Id3 upregulates BrdU incorporation associated with a DNA damage response, not replication, in human pancreatic β-cells

Abstract

Elucidating mechanisms of cell cycle control in normally quiescent human pancreatic β-cells has the potential to impact regeneration strategies for diabetes. Previously we demonstrated that Id3, a repressor of basic Helix-Loop-Helix (bHLH) proteins, was sufficient to induce cell cycle entry in pancreatic duct cells, which are closely related to β-cells developmentally. We hypothesized that Id3 might similarly induce cell cycle entry in primary human β-cells. To test this directly, adult human β-cells were transduced with adenovirus expressing Id3. Consistent with a replicative response, β-cells exhibited BrdU incorporation. Further, Id3 potently repressed expression of the cyclin dependent kinase inhibitor p57 (Kip2 ) , a gene which is also silenced in a rare β-cell hyperproliferative disorder in infants. Surprisingly however, BrdU positive β-cells did not express the proliferation markers Ki67 and pHH3. Instead, BrdU uptake reflected a DNA damage response, as manifested by hydroxyurea incorporation, γH2AX expression, and 53BP1 subcellular relocalization. The uncoupling of BrdU uptake from replication raises a cautionary note about interpreting studies relying solely upon BrdU incorporation as evidence of β-cell proliferation. The data also establish that loss of p57 (Kip2) is not sufficient to induce cell cycle entry in adult β-cells. Moreover, the differential responses to Id3 between duct and β-cells reveal that β-cells possess intrinsic resistance to cell cycle entry not common to all quiescent epithelial cells in the adult human pancreas. The data provide a much needed comparative model for investigating the molecular basis for this resistance in order to develop a strategy for improving replication competence in β-cells.

Figure 1. Id3 mediates cell p57^Kip2 downregulation and BrdU incorporation in human β-cells. (A–C) Adult human islets expressing insulin (green) were transduced with Ad-LacZ (A) or Ad-Id3 (B) and analyzed for p57^Kip2 (red) expression, quantified in (C) (*p < 0.0005, n = 5). (A) White arrows indicate representative insulin-positive cells expressing p57^Kip2. (D–G) Human β-cells (insulin, green) infected with Ad-Id3 and cultured for 48 h in the presence of BrdU alone (D), BrdU⁺ prolactin (E), BrdU⁺ exendin-4 (F), or BrdU⁺ caffeine (G), demonstrate pronounced BrdU incorporation [red, quantified in (H), *p < 0.005, **p < 0.001], in three independent islet cell preparations. Notably, these agents had no effect on BrdU incorporation in the absence of Id3. High power view of a typical nucleus from an Ad-Id3 infected BrdU-positive β-cell demonstrates a punctate, perinuclear pattern of BrdU uptake (I). Blue nuclear counterstain is DAPI. Scale bars, (A–C) = 100 μm, (D–G) = 50 μm, (I) = 10 μm.

Figure 2. BrdU incorporation proceeds in β-cells, but not exocrine and mesenchymal cells, in the presence of hydroxyurea, a proliferation inhibitor. (A–F) Islet cell cultures (insulin, green) were treated with BrdU (red). BrdU incorporation observed in Id3 infected β-cells (B) was retained in the presence of hydroxyurea (HU) (E and F), suggesting DNA damage, not replication. White arrows depict BrdU positive β-cells and inset (white box) in (E) is magnified in (F), quantified in (C) (*p < 0.005, n = 3) (G–J). In contrast, HU suppressed BrdU incorporation in Ad-Id3 infected exocrine cells (panCK, green) (H) vs. (J) and in mesenchymal cells (insulin and panCK negative, (D) vs. (A), and (G and H vs. I and J), evidence that mesenchymal cells and Id3 infected exocrine cells, are replicating, n = 3. Blue nuclear counterstain is DAPI. Scale bars, (A, B, D and E) = 50 μm, (F) = 10 μm, (G–J) = 100 μm.

Figure 3. Id3 induces the DNA damage response γH2AX and 53BP1 in human β-cells. Human islets, infected with Ad-LacZ (A, D, G and J) or Ad-Id3 (B, E, H and K) and cultured for 48 h in the presence of BrdU (n = 3), were immunostained for insulin (green) and the DNA damage marker γH2AX (red) in (A and B) and (G–I) (pseudocolored yellow). Greater than five γH2AX foci/nucleus were scored as positive, quantified in (C), *p < 0.0001. Triple immunostaining for insulin (green in D–I), BrdU (red in D–F) and γH2AX (Cy5, pseudocolored yellow in G–I), demonstrated colocalization of BrdU with γH2AX foci in β-cells. (F and I) are magnified from the white boxes in (E and H), respectively. (J–L) Immunostaining for total 53BP1 protein (green) and insulin (red) in control (Ad-LacZ) (J) and Ad-Id3 (K) infected β-cells. More than three foci/nucleus were scored as positive for 53BP1 protein redistribution, quantified in (L), *p < 0.001. Blue nuclear counterstain is DAPI. Scale bars, (A and B) = 100 μm, (D, E, G, H, J and K) = 50 μm, (F and I) = 10 μm.

Figure 4. DNA damage in β-cells in vivo. In order to measure DNA synthesis and damage in β-cells in vivo, mice were administered BrdU in drinking water for 3 d. Pancreata were harvested and immunostained for BrdU (green), insulin (blue), and γH2AX (red). Under normal conditions (A–C), approximately 7% of BrdU positive β-cells also exhibited γH2AX (red) expression. In the course of studying small molecules identified in high throughput screens, however, we found that intraparenchymal injection of DMSO in the pancreas resulted in expression of γH2AX (red) in over 25% of BrdU positive β-cells (D–F), quantified in (L). Thus, BrdU incorporation can represent DNA damage in a significant percentage of β-cells in vivo as well as in vitro (Fig. 3). Arrows in (D–F) mark region magnified in G–I respectively. *p < 0.01, **p < 0.001. Blue nuclear counterstain is DAPI. Scale bars (A–F) = 50 μm, (G–I) = 10μm.