Glucocorticoids induce G1 cell cycle arrest in human neoplastic thymic epithelial cells

Abstract

Purpose: Glucocorticoids exert anti-proliferative effects in various cell types and have long been known to induce apoptosis in thymocytes. Although a few reports have described the regression of human thymoma with glucocorticoid therapy, its effects on neoplastic thymic epithelial cells (TECs) have not been reported. In the present study, we investigated glucocorticoid receptor (GR) expression on neoplastic TECs and the effects of glucocorticoids in vitro on the cell cycle progression of tumor cells.

Patients and methods: Thymoma specimens were obtained during surgery from 21 patients. Three of the specimens with glucocorticoid therapy were examined using the TdT-mediated dUTP-biotin nick-end labeling method. Primary tumor specimens from ten untreated thymomas were examined for GR expression by immunohistochemistry. Isolated neoplastic TECs from the remaining eight untreated thymomas were examined using immunohistochemistry, flow cytometric and cell cycle analysis.

Results: GR are expressed on neoplastic TECs as well as on non-neoplastic thymocytes in thymomas, regardless of WHO histological classification. Glucocorticoids caused an accumulation of TEC in G0/G1 phase in all cases examined (n = 6), and also induced apoptosis in the three with the lowest levels of Bcl-2 expression.

Conclusions: Our results indicate that neoplastic TECs express GR and that glucocorticoids directly suppress their in vitro proliferation.

Fig. 1

TUNEL staining of pre-treatment (a) and glucocorticoid-treated thymoma samples (b) from Case 3. There were few positive epithelial cells in the pre-treated specimens, though some thymocytes were positive (short arrows). The number of epithelial cells with positive staining (long arrows) was increased after treatment (×400).

Fig. 2

Immunohistochemical staining of GR in thymoma tissues. WHO type A (a, b), AB (c, d), B1 (e, f), B2 (g, h), and B3 (i, j) thymoma tissues were stained with anti-GR (a, c, e, g, i), or anti-cytokeratin (b, d, f, h, j). GR expression was shown in both thymocytes and epithelial cells (×400).

Fig. 3

Confocal laser microscopic analysis of cytokeratin and GR in neoplastic TECs. Isolated neoplastic TECs were stained with anti-cytokeratin (a) or anti-GR (b). Confocal images are shown in (c). Cytokeratin-positive cells also expressed GR (Case 19) (×200).

Fig. 4

Flow cytometric analysis of the expression of cytokeratin (a) and GR (b) in neoplastic TECs. Isotype controls are shown by narrow lines. Two-color flow cytometric analysis of cytokeratin and GR are shown in (c). Cytokeratin-positive cells also expressed GR (Case 20).

Fig. 5

DNA-histograms of neoplastic TECs from the control culture (a), and cells incubated with 1×10^-6 M (b) and 1×10^-4 M (c) dexamethasone. After 48 h, whole cell propidium iodide staining was performed to determine cellular DNA content. Dexamethasone inhibited cell proliferation and caused an accumulation of cells with G0/G1 content of DNA (Case 16).

Fig. 6

Effect of dexamethasone on the cell cycle of neoplastic TECs. (a) Dexamethasone suppressed cell proliferation, resulting in an accumulation of cells with G1/G0 content of DNA. (b) The apoptotic population increased in neoplastic TECs of two cases. In Case 19, 1×10⁻⁴ M dexamethasone induced apoptosis in more than 30% of neoplastic TECs.

Fig. 7

Bcl-2 Expression in neoplastic TECs. (a) In the Case 19 specimen, in which dexamethasone induced apoptosis, the D-value of Bcl-2 expression was 0.09. (b) In the Case 17 specimen, in which dexamethasone did not induce apoptosis, that of Bcl-2 expression was 0.54. Isotype controls are shown by narrow lines.