Primary Cell Cultures Arising from Normal Kidney and Renal Cell Carcinoma Retain the Proteomic Profile of Corresponding Tissues

Abstract

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.

Primary Cell Cultures from Human Renal Cortex and Renal-Cell Carcinoma Evidence a Differential Expression of Two Spliced Isoforms of Annexin A3

“…For one-dimensional electrophoresis (1-DE) Western blotting, 30 g of protein lysates, obtained from primary cell cultures and from renal tissue homogenate as described, 5,16 quantified with a Bio-Rad microassay (Hercules, CA), were separated on 10% SDS-PAGE and on NuPage 4 to 12% Bis-Tris pre-cast gels using MOPS SDS running buffer (Invitrogen), with described conditions. 5 For two-dimensional electrophoresis (2-DE) Western blotting, 50 g of proteins were loaded onto 7-cm 3-10 NL IPG-strips (GE Health care, Uppsala, Sweden) and then separated on NuPage 4% to 12% pre-cast gels.…”

“…5 For two-dimensional electrophoresis (2-DE) Western blotting, 50 g of proteins were loaded onto 7-cm 3-10 NL IPG-strips (GE Health care, Uppsala, Sweden) and then separated on NuPage 4% to 12% pre-cast gels. 1-DE and 2-DE gels were blotted onto nitrocellulose membranes that were stained with Ponceau S (SigmaAldrich) to check transferred proteins.…”

“…Ϫ⌬⌬Ct , representing the expression fold respect to a calibrator 5 considered to be equal to 1. A pool of all normal cortex primary culture cDNA was chosen as the calibrator.…”

“…About 88% of the cells in cortex and 99% in RCC cultures were positive for vimentin ( Figure 1A), a mesenchymal marker known to be expressed by tubular cells during in vitro growth 21 and by RCC cells both in vivo and in vitro. 5 One third of our cultures have been also characterized with other markers. CD13, an antigen specific of the proximal brush border, 22 was positive in about 43% of cells in cortex cultures.…”

“…Moreover, this in vitro model retains the same phenotype of the corresponding original tissue during the first passages. 5 The reliability of data obtained with primary cultures is linked to the detailed characterization of their cellular composition, particularly regarding cellular con-tamination that can influence data interpretation. In the attempt to identify the differences between normal and renal tumor cells for the study of the molecular changes associated with the neoplastic status, we currently establish and characterize primary cell cultures.…”