Senescence Process in Primary Wilms' Tumor Cell Culture Induced by p53 Independent p21 Expression

Abstract

Wilms tumor (WT) is an embryonal tumor occurring in developing kidney tissue. WT cells showing invasive cancer characteristics, also retain renal stem cell behaviours. In-vitro culture of WT is hampered by limited replicative potential. This study aimed to establish a longterm culture of WT cells to enable the study of molecular events to attempt to explain its cellular senescence.

Methods: Primary cell cultures from fresh WT tumor specimen were established. Of 5 cultures tried, only 1 could be propagated for more than 7 passages. One culture, identified as PSU-SK-1, could be maintained > 35 passages and was then subjected to molecular characterization and evaluation for cancer characteristics. The cells consistently harbored concomitant mutations of CTNNB1 (Ser45Pro) and WT1 (Arg413Stop) thorough the cultivation. On Transwell invasion assays, the cells exhibited migration and invasion at 55% and 27% capability of the lung cancer cells, A549. On gelatin zymography, PSU-SK-1 showed high expression of the matrix metaloproteinase. The cells exhibited continuous proliferation with 24-hour doubling time until passages 28-30 when the growth slowed, showing increased cell size, retention of cells in G1/S proportion and positive β-galactosidase staining. As with those evidence of senescence in advanced cell passages, expression of p21 and cyclin D1 increased when the expression of β-catenin and its downstream protein, TCF, declined. There was also loss-of-expression of p53 in this cell line. In conclusion, cellular senescence was responsible for limited proliferation in the primary culture of WT, which was also associated with increased expression of p21 and was independent of p53 expression. Decreased activation of the Wnt signalling might explain the induction of p21 expression.

Keywords: Primary cell culture; Senescence.; Wilms tumor.

Figure 1

Morphology of a primary culture of PSU-SK-1. A-B) The early culture showed outgrowth cancer cells from the tumor tissue (20X magnification). C-D) After differential trypsinization and single clone selection. E) Histopathology of the original Wilms tumor tissue. F) Hematoxylin and Eosin stains of the cell culture at passage 10, showing polygonal cells with bipolar cell processes, ovoid nuclei and a prominent nucleolus (40X magnification).

Figure 2

Growth dynamics of PSU-SK-1 A) Cellular growth as shown by MTT assay. B) Cell progression in continuous culture.

Figure 3

Cell cycle studies of various passages of PSU-SK-1. The G1-S fraction increased from 49.7% in passage 5 to 83.0% in passage 30.

Figure 4

Immunohistochemistry of the original Wilms tumor and PSU-SK-1. The cells showed nuclear staining of β-catenin and cytokeratin (AE1/AE3). MyoD1 and myogenin gave weakly positive immunoreactivity.

Figure 5

Western blotting of Wilms tumor related proteins. PSU-SK-1 expressed only β-catenin and WT1.

Figure 6

Migration and invasion assays of PSU-SK-1 by the Transwell method. The figures on the left show cells that could migrate or invade to the opposite side of the filters. The graph shows comparisons of the migrating and invading cells between PSU-SK-1 and a lung cancer cell line A569.

Figure 7

Gelatin zymography of PSU-SK-1 with A569 as a control. The Mr 72,000 band in PSU-SK-1 has previously been shown to correspond to MMP-2 activity. M: marker, FBS: Fetal Bovine Serum (The serum had intrinsic enzymatic activity and was used as a positive control), SFM: serum free media.

Figure 8

Morphological changes of PSU-SK-1 in various passages (upper 4 Figures) and senescence-associated β-galactosidase (SA-β-Gal) stained for senescence cells (bottom 2 Figures), showing larger cell size and positive SA-β-Gal in passage 30.

Figure 9

Western blotting study of Wnt signaling related proteins A) and cell-cycle control proteins B). Late passage cells (passage 30) showed decreased β-catenin and TCF expression, while expression of p21 and cyclin D1 were increased.