Modifications of the radiosensitivity of a renal cancer cell line as a consequence of stable TIMP-1 overexpression

Abstract

Purpose: To investigate the potential effects of stable tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) overexpression on DNA damage and cell killing following low-dose γ-radiation and whether this up-regulation interfered with the activation of the matrix metalloproteinase – 2 (MMP-2) and -9 (MMP-9) in a highly metastatic renal carcinoma cell line.

Materials and methods: Stable transfections were carried out using the cytomegalovirus expression plasmid pRc/CMV carrying TIMP-1 cDNA and LIPOFECTAMINE reagent. TIMP-1 expression in selected clones was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Exponentially growing Caki-1 cells were treated with sub lethal doses of ionizing radiation (0 – 10Gy) either alone or following stable TIMP-1 transfection. DNA damage was assessed by the Alkaline Comet Assay and cell survival was determined by a clonogenic assay. Caki-1 cell cycle alterations following TIMP-1 transfection were assessed by fluorescence activated cell sorting (FACS) analysis of propidium iodide (PI)-stained cells. The interactions between TIMP-1 and MMP-2 and MMP-9 were analysed 24 hours post-irradiation by means of gelatin zymography.

Results: Three clones with varying degrees of TIMP-1 expression were selected and used for further analysis. TIMP-1 transfected Caki-1 cells displayed significantly higher mean tail moment values (p < 0.05) following irradiation at doses between 5 and 10 Gy relative to that seen with radiation alone. The TIMP-1 radiosensitizing effect was accompanied by large decreases in the survival fraction of the parental Caki-1 cell line and significant increases in the α-parameter of the linear-quadratic fit. These effects were directly correlated to the degree of TIMP-1 gene expression detected in the selected clones. Interestingly, elevated levels of TIMP-2 protein were detected in the three TIMP-1 clones compared to TIMP-2 levels present in Caki-1 cells. The three clones also displayed marked phenotypic alterations relative to their parental cell line. Significant increases in the percentage of cells arrested in the G₂/M phase of the cell cycle were detected in the three clones under normal growth conditions and reduced serum conditions (p < 0.05). When the TIMP-1 clones were assessed for their MMP-2 activity, a marked decrease in the MMP-2 mean protein levels was detected in clone T1-3 following irradiation at doses between 2 and 6 Grays (Gy) (p < 0.01) and clone T1-2 at 2-5Gy (p < 0.05). MMP-9 activity was differentially affected by ionizing radiation in the three TIMP-1 clones. T1-3 and T1-2 displayed significantly reduced MMP-9 levels at various dose points whereas T1-1 exhibited elevated levels of MMP-9 activity at higher doses of treatment (p < 0.05).

Conclusion: These results demonstrate a dual role for the TIMP-1 overexpression in this renal carcinoma cell line, both as radiosensitizing agents and effectors of MMP-2 and MMP-9 activity.

Keywords:

• TIMP-1
• MMP-2
• MMP-9
• ionizing radiation
• RCC
• DNA damage