dna damage does not cause brdu labeling of mouse or human ... · dna damage does not cause brdu...

DNA damage does not cause BrdU labeling of mouse or human beta cells

Rohit B. Sharma1, Christine Darko1, Xiaoying Zheng1, Brian Gablaski1 and Laura C. Alonso1

Diabetes Center of Excellence in the Department of Medicine1, University of Massachusetts Medical School, Worcester MA

Running titleDNA damage does not cause BrdU labeling

Corresponding authorLaura C. Alonso 774-455-3640 (phone)508-856-3803 (fax)AS7-2047, Division of Diabetes368 Plantation Street, Worcester, MA [email protected]

KeywordsPancreatic beta cell, replication, proliferation, mitosis, cell division, cell cycle, nucleoside analogs, bromodeoxyuridine, DNA damage

AbbreviationsBrdU, bromodeoxyuridineEdU, 5-ethinyl-2’-deoxyuridinegH2AX, gamma phosphorylated H2A histone family member X

Word count:Abstract: 193 (of 200)Main text: 3999 (of 4000)

of 35 Diabetes

Diabetes Publish Ahead of Print, published online March 4, 2019

mailto:[email protected]

ABSTRACT

Pancreatic beta cell regeneration, the therapeutic expansion of beta cell number to reverse diabetes, is an important goal. Replication of differentiated insulin-producing cells is the major source of new beta cells in adult mice and juvenile humans (1–3). Nucleoside analogs such as bromodeoxyuridine (BrdU), which are incorporated into DNA during S-phase, have been widely used to quantify beta cell proliferation. However, reports of beta cell nuclei labeling with both BrdU and gH2AX, 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-gH2AX double-labeling in mouse and human beta cells. BrdU-gH2AX co-labeling is neither an age-related phenomenon nor limited to human beta cells. DNA damage suppressed BrdU labeling and BrdU-gH2AX co-labeling. In dispersed islet cells, but not in intact islets or in vivo, pro-proliferative conditions promoted both BrdU and gH2AX labeling, which could indicate DNA damage, DNA replication stress, or cell-cycle related intrinsic H2AX phosphorylation. Strategies to increase beta 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.

of 35Diabetes

Beta cell replication, towards re-expansion of functional beta cell mass, is an important goal for diabetes

research. Incorporation of nucleoside analogs has been a high-value tool in quantifying cell proliferation

behavior for decades, allowing measurement of cumulative S-phase entry during a defined exposure

period, in vitro and in vivo. Nucleoside analogs such as BrdU and EdU (4) have been used not only in

beta cell biology but also broadly across developmental and cell biology.

Under certain conditions, BrdU incorporation in beta cells has been observed to co-localize with markers

of DNA damage (5,6). In other cell types, BrdU exposure has been shown to activate a DNA damage

response (7–9), but in beta cells the reasons for this co-localization are not well understood. The

observation has been widely discussed and rapidly incorporated into thinking in the field, with a range of

impacts. In the most-discussed work (5), the conclusion drawn by the originating authors was that some

BrdU-labeled human beta cells, particularly the subset with atypical punctate BrdU staining, fail to

complete S-phase, instead showing evidence of DNA damage, DNA re-replication, and failure to divide.

In other words, BrdU labeled cells that transitioned into S-phase, but BrdU-labeled cells could not be

assumed to progress through successful mitosis. However, concern in the field has extended beyond

this concept; in many quarters, the question has been raised as to whether BrdU labeling, counted as

evidence of S-phase entry, could in fact be due to a completely unrelated process, nucleotide

incorporation during DNA damage repair. If this hypothesis were true, then nuclei might label for BrdU in

the absence of S-phase entry, invalidating some prior results and diminishing the value of nucleoside

analogs in the study of cell replication.

The field of beta cell regeneration has been impacted by uncertainty of the correct interpretation of BrdU-

labeled nuclei. There is an urgent need to clarify the reasons for co-occurrence of BrdU and DNA damage

labeling in beta cells. The goals of this study were to explore possible causes of BrdU and gH2AX co-

localization in nuclei of mouse and human beta cells, and to specifically test whether there are conditions

in which BrdU labeling can be induced by DNA damage-related cellular processes rather than cell cycle

entry.

RESEARCH DESIGN AND METHODS

Mice for islet isolationAll mouse procedures were approved by the UMass Medical School Institutional Animal Care and Use

Committee. Young (10-12 weeks) or old (50-60 weeks) C57BL/6J male and female mice, from an in-

house breeding colony, had continuous access to normal mouse chow, on a 12-hour light/dark cycle.

Islets were isolated by pancreatic ductal collagenase injection and Ficoll (Histopaque-1077; Sigma)

of 35 Diabetes

gradient (10). Islets from multiple mice were pooled and mixed before experiments. Whenever possible,

all control and experimental comparisons were performed in parallel on islets from the same mice. Each

combined pool of islets was considered one biological replicate.

In vivo mouse experimentsTo study proliferative conditions in vivo, pancreas sections were analyzed from experiments previously

published on 10-12 week old male mice fed high fat diet for 7 days (11), or 10-12 week old male mice

with continuous hyperglycemia achieved by intravenous infusion of glucose (10,12).

Mouse islet cell cultureWhole islets were cultured overnight in islet medium (RPMI, 10% FBS, penicillin/streptomycin, 5.0

mmol/L glucose) after isolation. For dispersed-cell experiments, islets were hand-picked, digested with

single-use-apportioned 0.05% trypsin, and 50 IEQ per well were plated on uncoated glass coverslips

(Fisherbrand) in islet medium (12). Mouse islet cell experiments were 72 hours in duration, starting the

day after dispersion, with adenovirus, glucose and/or harmine (5 uM, Sigma #286044) exposure for 72

hours, BrdU (10ug/ml) added 24 hours prior to fixation. For whole islet experiments, islets were cultured

for 72 hours in islet medium with additives including glucose and/or Mitomycin C, with BrdU added for

the final 24 hours, fixed in 10% formalin for 30 minutes, washed with PBS and embedded in paraffin for

sectioning (13). For virus transduction, dispersed cells were transduced with adenovirus expressing

bacterial Cre recombinase (control virus) or human Cyclin D2 protein, at an MOI of 5, as described (12).

Mitomycin C (0.5 uM, Sigma) was added at the start of the 72-hour experiment. UV irradiation of islet cell

cultures was administered by placing the culture dish for 10 minutes on a UVP bench top transilluminator

equipped with a 302 nm filter (Cole-Parmer) after 24 hours of glucose treatment.

Human islet cultureHuman islets received from the IIDP (Supplemental Table 1) were cultured in islet medium overnight

after shipment, then dispersed and plated on coverslips as described above for mouse islets. After one

day recovery from plating, human islet cell experiments were performed exactly as described above,

except the experiment duration was 96 hours and BrdU was included for the entire 96 hours of culture.

Immunostaining and microscopyIslet cell cultures were fixed in 4% paraformaldehyde for 10 minutes at room temperature. Whole islets

(13) and pancreas sections were obtained from formalin-fixed, paraffin embedded tissue blocks as

described in the publications where these samples originated (10–12). For BrdU, gH2AX and pHH3

staining, fixed cells or rehydrated paraffin sections were submerged in 1N HCl for 20 minutes, blocked

of 35Diabetes

for 2 hours in goat serum-based block with 0.1% Tween-20, labeled with primary antibodies, then

secondary antibodies (Invitrogen), and mounted on glass slides with Fluoroshield mounting media

containing Dapi (Sigma). Primary antibodies were guinea pig anti-insulin (Dako A0564), rabbit anti-

gH2AX (Cell Signaling #9718), mouse anti-gH2AX (EMD Millipore #05-636), rat anti-BrdU (Abcam

Ab6326), rabbit anti-pHH3 (Cell Signaling #3377) and rabbit anti-Ki67 (Thermo Scientific RM-9106).

Fluorescent images were acquired using a Nikon fluorescent microscope or a Solamere CSU10 Spinning

Disk confocal system mounted on a Nikon TE2000-E2 inverted microscope. Image data were quantified

by an unbiased laboratory member (BG) using Cell Profiler automated counting software from the Broad

Institute (14,15) with manual image checking post-quantification. A cell was considered to be gH2AX

positive if the nucleus was labeled with multiple intense nuclear foci. The number of cells counted for

each experimental condition are included in Supplemental Table 2.

Statistical AnalysisData were analyzed and graphed using GraphPad Prism 7. In all column plots, each sample is

represented by one data point. Venn diagrams were generated using EulerAPE 3.0.0 from the University

of Kent (16). Data were compared using Student’s two-tailed T-test; p<0.05 was considered significant.

RESULTS

Possible explanations for BrdU and DNA damage co-labeling in the same beta cellConsistent with the reports of human beta cell co-labeling (5), we have observed BrdU-gH2AX co-labeling

in primary mouse islet cell cultures (unpublished, and Fig 1). We reasoned that these two markers could

label the same nucleus if: a) DNA damage leads to BrdU incorporation, b) BrdU incorporation leads to

DNA damage, c) an upstream process leads to both labels, or d) BrdU incorporation and DNA damage

occur stochastically and occasionally occur in the same cell but are unrelated (Supplemental Table 3).

BrdU-gH2AX co-labeling in mouse beta cells is not due to random chanceWe first tested the simplest explanation for co-localization: that BrdU labeling and DNA damage each

occur in a fraction of cultured primary beta cells, unrelated to one another, leading to occasional co-

occurrence in the same cell. If this model were true, gH2AX and BrdU labels should both be detectable

in standard culture conditions, and the presence of one label in a cell should not influence the likelihood

of presence of the other label. We cultured dispersed mouse islet cells under standard, unstimulated

conditions. When the cells were fixed and immunostained for insulin, BrdU and gH2AX, a small but

consistent fraction of insulin-containing cells labeled for each marker (Fig 1A-A’-B). To test whether co-

labeling could be due to random co-occurrence of two unrelated processes, we calculated the predicted

of 35 Diabetes

likelihood of random co-labeling based on the frequency of each individual label (predicted fraction co-

labeling was defined as the product of the observed fractions of each single label) and compared this

predicted frequency with the actual observed frequency of co-labeling. The observed frequency, although

low, was higher than the predicted random co-occurrence frequency (Fig 1C). Confocal microscopy

confirmed nuclear co-labeling in some beta cells (Fig 1D-D’). As reported (5), in some BrdU-gH2AX co-

labeled nuclei the BrdU labeling was punctate in appearance. Thus, in unstimulated culture conditions,

co-labeling of BrdU and gH2AX occurred more frequently in mouse beta cells than predicted by chance,

suggesting that one process influences the other or that both are influenced by a common upstream

process.

Proliferative stimuli increase BrdU-gH2AX double-labeling frequency Since overexpression of HNFA8 or Cyclin D3/Cdk6 in combination (5) increased co-labeled cells, we

reasoned that proliferation drivers increase the frequency of double-labeled cells. Since the originating

work was in human beta cells, which are considerably older than young-adult mouse beta cells, we also

considered that beta cells from older mice might be more vulnerable. We tested whether the mitogenic

stimuli frequently used in our lab, 15mM glucose and adenoviral-delivered Cyclin D2 (12,17), altered the

likelihood of BrdU and gH2AX co-labeling in beta cells from younger (10-12 weeks) and older (50-60

weeks) mice. In 15mM glucose, both BrdU and gH2AX (Fig 2A-B) labeling increased. Supporting the

concept that BrdU labeling reflects S-phase entry, beta cells from younger mice had a higher frequency

of BrdU incorporation than beta cells from older mice. Perhaps surprisingly, in high glucose beta cells

from older mice did not label more frequently for gH2AX, or both markers, than beta cells from younger

mice (Fig 2C).

To test whether directly forcing cell cycle entry increases BrdU-gH2AX co-labeling, we overexpressed

Cyclin D2. In normal 5mM glucose, Ad-cyclin D2 produced similar results to 15mM glucose stimulation

(Fig 2D-F): BrdU and gH2AX were individually modestly increased, the frequency of double-labeled cells

increased, and older mouse age did not increase the frequency of beta cells with gH2AX or double-

labeling. However, when Cyclin D2 overexpression was combined with 15mM glucose (Fig 2G-I) BrdU,

gH2AX, and BrdU-gH2AX labeled beta cells were markedly increased over baseline. In all proliferation-

stimulated conditions (Fig 2C, F and I), the observed frequency of co-labeled cells was significantly higher

than predicted if co-labeling was due to chance. To test a third, unrelated proliferative stimulus we treated

the cultures with harmine, a Dyrk1a inhibitor that increases mouse and human beta cell proliferation (18).

Harmine increased BrdU labeling synergistically with glucose, increased gH2AX labeling in both 5 and

15mM glucose, and similar to the other stimuli, the observed frequency of co-labeling was higher than

of 35Diabetes

predicted (Fig. 2J-L). Thus, the frequency of BrdU-gH2AX double-labeling is increased by proliferative

stimuli, especially when stimuli are combined in aging mouse beta cells.

DNA damage does not lead to BrdU incorporationTo explore whether DNA damage and BrdU incorporation influence each other, we next tested whether

BrdU incorporation was increased during conditions that induce DNA damage. These were perhaps the

most critical experiments in this study, because if BrdU is spuriously incorporated as part of a DNA

damage response, then BrdU labeling cannot be used to quantify S-phase cell cycle entry. To address

this question we cultured beta cells in DNA damage-inducing conditions and quantified BrdU- and double-

labeled cells. In islet cell cultures exposed to a sub-lethal dose of mitomycin C, which induces DNA

damage by crosslinking DNA (19–21), almost all beta cells had gH2AX labeling (Fig 3A), confirming DNA

damage. To test whether DNA damage caused spurious BrdU incorporation, we stained for BrdU. The

results showed the opposite; mitomycin C strongly suppressed BrdU labeling frequency in both young

and old beta cells, and the observed frequency of double-labeled cells was suppressed to the predicted

frequency if co-labeling were caused by random co-occurrence of unrelated processes (Fig 3B-C). These

results suggest that DNA damage repair following mitomycin C exposure did not cause spurious BrdU

incorporation in mouse beta cells that could be misconstrued as proliferation.

To thoroughly explore this important question, we repeated the experiment using a second DNA damage

insult: UV irradiation, which damages DNA by joining adjacent thymine nucleotides into pyrimidine dimers

(22–24). We selected a UV dose that caused moderate DNA damage based on trial experiments (data

not shown). Cellular response to UV was variable, with two experiments showing a high proportion of

cells labeling for gH2AX (circles in Fig 3D-F), and two experiments, despite being performed identically,

showing fewer cells labeling for gH2AX (triangles in Fig 3D-F). Similar to the mitomycin C result, UV-

induced DNA damage suppressed BrdU incorporation rather than increasing it, and reduced the

observed frequency of double-labeled beta cells to that predicted if co-labeling was due to random co-

occurrence of unrelated events (Fig 3E). Reduced BrdU incorporation even in the cultures with only

modest gH2AX labeling frequency (triangles) suggests that the suppression of BrdU label was not due

to overwhelming toxicity of the DNA damage insult. Taken together, these data suggest that BrdU label

in cultured mouse beta cells is not increased by DNA damage.

gH2AX labels a subset of BrdU-labeled beta cells in ex vivo cultureUnder most conditions, we noticed that although many BrdU-labeled cells were not gH2AX-labeled, the

converse was not true: most gH2AX-labeled cells were BrdU-labeled. Stated another way, the population

of gH2AX-labeling beta cells seemed to be a subset of the BrdU-labeled population. Expressing the data

using quantitative Venn diagrams confirmed this observation; especially under proliferation-stimulated

of 35 Diabetes

conditions, the majority of gH2AX-labeled beta cells were within the BrdU-labeled population (Fig 4A-B).

In contrast, under DNA damaging conditions, Venn analysis showed that the vast majority of gH2AX-

labeling beta cells did not label with BrdU (Fig 4C). We used confocal microscopy to identify the pattern

of BrdU label in double-labeled cells. We specifically asked whether BrdU labeled nuclei uniformly, as is

characteristic of S-phase DNA replication, or in punctate fashion overlapping with foci of the gH2AX DNA

damage label. Images showed that many of the double-labeled nuclei were uniformly labeled with BrdU

(Fig 4D-E), suggesting the possibility that gH2AX labeling occurred after DNA replication. Some nuclei

had punctate BrdU as described earlier (Fig 4F) (5). Consistent with the notion that DNA damage

prevents successful cell division, the few mitotic nuclei visualized had no gH2AX label (Fig 4G). On the

other hand, mitotic figures did label for BrdU (Fig 4G), and many BrdU-labeled nuclei co-stained for the

mitotic marker pHH3 or appeared as doublets (Suppl. Fig 1), confirming that some BrdU-labeled beta

cells do enter mitosis. We tested whether gH2AX-BrdU co-labeling of beta cells occurs in vivo under

conditions in which beta cell mass increases, such as high fat feeding (11) and hyperglycemia (10,12).

Careful examination of many islets in sections from multiple mice with elevated BrdU incorporation

revealed very rare gH2AX labeling in either condition: in eight pancreata from the HFD experiment only

four islet-related nuclei were found that stained for gH2AX (Suppl. Fig 2), and in four pancreata from the

hyperglycemia experiment only four islet-related nuclei stained for gH2AX (Suppl. Fig 3). Of the eight

islet nuclei labeling for gH2AX across both experiments, 6 were beta cell nuclei, but only two co-stained

for BrdU; interestingly, these two were found in the same islet (Suppl. Fig 2B). To assess whether gH2AX

labeling is induced by ex vivo islet culture in general, or of dispersed islet cell cultures specifically, whole

intact islets were cultured under proliferative conditions and assessed for BrdU and gH2AX co-labeling

(Suppl. Fig 4). Beta cells co-labeling for both markers were very rare in intact islets (Suppl. Fig 4D).

Taken together, these results suggest that in ex vivo dispersed islet cell culture, but not intact islet culture

or in vivo pancreas, conditions that promote cell cycle entry or BrdU incorporation increase beta cell risk

of gH2AX labeling, whereas conditions that promote DNA damage do not increase likelihood of BrdU

incorporation.

BrdU incorporation itself is a minor cause of beta cell DNA damage Since the data suggested that cell cycle entry, or possibly BrdU incorporation itself, increased the

likelihood of gH2AX labeling, and nucleoside analogs are known to induce a DNA damage response in

other cell types (7–9), we next asked whether BrdU exposure leads to DNA damage in beta cells. To test

this, we cultured mouse islet cells with or without BrdU and assessed the insulin (+) fraction labeling for

gH2AX. Omitting BrdU from the culture medium eliminated all BrdU labeling in the cultures (not shown).

In the absence of BrdU exposure, we observed a subtle reduction in gH2AX labeling in beta cells from

younger mice that was not observed in beta cells from older mice (Fig 5A). Hypothesizing that gH2AX

of 35Diabetes

labeling might increase over time following BrdU exposure, we compared cells exposed to BrdU during

the first 24 hours (condition X), the last 24 hours (condition Y) or the entire 72-hour glucose exposure

(condition Z). gH2AX labeling was not different between these conditions (Fig 5B-C). To test whether

BrdU exposure increased DNA damage under more pronounced proliferative stimuli, we cultured islet

cells with or without BrdU in the presence of Ad-cyclin D2 in low glucose (Fig 5D) or high glucose (Fig 5E). Intriguingly, BrdU exposure increased gH2AX labeling only in the low-glucose condition. To test

whether gH2AX labeling was more related to cycling beta cells or to BrdU incorporation itself, we stained

proliferation-stimulated cultures with pHH3 and gH2AX, predicting that if gH2AX labeling was due to BrdU

toxicity there would not be co-labeling of gH2AX with pHH3 in the absence of BrdU exposure (Fig 5F-H).

In fact, beta cell nuclei frequently co-labeled with both pHH3 and gH2AX, at levels much higher than

expected if co-labeling was due to random chance (Fig 5G-H). Similar results were observed with

experiments in which co-staining was performed for Ki67 and gH2AX (data not shown). These data

suggest that although BrdU exposure or BrdU incorporation into DNA under certain conditions can cause

double strand DNA breaks or nucleotide excision/repair that label with gH2AX (23), this toxicity is not the

major cause of BrdU-gH2AX double-labeled cells in islet cell cultures.

Human beta cells, resistant to proliferative stimuli, were less likely to co-label for BrdU and gH2AX than mouse beta cellsThe goal of studies in beta cell regeneration is to find approaches to expand human beta cell number. To

learn more about BrdU-gH2AX co-labeling in human beta cells (5,6), dispersed human islet cells were

cultured under the same conditions as the mouse experiments, and tested for BrdU incorporation, gH2AX

labeling, and co-labeling for both markers. Under basal 5mM glucose conditions, the frequency of human

beta cells labeling for BrdU was low, and the frequency of gH2AX labeling was also rare (Fig 6A-B). To

determine whether DNA damage increased in human beta cells during conditions of forced proliferation,

we compared basal conditions with pro-proliferative stimuli 15mM glucose without or with Ad-cyclin D2.

Similar to prior published results, insulin (+) cells in human islet cultures were relatively resistant to

proliferative stimuli, showing only a marginal increase in the fraction labeling with BrdU when exposed to

15mM glucose (Fig 6A-C), Ad-cyclin D2 (Fig 6D-F), or a combination of the two (Fig 6G-I). Surprisingly,

only a small fraction of human beta cells labeled for gH2AX under basal or stimulated conditions (Fig 6B, E, H). The fraction of human beta cells labeling for both labels remained low and was not significantly

higher than predicted by the random chance of co-occurrence of two rare events (Fig 6C, F, I). These

data suggest that under these growth conditions, human beta cells do not frequently co-label for gH2AX

and BrdU.

DNA damage did not increase BrdU labeling in human beta cells

of 35 Diabetes

To test whether human beta cells incorporate BrdU during DNA damage repair, DNA damaging

conditions were applied to human islet cultures. Mitomycin C exposure caused gH2AX labeling in the

majority of beta cells (Fig 7A). Despite widespread DNA damage in the cultures, the percent of insulin

(+) cells labeling for BrdU decreased (Fig 7B) and the observed fraction of double-labeled cells was

similar to predicted if co-labeling was by random chance. When human islet cultures were exposed to

UV irradiation (Fig 7D-F), the DNA damage response was variable (Fig 7D), but BrdU incorporation was

suppressed (Fig 7E) and there was no increase in the proportion of beta cells labeling for both markers

(Fig 7F). In sum, human beta cells behaved similarly to mouse beta cells: DNA damage did not cause a

spurious increase in BrdU labeling.

gH2AX and BrdU labeling were mostly independent in human beta cellsQuantitative Venn analysis of the human beta cell BrdU- and gH2AX-labeling populations revealed the

interesting result that under basal and proliferation-stimulated conditions, insulin (+) cells labeling for

BrdU were mostly not the same cells that labeled with gH2AX (Fig 8A). In fact, without Ad-cyclin D2, no

double-labeled cells were identified. Similar to mouse islet cultures, however, DNA damage stimuli

caused the majority of human beta cells to label for gH2AX, and only a tiny fraction of those cells also

labeled for BrdU (Fig 8B). Overall, fewer human beta cells labeled for gH2AX than mouse. Like mouse,

DNA damage conditions did not induce spurious BrdU incorporation in human beta cells. Neither BrdU

nor gH2AX were meaningfully increased by proliferative stimuli in human beta cells, and in these

conditions, the observed double-labeled fraction was not increased over that predicted by random

chance.

DISCUSSION

These studies explore the frequency and causes of mouse and human beta cell co-labeling for BrdU and

the gH2AX DNA damage marker. The results suggest that in mouse beta cells, co-labeling occurs more

frequently than predicted if the association is due to random chance. Evidence does not support the

hypothesis that co-labeling is due to BrdU incorporation during DNA repair. Instead, the results suggest

that gH2AX labeling can be triggered by something related to the proliferative process, either by an

upstream proliferation stimulus or by cellular events associated with the cell cycle itself.

These experiments are important, because uncertainty of the fidelity of BrdU as a label of S-phase in

beta cells has impacted the field. For some, conflating the concepts of cell cycle and DNA damage has

rekindled longstanding doubts that beta cells can replicate at all, despite a large quantity of evidence to

of 35Diabetes

the contrary, e.g. (3, 14–16). Investigators have been at times required to use multiple duplicative tools

to measure the same outcome, slowing progress. The results of these current studies are reassuring.

Labeling for gH2AX in human beta cells was rare, even under proliferation-stimulating conditions, and

most BrdU(+) human beta cells were not gH2AX(+). In vivo mouse pancreas rarely labeled with gH2AX,

even under conditions when many beta cells were BrdU(+). Overall, the results are consistent with the

conclusion that even in mouse islets ex vivo BrdU authentically labels beta cells that enter S-phase.

The data show that DNA damage does not cause BrdU labeling that could be misconstrued as S-phase

entry, in either mouse or human beta cells. Using two different DNA damage inducing conditions, one of

which caused widespread damage in essentially all beta cells (mitomycin C), and the other of which

caused more moderate damage (UV irradiation), BrdU incorporation was reduced rather than increased.

Furthermore, the observed frequency of double-labeled nuclei was suppressed in DNA damage

conditions to that expected if the occurrence was due to random chance. Thus, beta cell co-labeling for

BrdU and gH2AX in these cultures was not caused by spurious BrdU incorporation as part of the cellular

response to DNA damage.

The data show a clear association between gH2AX labeling and proliferative stimuli in mouse beta cells.

gH2AX labeling, and gH2AX-BrdU or gH2AX-pHH3 co-labeling, occurred occasionally under

unstimulated conditions but were markedly increased in 15mM glucose with or without overexpression of

cyclin D2 or addition of harmine. Intriguingly, the gH2AX-labeled cells were mostly a subset of the BrdU-

labeled population. Also, many gH2AX-labeled nuclei were evenly and completely BrdU labeled, giving

the impression that S-phase had occurred smoothly. Taken together with the observations that DNA

damage suppressed new S-phase entry and that gH2AX foci were absent from the few mitotic spindles

observed, we speculate that the gH2AX foci may mostly occur in a cell cycle window from late S-phase

through G2 phase, or in post-mitotic daughter cells. gH2AX is best known for labeling double strand

breaks or nucleotide excision repair, recruiting DNA repair effectors (23,24). gH2AX also labels DNA

replication stress, such as replication fork collapse, defects in chromatin assembly, cell cycle arrest, or

prolonged mitosis (26–29). This raises the possibility that beta cells forced into the cell cycle may have

weakened replication forks or other defects in completing mitosis. In some cell types, gamma

phosphorylation of H2AX may occur intrinsically during S- or G2-M phases of the cell cycle (30,31). It is

possible that chromatin handling during DNA replication or G2 may predispose beta cells to gH2AX

phosphorylation; whether this is a sign of damaged DNA or other process remains uncertain.

A toxic effect of nucleoside analogs, including both BrdU and EdU, has been reported (7–9). In the beta

cell field EdU is also commonly used, especially for high throughput screening; whether EdU performs

of 35 Diabetes

similarly to BrdU has not been carefully tested. The final concentration of BrdU used in our study (33 uM)

was similar to other published studies (10-100 uM) (5,7,8). BrdU exposure may or may not induce sister

chromatid exchanges, a marker of genome instability (32). Triphosphate nucleoside analogs such as 5-

fluorouracil act as replication fork blocks; however, these induce gH2AX not at the time of initial

incorporation, but rather during S-phase of a subsequent cell cycle event (33). Extensive co-labeling of

gH2AX and pHH3 in the absence of BrdU exposure argue against a primary role for BrdU toxicity in the

observed gH2AX population, but rather that gH2AX staining is associated with cycling cells. Beta cells

with smooth BrdU label and gH2AX foci may be cells that have re-entered the cell cycle following a

successful initial cell division.

Surprisingly, beta cells from older mice were not spontaneously more likely to label for gH2AX. However,

when both high glucose and cyclin D2 overexpression were combined, beta cells from older mice had a

higher frequency of gH2AX labeling, and a lower fraction of BrdU-labeled beta cells that did not show

evidence of DNA damage. This may be related to the fact that beta cells from older mice required a

combination of both glucose and cyclin D2 to enter the cell cycle, whereas beta cells from younger mice

responded to either stimulus. The human islet donors used for this study were from a narrow range of

ages, 41 to 56 years, precluding assessment of the impact of age on DNA damage markers in human

beta cells.

Also contrary to expectations, in these studies human beta cells did not have a high frequency of gH2AX

labeling. If anything, human beta cells were less likely than mouse beta cells to label for gH2AX,

especially in the presence of proliferative stimuli. However, the pro-proliferation stimuli used in these

studies did not result in a high rate of cell cycle entry in human beta cells. As such, the low rate of human

gH2AX labeling is consistent with the hypothesis that DNA damage occurs in later stages of the cell

cycle, or during cell cycle re-entry following successful mitosis. We have not tested conditions that induce

rapid proliferation in human beta cells.

This study does not address the extent to which BrdU, marking S-phase entry, predicts completion of the

cell cycle and production of two daughter cells. Evidence supporting the conclusion that some BrdU-

labeled beta cells do successfully complete cell division include the many studies showing that BrdU

labeling correlates with other proliferation markers such as Ki67, PCNA and pHH3, e.g. (17,34,35), the

documented increase in beta cell number under conditions when BrdU labeling is increased in vitro

(17,36,37) and in vivo, e.g. (33), and our current data showing BrdU+ nuclei in mitosis (Fig 4G) and co-

labeling with pHH3 (Suppl. Fig 1). We have not formally tested for beta cell endoreduplication, but similar

to prior observations (38) we did not detect systematically increased nuclear size in double-labeled cells.

of 35Diabetes

On the other hand, the fate of beta cells labeled for both BrdU and gH2AX remains unknown. Although

getting beta cells to traverse the G1-S transition is an important and challenging goal, studies are also

needed to find ways to increase the frequency of successful mitosis in beta cells that do enter the cell

cycle. Taken together, the results of the current studies support the use of BrdU labeling as a faithful

measure of S-phase entry and a useful tool in the overarching goal of the pursuit of beta cell regeneration

strategies as a therapy for diabetes.

ACKNOWLEDGMENTSLCA and RBS devised and planned the project. RBS performed and analyzed the experiments, with

assistance from XZ (immunostaining and microscopy) and BG (unbiased image analysis). LCA wrote the

manuscript; all authors had the opportunity to edit and approve of the manuscript. LCA is the guarantor

of this work and, as such, had full access to all the data in the study and takes responsibility for the

integrity of the data and the accuracy of the data analysis. The authors have no conflicts of interest with

the work contained in this manuscript. Human pancreatic islets were provided by the NIDDK-funded

Integrated Islet Distribution Program (IIDP) at City of Hope. We would like to thank the Beta Cell Biology

Group at the University of Massachusetts Medical School for helpful discussions. This work was

supported by NIH/NIDDK: R01DK114686 (LCA), R01 DK113300 (LCA), U2CDK093000 (MMPC

Analytical and Functional core, LCA), 2UC4DK098085 (IIDP), the American Diabetes Association grant

#1-18-IBS-233 (LCA) in collaboration with the Order of the Amaranth, and the George F. and Sybil H.

Fuller Foundation.

FIGURE LEGENDS

Figure 1. BrdU and gH2AX labeling co-occur in some beta cells in mouse islet cell cultures. Dispersed mouse islet cells were cultured for 72 hours in 5mM glucose (A, A’, B, C) or 15mM glucose (D, D’), with BrdU added for the final 24 hours. (A-A’) BrdU (A) and gH2AX (A’) each labeled a small fraction of beta cells under unstimulated culture conditions. (B-C) In mouse islet cells cultured in unstimulated (5mM glucose) conditions, a small proportion of beta cells labeled with BrdU or gH2AX (B). The observed fraction of beta cells co-labeled with both BrdU and gH2AX was low, but higher than that predicted by random chance (predicted fraction was defined by the product of the observed fractions of each single label) (C). (D-D’) Confocal microscopy of mouse islet cells cultured in 15mM glucose confirmed both labels occur in the same nuclei. For A, A’, B and B’, lines mark BrdU (+) gH2AX (-) nuclei, arrowheads mark BrdU (-) gH2AX (+) nuclei, and arrows mark BrdU (+) gH2AX (+) nuclei. *p<0.05, ns p>0.1.

Figure 2. Proliferative stimuli, especially in combination, increase BrdU-gH2AX co-localization frequency in mouse beta cells. Dispersed young (10-12 weeks) and old (50-60 weeks) mouse islet cells were cultured for 72 hours in the indicated conditions, with BrdU added for the final 24 hours. (A-C) 15mM glucose markedly increased BrdU incorporation (A), especially in young islets, and modestly increased gH2AX labeling (B) and BrdU-gH2AX co-labeling (C). (D-F) Ad-Cyclin D2 in 5mM glucose increased BrdU (D), gH2AX (E), and co-labeled cells (F). (G-I) Combined treatment with 15mM glucose

of 35 Diabetes

and Ad-Cyclin D2 markedly increased BrdU (G), gH2AX (H) and co-labeled cells (I). Exposure to a different beta cell mitogen, Harmine, also increased BrdU (J), gH2AX (K) and co-labeled cells (L). Note the variable y-axis scale in (A-L). (C, F, I and L) in all cases, the observed fraction of beta cells co-labeled for both BrdU and gH2AX was greater than that predicted if co-labeling occurred due to chance. Ad-cre was used as a control for Ad-cyclin D2. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3. DNA damage does not increase beta cell BrdU incorporation or BrdU-gH2AX co-labeling. Dispersed mouse islet cells were cultured for 72 hours in the indicated conditions, with BrdU added for the final 24 hours. (A-C). Mitomycin C treatment resulted in gH2AX labeling in the majority of beta cells (A), confirming DNA damage. (B) Contrary to the hypothesis that BrdU labeling might spuriously occur during DNA damage repair, mitomycin C treatment decreased, rather than increased, the percent of beta cells labeling for BrdU. (C) Double-labeled cells were not increased under conditions of DNA damage; mitomycin C treatment decreased the percent of beta cells co-labeling with both gH2AX and BrdU. In fact, the observed fraction with mitomycin C treatment was suppressed to the fraction predicted if co-labeling was due to chance. Mitomycin treatment was performed on the same biological samples and at the same time as the experiments in Figure 2; control data are repeated from Figure 2. (D-F) To test the mitomycin result in a different system, UV treatment (only performed on islets from old mice) increased the percent of beta cells labeling with gH2AX (D) but suppressed BrdU incorporation (E) and double-labeled cells (F). In (D-F), biological replicates with triangle labels and circle labels were treated identically but in the triangle samples a lower fraction labeled for gH2AX. The different labels are used to allow identification of the samples with lower DNA-damage across panels D-G. *p<0.05, **p<0.01, ***p<0.001.

Figure 4. gH2AX mostly labels a subset of BrdU-labeled cells. Quantitative Venn diagrams were used to generate a visual representation of the data shown in Figures 2-3. Diagrams represent the total number (sum of all replicates) of insulin (+) cells (white circles) that labeled with BrdU (green), gH2AX (red), and both labels (yellow) under different culture conditions. The total number of insulin (+) cells counted and number of biological replicates for each condition is included for each diagram (italics). (A) In young islet cells, 15mM glucose and Ad-cyclin D2 increased both the BrdU+ and gH2AX fractions. Insulin (+) nuclei labeled for gH2AX were mostly a subset of the BrdU-labeled nuclei, in both basal and stimulated conditions. (B) Beta cells from older mice behaved similarly to young beta cells, except that a higher fraction of BrdU (+) cells were also gH2AX(+)under 15mM glucose stimulation both with and without Ad-cyclin D2, and older beta cells required both glucose and Ad-cyclin D2 to meaningfully increase the fraction of BrdU-labeled cells. (C) Venn depiction of the data in Figure 3 demonstrates visually that DNA damage exposure did not increase, and in fact decreased, the frequency of BrdU (+) and co-labeled nuclei. (D-G): Confocal microscopy of cultures (15mM glucose) showed many examples of smoothly labeled BrdU (+) nuclei that also had gH2AX puncta (D-E) and some examples of nuclei with punctate labeling of both BrdU and gH2AX (F). Active mitoses generally had no gH2AX label (G).

Figure 5. BrdU-induced DNA damage does not explain most beta cell gH2AX labeling. Dispersed mouse islet cells were cultured with and without BrdU added to the culture medium, with 15mM glucose (A-C), Ad-cyclin D2 (D) or Ad-cyclin D2 +15mM glucose (E). (A) In 15mM glucose, the presence of BrdU caused a subtle increase in gH2AX labeling in young islets. (B-C) Shifting the timing of the BrdU exposure earlier in the culture to the first 24 hours of glucose exposure with BrdU washed out after 24 hours (exposure X), or for the entire 72 hours (exposure Z) did not increase the proportion of cells showing evidence of DNA damage compared with the standard exposure during the final 24 hours of the 72-hour culture (exposure Y). The time-course of exposures is diagrammed in (B), and the gH2AX quantification is shown in (C). Experiments in A-C were performed on the same biological samples; the Y data in (C) are the same as the BrdU+ data in (A). (D) With Ad-cyclin D2 stimulation, BrdU exposure (final 24 hours, similar to the rest of the experiments throughout this study) increased gH2AX labeling in 5mM glucose (B) but not in 15mM glucose (C). For (D-E), the BrdU labeling fraction in these experiments is shown for context, since these cultures had higher levels of stimulated proliferation than the experiments shown in Figure 2. (F-H): dispersed mouse islet cells cultured without BrdU in 15mM glucose with control or Ad-

of 35Diabetes

cyclin D2 had substantial co-labeling of gH2AX and pHH3, suggesting gH2AX is associated with cycling beta cells rather than BrdU incorporation itself. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6. Human beta cells did not increase DNA damage labeling after proliferative stimulation. Dispersed human islet cells were cultured for 96 hours in the described conditions, with BrdU included in the culture media for the entire 96 hours. (A-C) Human insulin (+) cells cultured in 15mM glucose showed a trend towards modestly increased BrdU incorporation (A) but did not increase gH2AX labeling (B) or BrdU-gH2AX double labeling (C). (D-F) Adenoviral overexpression of human cyclin D2 in 5mM glucose increased BrdU labeling fraction (D) but not the gH2AX-labeling fraction (E); double-labeled cells trended upwards (F). (G-I) Combining glucose and cyclin D2 proliferative stimuli did not further increase BrdU (G), gH2AX (H) or double-labeled nuclei (I). Adeno-cre was used as a control for Adeno-cyclin D2, at the same MOI. *p<0.05.

Figure 7. DNA damage does not cause spurious BrdU incorporation in human beta cells. Dispersed human islet cells were cultured for 96 hours in the described conditions, with BrdU included in the culture media for the entire 96 hours. (A-C): Human insulin (+) cells exposed to mitomycin C were nearly all labeled for gH2AX (A) but had suppressed BrdU labeling fraction (B) and no excess double-labeled cells beyond the fraction predicted by random chance (C). (D-F): Human beta cells exposed to UV irradiation showed variable induction of DNA damage label gH2AX (D), suppression of BrdU labeling (E) and no excess double-labeled cells (F). Note the scale differences in y-axes from left to right. *p<0.05, **p<0.01, ****p<0.0001.

Figure 8. In human beta cells, proliferation was infrequent even under stimulated conditions, and gH2AX labeling was mostly independent of BrdU-labeled cells. The data in Figures 6-7 are shown using quantitative Venn diagrams to illustrate the relationship between gH2AX-labeling and BrdU-labeling in these cultures. Diagrams represent the total number (sum of all replicates) of insulin (+) cells (white) that labeled with BrdU (green), gH2AX (red), and both labels (yellow) under different culture conditions. The total number of insulin (+) cells counted, summed for all biological replicates (n=3-5) is included for each diagram (italics). (A): Although BrdU labeling increased somewhat in proliferative conditions, the gH2AX-labeling index did not, and most gH2AX-labeled cells were not BrdU-labeled. (B): Mitomycin C and UV irradiation caused DNA damage in the majority of beta cells, but did not increase, in fact decreased, the BrdU-labeling fraction.

of 35 Diabetes

REFERENCES

1. Georgia S, Bhushan A. Beta cell replication is the primary mechanism for maintaining postnatal beta cell mass. J Clin Invest (2004) 114:963–968. doi:10.1172/JCI22098

2. Meier JJ, Butler AE, Saisho Y, Monchamp T, Galasso R, Bhushan A, Rizza RA, Butler PC. Beta-cell replication is the primary mechanism subserving the postnatal expansion of beta-cell mass in humans. Diabetes (2008) 57:1584–1594. doi:10.2337/db07-1369

3. Dor Y, Brown J, Martinez OI, Melton DA. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature (2004) 429:41–46. doi:10.1038/nature02520

4. Salic A, Mitchison TJ. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A (2008) 105:2415–2420. doi:10.1073/pnas.0712168105

5. Rieck S, Zhang J, Li Z, Liu C, Naji A, Takane KK, Fiaschi-Taesch NM, Stewart AF, Kushner JA, Kaestner KH. Overexpression of hepatocyte nuclear factor-4α initiates cell cycle entry, but is not sufficient to promote β-cell expansion in human islets. Mol Endocrinol Baltim Md (2012) 26:1590–1602. doi:10.1210/me.2012-1019

6. Lee S-H, Hao E, Levine F, Itkin-Ansari P. Id3 upregulates BrdU incorporation associated with a DNA damage response, not replication, in human pancreatic β-cells. Islets (2011) 3:358–366. doi:10.4161/isl.3.6.17923

7. Masterson JC, O’Dea S. 5-Bromo-2-deoxyuridine activates DNA damage signalling responses and induces a senescence-like phenotype in p16-null lung cancer cells. Anticancer Drugs (2007) 18:1053–1068. doi:10.1097/CAD.0b013e32825209f6

8. Kohlmeier F, Maya-Mendoza A, Jackson DA. EdU induces DNA damage response and cell death in mESC in culture. Chromosome Res Int J Mol Supramol Evol Asp Chromosome Biol (2013) 21:87–100. doi:10.1007/s10577-013-9340-5

9. Anda S, Boye E, Grallert B. Cell-Cycle Analyses Using Thymidine Analogues in Fission Yeast. PLOS ONE (2014) 9:e88629. doi:10.1371/journal.pone.0088629

10. Alonso LC, Yokoe T, Zhang P, Scott DK, Kim SK, O’Donnell CP, Garcia-Ocaña A. Glucose infusion in mice: a new model to induce beta-cell replication. Diabetes (2007) 56:1792–1801. doi:10.2337/db06-1513

11. Stamateris RE, Sharma RB, Hollern DA, Alonso LC. Adaptive β-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression. Am J Physiol - Endocrinol Metab (2013) 305:E149–E159. doi:10.1152/ajpendo.00040.2013

12. Stamateris RE, Sharma RB, Kong Y, Ebrahimpour P, Panday D, Ranganath P, Zou B, Levitt H, Parambil NA, O’Donnell CP, et al. Glucose Induces Mouse β-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor. Diabetes (2016) 65:981–995. doi:10.2337/db15-0529

13. Kong Y, Ebrahimpour P, Liu Y, Yang C, Alonso LC. Pancreatic Islet Embedding for Paraffin Sections. J Vis Exp JoVE (2018) doi:10.3791/57931

of 35Diabetes

14. Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH, Lindquist RA, Moffat J, et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol (2006) 7:R100. doi:10.1186/gb-2006-7-10-r100

15. Lamprecht MR, Sabatini DM, Carpenter AE. CellProfiler: free, versatile software for automated biological image analysis. BioTechniques (2007) 42:71–75.

16. Micallef L, Rodgers P. eulerAPE: drawing area-proportional 3-Venn diagrams using ellipses. PloS One (2014) 9:e101717. doi:10.1371/journal.pone.0101717

17. Sharma RB, O’Donnell AC, Stamateris RE, Ha B, McCloskey KM, Reynolds PR, Arvan P, Alonso LC. Insulin demand regulates β cell number via the unfolded protein response. J Clin Invest (2015) 125:3831–3846. doi:10.1172/JCI79264

18. Wang P, Alvarez-Perez J-C, Felsenfeld DP, Liu H, Sivendran S, Bender A, Kumar A, Sanchez R, Scott DK, Garcia-Ocaña A, et al. A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication. Nat Med (2015) 21:383–388. doi:10.1038/nm.3820

19. Dusre L, Covey JM, Collins C, Sinha BK. DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells. Chem Biol Interact (1989) 71:63–78.

20. Mladenov E, Tsaneva I, Anachkova B. Activation of the S phase DNA damage checkpoint by mitomycin C. J Cell Physiol (2007) 211:468–476. doi:10.1002/jcp.20957

21. Lee Y-J, Park S-J, Ciccone SLM, Kim C-R, Lee S-H. An in vivo analysis of MMC-induced DNA damage and its repair. Carcinogenesis (2006) 27:446–453. doi:10.1093/carcin/bgi254

22. Hanasoge S, Ljungman M. H2AX phosphorylation after UV irradiation is triggered by DNA repair intermediates and is mediated by the ATR kinase. Carcinogenesis (2007) 28:2298–2304. doi:10.1093/carcin/bgm157

23. Marti TM, Hefner E, Feeney L, Natale V, Cleaver JE. H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks. Proc Natl Acad Sci (2006) 103:9891–9896. doi:10.1073/pnas.0603779103

24. Oh K-S, Bustin M, Mazur SJ, Appella E, Kraemer KH. UV-induced histone H2AX phosphorylation and DNA damage related proteins accumulate and persist in nucleotide excision repair-deficient XP-B cells. DNA Repair (2011) 10:5–15. doi:10.1016/j.dnarep.2010.09.004

25. Levitt HE, Cyphert TJ, Pascoe JL, Hollern DA, Abraham N, Lundell RJ, Rosa T, Romano LC, Zou B, O’Donnell CP, et al. Glucose stimulates human beta cell replication in vivo in islets transplanted into NOD-severe combined immunodeficiency (SCID) mice. Diabetologia (2011) 54:572–582. doi:10.1007/s00125-010-1919-1

26. Gagou ME, Zuazua-Villar P, Meuth M. Enhanced H2AX Phosphorylation, DNA Replication Fork Arrest, and Cell Death in the Absence of Chk1. Mol Biol Cell (2010) 21:739–752. doi:10.1091/mbc.E09-07-0618

27. Ye X, Franco AA, Santos H, Nelson DM, Kaufman PD, Adams PD. Defective S phase chromatin assembly causes DNA damage, activation of the S phase checkpoint, and S phase arrest. Mol Cell (2003) 11:341–351.

of 35 Diabetes

28. Colin DJ, Hain KO, Allan LA, Clarke PR. Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins. Open Biol (2015) 5:140156. doi:10.1098/rsob.140156

29. Szüts D, Krude T. Cell cycle arrest at the initiation step of human chromosomal DNA replication causes DNA damage. J Cell Sci (2004) 117:4897–4908. doi:10.1242/jcs.01374

30. Huang X, Halicka HD, Traganos F, Tanaka T, Kurose A, Darzynkiewicz Z. Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis. Cell Prolif (2005) 38:223–243. doi:10.1111/j.1365-2184.2005.00344.x

31. MacPhail SH, Banáth JP, Yu Y, Chu E, Olive PL. Cell cycle-dependent expression of phosphorylated histone H2AX: reduced expression in unirradiated but not X-irradiated G1-phase cells. Radiat Res (2003) 159:759–767.

32. van Wietmarschen N, Lansdorp PM. Bromodeoxyuridine does not contribute to sister chromatid exchange events in normal or Bloom syndrome cells. Nucleic Acids Res (2016) 44:6787–6793. doi:10.1093/nar/gkw422

33. Tsuda M, Terada K, Ooka M, Kobayashi K, Sasanuma H, Fujisawa R, Tsurimoto T, Yamamoto J, Iwai S, Kadoda K, et al. The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C). Oncotarget (2017) 8:33457–33474. doi:10.18632/oncotarget.16508

34. Pascoe J, Hollern D, Stamateris R, Abbasi M, Romano LC, Zou B, O’Donnell CP, Garcia-Ocana A, Alonso LC. Free fatty acids block glucose-induced β-cell proliferation in mice by inducing cell cycle inhibitors p16 and p18. Diabetes (2012) 61:632–641. doi:10.2337/db11-0991

35. Fiaschi-Taesch NM, Kleinberger JW, Salim FG, Troxell R, Wills R, Tanwir M, Casinelli G, Cox AE, Takane KK, Srinivas H, et al. Cytoplasmic-Nuclear Trafficking of G1/S Cell Cycle Molecules and Adult Human β-Cell Replication A Revised Model of Human β-Cell G1/S Control. Diabetes (2013) 62:2460–2470. doi:10.2337/db12-0778

36. Tiwari S, Roel C, Wills R, Casinelli G, Tanwir M, Takane KK, Fiaschi-Taesch NM. Early and Late G1/S Cyclins and Cdks Act Complementarily to Enhance Authentic Human Beta-cell Proliferation and Expansion. Diabetes (2015)db141885. doi:10.2337/db14-1885

37. Wang P, Karakose E, Liu H, Swartz E, Ackeifi C, Zlatanic V, Wilson J, González BJ, Bender A, Takane KK, et al. Combined Inhibition of DYRK1A, SMAD, and Trithorax Pathways Synergizes to Induce Robust Replication in Adult Human Beta Cells. Cell Metab (2018) doi:10.1016/j.cmet.2018.12.005

38. Pechhold K, Koczwara K, Zhu X, Harrison VS, Walker G, Lee J, Harlan DM. Blood Glucose Levels Regulate Pancreatic β-Cell Proliferation during Experimentally-Induced and Spontaneous Autoimmune Diabetes in Mice. PLoS ONE (2009) 4: doi:10.1371/journal.pone.0004827

of 35Diabetes

B C

0

1

2

3

4

5

% b

eta

cells

labe

ling

ns

Brd

U

gH2A

X

012345

% b

eta

cells

with

bot

h Br

dU a

nd g

H2A

X la

bels *

Pre

dict

ed

Obs

erve

d

BrdU + gH2AX BrdU only gH2AX only

Insulin BrdU Dapi gH2AX Dapi

BrdU gH2AXDapi

BrdU InsulinDapi

A

D D’

A’

of 35 Diabetes

A B C

D E F

G H I

**** * * **** ******** ****

****

5 15mM glucYoung

5 15Old

5 15Young

5 15Old

5 15Predict

5 15Obs

Young

5 15Predict

5 15Obs

Old

5 5mM glucAdeno cre D2 cre D2

Young

5 5

Old

5 5cre D2 cre D2

Young

5 5

Old

cre D2 cre D2 cre D2 cre D25 5 5 5 5 5 5 5

Predict ObsYoung

Predict ObsOld

15 15mM glucAdeno cre D2 cre D2

Young

15 15

Old

cre D2 cre D2

Young Old

15 15 15 15cre D2 cre D2 cre D2 cre D2

Predict ObsYoung

Predict ObsOld

15 15 15 15 15 15 15 15

** ** * * ** * ns**** 0.05

**** *** *** *******

** ****

0

5

10

15%

BrdU

+ be

ta c

ells

0

5

10

15

%gH

2AX+

bet

a ce

lls

0

1

2

3

4

5

% b

eta

cells

with

bot

h Br

dU a

nd g

H2A

X la

bels

0

5

10

15

%Br

dU+

beta

cel

ls

0

5

10

15

%gH

2AX+

bet

a ce

lls

0

2

4

6

8

10

% b

eta

cells

with

bot

h Br

dU a

nd g

H2A

X la

bels

0

10

20

30

40

50

%Br

dU+

beta

cel

ls

0

10

20

30

40

50

%gH

2AX+

bet

a ce

lls

0

10

20

30

40

50

% b

eta

cells

with

bot

h Br

dU a

nd g

H2A

X la

bels

J K L

veh HAR veh HAR5mM 15mM

veh HAR veh HAR5mM 15mM Predict Obs

5mMPredict Obs

15mM

ns ns ****** ** *****

***

0

10

20

30

%Br

dU+

beta

cel

ls

0

10

20

30

%gH

2AX+

bet

a ce

lls

0

10

20

30

% b

eta

cells

with

bot

h Br

dU a

nd g

H2A

X la

bels

veh HAR veh HAR veh HAR veh HAR

of 35Diabetes

15 15mM glucctrl Mito ctrl Mito

Young

15 15

Old

15 15ctrl Mito ctrl Mito

Young

15 15

Old

A B C

D E F

*** *** ** nsns ns

ns*** ******

**ns

*** *

ctrl

Predict Obs

Young

Predict Obs

Old

15 15 15 15 15 15 15 15ctrl Mito ctrl Mitoctrl MitoMito

0246

20

60

100

%gH

2AX+

bet

a ce

lls

0246

20

60

100

%Br

dU+

beta

cel

ls

ctrl UV15 15

Old

ctrl UV15 15

Old Predict Obs

Old

15 15 15 15ctrl UV ctrl UV

0

10

20406080

100

%gH

2AX+

bet

a ce

lls

0

10

20406080

100

%Br

dU+

beta

cel

ls

0

1

2

3

4

5

% b

eta

cells

with

bot

h Br

dU a

nd g

H2A

X la

bels

0

1

2

3

4

5%

bet

a ce

lls w

ith b

oth

BrdU

and

gH

2AX

labe

ls

of 35 Diabetes

Young

10141 cells; (n=4, 2535±278)

Old 11009 cells(n=4, 2752±642)

11566 cells(n=4, 2892±342)

8937 cells(n=4, 2234±245)

10341 cells(n=4, 2585±625)

5mM glu 15mM glu 5mM + AdcycD2 15mM + AdcycD2

Insulin(+) cellsBrdU(+) nuclei

BrdU(+) gH2AX(+) nucleigH2AX(+) nuclei

A

C

B

Mitomycin UV

D E

F G

BrdU gH2AX Dapi Insulin Dapi BrdU gH2AX Dapi Insulin Dapi

9134 cells(n=5, 1827±134)

18473 cells(n=10, 1847±290)

9589 cells(n=5, 1918±367)

12306 cells(n=5, 2461±434)


10192 cells; (n=4, 2548±400)

of 35Diabetes

D

A B C

E

* ns

15 15+BrdU: - + -

Young

15 15

Old

0

1

2

3

4

5%

gH2A

X+ b

eta

cells

** **** **

5 5

+BrdU: - + -+D2:

Gluc:+ + +

Young

5 5

Old

5 5

+ - + -+ + + +

Young

5 5

Old

0

20

40

60

%Br

dU+

beta

cel

ls

0

20

40

60

%gH

2AX+

bet

a ce

lls

*** *** nsns

15 15

+BrdU: - + -+D2:

Gluc:+ + +

Young

15 15

Old

15 15

+ - + -+ + + +

Young

15 15

Old

0

20

40

60

%Br

dU+

beta

cel

ls0

20

40

60

%gH

2AX+

bet

a ce

lls

15mMglucose

BrdU: X1st 24h

only

BrdU: Z (all 72 hours)

BrdU: Ylast 24h

only

0h 48h24h 72h

Fix

15 15XBrdU: Y

15Z

15 15X Y

15Z

Young Old

nsns

0

2

4

6

8

10

%gH

2AX+

bet

a ce

lls

F HG***

0

5

10

15

%pH

H3+

bet

a ce

lls

lacZ D215 15

*

0

5

10

15

%gH

2AX+

bet

a ce

lls

lacZ D215 15

*******

***

0

1

2

3

4

5

% b

eta

cells

with

bot

h pH

H3

and

gH2A

X la

bels

pred obs15 15

pred obs15 15

lacZ D2

8366 cells(n=4, 2092±214)

8244 cells(n=4, 2061±302)

15mM + AdlacZ 15mM + AdcycD2

Insulin(+)pHH3(+) pHH3(+) gH2AX(+)

gH2AX(+)

Young Young

of 35 Diabetes

0

1

2

3

4

5

%Br

dU+

beta

cel

ls

5 15mM gluc

p=0.09

0

1

2

3

4

5

%gH

2AX+

bet

a ce

lls

5 15

ns

0

1

2

3

4

5

%gH

2AX+

bet

a ce

lls

5 5cre D2

ns

0

1

2

3

4

5

%Br

dU+

beta

cel

ls

5 5mM glucAdeno cre D2

*

0.0

0.5

1.0

1.5

2.0

% b

eta

cells

with

bot

h Br

dU a

nd g

H2A

X la

bels

5 15Predict

5 15Obs

ns nsns

cre D2 cre D2

Predict Obs

15 15 15 150.0

0.5

1.0

1.5

2.0

% b

eta

cells

with

bot

h Br

dU a

nd g

H2A

X la

bels

ns nsns

cre D2 cre D25 5 5 5

Predict Obs

0.0

0.5

1.0

1.5

2.0

% b

eta

cells

with

bot

h Br

dU a

nd g

H2A

X la

bels

ns 0.090.06

0

1

2

3

4

5

%Br

dU+

beta

cel

ls

15 15mM glucAdeno cre D2

ns

0

1

2

3

4

5

%gH

2AX+

bet

a ce

lls

15 15cre D2

ns

A B C

D E F

(A-C) Human islet cells: e�ect of high (15mM) glucose

G H I

(D-F) Human islet cells: e�ect of Ad-cyclin D2 in standard (5mM) glucose

(G-I) Human islet cells: e�ect of Ad-cyclin D2 in high (15mM) glucose

of 35Diabetes

15 15ctrl Mito

0

1

2

3

4

5

%Br

dU+

beta

cel

ls

*

15 15ctrl Mito

0

20

40

60

80

100

%gH

2AX+

bet

a ce

lls

****

ctrl

Predict Obs

15 15 15 15ctrl MitoMito

0.07 0.09ns

0.0

0.5

1.0

1.5

2.0

% b

eta

cells

with

bot

h Br

dU a

nd g

H2A

X la

bels

Predict Obs

15 15 15 15ctrl UV ctrl UV

ns nsns

0.0

0.5

1.0

1.5

2.0

% b

eta

cells

with

bot

h Br

dU a

nd g

H2A

X la

bels

ctrl UV15 15

0

20

40

60

80

100

%gH

2AX+

bet

a ce

lls

**

ctrl UV15 15

0

1

2

3

4

5

%Br

dU+

beta

cel

ls

0.06

A B C

D E F

of 35 Diabetes

4729 cells(n=4, 1182±301)

3817 cells(n=5, 763±162)

3207 cells(n=5, 641±303)

2971 cells(n=3, 990±247)

3571cells(n=3, 1190±196)

4271 cells(n=3, 1424±261)


Insulin(+) cellsBrdU(+) nuclei

BrdU(+) gH2AX(+) nucleigH2AX(+) nuclei

A

B

Mitomycin UV

of 35Diabetes

Donor number

Diagnosed diabetes Sex Age

(years) BMI Ethnicity Cause of Death Experiments performed

1 No F 45 26.6 White Cerebrovascular/stroke Fig 6 A-C

2 No F 56 33.4Black or African

AmericanCerebrovascular/stroke Fig 6 A-C

3 No M 55 28.5 White Anoxia Fig 6 A-I, Fig 7 A-F

4 No F 51 22.5 Asian Cerebrovascular/stroke Fig 6 A-I, Fig 7 A-F

5 No M 54 21.7 White Anoxia Fig 6 A-I, Fig 7 A-F

Supplemental Table 1. Characteristics of human islet donors used in these studies

of 35 Diabetes

Figure panels

Type of

cellsExperimental

conditions Outcomes number

of biological replicates

Total number of cells counted

Number of cells

counted (mean +/-

SE)

1B-1C mouse 5mM gluBrdU, gH2AX,

predicted, observed

6 14295 2382 +/- 471

2A-2C mouse 5mM, youngBrdU, gH2AX,

predicted, observed

5 9134 1827 +/- 134

2A-2C mouse 15mM, youngBrdU, gH2AX,

predicted, observed

10 18473 1847 +/- 290

2A-2C mouse 5mM, oldBrdU, gH2AX,

predicted, observed

4 11009 2752 +/- 642

2A-2C mouse 15mM, oldBrdU, gH2AX,

predicted, observed

4 11566 2892 +/- 342

2D-2F mouse 5mM, cre, young

BrdU, gH2AX, predicted, observed

5 9203 1841 +/- 181

2D-2F mouse 5mM, D2, young


5 9589 1918 +/- 367

2D-2F mouse 5mM, cre, oldBrdU, gH2AX,

predicted, observed

4 8928 2232 +/- 583

2D-2F mouse 5mM, D2, oldBrdU, gH2AX,

predicted, observed

4 8937 2234 +/- 245

2G-2I mouse 15mM, cre, young


5 9653 1931 +/- 302

2G-2I mouse 15mM, D2, young


5 12306 2461 +/- 434

2G-2I mouse 15mM, cre, old


4 10947 2737 +/- 360

2G-2I mouse 15mM, D2, old


4 10341 2585 +/- 625

2J-2L mouse 5mM, vehBrdU, gH2AX,

predicted, observed

3 5347 1782 +/- 280

2J-2L mouse 5mM, harmine


3 3626 1209 +/- 193

of 35Diabetes

2J-2L mouse 15mM, vehBrdU, gH2AX,

predicted, observed

3 4696 1565 +/- 19

2J-2L mouse 15mM, harmine


3 3737 1246 +/- 105

3A-3C mouse 15mM, ctrl, young


7 9978 1425 +/- 233

3A-3C mouse 15mM, mito, young


6 4239 707 +/- 378

3A-3C mouse 15mM, ctrl, old


4 11566 2892 +/- 342

3A-3C mouse 15mM, mito, old


4 10192 2548 +/- 400

3D-3F mouse 15mM, ctrl, old


4 11566 2892 +/- 342

3D-3F mouse 15mM, UV, old


4 10141 2535 +/- 278

5A mouse 15mM, +BrdU, young gH2AX 7 9978 1425 +/-

233

5A mouse 15mM, no BrdU, young gH2AX 4 8606 2152 +/-

595

5A mouse 15mM, +BrdU, old gH2AX 4 11566 2892 +/-

342

5A mouse 15mM, no BrdU, old gH2AX 4 14538 3635 +/-

926

5C mouse 15mM, X, young gH2AX 5 7570 1514 +/-

365

5C mouse 15mM, Y, young gH2AX 7 9978 1425 +/-

233

5C mouse 15mM, Z, young gH2AX 5 7515 1503 +/-

371

5C mouse 15mM, X, old gH2AX 4 11203 2801 +/- 355

5C mouse 15mM, Y, old gH2AX 4 11566 2892 +/- 342

5C mouse 15mM, Z, old gH2AX 4 10111 2528 +/- 365

5D mouse 5mM, D2, + BrdU, young BrdU, gH2AX 3 5423 1808 +/-

286

5D mouse 5mM, D2, no BrdU, young BrdU, gH2AX 3 3663 1221 +/-

208

of 35 Diabetes

5D mouse 5mM, D2, + BrdU, old BrdU, gH2AX 3 12684 4228 +/-

196

5D mouse 5mM, D2, no BrdU, old BrdU, gH2AX 3 10600 3533 +/-

252

5E mouse 15mM, D2, + BrdU, young BrdU, gH2AX 3 3700 1233 +/-

202

5E mouse 15mM, D2, no BrdU, young BrdU, gH2AX 3 5762 1921 +/-

228

5E mouse 15mM, D2, + BrdU, old BrdU, gH2AX 3 8296 2765 +/-

102

5E mouse 15mM, D2, no BrdU, old BrdU, gH2AX 3 8291 2764 +/-

279

5F-5G mouse 15mM, lacZ, young pHH3 4 8366 2092 +/-

214

5F-5G mouse 15mM, D2, young pHH3 4 8244 2061 +/-

302

6A-6C human 5mMBrdU, gH2AX,

predicted, observed

4 4729 1182 +/- 301

6A-6C human 15mMBrdU, gH2AX,

predicted, observed

5 3817 763 +/- 162

6D-6F human 5mM, creBrdU, gH2AX,

predicted, observed

3 2651 884 +/- 227

6D-6F human 5mM, D2BrdU, gH2AX,

predicted, observed

3 3571 1190 +/- 196

6G-6I human 15mM, creBrdU, gH2AX,

predicted, observed

3 2615 872 +/- 208

6G-6I human 15mM, D2BrdU, gH2AX,

predicted, observed

3 4271 1424 +/- 261

7A-7C human 15mM, ctrlBrdU, gH2AX,

predicted, observed

5 3817 763 +/- 162

7A-7C human 15mM, mitoBrdU, gH2AX,

predicted, observed

5 3207 641 +/- 303

7D-7F human 15mM, ctrlBrdU, gH2AX,

predicted, observed

5 3817 763 +/- 162

7D-7F human 15mM, UV BrdU, gH2AX,

predicted, observed

3 2971 990 +/- 247

Supplemental Table 2. Number of cells counted for each experiment

of 35Diabetes

Explanation Possible mechanism if true Implications if true How to test

DNA damage causes BrdU incorporation

DNA damage repair results in incorporation of BrdU

nucleotide

BrdU incorporation does not reflect cell cycle

entry in cells with DNA damage.

Induce DNA damage, test for increased frequency of BrdU labeling. Observe

pattern of BrdU label.

Brdu exposure causes DNA

damage

Incorporation of the BrdU nucleotide leads to a DNA

damage response

BrdU incorporation reflects cell cycle entry but incurs toxicity. BrdU toxicity may impact cell

cycle completion.

Test for increased DNA damage in the presence of BrdU compared with

DNA damage in the absence of BrdU

An upstream process causes

both DNA damage and BrdU

incorporation

Many possible mechanisms. Most likely is that proliferative

signals, DNA replication or other cell cycle related

process also induces DNA damage

BrdU incorporation reflects cell cycle entry. Cells may not complete

the cell cycle. Proliferation is a

dangerous process for beta cells.

Test whether proliferative conditions increase the fraction of gH2AX-labeled cells.

DNA damage and BrdU incorporation are unrelated but occasionally co-

occur stochastically

Cells occasionally co-label with BrdU and gH2AX by

random chance

BrdU incorporation reflects cell cycle entry.

Test whether the observed BrdU-gH2AX co-labeling frequency matches the predicted frequency based on

prevalence of the individual labels

Supplemental Table 3. Putative explanations for beta cell co-labeling with BrdU and DNA damage marker gH2AX, implications, and an approach to testing each hypothesis.

of 35 Diabetes

Supplemental Figure 1. Many BrdU+ beta cell nuclei co-label with pHH3, suggesting progression of BrdU-labeled cells to the mitotic phase of the cell cycle. Mouse islet cells were dispersed and cultured in 15mM glucose for 72 hours, with BrdU present for the �nal 24 hours, then �xed and stained for insulin, BrdU, pHH3 and dapi. Many BrdU-labeled beta cell nuclei co-stain for pHH3 (arrows). Given the long duration of BrdU exposure, some of the BrdU(+) pHH3(-) cells may have progressed through the cell cycle entirely; note the occasional BrdU doublets, which are negative for pHH3.

pHH3 pHH3DapiInsulin DapiInsulinBrdU DapiBrdU

A

of 35Diabetes

A

C

B

Supplemental Figure 2. In vivo HFD-stimulated beta cell proliferative expansion leads to very few gH2AX-labeled beta cell nuclei. Pancreas sections from control diet (A) or high fat diet (HFD; B-C) fed mice were stained for insulin, BrdU, gH2AX and dapi. All gH2AX-stained islet nuclei identi�ed in this experiment are shown above; the vast majority of islets imaged contained no detectable gH2AX-labeled cells. Although many insulin+ cells labeling with BrdU were found (white lines), only four gH2AX+ nuclei (white arrows) were found in the 8 sections. The gH2AX+ nucleus in (A) appears to be a non-insulin-positive cell, whereas the gH2AX+ nuclei in (B-C) appear to belong to insulin+ cells. The gH2AX+ nuclei in (B) are also BrdU+, but those in (A) and (C) are BrdU-negative.

gH2AX gH2AXDapi Dapi Dapi

Insulin InsulinBrdU BrdU

of 35 Diabetes

A

C

D

B

Supplemental Figure 3. In vivo hyperglycemia-stimulated beta cell proliferative expansion leads to very few gH2AX-labeled beta cell nuclei. Pancreas sections from mice rendered continuously hyperglyce-mic for 4 days by intravenous glucose infusion were stained for insulin, BrdU, gH2AX and dapi. As with the HFD experiment shown in Suppl. Fig 2, all gH2AX-stained islet nuclei identi�ed in this experiment are shown above. Again, the vast majority of islets imaged contained no detectable gH2AX-labeled cells. Although many insulin+ cells labeling with BrdU were found (white lines), only four gH2AX+ nuclei (white arrows) were found in pancreas sections from four mice. The gH2AX+ nucleus in (D) appears to be a non-insulin-positive cell, whereas the gH2AX+ nuclei in (A-C) appear to belong to insulin+ cells. In this hyperglycemia experiment, none of the gH2AX+ nuclei were also BrdU+.

gH2AXDapi

InsulinDapi

InsulinBrdU gH2AXDapi

BrdU

of 35Diabetes

A

C DB

5mM

glu

cose

15m

M g

luco

se15

mM

glu

cose

+ M

itom

ycin

C

Supplemental Figure 4. Ex vivo cultured intact islets labeled infrequently for gH2AX unless exposed to DNA damaging agent Mitomycin C, and very rarely labeled for both BrdU and gH2AX. Whole mouse islets were cultured for 72 hours in islet medium containing 5mM glucose, 15mM glucose or 15mM glucose with Mitomycin C, with BrdU added for the �nal 24 hours of culture. The islets were then �xed, embedded in para�n, sectioned and stained for insulin, BrdU, gH2AX and dapi (A). Some insulin+ cells labeled with BrdU (white lines, B) or gH2AX (white arrows, C) but very few of the gH2AX+ nuclei were also BrdU+ (D).

gH2AXDapi

InsulinDapi

InsulinBrdU gH2AXDapi

BrdU

024

204060

%gH

2AX+

bet

a ce

lls

0

1

2

3

4

5

%Br

dU+

beta

cel

ls

5 15veh veh

15Mito

5 15veh veh

15Mito

5 15veh

pred ob

s

pred ob

s

pred ob

s

veh15

Mito

ns ns ns ns ns* *

0

1

2

3

4

5

% b

eta

cells

with

bot

h Br

dU a

nd g

H2A

X la

bels

of 35 Diabetes

dna damage does not cause brdu labeling of mouse or human ... · dna damage does not cause brdu...

Documents