Bussolati O, Belletti S, Uggeri J, Gatti R, Orlandini G, Dall'Asta V, Gazzola G C
Istituti di Patologia Generale, Università degli Studi di Parma, Italy.
Exp Cell Res. 1995 Oct;220(2):283-91. doi: 10.1006/excr.1995.1317.
The treatment of NIH3T3 cells with L-asparaginase causes a complete and reversible growth arrest with a decrease of cell number in the first 2 days. The enzyme induces impressive morphological changes that have been studied exploiting eosin in fixed cells and calcein in intact cells as sources of fluorescence for confocal microscopy. The first changes are observed after 12 h of treatment and the process is complete after 48 h. Both nucleus and cytoplasm shrink, while cells round and lose processes. Eventually most cells break; several debris include strongly hematoxylinic bodies negative for eosin fluorescence. Some cells neither round nor break in fragments. Throughout the process cells and fragments retain calcein fluorescence, thus indicating the integrity of the cell membrane. A rapid depletion of the intracellular pools of both glutamine and glutamate occurs in treated cells, followed by a decrease in DNA and protein syntheses, while the cell content of ATP, the transmembrane gradient of sodium, and the active transport of amino acids are scarcely affected. It is concluded that (i) L-asparaginase induces an apoptotic process in NIH3T3 cells that is forerun by a marked intracellular depletion of glutamate and glutamine; and (ii) although the enzyme completely suppresses cell proliferation, only a subset of cells undergoes apoptosis upon treatment. These findings provide a model for the characterization of factors that determine cell sensitivity to the effects of L-asparaginase.
用L-天冬酰胺酶处理NIH3T3细胞会导致完全且可逆的生长停滞，在最初2天细胞数量减少。该酶会诱导显著的形态变化，利用固定细胞中的伊红和完整细胞中的钙黄绿素作为共聚焦显微镜的荧光来源对这些变化进行了研究。处理12小时后观察到最初的变化，48小时后过程完成。细胞核和细胞质都会收缩，同时细胞变圆并失去突起。最终大多数细胞破裂；一些碎片包括对伊红荧光呈阴性的强苏木精小体。一些细胞既不变圆也不破碎成碎片。在整个过程中，细胞和碎片都保留钙黄绿素荧光，因此表明细胞膜的完整性。处理后的细胞中谷氨酰胺和谷氨酸的细胞内池迅速耗尽，随后DNA和蛋白质合成减少，而细胞内ATP含量、钠的跨膜梯度以及氨基酸的主动转运几乎不受影响。得出的结论是：（i）L-天冬酰胺酶在NIH3T3细胞中诱导凋亡过程，该过程由谷氨酸和谷氨酰胺在细胞内的显著耗竭引发；（ii）尽管该酶完全抑制细胞增殖，但处理后只有一部分细胞发生凋亡。这些发现为确定细胞对L-天冬酰胺酶作用敏感性的因素的表征提供了一个模型。
