Identification of differentially expressed genes in a renal cell carcinoma tumor model after endostatin-treatment.
Endostatin is a cleavage product of collagen XVIII that has shown to inhibit tumor-angiogenesis in experimental tumor models. At present, the exact molecular mechanism of action of endostatin is not completely elucidated. In this study, we wanted to identify specific target genes of endostatin. For this purpose, the human renal cell carcinoma RC-9 was subcutaneously implanted in nude mice and treated with endostatin. Tumor growth was inhibited by endostatin after 4 days of treatment. Using immunohistochemistry and the hypoxia marker pimonidazole, we demonstrate disintegration of blood vessels and hypoxia and anoxia as a result of the treatment. Hereafter, we applied the polymerase chain reaction (PCR)-based subtractive suppression hybridization (SSH) method, together with the mirror orientation selection (MOS) technique to identify specifically induced and suppressed genes after endostatin-treatment. We found eight genes to be specifically induced and 11 to be suppressed by the endostatin-treatment. Among other genes, core binding factor a-1/osteoblast-specific factor-2 (cbfa1/osf2) was found to be specifically suppressed by endostatin. Unexpectedly, cbfa1/osf2 was found to be specifically expressed in granulocytes in the tumor, not only in the experimental RC-9 tumor model, but in sections of human breast cancer as well. Since an effect of antiangiogenic therapy on granulocytes has been reported before, this might lead to new insights in the role of granulocytes in antiangiogenic therapy in general. In conclusion, the SSH-PCR implemented with the MOS-technique is a powerful tool to identify differentially expressed genes. Using these techniques, we have identified several target genes of endostatin, of which cbfa1/osf2 was found to be specifically expressed in granulocytes in the tumor.
Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands.
Jens
J
van Wijngaarden
Karien
K
de Rooij
Ermond
E
van Beek
Hans
H
Bernsen
Ivo
I
Que
Victor W M
VW
van Hinsbergh
Clemens
C
Löwik
D020533:Angiogenesis Inhibitors / Q000627:therapeutic use*; D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002292:Carcinoma, Renal Cell / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D050659:Core Binding Factor Alpha 1 Subunit; D050660:Core Binding Factors; D043169:Endostatins / Q000627:therapeutic use*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007680:Kidney Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000188:drug therapy / Q000473:pathology / Q000517:prevention & control; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D020533:Angiogenesis Inhibitors; D000970:Antineoplastic Agents; D050659:Core Binding Factor Alpha 1 Subunit; D050660:Core Binding Factors; D043169:Endostatins; D009363:Neoplasm Proteins; D014157:Transcription Factors