Scott D K, Weaver W R, Clohisy J C, Brakenhoff K D, Kahn A J, Partridge N C
Pediatric Research Institute, Cardinal Glennon Children's Hospital, St. Louis University School of Medicine, Missouri 63110.
Mol Endocrinol. 1992 Sep;6(9):1425-32. doi: 10.1210/mend.6.9.1359401.
The rat osteosarcoma cell line UMR 106-01 is a commonly used model system for the study of osteoblast function. However, it also expresses a phenotype characteristic of transformed cells. To test whether the latter could be accounted for by aberrant oncogene expression, we probed Northern blots of UMR and other osteoblastic cells with a panel of oncogene probes. These blots, when probed with a cDNA specific for v-H-ras, revealed a 7.0-kilobase (kb) H-ras-related transcript (designated HRRT) in UMR 106-01 cells that was not expressed in other osteoblastic cells. Osteoblast-enriched calvarial cells expressed the typical 1.1-kb H-ras mRNA, which was absent in UMR cells. Additionally, Western blots of lysates of UMR cells documented the presence of three proteins immunologically related to H-rasp21. To determine whether HRRT represented a recombinant retrovirus product, Northern blots were probed with a cDNA specific for the highly conserved gag-pol region of Moloney murine leukemia virus. These blots showed parallel cross-reactivity with an apparently identical transcript of 7.0 kb. The 7.0-kb transcripts detected by both v-H-ras and gag-pol probes declined to the same extent after treatment with concentrations of PTH known to inhibit proliferation of these cells. PTH regulated the abundance of HRRT in a time- and dose-dependent manner, with greatest repression of the transcript after 8 h of treatment with 10(-8) M PTH. The decrease in HRRT could not be completely accounted for by changes in transcriptional activity, as determined by nuclear run-on assays.(ABSTRACT TRUNCATED AT 250 WORDS)
大鼠骨肉瘤细胞系UMR 106 - 01是研究成骨细胞功能常用的模型系统。然而，它也表现出转化细胞的表型特征。为了测试后者是否可由异常的癌基因表达来解释，我们用一组癌基因探针探测UMR和其他成骨细胞的Northern印迹。当用v - H - ras特异的cDNA探测这些印迹时，在UMR 106 - 01细胞中发现了一个7.0千碱基(kb)的H - ras相关转录本(命名为HRRT)，而在其他成骨细胞中未表达。富含成骨细胞的颅骨细胞表达典型的1.1 kb H - ras mRNA，而UMR细胞中没有。此外，UMR细胞裂解物的Western印迹证明存在三种与H - rasp21免疫相关的蛋白质。为了确定HRRT是否代表重组逆转录病毒产物，用莫洛尼鼠白血病病毒高度保守的gag - pol区域特异的cDNA探测Northern印迹。这些印迹显示与一个明显相同的7.0 kb转录本有平行交叉反应。在用已知可抑制这些细胞增殖的甲状旁腺激素(PTH)浓度处理后，v - H - ras和gag - pol探针检测到的7.0 kb转录本下降程度相同。PTH以时间和剂量依赖的方式调节HRRT的丰度，在用10(-8)M PTH处理8小时后转录本的抑制作用最大。如通过核转录分析所确定的，HRRT的减少不能完全由转录活性的变化来解释。(摘要截断于250字)
