Hypoxic-inducible factor-1α might up-regulate matrix metalloproteinase in subclones that survive 10-Gy X-irradiation

Abstract

To the best of our knowledge, most studies of irradiation effects on cells have focused on early responses, such as RNA expression and protein levels a few hours later or days after irradiation. Of course, it is important to understand how cells respond to irradiation immediately after exposure. However, most cells die if a large dose is given (e.g., 10 Gy, which is now commonly used for lung cancer). To understand the nature of the surviving cells is probably much more relevant to the clinical situation in terms of residual disease or local recurrence. Here we discuss matrix metalloproteinase 1 (MMP1) in relation with hypoxic inducible factor 1α (HIF-1α). The reasons we focused on these molecules were (1) in our previous experiments using lung carcinoma cell lines, MMP1 mRNA was up-regulated in subclones that survived 10 Gy (data not shown); and (2) recently, several studies have shown that HIF-1α up-regulates MMP1 expression. In the present experiment, we fi rst established irradiation-surviving H1299 cells (referred to as H1299-IR). Semi-confl uent H1299 cells were irradiated with 10 Gy and were soon seeded onto a 3-cm culture dish. Twelve days later, all the colonies were harvested, and the cells were stored at −80°. The parent cells were derived from the lung adenocarcinoma cell line H1299. To examine the invasiveness of each cell line, a collagen overlay assay was performed. For this, 1 × 10 cells were seeded into a collagen-fi lled cultured dish. The details of the threedimensional (3D) cell culture are described elsewhere. Cells were allowed to proliferate sterically for 12 days. In this experiment, 95 colonies were observed for H1299 and 116 for H1299-IR. Colonies were classifi ed into “spheroid” and “nonspheroid” (Fig. 1). Of the 95 H1299 colonies, 25 (26.3 %) took on a nonspheroid shape, whereas 87 (75.0 %) the 116 H1299-IR colonies were nonspheroid-like (chisquared test, P < 0.01). This result is consistent with the report showing 10 Gy-surviving malignant cells had a tendency to take on an irregular shape and had a highly invasive nature. We then performed the MMP1 luciferase assay. The promoter regions included E-box, Ets, and AP1 sites so the promoter activities of pcDNA (as a control) E1AF, Jun/Fos, and HIF-1α could be measured. The construct is shown in Fig. 2a. The luciferase assay for E1AF, Jun/ Fos, and HIF-1α revealed the following fold changes, with the SD, compared to the control, pcDNA, for H1299 cells: E1AF (1.9 ± 0.1), Jun/Fos (5.6 ± 0.2), and HIF-1α (0.8 ± 0.2), respectively. A statistically signifi cant value was observed for Jun/Fos (Student’s t-test, P < 0.01). On the other hand, the respective values for H1299-IR were E1AF (2.2 ± 0.1), Jun/Fos (2.7 ± 0.3), and HIF-1α (3.3 ± 0.2). These results are shown in Fig. 2b. Statistically signifi cant values of P ≤ 0.01 were seen for all transcriptional factors for H1299-IR. Paying attention to their initial (i.e., control) status, 4.6-fold higher MMP1 promoter activity was observed for H1299-IR. On cDNA array analysis, MMP1 mRNA was up-regulated 4.4-fold, similar to the value on the luciferase assay. Silva et al. performed an immunohistochemical study on oropharyngeal carcinoma and found that high HIF-1α staining was well correlated with a poor Received: June 3, 2010 / Accepted: October 7, 2010 © Japan Radiological Society 2011