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


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 alpha-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-l 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 TI-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).


CONCLUSIONS
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.
Smyth
A
A
Cancer & Ageing Research Group, School of Biomedical Sciences, University of Ulster, County Londonderry, Northern Ireland.
Reid
H M
HM
Baker
A H
AH
McGlynn
H
H
D002453:Cell Cycle / Q000528:radiation effects*; D045744:Cell Line, Tumor / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects; D004249:DNA Damage / Q000502:physiology / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007680:Kidney Neoplasms; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D010957:Plasmids / Q000528:radiation effects*; D011836:Radiation Tolerance / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; D020780:Matrix Metalloproteinase 9