Chromosomal and proteome analysis of a new T24‐based cell line model for aggressive bladder cancer

Abstract

Cell line models aid in understanding cancer aggressiveness. The aim of this study was the establishment of a metastatic variant (T24M) of the T24 bladder cancer cell line and its initial characterization at chromosomal and proteomic levels. T24M were spontaneously developed in mice from T24 cells, following cycles of subcutaneous injections and culture in vitro. Transwell migration assays and injections in mice revealed increased migration and tumorigenic properties of T24M compared to the T24 cells. Cytogenetic analysis demonstrated that T24M retained several karyotypic characteristics of the parental cells and also acquired novel chromosomal aberrations related to aggressive bladder cancer. Proteomic analysis of the T24 and T24M cells by 2-DE and MS led to the generation of their 2-DE proteomic map and revealed differences in multiple proteins. These include proteases of the lysosomal and proteasome degradation pathways, mitochondrial and cytoskeletal proteins. The 2-DE findings were confirmed by immunoblotting of cell lysates and immunohistochemistry of bladder cancer tissue sections for cathepsin D and activity assays for proteasome. Collectively, our results suggest that the T24M cells reflect many known chromosomal and proteomic aberrations encountered in aggressive bladder cancers but also provide access to novel findings with potentially clinical applications.

ATF3 Suppresses Metastasis of Bladder Cancer by Regulating Gelsolin-Mediated Remodeling of the Actin Cytoskeleton

“…Our findings support a role for ATF3 suppression of bladder cancer cell motility and metastasis via a GSN-mediated mechanism. Several studies, including our own, have previously developed tumorigenic and metastatic isogenic variants of T24 cells (33,(36)(37)(38). T24-L established in this study was confirmed by variation analysis to have an identical origin to the parental T24 line and these 2 lines do not differ in their proliferative capacity in vitro.…”

Profilin 1 is a Potential Biomarker for Bladder Cancer Aggressiveness

“…Samples were stored at Ϫ20°C until use. For the preparation of cell extracts, cells were collected following trypsinization and cell pellets were washed 3 times in PBS and dissolved in isoelectric focusing sample buffer by bath sonication (14). The suspension was centrifuged at 13,000 rpm for 20 min, supernatants were collected, aliquoted, and stored at Ϫ20°C until used.…”

“…Cell Culture and Sample Preparation for Western Blot Analysis-T24 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen-Invitrogen, Grand Island, New York) at 37°C, 5% CO 2 as previously described (14). When the cells reached a concentration of 10 6 cells per ml, the medium was removed and the cell layer was washed 3 times with phosphate-buffered saline (Invitrogen) and 1 time with Serum and Phenol Red Free Medium (Invitrogen).…”

High Resolution Proteomic Analysis of the Cervical Cancer Cell Lines Secretome Documents Deregulation of Multiple Proteases

“…SiHa, HeLa and C33A cells (ATCC, Manassas, VA, USA) were cultured in DMEM, supplemented with 10% FBS, 1% P/S, supplied by Gibco-Invitrogen (Waltham, MA, USA) at 37˚C and 5% CO 2 , as previously described (14). ΗCK1T cells were a kind gift of Dr. Tohru Kiyono (13), and were cultured as proposed (13), in Defined Keratinocyte Serum-Free Medium (Gibco BRL, San Francisco, CA, USA), supplemented with 5 ng/ml Epidermal Growth Factor (Gibco BRL) and 50 μg/ml of Bovine Pituitary Extract (Gibco BRL).…”