Diversity in secreted PLA2-IIA activity among inbred mouse strains that are resistant or susceptible to ApcMin/+ tumorigenesis
Diversity in secreted PLA2-IIA activity among inbred mouse strains that are resistant or susceptible to ApcMin/+ tumorigenesis
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DOI：
10.1038/sj.onc.1208791
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发表时间：
2005-09-22
期刊：
影响因子：
8
通讯作者：
Farber, SA
中科院分区：
文献类型：
--
作者：
Markova, M;Koratkar, RA;Farber, SA
分泌型磷脂酶 A(2) IIA 型 (Pla2g2a) 基因先前被鉴定为 Apc(Min/+) 小鼠肠道腺瘤多重性的调节因子。为了确定易感菌株中肠道分泌的磷脂酶 A(2) (sPLA(2)) 活性是否也减弱，我们开发了一种灵敏的检测方法，利用荧光 BODIPY 标记的磷脂底物直接定量小鼠肠道中 sPL2 的活性。在这里，我们报告了区分肠组织提取物中分泌型和胞质 PLA(2) 酶活性的测定条件。小肠表现出比大肠更高的活性水平。与近交系中 sPLA(2)-IIA 基因序列的预测一致，我们检测到含有 sPLA(2)-IIA 突变的近交系中酶活性水平较低；这些菌株还与更多的肠息肉有关。此外，该测定能够区分肿瘤抗性菌株之间 sPLA(2) 活性水平的差异，然后通过测序显示这些菌株携带变异的野生型 sPLA(2)-IIA 等位基因。肠道组织的免疫组织化学分析与 sPLA(2)-IIA 活性水平一致。这种方法能够进一步研究 sPLA(2) 作用影响小鼠和人类小肠和结肠发育和肿瘤发生的机制。
The secreted phospholipase A(2) type IIA (Pla2g2a) gene was previously identified as a modifier of intestinal adenoma multiplicity in Apc(Min/+) mice. To determine if intestinal secreted phospholipase A(2) (sPLA(2)) activity was also attenuated in susceptible strains, we developed a sensitive assay to directly quantitate sPL2 activity in the murine intestinal tract utilizing a fluorescent BODIPY-labeled phospholipid substrate. Here, we report assay conditions that distinguish between secreted and cytosolic PLA(2) enzyme activities in extracts of intestinal tissue. The small intestine exhibited higher activity levels than the large intestine. Consistent with predictions from the sPLA(2)-IIA gene sequence in inbred strains, we detected low levels of enzyme activity in inbred strains containing sPLA(2)-IIA mutations; these strains were also associated with greater numbers of intestinal polyps. Additionally, the assay was able to distinguish differences in levels of sPLA(2) activity between neoplasia-resistant strains, which were then shown by sequencing to carry variant wild-type sPLA(2)-IIA alleles. Immunohistochemical analyses of intestinal tissues were consistent with sPLA(2)-IIA activity levels. This approach enables further studies of the mechanisms of sPLA(2) action influencing the development and tumorigenesis of the small intestine and colon in both mice and humans.