Levano Kelly, Sobocki Tomasz, Jayman Farah, Debata Priya Ranjan, Sobocka Malgorzata B, Banerjee Probal
CUNY Doctoral Program in Biochemistry, City University of New York at the College of Staten Island, Staten Island, NY 10314, USA.
Glycoconj J. 2009 Aug;26(6):739-48. doi: 10.1007/s10719-009-9233-1. Epub 2009 Mar 12.
The most common therapeutic strategy for the treatment of cancer uses antimetabolites, which block uncontrolled division of cancer cells and kill them. However, such antimetabolites also kill normal cells, thus yielding detrimental side effects. This emphasizes the need for an alternative therapy, which would have little or no side effects. Our approach involves designing genetic means to alter surface lipid determinants that induce phagocytosis of cancer cells. The specific target of this strategy has been the enzyme activity termed aminophospholipid translocase (APLT) or flippase that causes translocation of phosphatidylserine (PS) from the outer to the inner leaflet of the plasma membrane in viable cells. Efforts to identify the enigmatic, plasma membrane APLT of mammalian cells have led investigators to some P-type ATPases, which have often proven to be the APLT of internal membranes rather than the plasma membrane. By measuring kinetic parameters for the plasma membrane APLT activity, we have shown that the P-type ATPase Atp8a1 is the plasma membrane APLT of the tumorigenic N18 cells, but not the non-tumorigenic HN2 (hippocampal neuron x N18) cells. Targeted knockdown of this enzyme causes PS externalization in the N18 cells, which would trigger phagocytic removal of these cells. But how would we specifically express the mutants or antisense Atp8a1 in the cancer cells? This has brought us to a glycosyltransferase, GnT-V, which is highly expressed in the transformed cells. By using the GnT-V promoter to drive a luciferase reporter gene we have demonstrated a dramatic increase in luciferase expression selectively in tumor cells. The described strategy could be tested for the removal of cancer cells without the use of antimetabolites that often kill normal cells.
治疗癌症最常用的策略是使用抗代谢物，这类物质能阻止癌细胞的无节制分裂并将其杀死。然而，此类抗代谢物也会杀死正常细胞，从而产生有害的副作用。这凸显了开发一种副作用很小或几乎没有副作用的替代疗法的必要性。我们的方法是设计基因手段来改变诱导癌细胞吞噬作用的表面脂质决定因素。该策略的具体靶点是一种名为氨基磷脂转位酶（APLT）或翻转酶的酶活性，它会导致磷脂酰丝氨酸（PS）在活细胞中从质膜的外侧小叶转运到内侧小叶。鉴定哺乳动物细胞质膜上神秘的APLT的努力，使研究人员找到了一些P型ATP酶，但事实证明这些酶往往是内膜而非质膜的APLT。通过测量质膜APLT活性的动力学参数，我们发现P型ATP酶Atp8a1是致瘤性N18细胞的质膜APLT，但不是非致瘤性HN2（海马神经元x N18）细胞的质膜APLT。靶向敲低这种酶会导致N18细胞中的PS外化，这将触发对这些细胞的吞噬清除。但是，我们如何在癌细胞中特异性地表达Atp8a1的突变体或反义基因呢？这使我们想到了一种糖基转移酶——GnT-V，它在转化细胞中高度表达。通过使用GnT-V启动子驱动荧光素酶报告基因，我们已经证明荧光素酶表达在肿瘤细胞中选择性地显著增加。可以对所描述的策略进行测试，以在不使用通常会杀死正常细胞的抗代谢物的情况下清除癌细胞。
