Prezioso J A, Shields D, Wang N, Rosenstein M
Department of Radiation Oncology, University of Pittsburgh School of Medicine, PA 15213.
Int J Radiat Oncol Biol Phys. 1994 Sep 30;30(2):373-81. doi: 10.1016/0360-3016(94)90017-5.
To elucidate the role of gamma-glutamyltranspeptidase-mediated glutathione transport on the radiosensitivity of B16 melanoma variant cell lines.
B16 melanoma variant cell lines were examined for their levels of gamma-glutamyltranspeptidase (GGTP; E.C. 2.3.2.2), a plasma membrane-associated ectoenzyme that is involved in the transport of extracellular glutathione, by flow cytometric and biochemical analysis. B16 cell lines were examined for rates of de novo glutathione synthesis from extracellular glutathione and for their sensitivity to gamma-irradiation and glutathione synthesis inhibition. The GGTP inhibitors were examined for their effect on the radiosensitivity of B16 melanoma cells.
B16-F10-BL6 (BL6) melanoma cells were shown to express a 20-fold higher level of GGTP than the B16-F1 melanoma variant cells. Cultures of BL6 and B16-F1 cells depleted extracellular glutathione at rates of 2.4 and < 0.1 nmol glutathione/10(6) cells/h, respectively, and BL6 cells exported glutathione at a rate 7.2-fold higher than B16-F1 cells (710 and 98 pmol glutathione/10(6) cells/h, respectively). BL6 melanoma cells replenished exhausted intracellular glutathione levels from an extracellular glutathione source at a rate of 1.21 nmol glutathione/h (18% basal glutathione/h); however, B16-F1 cells lacked the capacity to replenish intracellular glutathione despite the presence of exogenous glutathione in the culture medium. BL6 melanoma cells were radioresistant compared to the B16-F1 cell line, exhibiting extrapolation numbers (ñ) of 14.9 and 1.0, respectively, and a lower surviving fraction to a wide range of radiation doses. The GGTP inhibitor combination of L-serine and sodium borate blocked the repletion of intracellular glutathione and in the presence or absence of buthionine sulfoximine-mediated depletion of glutathione reverses the radiation resistance in BL6 melanoma cells to near baseline levels observed with the B16-F1 parent clone. Serine-borate treatment of low-GGTP expressing B16-F1 cells had no effect on the ñ value or the surviving fraction of cells to a range of ionizing irradiation doses.
These results suggest that GGTP plays an important role in the extracellular metabolism and transport of glutathione, which also provides radioresistance to BL6 melanoma cells in vitro.
阐明γ-谷氨酰转肽酶介导的谷胱甘肽转运在B16黑色素瘤变异细胞系放射敏感性中的作用。
通过流式细胞术和生化分析，检测B16黑色素瘤变异细胞系中γ-谷氨酰转肽酶（GGTP；E.C. 2.3.2.2）的水平，该酶是一种与质膜相关的胞外酶，参与细胞外谷胱甘肽的转运。检测B16细胞系从细胞外谷胱甘肽合成新的谷胱甘肽的速率，以及它们对γ射线照射和谷胱甘肽合成抑制的敏感性。检测GGTP抑制剂对B16黑色素瘤细胞放射敏感性的影响。
显示B16-F10-BL6（BL6）黑色素瘤细胞表达的GGTP水平比B16-F1黑色素瘤变异细胞高20倍。BL6和B16-F1细胞培养物分别以2.4和<0.1 nmol谷胱甘肽/10⁶细胞/小时的速率消耗细胞外谷胱甘肽，并且BL6细胞输出谷胱甘肽的速率比B16-F1细胞高7.2倍（分别为710和98 pmol谷胱甘肽/10⁶细胞/小时）。BL6黑色素瘤细胞以1.21 nmol谷胱甘肽/小时的速率（基础谷胱甘肽的18%/小时）从细胞外谷胱甘肽源补充耗尽的细胞内谷胱甘肽水平；然而，尽管培养基中存在外源性谷胱甘肽，B16-F1细胞仍缺乏补充细胞内谷胱甘肽的能力。与B16-F1细胞系相比，BL6黑色素瘤细胞具有放射抗性，外推数（ñ）分别为14.9和1.0，并且在广泛的辐射剂量下存活分数较低。L-丝氨酸和硼酸钠的GGTP抑制剂组合阻断了细胞内谷胱甘肽的补充，并且在存在或不存在丁硫氨酸亚砜胺介导的谷胱甘肽耗竭的情况下，将BL6黑色素瘤细胞的放射抗性逆转至与B16-F1亲本克隆观察到的接近基线水平。用丝氨酸-硼酸盐处理低GGTP表达的B16-F1细胞对ñ值或细胞在一系列电离辐射剂量下的存活分数没有影响。
这些结果表明GGTP在谷胱甘肽的细胞外代谢和转运中起重要作用，这也在体外赋予BL6黑色素瘤细胞放射抗性。
