Identification of drug-regulated genes in osteosarcoma cells.
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.
Stiftung Orthopädische Universitätsklinik, Schlierbacher Landstrasse 200a, 69118 Heidelberg, Germany. joerg.fellenberg@ok.uni-heidelberg.de
Jörg
J
Fellenberg
Markus J
MJ
Dechant
Volker
V
Ewerbeck
Hans
H
Mau
D000185:Actinin / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015152:Blotting, Northern; D001859:Bone Neoplasms / Q000235:genetics / Q000473:pathology*; D002945:Cisplatin / Q000494:pharmacology*; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D005293:Ferritins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D007168:IMP Dehydrogenase / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D008727:Methotrexate / Q000494:pharmacology*; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D009693:Nucleic Acid Hybridization; D012516:Osteosarcoma / Q000235:genetics / Q000473:pathology*; D000091402:Prohibitins; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000502:physiology; D012097:Repressor Proteins; D012264:Ribonucleotide Reductases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D013382:Subtraction Technique; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology
D000970:Antineoplastic Agents; D007527:Isoenzymes; D009363:Neoplasm Proteins; D000091402:Prohibitins; D011506:Proteins; D012097:Repressor Proteins; D000185:Actinin; D004317:Doxorubicin; D005293:Ferritins; D007168:IMP Dehydrogenase; D012264:Ribonucleotide Reductases; D020742:rhoA GTP-Binding Protein; D002945:Cisplatin; D008727:Methotrexate