DNA Damage Does Not Cause BrdU Labeling of Mouse or Human β-Cells

Abstract

Pancreatic β-cell regeneration, the therapeutic expansion of β-cell number to reverse diabetes, is an important goal. Replication of differentiated insulin-producing cells is the major source of new β-cells in adult mice and juvenile humans. Nucleoside analogs such as BrdU, which are incorporated into DNA during S-phase, have been widely used to quantify β-cell proliferation. However, reports of β-cell nuclei labeling with both BrdU and γ-phosphorylated H2A histone family member X (γH2AX), a DNA damage marker, have raised questions about the fidelity of BrdU to label S-phase, especially during conditions when DNA damage is present. We performed experiments to clarify the causes of BrdU-γH2AX double labeling in mouse and human β-cells. BrdU-γH2AX colabeling is neither an age-related phenomenon nor limited to human β-cells. DNA damage suppressed BrdU labeling and BrdU-γH2AX colabeling. In dispersed islet cells, but not in intact islets or in vivo, pro-proliferative conditions promoted both BrdU and γH2AX labeling, which could indicate DNA damage, DNA replication stress, or cell cycle–related intrinsic H2AX phosphorylation. Strategies to increase β-cell number must not only tackle the difficult challenge of enticing a quiescent cell to enter the cell cycle, but also achieve safe completion of the cell division process.

Human Beta Cell Regenerative Drug Therapy for Diabetes: Past Achievements and Future Challenges

“…Understanding the timeline and cause of the reduced beta cell mass in T2D is difficult define in precise terms, since human beta cell mass can only be assessed at autopsy. The relative reduction in beta cell mass in many people with T2D likely reflects combinations of: 1) genetic predisposition to lower beta cell mass and/or reduced beta cell function, illustrated by GWAS studies (11,12); 2) inadequate attainment of beta cell mass during fetal life and childhood (13,14); 3) beta cell dedifferentiation induced by glucotoxicity, lipotoxicity and/or endoplasmic reticulum (ER) stress, all driven by excessive demand for insulin resulting from insulin resistance and excessive caloric intake (15)(16)(17)(18); and/or combinations of the above. Importantly in the current context, most people with T2D retain substantial beta cell mass, and most produce substantial quantities of insulin (2,(4)(5)(6)10).…”

“…On the other hand, beta cell replacement for T2D is not widely performed, reflecting the facts that there is a broad range of drug therapies for T2D, and that beta cell replacement is simply impractical because of the sheer number of affected individuals. In addition, since beta cells are de-differentiated in T2D (10,(15)(16)(17), optimal drug therapies for T2D will need to induce human beta cells to re-differentiate from their de-differentiated state.…”

“…Analogously, since these same markers are activated in DNA damage and DNA repair pathways, expression of these "cell cycle markers" may instead reflect cell damage and impending death rather than true cell cycle completion. These events are commonly explored through immunolabeling with cell death markers or DNA damage markers such as TUNEL and gH2AX, respectively (15).…”

“…This practice makes it unlikely that authentic cell cycle entry does not occur in response to DYRK1A inhibitors. For those interested in more details, Alonso et al have provided convincing evidence that DNA damage cannot account for observed increases in beta cell labeling with BrdU (15). Some have used flow cytometric assessment of cell cycle stage.…”

Intersection of the ATF6 and XBP1 ER stress pathways in mouse islet cells

“…Islets were isolated by pancreatic ductal collagenase (Roche, Germany) injection and Ficoll gradient (Histopaque-1077; Sigma) as described previously (32,33). Whole islets were cultured at 37°C in 5% CO2 in islet complete medium (ICM; RPMI 1640 medium containing 10% FBS, 5 mM glucose, 2 mM glutamine, and 1% penicillin-streptomycin) for 24 hours after isolation to allow recovery.…”