Peleg S, Liu Y Y, Reddy S, Horst R L, White M C, Posner G H
Department of Medical Specialties, University of Texas M.D. Anderson Cancer center, Houston 77030, USA.
J Cell Biochem. 1996 Nov 1;63(2):149-61. doi: 10.1002/(sici)1097-4644(19961101)63:2<149::aid-jcb3>3.0.co;2-y.
1 alpha-hydroxymethyl-25-hydroxyvitamin D3 and 1 beta-hydroxymethyl -3 alpha,25-hydroxyvitamin D3, two analogs with modifications restricted to the A ring, bind poorly to vitamin D receptor (VDR). The effective doses required for 50% of maximal binding activity (ED50) are 7 x 10(-7) M for the former and 8 x 10(-8) M for the latter, and the ED50 for their growth-inhibitory activities is greater than 10(-6) M. Unexpectedly, a hybrid analog with 20-epi configuration at its side chain and a 1 beta-hydroxymethyl group but not a 1 alpha-hydroxymethyl group inhibits malignant cell growth with an ED50 of 7 x 10(-9) M. To determine if the restored biological activity of the hybrid analog is associated with better binding to VDR, we performed competitive binding assays in vitro with calf thymus VDR and in vivo with recombinant human VDR. We found that the 20 epi side chain reduced the affinity of the 1 beta- and the 1 alpha-hydroxymethyl hybrid analogs for VDR in vitro and in vivo fourfold to tenfold. To determine whether the 1 beta-hydroxymethyl analogs induced a VDR-mediated transcription, we tested the induction of reporter gene expression through the osteocalcin vitamin D response element (VDRE) in ROS 17/2.8 cells and the induction of binding activity of VDR to VDRE in COS-1 cells. We found that the ED50 for transcriptional activity of 1 beta-hydroxymethyl-3 alpha,25-hydroxyvitamin D3 was greater than 10(-6) M, but its 1 alpha diastereomer had barely detectable transcriptional activity. The 20-epi side chain preferentially increased the transcriptional activity of the 1 beta-hydroxymethyl hybrid analog to an ED50 of 10(-8) M, but the 1 alpha-hydroxymethyl hybrid analog remained inactive. To confirm that this transcriptional activity was dependent on the VDR, we repeated the assay in VDR-negative CV-1 cells and compared ligand-dependent expression of the VDRE/growth hormone reporter in the presence of either wild-type or transcriptionally inactive mutant VDR expression vectors. Transcription was induced by the 1 beta-hydroxymethyl compounds only in the presence of wild-type VDR. Thus, we conclude that it is possible, by adding a 20 epi side chain, to restore growth-inhibitory and VDR-mediated transcriptional activities without increasing binding to the VDR of A ring-modified analogs.
1α-羟甲基-25-羟基维生素D3和1β-羟甲基-3α,25-羟基维生素D3是两种仅在A环有修饰的类似物，它们与维生素D受体（VDR）的结合能力很差。前者达到最大结合活性的50%所需的有效剂量（ED50）为7×10−7M，后者为8×10−8M，其生长抑制活性的ED50大于10−6M。出乎意料的是，一种在侧链具有20-表位构型且含有1β-羟甲基而非1α-羟甲基的杂合类似物，其抑制恶性细胞生长的ED50为7×10−9M。为了确定杂合类似物恢复的生物学活性是否与更好地结合VDR有关，我们用小牛胸腺VDR进行了体外竞争性结合试验，并用重组人VDR进行了体内试验。我们发现，20-表位侧链在体外和体内使1β-和1α-羟甲基杂合类似物对VDR的亲和力降低了4至10倍。为了确定1β-羟甲基类似物是否诱导VDR介导的转录，我们在ROS 17/2.8细胞中通过骨钙素维生素D反应元件（VDRE）测试了报告基因表达的诱导情况，并在COS-1细胞中测试了VDR与VDRE结合活性的诱导情况。我们发现，1β-羟甲基-3α,25-羟基维生素D3转录活性的ED50大于10−6M，但其1α非对映异构体的转录活性几乎检测不到。20-表位侧链优先将1β-羟甲基杂合类似物的转录活性提高到ED50为10−8M，但1α-羟甲基杂合类似物仍然无活性。为了证实这种转录活性依赖于VDR，我们在VDR阴性的CV-1细胞中重复了试验，并在存在野生型或转录无活性的突变VDR表达载体的情况下比较了VDRE/生长激素报告基因的配体依赖性表达。仅在存在野生型VDR时，1β-羟甲基化合物诱导了转录。因此，我们得出结论，通过添加20-表位侧链，可以恢复生长抑制和VDR介导的转录活性，而不增加与A环修饰类似物的VDR的结合。
