mM PDP solution (pH 6.5), but diminished within one week in 100 mM PDP solution (pH 7.4). Anti-tumor activity of the purified rADI was estimated to be 0.036 U/ml as 50% growth inhibitory activity against human melanoma cell line G-361. Arginine dehydrolysing mycoplasmas convert L-arginine into L-citrulline and ammonia by the arginine deiminase (ADI; EC 3.5.3.6) activity [1]. ADI gene of Mycoplasma hominis PG21 has been cloned in Escherichia coli by using plasmid pUC118 [7]. ADI has been shown to affect apoptosis and inhibit NO synthesis (i.e., anti-angiogenic effects), and exert effects against tumor necrosis factor- (TNF-) and neutralize endotoxin. [2,6,11,16,23,27]. ADI has also been known to inhibit the growth of melanoma and hepatocellular carcinoma cells in vitro as well as in vivo [19,20,24,26]. This anti-tumor activity of ADI has been attributed to the depletion of L-arginine, which is essential in these tumor cells [20,25]. Normal cells are not sensitive to external addition of ADI since they produce L-arginine by themselves. Expression of the ADI gene of M. homins PG21 in E. coli has been hampered since the ADI gene of M. hominis has four opal nonsense codons, which are known to encode tryptophan in mycoplasma cells. We have changed these opal codons to universal tryptophan codons and successfully expressed the recombinant ADI (rADI) gene of M. homins in E. coli, and demonstrated the enzymatic activity of purified rADI. MATERIALS AND METHODS Construction of ADI expression vector:Four stop codons TGA in the ADI gene cloned in pUC118 was changed into TGG by site-directed mutagenesis mediated by DpnImethod in order to express in E. coli [21]. Correct introductions of the mutation were confirmed by DNA sequencing. The mutated ADI gene, designated as SYN1903, was recloned into pET-47b (Novagen, Darmstadt, Germany) expression vector.Expression and purification of ADI: Recombinant pET47b plasmids encoding mycoplasmal ADI gene were transformed into competent E. coli BL21 (DE3) cells. The transformant was grown in 600 ml Luria broth (Sigma-Aldrich, St. Louis, MO, U.S.A.) at 30C, and isopropyl 1-thio--Dgalactoside was added at final concentration of 0.1 mM for induction of t...

[Background] Anti-human EGFR monoclonal antibody (cetuximab) has been widely used in the treatment of colorectal cancer (CRC). However it is thought that a reliable predictor for therapeutic efficacy should be established because the benefit of cetuximab treatment is often still uncertain. Our preliminary study showed that the prediction of therapeutic efficacy using a conventional chemosensitivity test using growth suppression assay is generally not informative because cetuximab does not seem to have as strong a cytotoxicity as other anticancer drugs. KRAS mutation status has been used as a biomarker to predict therapeutic efficiency but it is not sensitive enough to determine the absolute indication. [Purpose] We hypothesized that we could predict therapeutic efficacy by evaluating expected molecular reaction when cetuximab is administrated in vitro. In the present study, we build up an in vitro EGFR signaling model for a new chemosensitivity test based on protein monitoring in response to cetuximab administration. [Materials and Methods] Three growth factors (EGF, TGF-α, and IGF) and the inhibitor (cetuximab) were added to HT29 (KRAS wild type, EGFR positive) in six combinations. Cell pellets were harvested at 5 time points over a 15min time course, each of which was processed for cell lysate. The lysates were analyzed by Western blotting to see if the proteins involved in the EGFR signaling pathway responded over time. [Results] We confirmed the activation of signaling stimulated by EGF, TGF-α, and IGF; and the effect on signaling by cetuximab in HT29 with Western blotting. Intriguingly, the phosphorylation level of EGFR was rapidly elevated and declined by EGF stimulating in the absence of cetuximab whereas dephosphorylation was prolonged in the presence of cetuximab. In the administration of either TGF-α or IGF, the phosphorylation pattern of EGFR, c-RAF, MEK, MAPK, ERK, PI3K, AKT, PTEN, and STAT was different from that of EGF alone. [Conclusions] These results shows that:(i) EGF is not EGFR's only ligand; (ii) cetuximab can't inhibit every signaling pathway starting from EGFR; and (iii) TGF-α and IGF activate signaling pathway starting from EGFR partially but not thoroughly. An additional reliable predictor of cetuximab efficacy should be identified based on functional protein characteristics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4146.