Primary Cell Cultures Arising from Normal Kidney and Renal Cell Carcinoma Retain the Proteomic Profile of Corresponding Tissues Roberto A. Perego,*,† Cristina Bianchi,† Matteo Corizzato,† Barbara Eroini,† Barbara Torsello,† Cristina Valsecchi,‡ Andrea Di Fonzo,† Nicoletta Cordani,§ Paolo Favini,| Stefano Ferrero,§ Marina Pitto,† Cecilia Sarto,‡ Fulvio Magni,† Francesco Rocco,| and Paolo Mocarelli†,‡ Milano-Bicocca University, Department of Experimental, Environmental Medicine and Medical Biotechnologies, Monza, Italy, Milano-Bicocca University, Department of Laboratory Medicine, Hospital of Desio, Desio, Italy, Milano University, Department of Medicine, Surgery, and Dentistry, Pathological Anatomy, S. Paolo Hospital, Milano, Italy, and Milano University, Institute of Urology, IRCCS Policlinico, Milano, Italy Received January 14, 2005
Renal cell carcinoma (RCC) tissue is composed of a mixture of neoplastic and normal cells, which complicate proteome analysis. The aim of our study was to investigate whether it is feasible to establish primary cell cultures of RCC and of renal cortex maintaining the tissue phenotype along with a more homogeneous and enriched cytological material. Fourteen (82.3%) primary cultures from 17 surgical cases were established and characterized by morphology, growth rate, immunocytochemistry, and molecular analysis performed by Real-time PCR, Western blotting, two-dimensional electrophoresis (2-DE), and mass spectrometry. Cultures showed >90% cytokeratine-positive epithelial cells. In primary tumor cultures, the molecular phenotype of manganese superoxide dismutase and heat shock protein 27 was the same as that found in tumor tissues with overexpression and increased number of isoforms. Moreover, 27 out 28 specific proteins and their isoforms, present in spots excised from 2-DE gel of cortex or RCC cultures, corresponded to those identified on the 2-DE tissue cortex reference map, suggesting that these primary cultures retain the proteomic profile of the corresponding tissues. Keywords: renal cell carcinoma • primary cultures • proteome • real-time PCR • two-dimensional gel electrophoresis • mass spectrometry • Western blotting • immunocytochemistry • manganese superoxide dismutase • heat shock protein 27
Introduction Renal cell carcinomas (RCCs) are the most frequent tumors of the kidney. They are characterized by different subtypes and account for about 3% of all human adult malignant diseases. The etiology of RCC is obscure and although the majority of renal tumors develop as sporadic forms rare familial forms were described1 and distinct genetic abnormalities in the different subtypes were observed.2 These tumors that differ also for natural history and prognosis may be adequately defined by a systematic investigation of gene expression and/or proteomic profile that allows for the identification of protein changes caused by the disease process.3-5 Clear cell carcinoma, the most frequent subtype of RCC, originates from the proximal tubular epithelium in the renal cortex. Molecular analysis and proteome profile of this solid tumor is however complex and complicated due to the mixture of tumor cells and normal cells, such as leukocytes and connective tissue cells,6 which compose * To whom correspondence should be addressed. Phone: +39 02 6448 8303. Fax: +39 02 6448 8450. E-mail:
[email protected]. † Milano-Bicocca University, Department of Experimental, Environmental Medicine and Medical Biotechnologies. ‡ Milano-Bicocca University, Department of Laboratory Medicine, Hospital of Desio. § Milano University, Department of Medicine, Surgery, and Dentistry, Pathological Anatomy, S. Paolo Hospital. | Milano University, Institute of Urology, IRCCS Policlinico. 10.1021/pr050002o CCC: $30.25
2005 American Chemical Society
it. To overcome this problem, the tumor cells may be adapted to grow in vitro, as primary cell cultures, to provide a more homogeneous cellular material for studying the biochemical and molecular changes associated to the neoplastic status. The disadvantage of establishing primary cell cultures is that the cells tend to quickly dedifferentiate as they are maintained in culture. Therefore, it may be difficult to observe altered marker expression which can be unambiguously assigned to the respective ancestor cell.7 The aim of the present study was to investigate whether it is feasible to establish primary cell cultures of RCC and normal renal cortex which maintain the same phenotype as seen in the tissue, and obtain a more homogeneous and enriched cytological material. The RCC and normal cortex primary cell cultures were characterized by morphology, growth rate, immunocytochemistry, and molecular analysis performed by Real-time PCR, western blotting, two-dimensional electrophoresis (2-DE), and mass spectrometry (MS). The molecular phenotype of these primary cell cultures was then compared to the proteomic phenotype of normal cortex tissue and RCC tissue previously described.8,9
Materials and Methods Patients. Seventeen consecutive non selected patients affected by RCC clear cell (RCCcc, 15 cases) or papillary (RCCpap, 2 cases) subtypes were diagnosed and treated by surgery. The Journal of Proteome Research 2005, 4, 1503-1510
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Published on Web 08/25/2005
research articles
Perego et al.
Table 1. Clinical Characteristics of Patients with RCC and the Primary Cell Cultures Obtained primary culturesd patient
sex
age Dxa
Dxa
Gb
01A 3CCS 4CCL 7ZG 11DP 12BGs 13SV 16CP 17QA 20GMa 21LA 22VP 24MO 25MI 27CG 28RA 29CV
M F F M M M M F M F F M M F M M M
43 70 50 66 77 62 66 58 58 53 69 44 70 85 59 63 52
RCCcc RCCcc RCCcc RCCcc RCCcc RCCcc RCCcc RCCcc RCCpap RCCcc RCCcc RCCcc RCCpap RCCcc RCCcc RCCcc RCCcc
2 2 1 2 2 3 1 2
a
2 2 2 2 2 2 2
φ maxc (cm)
necrotic areas (%)
tumor stage
tumor
cortex
4,0 5,5 2,0 6,5 2,1 11,0 1,5 8,0 2,0 6,7 7,0 3,5 2,2 9,0 8,0 4,5 4,5
12 0 0 70 10 50 0 0 0
