Fellenberg Jörg, Dechant Markus J, Ewerbeck Volker, Mau Hans
Stiftung Orthopädische Universitätsklinik, Schlierbacher Landstrasse 200a, 69118 Heidelberg, Germany.
Int J Cancer. 2003 Jul 10;105(5):636-43. doi: 10.1002/ijc.11135.
The introduction of systemic chemotherapy improved significantly the prognosis of osteosarcoma. Despite this success, approximately 30-40% of patients will relapse. Cytotoxic drugs have been shown to induce apoptosis in the target cells independent of their primary effects. The underlying molecular mechanisms and the intracellular mediators, however, are still largely unknown. Therefore, the purpose of our study was to identify drug-regulated genes in osteosarcoma cells useful as prognostic factors and for the development of new therapeutic strategies. Using suppressive subtractive hybridization (SSH) the gene expression pattern of untreated Saos-2 cells was compared to cells treated with cisplatin, methotrexate and doxorubicin, respectively. We identified 8 genes that are regulated >2-fold in drug-treated osteosarcoma cell lines. Expression of ferritin light chain, rhoA, inosine monophosphatdgehydrogenase II, ribonucleotide reductase M2, pro2000 and pro1859 were increased after drug treatment, whereas prohibitin and alpha-actinin expressions were significantly downregulated. Differential expression of the identified genes was verified by Northern blot analysis of 3 different osteosarcoma cell lines. In addition, the effects on chemosensitivity of 4 selected genes was analyzed by overexpression of recombinant constructs in Saos-2 cells and subsequent quantification of drug-induced apoptosis. Overexpression of prohibitin and rhoA reduced significantly drug sensitivity to approximately 52% and 59% indicating a crucial role in the modulation of drug-induced cell death.
全身化疗的引入显著改善了骨肉瘤的预后。尽管取得了这一成功，但仍有大约30%-40%的患者会复发。细胞毒性药物已被证明可在与其主要作用无关的情况下诱导靶细胞凋亡。然而，其潜在的分子机制和细胞内介质仍 largely 未知。因此，我们研究的目的是在骨肉瘤细胞中鉴定出可作为预后因素和用于开发新治疗策略的药物调节基因。使用抑制性消减杂交（SSH），将未处理的Saos-2细胞的基因表达模式分别与用顺铂、甲氨蝶呤和阿霉素处理的细胞进行比较。我们鉴定出8个在药物处理的骨肉瘤细胞系中调节倍数>2倍的基因。药物处理后，铁蛋白轻链、rhoA、肌苷单磷酸脱氢酶II、核糖核苷酸还原酶M2、pro2000和pro1859的表达增加，而抑制素和α-肌动蛋白的表达显著下调。通过对3种不同骨肉瘤细胞系的Northern印迹分析验证了所鉴定基因的差异表达。此外，通过在Saos-2细胞中过表达重组构建体并随后定量药物诱导的凋亡，分析了4个选定基因对化学敏感性的影响。抑制素和rhoA的过表达显著降低了药物敏感性，分别降至约52%和59%，表明它们在调节药物诱导的细胞死亡中起关键作用。
