Effect of p16 on glucocorticoid response in a B-cell lymphoblast cell line

Abstract

Purpose: It has been suggested that p16 has a role in glucocorticoid (GC)-related apoptosis in leukemic cells, but the exact mechanisms have yet to be clarified. We evaluated the relationship between the GC response and p16 expression in a lymphoma cell line.

Methods: We used p16 siRNA transfection to construct p16-inactivated cells by using the B-cell lymphoblast cell line NC-37. We compared glucocorticoid receptor (GR) expression, apoptosis, and cell viability between control (p16+ NC-37) and p16 siRNA-transfected (p16- NC-37) cells after a single dose of dexamethasone (DX).

Results: In both groups, there was a significant increase in cytoplasmic GR expression, which tended to be higher for p16+ NC-37 cells than for p16- NC37 cells at all times, and the difference at 18 h was significant (P<0.05). Similar patterns of early apoptosis were observed in both groups, and late apoptosis occurred at higher levels at 18 h when the GR had already been downregulated (P<0.05). Cell viability decreased in both groups but the degree of reduction was more severe in p16+ NC-37 cells after 18 h (P<0.05).

Conclusion: These results suggest a relationship between GR expression and cell cycle inhibition, in which the absence of p16 leads to reduced cell sensitivity to DX.

Keywords: Glucocorticoid; Lymphoblast; p16.

Fig. 1

Western blot analysis of p16 compared with β-actin in NC-37 cells. Wild-type, control siRNA-transfected, and p16 siRNA-transfected NC-37 cells were immunoblotted with p16 antibody. The p16 siRNA-transfected NC-37 cells did not express p16 protein. The bar graphs express the ratio of p16 to β-actin calculated from densitometry measurements. ^*P<0.05.

Fig. 2

Cytoplasmic glucocorticoid receptor (GR) expression levels as measured by flow cytometry. Time-dependent changes in GR expression after dexamethasone (DX) treatment are shown for control and p16 siRNA-transfected NC-37 (A) along with the results of flow cytometry (B). GR expression peaked at 18 h and decreased sharply at 24 h. The control NC-37 cells expressed higher glucocorticoid receptor (GR) levels compared to the p16 siRNA-transfected NC-37 cells at 18 h. ^*P<0.05.

Fig. 3

Apoptotic cells stained with annexin V and propidium iodide (PI) as assessed by flow cytometry in both groups. Annexin V single-positive cells were regarded as early apoptotic cells (A) and double-positive cells were regarded as late apoptotic cells (B). There were no statistical differences between the 2 groups for early apoptosis (A). Late apoptotic cells increased in a time-dependent manner and peaked at 18 h in the control NC-37 cells. ^*P<0.05.

Fig. 4

Alamar blue (AB) assay estimating time-dependent cell viability. Cell viability decreased after dexamethasone (DX) treatment. After 12 h, viability of the control NC-37 cells decreased more rapidly than that of the p16 siRNA-transfected NC-37 cells. ^*P<0.05.