H-ras-transformed NRK-52E renal epithelial cells have altered growth, morphology, and cytoskeletal structure that correlates with renal cell carcinoma in vivo

Abstract

We studied the effect of the ras oncogene on the growth kinetics, morphology, cytoskeletal structure, and tumorigenicity of the widely used NRK-52E rat kidney epithelial cell line and two H-ras oncogene-transformed cell lines, H/1.2-NRK-52E (H/1.2) and H/6.1-NRK-52E (H/6.1). Population doubling times of NRK-52E, H/1.2, and H/6.1 cells were 28, 26, and 24 h, respectively, with the transformed cells reaching higher saturation densities than the parent cells. NRK-52E cells had typical epithelial morphology with growth in colonies. H/1.2 and H/6.1 cell colonies were more closely packed, highly condensed, and had increased plasma membrane ruffling compared to parent cell colonies. NRK-52E cells showed microfilament, microtubule, and intermediate filament networks typical of epithelial cells, while H/1.2 and H/6.1 cells showed altered cytoskeleton architecture, with decreased stress fibers and increased microtubule and intermediate filament staining at the microtubule organizing center. H/1.2 and H/6.1 cells proliferated in an in vitro soft agar transformation assay, indicating anchorage-independence, and rapidly formed tumors in vivo with characteristics of renal cell carcinoma, including mixed populations of sarcomatoid, granular, and clear cells. H/6.1 cells consistently showed more extensive alterations of growth kinetics, morphology, and cytoskeleton than H/1.2 cells, and formed tumors of a more aggressive phenotype. These data suggest that analysis of renal cell characteristics in vitro may have potential in predicting tumor behavior in vivo, and significantly contribute to the utility of these cell lines as in vitro models for examining renal epithelial cell biology and the role of the ras proto-oncogene in signal transduction involving the cytoskeleton.

Comparative Analysis of Caspase Activation and Apoptosis in Renal Tubular Epithelial Cells and Renal Cell Carcinomas

“…RCCs are extremely resistant to conventional cancer therapies, and no effective systemic therapy presently exists for patients with metastatic RCCs. Although some apoptosis-related genes have altered expression in RCCs, this appears to relate largely to genes that have functions in the broader context of apoptosis induction, such as H-Ras [29], rather than genes whose products make up the apoptosis pathway itself, for example, the Bcl-2-gene family. Kolenko et al [22]have demonstrated in some human-derived RCC cell lines that caspase-3 expression is low or lacking.…”

Inhibition of c-Jun N-Terminal Kinase Ameliorates Apoptosis Induced by Hydrogen Peroxide in the Kidney Tubule Epithelial Cells (NRK-52E)

“…This cell line, derived from the proximal tubule epithelium (PTE) of the rat kidney, is non-tumorigenic, and retains many morphological and physiological characteristics of PTE in vivo [18, 19]. Cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin) at 37°C in 95% O 2 /5% CO 2 .…”

Identification of thrombospondin 1 (TSP-1) as a novel mediator of cell injury in kidney ischemia

“…TSP-1 at 1 µg/ml was added to cultured kidney proximal tubule (NRK) cells and studied at 24 and 48 hours for evidence of apoptosis by cytochrome c release and caspase-3 activity. The NRK-52E normal kidney cell line is derived from the proximal tubule epithelium (PTE) of the rat kidney, is nontumorigenic, retains many morphological and physiological characteristics of PTE, and has been used to mimic changes in vivo (31)(32)(33). Cytochrome c apoptosis detection kit (Alexa Fluor 488 assay; Invitrogen Corp.) was used to perform the apoptosis assay.…”