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Proteomelab™ PF 2D Analysis of Gliomas Treated with Novel ProteomeLab PF 2D Analysis of Gliomas Treated with Novel Platinum Compounds Chris Billecke1, Victoria Ioffe1, Ashken Movsisyan1, Nick Farrell2 and Oliver Bögler1 1 William and Karen Davidson Laboratory, Hermelin Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital, Detroit MI 48202 2 Department of Chemistry, Virginia Commonwealth University, Richmond VA 23298 INTRODUCTION Figure 1. UV/pI Maps of Proteomes of Figure 2. Detail comparison of the Proteomes for SUMMARY LNZ308 Glioma Cells Either Untreated or LNZ308 Cells Treated with Cisplatin or BBR3464 Platinum compounds are amongst the most widely used chemotherapeutics, Treated with Cisplatin or with BBR3464. Proteomic analysis of platinum drug treated tumor finding application in a wide spectrum of human malignancies. However the A samples with the ProteomeLab PF2D system most commonly used platinum compounds, cisplatin, carboplatin and oxaliplatin allows rapid and high sensitivity resolution of are not effective in the treatment of glioma. Indeed gliomas remain largely A incurable making the search for effective treatments urgent. Here we investigate proteins, thereby facilitating the identification of the first member of a new series of platinum compounds, BBR3464,(1) by testing molecular differences, and accelerating their its impact on the proteomes of glioma cells. exploitation by downstream technologies. The biological basis of the antitumor effect of cisplatin is not well understood and that of the recently developed BBR compounds even less so. While one might expect there to be some common elements in their mode of action, which in all cases involves the crosslinking of DNA,(2,3) the observation that there is little cross-resistance between cisplatin and BBR3464(4) suggests that there are also important differences. It is unlikely that the effectiveness of platinum compounds relates directly to the amount of DNA damage they cause. For example, it has been shown that oxaliplatin, which is more potent and shows little cross-resistance with than cisplatin, forms fewer DNA adducts and intrastrand crosslinks than cisplatin.(5) This suggests that platinum compounds do B not act solely by interfering with the genome and preventing replication of transcription by physical obstruction of the responsible enzyme complexes. Platinum compounds can influence the proteome in two ways: by altering the repertoire of genes expressed in a cell,(2) or by the direct interaction with B proteins. This latter aspect is under studied. In the case of cisplatin, it has been reported that over 75% of the adducts formed are on protein.(6-8) In this paper, we analyze the proteome of glioma cells treated with cisplatin and the novel compound BBR3464 using the ProteomeLab platform, which will capture both changes in gene expression at the proteome level and structural alteration of proteins themselves. * ProteoVue and DeltaVue are trademarks of Eprogen, Inc. All other trademarks are property of their The proteomes for cisplatin or BBR3464 treated LNZ308 cells shown respective owners. in Figure 1 were compared using DeltaVue* software. Shown here is the region between 14.5 and 18 minutes retention times of Lanes 17 to 20. The center portion shows the qualitative and quantitative differences between the samples. The far outside panels show peak C traces of the lane being analyzed. (A) Comparison of Lane 18. (B) Comparison of Lane 19. MATERIALS & METHODS Treatment of Cell Cultures References 1. Manzotti, C., Pratesi, G., Menta, E., Di Domenico, R., Cavalletti, E., Q Glioma cell line LNZ308 was grown in standard Fiebig, H. H., Kelland, L. R., Farrell, N., Polizzi, D., Supino, R., Pezzoni, Figure 3. Peak Picking Feature of DeltaVue G., and Zunino, F. BBR 3464: a novel triplatinum complex, exhibiting a tissue culture conditions. preclinical profile of antitumor efficacy different from cisplatin. Clin.Cancer Res. 6, 2626-2634. 2000. Q Cells were treated with cisplatin or BBR3464 at 2. Brabec, V. Chemistry and Structural Biology of 1,2-Interstrand Adducts of Cisplatin. Kelland, L. R. and Farrell, N. Platinum-Based Drugs in Cancer concentrations known to induce killing 90% of Therapy. 37-61. 2002. Totowa, NJ, Humana Press Inc. cells as measured using a clonogenic assay. 3. Berners-Price, S. J. and Appleton, T. G. The Chemistry of Cisplatin in Aqueous Solution. Kelland, L. R. and Farrell, N. Platinum-Based Drugs in Cancer Therapy. 3-35. 2002. Totowa, NJ, Humana Press Inc. Q Proteins were lysed from approximately 10 million LNZ308 glioma cells untreated (A), or treated with cisplatin (B) 4. Servidei, T., Ferlini, C., Riccardi, A., Meco, D., Scambia, G., Segni, G., cells 24 hours later according to the protocol Manzotti, C., and Riccardi, R. The novel trinuclear platinum complex provided with the ProteomeLab PF2D chemistry or BBR3464 (C) were fractionated with the ProteomeLab PF2D BBR3464 induces a cellular response different from cisplatin. Eur.J.Cancer 37, 930-938. 2001. kit. system and are shown in the ProteoVue* application. This 5. Raymond, E., Faivre, S., Chaney, S., Woynarowski, J., and Cvitkovic, E. application displays the pI fractions as lanes and the 2nd dimension Cellular and Molecular Pharmacology of Oxaliplatin. Mol Cancer Therap 1, ProteomeLab PF 2D Fractionation retention time as vertical position in individual lanes. The intensity 227-235. 2002. of the bands in the map corresponds to absorbance at 214 nm. 6. Akaboshi, M., Kawai, K., Maki, H., Akuta, K., Ujeno, Y., and Miyahara, T. The number of platinum atoms binding to DNA, RNA and protein Shown are the retention times between 6 and 26 minutes in the molecules of HeLa cells treated with cisplatin at its mean lethal Q 1st Dimension chromatofocusing between pH 8.5- 2nd dimension where most protein peaks are found. The results concentration. Jpn.J.Cancer Res 83, 522-526. 1992. 4.0, pH and 280nm absorbance detection, fraction show that overall the proteomes of these cells are very similar, The proteomes for cisplatin (left, red) or BBR3464 (right, green) treated 7. Akaboshi, M., Kawai, K., Ujeno, Y., Takada, S., and Miyahara, T. collection by pH or time. Binding characteristics of (-)-(R)-2-aminomethylpyrrolidine(1,1- which suggests that the number of molecular changes induced by LNZ308 cells were compared using the peak pick feature of DeltaVue. Shown cyclobutanedicarboxylato)-2-platin um(II) to DNA, RNA and protein molecules in HeLa cells and its lethal effect: comparison with cis- and trans- Q 2nd Dimension reversed-phase chromatography treatment with different chemotherapeutics is limited. here is Lane 18 between 13 and 19 minutes. The center panel shows the diamminedichloroplatinums(II). Jpn.J.Cancer Res 85, 106-111. 1994. with 0.8% TFA in acetonitrile gradient from 0- qualitative and quantitative differences between the samples. The far outside 8. Akaboshi, M., Kawai, K., Kinashi, Y., Masunaga, S., and Ono, K. panels show the traces with the numbered picked peaks. Quantitative analysis of Relationship between cell-killing efficiency and number of platinum atoms 100% in 30 minutes at 50ºC and 214nm binding to DNA, RNA, and protein molecules in HeLa cells treated with cis- absorbance detection. individual peaks showed that Peak 5 in Lane 18 was more than 36-fold higher in diamine(glycolato)platinum(II). Jpn.J.Cancer Res 87, 178-183. 1996. cisplatin treated cells than in BBR3464 treated cells..
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