Analysis of central regulatory pathways in p53-deficient primary cultures of malignant fibrous histiocytoma exposed to ifosfamide.
Soft tissue sarcomas frequently carry p53 mutations reducing chemotherapeutical response. Especially malignant fibrous histiocytoma (MFH) reveals a reduced ifosfamide (IF) chemosensitivity when compared to other sarcoma entities. This is the first study to analyze MFH cells for the effects of IF on the expression of the pathways P16-CDK4-Rb and P14ARF-MDM2-P73 regulating cell cycle. The aim was to identify candidate genes possibly involved in the anti-apoptotic response of p53-deficient MFH cells during chemotherapy. PCR, real-time RT-PCR and confocal laser scanning microscopy were applied on primary cultures of MFH cells containing defective p53 genes. The cultures were treated with different concentrations of IF. A non-treated MFH culture served as negative control. A threshold concentration of IF (100 microM) was determined sparing the majority of the cells (99%), whereas higher IF quantities caused complete apoptosis. Data collected over a period of 48 h showed that the MFH cells surviving 100 microM IF overexpressed the kinase gene CDK4 and oncogene MDM2 by a factor of 63. A similar strong increase was observed at the protein level for both proteins. In contrast, the other proteins analyzed were not detectable. Additionally, the MFH cells induced complex patterns of MDM2 mRNA splicing and an abnormal mRNA transcript carrying a novel MDM2 missense mutation. These effects were neither observed in the non-treated culture nor in cultures completely inducing spontaneous apoptosis. Therefore, we speculate that the induction of the gene CDK4, and especially of MDM2, is involved in anti-apoptotic mechanisms of p53-negative MFH cells tolerating IF in vitro. Further experiments are necessary to test whether the novel candidate genes favor development of chemoresistance and whether MDM2 mRNA splicing variants contribute to this process in vivo.
Schlott
Thilo
T
Department of Pathology, Center for Pathology, Georg-August-University, D-37075 Goettingen, Germany. tschlott@med.uni-goettingen.de
Taubert
Helge
H
Fayyazi
Afshin
A
Schweyer
Stefan
S
Bartel
Frank
F
Korabiowska
Monika
M
Brinck
Ulrich
U
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D017398:Alternative Splicing / Q000187:drug effects; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis / Q000235:genetics; D018844:Cyclin-Dependent Kinases / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D016147:Genes, Tumor Suppressor; D018219:Histiocytoma, Benign Fibrous / Q000188:drug therapy* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007069:Ifosfamide / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D016160:Retinoblastoma Protein / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D000072160:Tumor Protein p73; D025803:Tumor Suppressor Protein p14ARF / Q000096:biosynthesis / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency* / Q000235:genetics; D025521:Tumor Suppressor Proteins
D000199:Actins; D018906:Antineoplastic Agents, Alkylating; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; C000606460:TP73 protein, human; D000072160:Tumor Protein p73; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D007069:Ifosfamide