Aspergillus fumigatus IFO 5840이 생산하는 cytosine deaminase에 미치는 cytosine analogue의 영향을 조사하여 다음의 결과를 얻었다. 1.Cytosine deaminase의 강한 저해를 나타내는 cytosine analogue는 2-thiouracil, 2-thiocytosine, 2-mercaptopyrimidine, 및 6-azacytosine이었다. 2.본 효소에 대한 2-thiocytosine 및 6-azacytosine의 50% 효소활성 저해농도(HIC)는 각각 0.80mM 과 1.15mM 이었다. 3. 2-thiocytosine은 일정한 수준에서 본 효소의 반응을 정지시키는 반면, 6-azacytosine은 반응시간에 비례하여 일정한 비율로 저해하였다. 4. 2-thiocytosine과 6-azacytosine은 cytosine이나 5-fluorocytosine의 기질 종류에 관계없이 본 효소의 저해제로 작용하였으며 2-thiocytosine이 6-azacytosine보다 약 2배 정도의 높은 저해를 나타내었다. 5. cytosine을 기질로 하였을 경우, 2-thiocytosine과 6-azacytosine에 의해 경쟁적 저해를 나타내며 이들에 대한 K_i값은 각각 4.5 × 10^-4M 과 1.756 × 10^-3M 이었으며 cytosine, 2-thiocytosine 및 6-azacytosine에 대한 Hill 게수(Hn)는 각각 1.80, 1.81, 2.45로 나타났다.

Mesenchymal stem cells (MSCs) are considered to be attractive approaching in gene or drug delivery for cancer therapeutic strategies. In this study, the ability and feasibility of human bone marrow derived MSCs expressing the cytosine deaminase (CD)/5-Fluorocytosin (5-FC) prodrug was evaluated to target human osteosarcoma cell line Cal-72. At first, the fibroblast-like cells were successfully obtained from human bone marrow and demonstrated that they contained full of stem characteristics by the ability of differentiation into adipocyte/osteocyte and expression of typical mesenchymal markers CD90, CD44, while negative for CD34 and CD133 markers. We established the stable CD-expressing MSCs cell line (CD-MSCs) by transfection of pEGFP-C3 containing cytosine deaminase::uracil phos-phoribosyltransferase (CD::UPRT) gene into MSCs, and confirmed that the manipulated MSCs still remained full characteristics of multipotent cells and shown migration toward human osteosarcoma cancer cells Cal-72 as high as origin MSCs. Based on bystander effect, the therapeutic CD-MSCs significantly augmented the cytotoxicity on cancer cell Cal72 in either direct co-culture or conditioned medium in the presence of 5-FC. Moreover, in osteosarcoma cancer- bearing mice, the therapeutic CD/5-FC MSCs showed the inhibition of tumor growth compared with control mice which was s.c injected with only Cal72. Our findings suggest that these therapeutic CD-MSCs may be suitable and viable cellular vehicles for targeting human osteosarcoma cancer.