![Effect of race on basal and damage susceptibility of BLM-induced double-strand break in NHB and NHW breast cancer survivors. Basal (A) and induction/susceptibility (B) of BLM-induced double-strand breaks were measured by neutral assay. Differences in DNA damage (Mean ± SD) between NHB and NHW (A,B) were assessed using unpaired, two-tail Mann-Whitney test. The red lines denote the mean value. DNA damage induction in (panel B) was calculated using the formula: [DNA damage (moment) after BLM treatment − basal DNA damage (Moment) before BLM treatment]/basal DNA damage (moment) before BLM treatment.](https://www.researchgate.net/publication/380480181/figure/fig4/AS:11431281247066267@1716572145186/Effect-of-race-on-basal-and-damage-susceptibility-of-BLM-induced-double-strand-break-in_Q320.jpg)
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Citation: Divekar, S.; Kritzer, R.; Shu,
H.; Thakkar, K.; Hicks, J.; Mills, M.G.;
Makambi, K.; Dash, C.; Roy, R.
Systemic DNA Damage and Repair
Activity Vary by Race in Breast Cancer
Survivors. Cancers 2024,16, 1807.
https://doi.org/10.3390/
cancers16101807
Academic Editor: Kekoa Taparra
Received: 7 April 2024
Revised: 30 April 2024
Accepted: 3 May 2024
Published: 9 May 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
cancers
Article
Systemic DNA Damage and Repair Activity Vary by Race in
Breast Cancer Survivors
Shraddha Divekar, Ryan Kritzer, Haokai Shu, Keval Thakkar , Jennifer Hicks, Mary G. Mills, Kepher Makambi,
Chiranjeev Dash * and Rabindra Roy *
Georgetown University’s Lombardi Comprehensive Cancer Center, Georgetown University Medical Center,
Washington, DC 20057, USA; sd1292@georgetown.edu (S.D.); rykritzer@gmail.com (R.K.);
hs1052@georgetown.edu (H.S.); kevalvthakkar@gmail.com (K.T.); js936@georgetown.edu (J.H.);
mg266@georgetown.edu (M.G.M.); khm33@georgetown.edu (K.M.)
*Correspondence: cd422@georgetown.edu (C.D.); rr228@georgetown.edu (R.R.); Tel.: +1-202-687-0100 (C.D.);
+1-202-687-7390 (R.R.)
Simple Summary: Non-Hispanic Black breast cancer survivors have poorer outcomes than White
survivors, but the biological mechanisms underlying these disparities are unclear. We discovered
novel race-based differences in systemic DNA damage and repair activity among breast cancer
survivors. This finding suggests DNA damage and repair are important basic science mechanisms in
cancer disparities.
Abstract: Non-Hispanic Black breast cancer survivors have poorer outcomes and higher mortality
rates than White survivors, but systemic biological mechanisms underlying these disparities are
unclear. We used circulating leukocytes as a surrogate for measuring systemic mechanisms, which
might be different from processes in the target tissue (e.g., breast). We investigated race-based
differences in DNA damage and repair, using a novel CometChip assay, in circulating leukocytes
from breast cancer survivors who had completed primary cancer therapy and were cancer free.
We observed novel race-based differences in systemic DNA damage and repair activity in cancer
survivors, but not in cells from healthy volunteers. Basal DNA damage in leukocytes was higher in
White survivors, but Black survivors showed a much higher induction after bleomycin treatment.
Double-strand break repair activity was also significantly different between the races, with cells from
White survivors showing more sustained repair activity compared to Black leukocytes. These results
suggest that cancer and cancer therapy might have long-lasting effects on systemic DNA damage and
repair mechanisms that differ in White survivors and Black survivors. Findings from our preliminary
study in non-cancer cells (circulating leukocytes) suggest systemic effects beyond the target site, with
implications for accelerated aging-related cancer survivorship disparities.
Keywords: racial disparity; CometChip assay; double-strand break repair; single-strand break repair
1. Introduction
Genome stability, DNA damage, and DNA repair are associated with cellular aging
and are major hallmarks of breast cancer and other cancers [
1
–
4
]. Accelerated cellular aging
results from adverse social and metabolic risk factors over the life course. It is recognized
as a central mechanism leading to cancer, and adverse outcomes in cancer survivorship [
5
].
Standard cancer treatments, particularly radiation therapy and chemotherapy, often in-
volve agents or strategies that damage the DNA in non-cancerous tissues, resulting in
systemic effects associated with accelerated aging processes [
5
,
6
]. Previous studies on cells
obtained from breast cancer patients have shown a higher oxidative stress level, suscep-
tibility to DNA damage, and a decreased DNA repair capacity, than cells from healthy
controls [
3
,
4
,
7
,
8
]. Several specific repair mechanisms repair single-strand and double-strand
breaks in DNA [
9
]. However, it is unclear whether these changes are also seen systemically,
Cancers 2024,16, 1807. https://doi.org/10.3390/cancers16101807 https://www.mdpi.com/journal/cancers
Cancers 2024,16, 1807 2 of 14
e.g., in circulating leukocytes and other non-cancer cells, as the characterization of these
repair mechanisms at the systemic level during the cancer survivorship period is still not
well defined.
The incidence of breast cancer is slightly higher in non-Hispanic White (NHW) women
compared to non-Hispanic Black (NHB) women, but mortality from breast cancer is 27%
higher in NHB than NHW women [
10
]. There are currently over 4 million breast cancer
survivors in the U.S. who, despite a generally favorable prognosis, face lifelong risks of
clinically important symptoms related to poor quality of life and adverse health effects, such
as obesity, metabolic syndrome, and diabetes [
10
]. There are well-documented disparities
in the prevalence of these symptoms and metabolic comorbidities, with NHB breast cancer
survivors bearing a higher burden than NHW women [
10
]. Systemic mechanistic pathways
related to accelerated cellular aging possibly underlie persistent race/ethnic differences in
cancer mortality, due to disparities in these clinical symptoms and adverse metabolic health
effects, but have been understudied. Recent studies have shown that like NHW women,
NHB women carrying germline pathogenic mutations in DNA damage response (ATM,
CHEK2) and repair genes (BRCA1,BRCA2,PALB2,RAD51D,XPB,FANCC and RECQL)
are at moderate to high risk for breast cancer [
11
]. However, the expression of DNA repair
genes, primarily in single strand break/base excision repair (SSBR/BER) and double strand
break repair (DSBR), differed in breast tumor tissue by race [
12
]. Given the integral role of
DNA damage and repair in accelerated aging, [
13
] and recent findings of race differences
in tumor tissue [
11
,
12
], systemic differences in these mechanisms should be investigated in
NHB and NHW breast cancer survivors.
Alkaline single cell gel electrophoresis (SCGE), also known as Comet Assay, is rou-
tinely used to measure DNA damage and repair activity due to its high sensitivity and
simplicity [
14
–
16
]. However, an issue with the standard comet assay is its reproducibil-
ity [
17
]. CometChip Assay employs the same fundamental concepts as SCGE; but utilizes a
microwell system that traps ~300, non-overlapping, single cells within each well of a 96-well
plate [
17
,
18
]. Thereby, this innovative CometChip assay provides a high-content, highly
sensitive, quantitative, and high-throughput DNA damage assay platform, with high repro-
ducibility. The use of the CometChip assay to measure DNA damage has potential in breast
cancer translational research [
19
,
20
]. The versatile nature of the CometChip assay allows
the detection of specific DNA damage types by modifying the alkaline CometChip assay, to
detect and quantify diverse damage types, such as single-strand breaks, alkali–labile abasic
sites, and double-strand breaks, whereas the neutral CometChip assay predominantly de-
tects double-strand breaks [
21
–
24
]. Double-strand breaks are repaired by non-homologous
end joining/homologous recombination (NHEJ/HR) pathway, whereas the oxidized bases
are repaired by BER pathway, and the bulky adducts, induced by UV-light and polyaro-
matic hydrocarbons, are repaired by nucleotide excision repair (NER) pathway [
22
,
25
–
27
].
Therefore, CometChip assays can be used to detect a variety of damage types, as well as to
identify specific DNA repair mechanisms and pathways in clinical samples.
We used the CometChip assay to measure global double-strand damage and repair
capacity in breast cancer survivors, and participants without a history of cancer (non-cancer
participants). We also compared DNA damage and repair by race among breast cancer
survivors to investigate potential mechanistic pathways for cancer mortality disparities
between NHB and NHW women.
2. Materials and Methods
2.1. Overview
Figure 1describes the study workflow schematic. CometChip assays were performed
on buffy coat cells isolated from peripheral blood from non-Hispanic Black (NHB) and non-
Hispanic White (NHW) cancer-free participants and breast cancer survivors, to measure
DNA damage at a basal level, after bleomycin (BLM) treatment and in the recovery/repair
period.
Cancers 2024,16, 1807 3 of 14
Cancers 2024, 16, x FOR PEER REVIEW 3 of 15
(NHB) and non-Hispanic White (NHW) cancer-free participants and breast cancer survi-
vors, to measure DNA damage at a basal level, after bleomycin (BLM) treatment and in
the recovery/repair period.
Figure 1. Study Schematic. For details, see Materials and Methods. Created with Biorender.com (ac-
cessed on 6 May 2024).
2.2. Patient Recruitment and Sample Collection
Adult non-Hispanic Black (NHB, n = 13) and White (NHW, n = 12) invasive breast
cancer survivors, with no history of breast cancer recurrence or known breast cancer-re-
lated germline mutations, who had completed primary treatment (surgery, chemother-
apy, radiation therapy) at least six months, and at most three years, before study entry
(women currently on hormone therapy were eligible) were recruited through community-
based approaches, and identification and screening using the Survey, Recruitment, and
Biospecimen Collection Shared Resource (SRBSR) at Georgetown University’s Lombardi
Comprehensive Cancer Center. After providing wrien informed consent, all participants
completed a demographic and medical history questionnaire, and anthropometrics meas-
urements to determine weight and height. Notably, the majority of breast cancer patients
undergo testing for germline mutations, particularly focusing on BRCA mutations. We
identified non-BRCA patients from the electronic medical record (EMR) database and in-
cluded them in this study. It is also worth noting that patients who reported non-BRCA
mutations may not have undergone formal genetic testing. Venous blood (20 mL) was
collected into sodium heparin tubes. All data collection activities were conducted at the
Lombardi Cancer Center’s Office of Minority Health and Health Disparities Research. The
Georgetown MedStar Institutional Review Board approved the study protocol
(#STUDY00003904).
All participant blood specimens were transported to Lombardi Cancer Center’s Tis-
sue Culture and Bio-banking Shared Resource (TCBSR) and processed within two hours
of collection. Processing was performed under the National Cancer Institute’s (NCI) Best
Practices for Biospecimen Resources guidelines. Aliquots were stored, and viable leuko-
cytes were isolated in the form of buffy coat by centrifugation and cryopreserved in ali-
quots at −80 °C freezers with emergency power backup. We also procured buffy coat sam-
ples of 6 NHB and 6 NHW cancer-free participants, matched on age-intervals from SRBSR
at Georgetown University’s Lombardi Comprehensive Cancer Center. Previous studies
have demonstrated the reliability and validity of Comet assays on stored samples [28,29].
2.3. CometChip Assay for DNA Damage Detection and Repair Kinetics Evaluation
Alkaline and Neutral CometChip assays were conducted following previously pub-
lished procedure [17] and using a polydimethylsiloxane (PDMS) stamp, with an array of
micro pegs (a kind gift from Dr. Engelward’s lab at MIT, Cambridge, MA, USA), to form
an array of about 300 microwells in each of 96 macro wells on the agarose chip. CometAs-
say Alkaline (biotechne, catalog no. 4256-010-CC, Minneapolis, MN, USA) and Neutral
(biotechne, catalog no. 4257-010-NC) reference cells served as baseline controls
Figure 1. Study Schematic. For details, see Materials and Methods. Created with Biorender.com
(accessed on 2 May 2024).
2.2. Patient Recruitment and Sample Collection
Adult non-Hispanic Black (NHB, n= 13) and White (NHW, n= 12) invasive breast
cancer survivors, with no history of breast cancer recurrence or known breast cancer-related
germline mutations, who had completed primary treatment (surgery, chemotherapy, ra-
diation therapy) at least six months, and at most three years, before study entry (women
currently on hormone therapy were eligible) were recruited through community-based ap-
proaches, and identification and screening using the Survey, Recruitment, and Biospecimen
Collection Shared Resource (SRBSR) at Georgetown University’s Lombardi Comprehensive
Cancer Center. After providing written informed consent, all participants completed a
demographic and medical history questionnaire, and anthropometrics measurements to
determine weight and height. Notably, the majority of breast cancer patients undergo
testing for germline mutations, particularly focusing on BRCA mutations. We identified
non-BRCA patients from the electronic medical record (EMR) database and included them
in this study. It is also worth noting that patients who reported non-BRCA mutations
may not have undergone formal genetic testing. Venous blood (20 mL) was collected into
sodium heparin tubes. All data collection activities were conducted at the Lombardi Cancer
Center’s Office of Minority Health and Health Disparities Research. The Georgetown
MedStar Institutional Review Board approved the study protocol (#STUDY00003904).
All participant blood specimens were transported to Lombardi Cancer Center’s Tissue
Culture and Bio-banking Shared Resource (TCBSR) and processed within two hours of
collection. Processing was performed under the National Cancer Institute’s (NCI) Best Prac-
tices for Biospecimen Resources guidelines. Aliquots were stored, and viable leukocytes
were isolated in the form of buffy coat by centrifugation and cryopreserved in aliquots
at
−
80
◦
C freezers with emergency power backup. We also procured buffy coat samples
of 6 NHB and 6 NHW cancer-free participants, matched on age-intervals from SRBSR at
Georgetown University’s Lombardi Comprehensive Cancer Center. Previous studies have
demonstrated the reliability and validity of Comet assays on stored samples [28,29].
2.3. CometChip Assay for DNA Damage Detection and Repair Kinetics Evaluation
Alkaline and Neutral CometChip assays were conducted following previously pub-
lished procedure [
17
] and using a polydimethylsiloxane (PDMS) stamp, with an array
of micro pegs (a kind gift from Dr. Engelward’s lab at MIT, Cambridge, MA, USA), to
form an array of about 300 microwells in each of 96 macro wells on the agarose chip.
CometAssay Alkaline (biotechne, catalog no. 4256-010-CC, Minneapolis, MN, USA) and
Neutral (biotechne, catalog no. 4257-010-NC) reference cells served as baseline controls
representing DNA damage at various levels in alkaline and neutral CometChip assays.
These standard control cells are a reference for monitoring the variation in day-to-day
CometChip assay procedures for evaluating patient samples. This was based on recom-
mendations from consensus statements published in 2020, and updated in 2023 [
30
,
31
], by
an international group of Comet assay users. We added these reference undamaged and
Cancers 2024,16, 1807 4 of 14
damaged cells as internal controls for every alkaline and neutral assay run and for every
human sample we analyzed. This was to make sure that the observed DNA damage and
repair activity variation is actually inherent to the sample, and not due to assay variability.
The reproducibility of the experimental protocol across seven runs for alkaline assays and
four runs for neutral assays is presented in Figure S1A. The mean moments of four internal
controls from the experimental runs were within
±
3 SD of the mean moment of all indi-
vidual experiments for CC0 (0.48
±
0.31), CC2 (1.23
±
1.26), NC0 (0.97
±
0.26), and NC2
(9.53
±
5.18), suggesting high reproducibility. Alkaline and Neutral CometChip assays
were conducted with few modifications from previously published protocols, and are
described below in the appropriate sections.
2.3.1. Cell Recovery and Preparation for CometChip Assays
On the day of the experiment, the aliquoted Buffy coat cells and control cells were
thawed in a 37
◦
C water bath and kept on ice. The Buffy coat cells and the control cells were
then resuspended in 2.5 mL and 0.5 mL cold Phosphate Buffered Saline (PBS), respectively.
HCT 116 cells were freshly harvested from cell culture plates on the day of CometChip
assay, washed with cold PBS, resuspended in 2.5 mL cold PBS, and stored on ice until
loaded into the CometChip.
2.3.2. CometChip Preparation
For the Comet Chip assay, 96-well agarose gels were prepared by using molten 1%
normal melting point (NMP) agarose (ThermoFisher, Waltham, MA, USA) in PBS. The
molten agarose was poured onto the hydrophilic side of a Gel Bond
®
film (Lonza, Walk-
ersville, MD, USA), which was placed up in a rectangular petri dish lid and evenly spread
across the lid. The polydimethylsiloxane (PDMS) stamp, with an array of micropegs, was
gently placed on top of the gel. The gel was allowed to solidify for 15 min. The PDMS was
removed, and 10 mL PBS was added to the dish. The micropore formation was confirmed
by observing the gel on an inverted microscope with 10
×
magnification. The excess gel
from the lid was removed, and the gel attached to the Bond
®
film was submerged in PBS
and stored before use at 4 ◦C for no longer than a week.
2.3.3. CometChip Cell Loading
The whole CometChip experiment was carried out in yellow light to minimize spuri-
ous DNA damage to the cells. The agarose gel attached to the Bond
®
film was placed on a
glass plate, and a bottomless 96-well plate (VWR) was pressed on the agarose chip to form
96 macrowells. The whole sandwich was then secured with the binder clips (staples) on four
sides. Each macrowell contained an array of about 300 microwells. Each microwell (30
µ
m)
receives mostly a single cell. Rarely, more than one cell is deposited in a microwell. Two or
more cells, if deposited, are discarded manually during Comet analysis by Comet Assay
Software (comet analysis is described below in Section 2.3.6). One hundred microliters
of single cell suspension (~2000 or more cells) were added into each macrowell, and the
sandwiches with the chip were incubated in a 37
◦
C cell culture incubator in the presence
of 5% CO
2
for 30 min. After incubation, the media from the wells was aspirated and the
agarose chips were washed with 10 mL PBS to remove excess cells. This process results in
generation of array of single cells in microwells (Figure 2A). Molten 1% low melting point
(LMP) agarose (ThermoFisher; kept at 46
◦
C until use) was poured on the chips. To ensure
complete solidification, the gels were kept at room temperature for 3 min and then at 4
◦
C
for 5 min.
Cancers 2024,16, 1807 5 of 14
Cancers 2024, 16, x FOR PEER REVIEW 5 of 15
Figure 2. (A) Cell loading and single cell array generation. (B) A representative CometChip image
shows undamaged and damaged nucleoids and the mode of analys is used in this study. (C) Alkaline
(global DNA damage) and neutral (double-strand break) CometChip assay optimization for detec-
tion of double-strand breaks (DSB), and single-strand breaks (SSB). HCT116 cells were treated with
methylmethane sulfonate (MMS) and bleomycin (BLM) and analyzed in alkaline and neutral
CometChip assays. Tail Moment is used as a DNA damage parameter. p < 0.05 was considered sig-
nificant. Created with Biorender.com (Accessed on 6 May 2024).
2.3.4. Bleomycin and Methyl Methanesulfonate Treatment for Repair Assay
Bleomycin (BLM; Cayman Chemical Company, catalog no. 13877, Ann Arbor, MI,
USA) is a radiomimetic agent that induces double-strand breaks along with single-strand
breaks containing 3′-phosphoglycolate/5′-phosphate ends or 4′-oxidized abasic sites in
DNA [32,33]. Doses of BLM (2.5–10 µg/mL for alkaline and 1.25–5 µg/mL for neutral as-
says) were prepared in RPMI 1640 media, and the patient cells embedded in CometChips
were submerged in BLM-containing media and incubated for 15 min in a 37 °C incubator
to induce DNA damage. Optimization of BLM dose and repair time for alkaline and neu-
tral assays are shown in Figure S1B. Based on the normal repair response of the cells where
the damage level reached the basal or near basal level, we selected 2.5 µg/mL BLM for
damage induction and a maximum of 60 min for repair kinetics under alkaline conditions
to analyze all survivors’ samples. Similarly, we chose 5 µg/mL BLM for damage induction
and a full 120 min for repair kinetics for neutral conditions.
The CometChips were cut into pieces for the convenience of their use for multiple
BLM doses and different repair time points. Post-BLM treatment, the chips were washed
briefly three times by submerging them in PBS. After optimization of the doses and the
repair time points, patient samples were analyzed routinely for damage induction and
repair kinetics by incubating them with BLM concentrations of 2.5 µg/mL and 5.0 µg/mL
for alkaline and neutral assays, respectively. For repair kinetics, 0–60 min and 0–120 min
were chosen for alkaline and neutral assays, respectively. Basal DNA damage in patient
Figure 2. (A) Cell loading and single cell array generation. (B) A representative CometChip image
shows undamaged and damaged nucleoids and the mode of analysis used in this study. (C) Alka-
line (global DNA damage) and neutral (double-strand break) CometChip assay optimization for
detection of double-strand breaks (DSB), and single-strand breaks (SSB). HCT116 cells were treated
with methylmethane sulfonate (MMS) and bleomycin (BLM) and analyzed in alkaline and neutral
CometChip assays. Tail Moment is used as a DNA damage parameter. p< 0.05 was considered
significant. Created with Biorender.com (accessed on 2 May 2024).
2.3.4. Bleomycin and Methyl Methanesulfonate Treatment for Repair Assay
Bleomycin (BLM; Cayman Chemical Company, catalog no. 13877, Ann Arbor, MI,
USA) is a radiomimetic agent that induces double-strand breaks along with single-strand
breaks containing 3
′
-phosphoglycolate/5
′
-phosphate ends or 4
′
-oxidized abasic sites in
DNA [
32
,
33
]. Doses of BLM (2.5–10
µ
g/mL for alkaline and 1.25–5
µ
g/mL for neutral
assays) were prepared in RPMI 1640 media, and the patient cells embedded in CometChips
were submerged in BLM-containing media and incubated for 15 min in a 37
◦
C incubator to
induce DNA damage. Optimization of BLM dose and repair time for alkaline and neutral
assays are shown in Figure S1B. Based on the normal repair response of the cells where
the damage level reached the basal or near basal level, we selected 2.5
µ
g/mL BLM for
damage induction and a maximum of 60 min for repair kinetics under alkaline conditions
to analyze all survivors’ samples. Similarly, we chose 5
µ
g/mL BLM for damage induction
and a full 120 min for repair kinetics for neutral conditions.
The CometChips were cut into pieces for the convenience of their use for multiple BLM
doses and different repair time points. Post-BLM treatment, the chips were washed briefly
three times by submerging them in PBS. After optimization of the doses and the repair
time points, patient samples were analyzed routinely for damage induction and repair
kinetics by incubating them with BLM concentrations of 2.5
µ
g/mL and 5.0
µ
g/mL for
alkaline and neutral assays, respectively. For repair kinetics, 0–60 min and 0–120 min were
chosen for alkaline and neutral assays, respectively. Basal DNA damage in patient cells
Cancers 2024,16, 1807 6 of 14
and damage in reference Trevigen control cells were analyzed immediately after loading
cells and overlaid with LMP agarose in a cold lysis solution (10 mM Tris-HCl, 2.5 M NaCl,
100 mM Na
2
EDTA, 1% v/vTriton X-100 at pH 10) for 18 h at 4
◦
C for alkaline CometChip
assay, whereas the chips were submerged in 43
◦
C pre-warmed lysis solution (10 mM
Tris-HCl, 2.5 M NaCl, 100 mM Na
2
EDTA, 1% N-Lauroylsarcosine, 10% DMSO, 0.5% v/v
Triton X-100 at pH 9.5) and incubated for 18 h at 43
◦
C for the neutral CometChip assay. To
analyze induced DNA damage, the post-BLM treated CometChips were washed with PBS
and placed immediately in either a cold lysis buffer or 43
◦
C pre-warmed lysis solution,
and processed following the remaining steps on alkaline or neutral CometChip assays. To
evaluate repair kinetics, the post-BLM treated CometChips were washed with PBS, placed
immediately in a growth medium containing RPMI 1640 and 10% FBS, and incubated for
aforementioned repair time points for alkaline and neutral assays, respectively, in a 37
◦
C
cell culture incubator in the presence of 5% CO
2
. At the completion of each repair time
point, the growth media was aspirated and the CometChip or its fragments were placed in
CometChip assay-specific lysis buffer and processed as described above.
To assess the damage type, SSB, or DSB detection by alkaline and neutral CometChip
assays under our assay conditions, we used an alkylating agent, methyl methanesulfonate
(MMS; ThermoFisher, catalog no. H55120.06), as it does not form DSBs, but forms SSBs
indirectly. It induces methylated bases such as 3-methyl adenine and 7-methyl guanine in
DNA. SSBs are formed upon spontaneous hydrolysis of 7-methylguanine and cleavage of
the resulting alkali labile abasic sites in alkaline CometChip assay. We used HCT116 (ATCC
catalog no. CCL-247, RRID: CVCL_0291) cells for these optimization experiments, because
they are a well-established model for studying DNA damage and repair processes, and
they have been widely used in previous studies, including ours [
34
–
37
]. The HCT116 cell
line was obtained directly from Lombardi Cancer Center’s Tissue Culture and Biobanking
Shared Resource (TCBSR) and fingerprinted for identity confirmation. The cell line was
passaged for 3 months after receipt for use in the described experiments. The cell line was
routinely tested for the presence of mycoplasma, with the latest test on 18 January 2024,
using the MycoFluor Mycoplasma Detection Kit according to manufacturer’s instructions
(Molecular Probes, catalog no. M-7006). The HCT116 cells embedded in CometChips
were submerged in DMEM media containing 1 mM MMS and incubated for 1 h in a
37
◦
C incubator to induce DNA damage. Post-MMS treated CometChips were washed
with PBS, placed immediately in either cold lysis buffer or 43
◦
C pre-warmed lysis solution,
and processed following the remaining steps on alkaline or neutral CometChip assays.
2.3.5. Alkaline and Neutral Electrophoresis
For the alkaline assay, the gel was washed with cold distilled water and denatured
to unwind nuclei in an alkaline unwinding buffer (0.3 M NaOH and 1 mM Na
2
EDTA at
pH 14) for 40 min at 4
◦
C, followed by electrophoresis in the same alkaline unwinding
buffer at 4
◦
C for 30 min at a constant 21 V and ~300 mA, using the Trevigen Comet Assay
®
Electrophoresis System II (Biotechne, catalog no. 4250-050-ES). After electrophoresis, the
gel was washed briefly by submerging it in the neutralization buffer (0.4 M Tris-HCl at pH
7.5) for 5 min at room temperature, it was then kept in the same buffer for 15 min at 4
◦
C,
and finally stored in the same buffer at 4
◦
C for up to 2 days. For the neutral assay, the gel
was washed and incubated in cold neutral electrophoresis TBE buffer (90 mM Tris-HCl,
90 mM Boric acid, 2 mM Na
2
EDTA at pH 8.5) for 60 min at 4
◦
C, followed by electrophoresis
in the same TBE buffer at 4
◦
C for 15–16 min at a constant 21 V and 8–12 mA, using the
same Trevigen Comet Assay®Electrophoresis System II.
2.3.6. Gel Staining, Imaging and Comet Analysis
The gels were stained for 20 min on a shaker with 1
×
SYBR Gold (ThermoFisher,
catalog # S11494) in TE buffer, and, followed by brief washing, imaged using an automated
fluorescent microscope (Keyence BZ-X710 series in one) at 4
×
magnification in the GFP
channel (488 nm). The images of the comets were captured by automatic scanning, com-
Cancers 2024,16, 1807 7 of 14
pressed, and stitched for analysis and necessary editing by the Comet Assay Software
(Trevigen, catalog no. 4260-000-CS), and a .csv file was generated for graphical and statisti-
cal analysis. Figure 2B shows an example of the damaged and undamaged cells during
imaging and analysis.
2.4. Data Analysis, Statistics, and Reproducibility
The graphs were generated using GraphPad Prism v10. Differences in DNA damage
(Mean
±
SD) between untreated and MMS/BLM treatment (Figure 2C), and between NHB
and NHW (Figures 4A,B and 5A,B), were assessed using unpaired two-tailed t-test with
Welch’s correction and unpaired, two-tailed Mann–Whitney test, respectively (significance
level p< 0.05). A two-way repeated measures ANOVA (significance level p< 0.05) was
used to assess the impact of disease (Figure 3A,B) and race (Figures 6A–D and 7A–D)
across different repair time points. Further analyses, to investigate the role of covariates
on DNA damage and repair activity, were conducted using generalized linear models,
adjusted for BMI, age, cancer stage, and treatment. All statistical analyses were performed
using GraphPad Prism v10 and R v4.2. No statistical methods were used to pre-determine
sample sizes for this pilot study, and the normality assumption was not formally tested.
Data collection and analysis were performed blinded to the experimental conditions. Pre-
determined criteria guided participants’ eligibility for this study. We did not exclude any
data points from our analyses. All analyses are from five replicates of each measurement
for each participant’s sample.
3. Results
3.1. CometChip Assay Optimization for Global DNA Damage and Double-Strand Break,
Bleomycin Dose, and Repair Kinetics
This pilot study investigated race-based differences in systemic basal DNA damage,
damage induction, and repair after bleomycin (BLM) treatment using a high-sensitivity,
high-throughput novel CometChip assay in circulating leukocytes from cancer-free par-
ticipants and breast cancer survivors [
17
] (Figure 1). All cancer survivors had completed
primary treatment at least six months before sample collection and were cancer-free at
enrollment. Buffy coat cells isolated from venous blood were collected from 13 NHB and
12 NHW breast cancer survivors. Participants reported a mean age of 58 years at enrollment
and a mean BMI of 30.6 kg/m
2
. Distributions of breast cancer stage, histology, smoking
status, prior history of other cancers, and treatment were well-matched between the two
races, with 67% of NHW and 62% of NHB women having an early-stage diagnosis. All
participants were non-smokers and had invasive cancer. None of the participants had prior
history of other cancers (Table S1).
Through optimization, we confirmed that the CometChip assay, under alkaline con-
ditions detects, global DNA damage, including alkali-labile sites, double-strand breaks
(DSB), and single-strand breaks (SSB). In contrast, under the neutral condition, only DSBs
are detected (Figure 2C). Both alkaline and neutral comet assays were used to investigate
cancer-free controls vs. cancer survivors and race-based differences in moment, %DNA in
tail, and proportion of undamaged cells at baseline and different repair time points (0, 15,
30, 60, 120 min) following BLM treatment.
3.2. Repair of BLM-Induced Double-Strand Break and Global DNA Damage in Cancer-Free
Women and Breast Cancer Survivors
We observed differences in the DNA repair capacity of DSBs in leukocytes between
cancer-free women and breast cancer survivors (Figure 3A). Although most cellular damage
from BLM has been repaired by the 15-min repair point for cancer-free women and breast
cancer survivors, there are significant differences in repair kinetics and residual damage
between the two groups. Compared to cancer survivors, there seemed to be increased
sustained damage and subsequent repair activity 15 min post-BLM among cancer-free
participants. Repair activity was different between cancer-free women and breast cancer
survivors, as measured by the interaction of two groups with repair activity (mean moment;
Cancers 2024,16, 1807 8 of 14
overall (p< 0.01), Figure 3A). Global DNA damage and repair differences between these two
groups were similar to DSB. However, the differences in magnitude between cancer-free
participants and cancer survivors were lower, with less likelihood of statistical significance
across the four measures (Figure 3B).
Cancers 2024, 16, x FOR PEER REVIEW 8 of 15
3.2. Repair of BLM-Induced Double-Strand Break and Global DNA Damage in Cancer-free
women and Breast Cancer Survivors
We o bserved differences in the DNA repair capacity of DSBs in leukocytes between
cancer-free women and breast cancer survivors (Figure 3A). Although most cellular dam-
age from BLM has been repaired by the 15-min repair point for cancer-free women and
breast cancer survivors, there are significant differences in repair kinetics and residual
damage between the two groups. Compared to cancer survivors, there seemed to be in-
creased sustained damage and subsequent repair activity 15 min post-BLM among cancer-
free participants. Repair activity was different between cancer-free women and breast can-
cer survivors, as measured by the interaction of two groups with repair activity (mean
moment; overall (p < 0.01), Figure 3A). Global DNA damage and repair differences be-
tween these two groups were similar to DSB. However, the differences in magnitude be-
tween cancer-free participants and cancer survivors were lower, with less likelihood of
statistical significance across the four measures (Figure 3B).
UT 0 15 30 60 120
0
5
10
15
15
20
25
30
All cell types: undamaged and damaged
Repair Time (minutes)
Cancer survivor (individual)
Cancer-free (in dividual)
Cancer survivor (Mean)
Cancer-free (Mean)
p<0.01
UT 0 153060
0
2
4
6
8
10
20
Repair Time (minutes)
Cancer survivor (individual)
Cancer-free (in dividual)
Cancer survivor (M ean)
Cancer-free (Mean)
p=0.22
All cell types: undamaged and damaged
Figure 3. Repair of BLM-induced double-strand Break and global DNA damage in cancer-free
women and breast cancer survivors. The repair kinetics of BLM-induced DNA double-strand breaks
was measured by neutral (A) and alkaline (B) assays. DNA double-strand breaks (A) and global
damage (B) were measured in cells after BLM treatment during their recovery period at different
time points. A repeated measures ANOVA was conducted to assess the impact of disease (cancer)
on repair kinetics. p-values represent the statistical significance of the interaction of disease with
repair activity at different time points. p < 0.05 is considered significant.
3.3. Race Effect on Basal and Damage Susceptibility and Repair of BLM-Induced Double-Strand
Break and Global DNA Damage in NHB and NHW Breast Cancer Survivors
Mean (± SD) basal DNA damage in leukocytes was higher in NHW women compared
to NHB women for both DSB (3.7 ± 1.43 au for NHW vs. 1.26 ± 0.81 au for NHB, p < 0.01;
Figure 4A) and global damage (2.24 ± 0.66 au for NHW vs. 1.06 ± 1.2 au for NHB, p < 0.01;
Figure 5A). However, NHB-derived cells showed a much higher induction (normalized to
basal levels) in response to BLM than NHW cells for both damage types (DSB: 3.34 ± 2.95
au for NHW cells vs. 8.12 ± 5.6 au for NHB cells, p = 0.02; Figure 4B; global damage: 0.94 ±
0.76 au for NHW vs. 7.64 ± 6.6 au for NHB, p = 0.01; Figure 5B). In addition, variability in
basal damage and damage induction was higher for NHB than NHW individuals.
Figure 3. Repair of BLM-induced double-strand Break and global DNA damage in cancer-free women
and breast cancer survivors. The repair kinetics of BLM-induced DNA double-strand breaks was
measured by neutral (A) and alkaline (B) assays. DNA double-strand breaks (A) and global damage
(B) were measured in cells after BLM treatment during their recovery period at different time points.
A repeated measures ANOVA was conducted to assess the impact of disease (cancer) on repair
kinetics. p-values represent the statistical significance of the interaction of disease with repair activity
at different time points. p< 0.05 is considered significant.
3.3. Race Effect on Basal and Damage Susceptibility and Repair of BLM-Induced Double-Strand
Break and Global DNA Damage in NHB and NHW Breast Cancer Survivors
Mean (
±
SD) basal DNA damage in leukocytes was higher in NHW women com-
pared to NHB women for both DSB (3.7
±
1.43 au for NHW vs. 1.26
±
0.81 au for NHB,
p< 0.01; Figure 4A) and global damage (2.24
±
0.66 au for NHW vs. 1.06
±
1.2 au for NHB,
p< 0.01; Figure 5A). However, NHB-derived cells showed a much higher induction (nor-
malized to basal levels) in response to BLM than NHW cells for both damage types (DSB:
3.34
±
2.95 au for NHW cells vs. 8.12
±
5.6 au for NHB cells, p= 0.02; Figure 4B; global
damage: 0.94
±
0.76 au for NHW vs. 7.64
±
6.6 au for NHB, p= 0.01; Figure 5B). In
addition, variability in basal damage and damage induction was higher for NHB than
NHW individuals.
Cancers 2024, 16, x FOR PEER REVIEW 9 of 15
Figure 4. Effect of race on basal and damage susceptibility of BLM-induced double-strand break in
NHB and NHW breast cancer survivors. Basal (A) and induction/susceptibility (B) of BLM-induced
double-strand breaks were measured by neutral assay. Differences in DNA damage (Mean ± SD)
between NHB and NHW (A, B) were assessed using unpaired, two-tail Mann-Whitney test. The red
lines denote the mean value. DNA damage induction in (panel B) was calculated using the formula:
[DNA damage (moment) after BLM treatment − basal DNA damage (Moment) before BLM treat-
ment]/basal DNA damage (moment) before BLM treatment.
Figure 5. Effect of race on basal and damage susceptibility of BLM-induced global DNA damage in
NHB and NHW breast cancer survivors. Basal (A) and induction/susceptibility (B) of BLM-induced
global damage were measured by alkaline assay. Differences in DNA damage (Mean± SD) between
NHB and NHW survivors (A, B) were assessed using an unpaired, two-tail Mann–Whitney test. The
red lines denote the mean value. DNA damage induction in panel B was calculated using the for-
mula described in Figure 4 legends.
We o bs erved differences in the DNA repair capacity of DSBs in leukocytes between
NHB and NHW groups (Figure 6A–D). Although most cellular damage from BLM has
been repaired by the 15-min repair point for NHW and NHB groups, there are significant
differences in repair kinetics and residual damage between the two races. Among the
NHB group, there did not seem to be any measurable ongoing damage or repair activity
after 15 min, compared to the sustained damage and subsequent repair activity among
the NHW group, even at 120 min post-BLM. Repair activity was different between the
NHB and NHW groups, as measured by the interaction of race with repair activity (mean
moment; overall (p = 0.01) and in damaged cells (p = 0.01), Figure 6A,C). Consequently,
the proportion of undamaged cells across repair time points was higher in NHB cells than
in NHW cells (p < 0.01, Figure 6D). However, at 120 min post-BLM, the proportion of un-
damaged cells in the NHW group was similar to basal levels. Still, among the NHB group,
it did not recover to the basal levels (Figure 6D). In generalized linear models, race differ-
ences remained significant after adjusting for age and BMI. However, repair differences
Figure 4. Effect of race on basal and damage susceptibility of BLM-induced double-strand break in
NHB and NHW breast cancer survivors. Basal (A) and induction/susceptibility (B) of BLM-induced
double-strand breaks were measured by neutral assay. Differences in DNA damage (Mean
±
SD)
between NHB and NHW (A,B) were assessed using unpaired, two-tail Mann-Whitney test. The
red lines denote the mean value. DNA damage induction in (panel B) was calculated using the
formula: [DNA damage (moment) after BLM treatment
−
basal DNA damage (Moment) before BLM
treatment]/basal DNA damage (moment) before BLM treatment.
Cancers 2024,16, 1807 9 of 14
Cancers 2024, 16, x FOR PEER REVIEW 9 of 15
Figure 4. Effect of race on basal and damage susceptibility of BLM-induced double-strand break in
NHB and NHW breast cancer survivors. Basal (A) and induction/susceptibility (B) of BLM-induced
double-strand breaks were measured by neutral assay. Differences in DNA damage (Mean ± SD)
between NHB and NHW (A, B) were assessed using unpaired, two-tail Mann-Whitney test. The red
lines denote the mean value. DNA damage induction in (panel B) was calculated using the formula:
[DNA damage (moment) after BLM treatment − basal DNA damage (Moment) before BLM treat-
ment]/basal DNA damage (moment) before BLM treatment.
Figure 5. Effect of race on basal and damage susceptibility of BLM-induced global DNA damage in
NHB and NHW breast cancer survivors. Basal (A) and induction/susceptibility (B) of BLM-induced
global damage were measured by alkaline assay. Differences in DNA damage (Mean± SD) between
NHB and NHW survivors (A, B) were assessed using an unpaired, two-tail Mann–Whitney test. The
red lines denote the mean value. DNA damage induction in panel B was calculated using the for-
mula described in Figure 4 legends.
We o bserved di fferences in the DNA repair capacity of DSBs in leukocytes between
NHB and NHW groups (Figure 6A–D). Although most cellular damage from BLM has
been repaired by the 15-min repair point for NHW and NHB groups, there are significant
differences in repair kinetics and residual damage between the two races. Among the
NHB group, there did not seem to be any measurable ongoing damage or repair activity
after 15 min, compared to the sustained damage and subsequent repair activity among
the NHW group, even at 120 min post-BLM. Repair activity was different between the
NHB and NHW groups, as measured by the interaction of race with repair activity (mean
moment; overall (p = 0.01) and in damaged cells (p = 0.01), Figure 6A,C). Consequently,
the proportion of undamaged cells across repair time points was higher in NHB cells than
in NHW cells (p < 0.01, Figure 6D). However, at 120 min post-BLM, the proportion of un-
damaged cells in the NHW group was similar to basal levels. Still, among the NHB group,
it did not recover to the basal levels (Figure 6D). In generalized linear models, race differ-
ences remained significant after adjusting for age and BMI. However, repair differences
Figure 5. Effect of race on basal and damage susceptibility of BLM-induced global DNA damage in
NHB and NHW breast cancer survivors. Basal (A) and induction/susceptibility (B) of BLM-induced
global damage were measured by alkaline assay. Differences in DNA damage (Mean
±
SD) between
NHB and NHW survivors (A,B) were assessed using an unpaired, two-tail Mann–Whitney test. The
red lines denote the mean value. DNA damage induction in panel B was calculated using the formula
described in Figure 4legends.
We observed differences in the DNA repair capacity of DSBs in leukocytes between
NHB and NHW groups (Figure 6A–D). Although most cellular damage from BLM has
been repaired by the 15-min repair point for NHW and NHB groups, there are significant
differences in repair kinetics and residual damage between the two races. Among the NHB
group, there did not seem to be any measurable ongoing damage or repair activity after
15 min, compared to the sustained damage and subsequent repair activity among the NHW
group, even at 120 min post-BLM. Repair activity was different between the NHB and NHW
groups, as measured by the interaction of race with repair activity (mean moment; overall
(p= 0.01) and in damaged cells (p= 0.01), Figure 6A,C). Consequently, the proportion of
undamaged cells across repair time points was higher in NHB cells than in NHW cells
(p< 0.01, Figure 6D). However, at 120 min post-BLM, the proportion of undamaged cells in
the NHW group was similar to basal levels. Still, among the NHB group, it did not recover
to the basal levels (Figure 6D). In generalized linear models, race differences remained
significant after adjusting for age and BMI. However, repair differences were attenuated
after further adjustment for cancer stage and treatment, suggesting a systemic effect of
cancer and cancer therapy that persists beyond the treatment period.
Cancers 2024, 16, x FOR PEER REVIEW 10 of 15
were aenuated after further adjustment for cancer stage and treatment, suggesting a sys-
temic effect of cancer and cancer therapy that persists beyond the treatment period.
Figure 6. Effect of race on repair of BLM-induced double-strand break in NHB and NHW breast
cancer survivors. The repair kinetics of the BLM-induced DNA double-strand breaks were meas-
ured by neutral (A–D) assay. DNA double-strand breaks were measured in cells after BLM treat-
ment during their recovery period at different time points. A repeated measures ANOVA was con-
ducted to assess the impact of race on repair kinetics. p-values represent the statistical significance
of the interaction of race with repair activity at different time points. Upon segregation of all cells,
damaged and undamaged cell populations were assessed by excluding cells with zero values and
including cells only with zero values, respectively. p < 0.05 was considered significant.
Global DNA damage and repair differences between NHB and NHW survivors were
similar to DSB. However, the magnitude of race differences was much lower and less
likely to be statistically significant across the four measures (Figure 7A–D). We also inves-
tigated DSB damage and repair in 12 cancer-free women (6 NHW women and 6 NHB
women) to determine whether the differences in cancer patients might result from race
differences, irrespective of cancer status. We found no differences in DNA damage or re-
pair across time points among NHB and NHW women without cancer (Figure S2). How-
ever, the sample size is small to draw any definitive conclusions.
Figure 6. Effect of race on repair of BLM-induced double-strand break in NHB and NHW breast
cancer survivors. The repair kinetics of the BLM-induced DNA double-strand breaks were measured
Cancers 2024,16, 1807 10 of 14
by neutral (A–D) assay. DNA double-strand breaks were measured in cells after BLM treatment
during their recovery period at different time points. A repeated measures ANOVA was conducted
to assess the impact of race on repair kinetics. p-values represent the statistical significance of the
interaction of race with repair activity at different time points. Upon segregation of all cells, damaged
and undamaged cell populations were assessed by excluding cells with zero values and including
cells only with zero values, respectively. p< 0.05 was considered significant.
Global DNA damage and repair differences between NHB and NHW survivors were
similar to DSB. However, the magnitude of race differences was much lower and less likely
to be statistically significant across the four measures (Figure 7A–D). We also investigated
DSB damage and repair in 12 cancer-free women (6 NHW women and 6 NHB women) to
determine whether the differences in cancer patients might result from race differences,
irrespective of cancer status. We found no differences in DNA damage or repair across time
points among NHB and NHW women without cancer (Figure S2). However, the sample
size is small to draw any definitive conclusions.
Cancers 2024, 16, x FOR PEER REVIEW 11 of 15
Figure 7. Effect of race on repair of BLM-induced global DNA damage in NHB and NHW breast
cancer survivors. The repair kinetics of BLM-induced global DNA damage was measured by alka-
line (A–D) assay. DNA damage was measured in cells after BLM treatment during their recovery
period at different time points. A repeated measures ANOVA was conducted to assess the impact
of race on repair kinetics. p-values represent the statistical significance of the interaction of race with
repair activity at different time points. Upon segregation of all cells, damaged and undamaged cell
populations were assessed by excluding cells with zero values and including cells only with zero
values, respectively. p < 0.05 is considered significant.
4. Discussion
Our study using the novel CometChip assay is one of the first to show novel differ-
ences in systemic DNA damage and repair, primarily DSB repair, between NHB and
NHW breast cancer survivors. Although some studies have reported race-based differ-
ences in systemic DNA damage and repair activity in healthy volunteers, none have fo-
cused on cancer survivors [38]. It is also important to note that prior molecular mechanism
studies investigating racial differences were either not focused on DNA repair mecha-
nisms, or were conducted only among cancer patients, not in cancer survivors [12,39–42].
Our results on systemic DNA damage and repair activity between NHB and NHW
survivors add to recent literature that showed differences in the expression of DNA repair
genes, especially those involved in DSBR, in breast tumor tissue by race [12]. Our results
of comparing breast cancer survivors to cancer-free women also support findings from
Scuric et al., who used DNA damage assays on circulating WBCs that suggest the persis-
tence of systemic effects of cancer and cancer treatment, primarily radiation therapy and
chemotherapy, well beyond the end of treatment [43]. Contrary to our preliminary hy-
pothesis, NHB survivors had lower basal DNA damage and lower measured DNA dam-
age post-BLM. They also had less sustained DSB repair activity after the BLM challenge
than NHW cells. NHW cells returned to basal proportions of undamaged cells after post-
BLM repair activity, but NHB cells did not (Figure 6D). The observed low repair activity
could be due to mutations in BRCA genes in those survivors. However, it is important to
note that we selected BRCA mutation-free survivor samples for this study. Moreover, we
did not force the leukocytes in our assay to proliferate by treatment with any mitogen,
such as phytohemagglutinin [44], and we speculate that NHEJ, which does not require
BRCA, was possibly the predominant DSBR pathway in our BLM-treated cells.
Figure 7. Effect of race on repair of BLM-induced global DNA damage in NHB and NHW breast
cancer survivors. The repair kinetics of BLM-induced global DNA damage was measured by alkaline
(A–D) assay. DNA damage was measured in cells after BLM treatment during their recovery period
at different time points. A repeated measures ANOVA was conducted to assess the impact of
race on repair kinetics. p-values represent the statistical significance of the interaction of race with
repair activity at different time points. Upon segregation of all cells, damaged and undamaged cell
populations were assessed by excluding cells with zero values and including cells only with zero
values, respectively. p< 0.05 is considered significant.
4. Discussion
Our study using the novel CometChip assay is one of the first to show novel differences
in systemic DNA damage and repair, primarily DSB repair, between NHB and NHW
breast cancer survivors. Although some studies have reported race-based differences in
systemic DNA damage and repair activity in healthy volunteers, none have focused on
cancer survivors [
38
]. It is also important to note that prior molecular mechanism studies
investigating racial differences were either not focused on DNA repair mechanisms, or
were conducted only among cancer patients, not in cancer survivors [12,39–42].
Our results on systemic DNA damage and repair activity between NHB and NHW
survivors add to recent literature that showed differences in the expression of DNA repair
Cancers 2024,16, 1807 11 of 14
genes, especially those involved in DSBR, in breast tumor tissue by race [
12
]. Our results
of comparing breast cancer survivors to cancer-free women also support findings from
Scuric et al., who used DNA damage assays on circulating WBCs that suggest the persis-
tence of systemic effects of cancer and cancer treatment, primarily radiation therapy and
chemotherapy, well beyond the end of treatment [
43
]. Contrary to our preliminary hypoth-
esis, NHB survivors had lower basal DNA damage and lower measured DNA damage
post-BLM. They also had less sustained DSB repair activity after the BLM challenge than
NHW cells. NHW cells returned to basal proportions of undamaged cells after post-BLM
repair activity, but NHB cells did not (Figure 6D). The observed low repair activity could
be due to mutations in BRCA genes in those survivors. However, it is important to note
that we selected BRCA mutation-free survivor samples for this study. Moreover, we did
not force the leukocytes in our assay to proliferate by treatment with any mitogen, such as
phytohemagglutinin [
44
], and we speculate that NHEJ, which does not require BRCA, was
possibly the predominant DSBR pathway in our BLM-treated cells.
Race-based differences in systemic DNA damage and repair activity could also be
related to signaling pathways involved in accelerated aging, such as inflammation and ox-
idative stress, associated with cancer and cancer treatment [
45
]. It is noteworthy that some
leukocytes damaged by chemo or radiotherapy will not necessarily survive for extended
periods, but some do. In addition, the treatment-related changes in systemic mechanisms
(e.g., inflammation and oxidative stress) might be long-lasting, and continuously damage
healthy cells, including new leukocytes [
43
]. Persistent treatment-associated changes may
also alter immune functions and cause post-treatment health complications in the survivors.
In a mouse study, restraint-induced stress activated genes responsible for priming the T
cells to either undergo apoptosis or proliferation, the major function needed for cellular
immunity [46].
The strengths of our study include the quantitative measurement of different damage
types and repair pathways, rather than only measuring global damage (alkaline comet
assays), as is commonly reported [
8
,
20
]; innovative data analyses, such as segregating cells
into undamaged (zero damage) and damaged; and the focus on systemic DNA damage and
repair in the survivorship period, rather than on tumor tissue/cells. Our study is the first
of its kind in systemic disparities in cancer survivorship, but is limited by a relatively small
sample size, lack of breast cancer subtypes and detailed cancer treatment data, and lack of
survivorship-related outcome data. Additionally, although we looked at DSB, global DNA
damage, and their repair, we did not measure other DNA damage and activities, such as
base damage and its repair.
5. Conclusions
In conclusion, we report novel differences in systemic DNA damage and repair by
race in breast cancer survivors. Future confirmatory studies in diverse cancer survivor
populations are needed to validate our findings and to investigate the association of
underlying inflammation and aging-related pathways using gene expression and mutation
data, socio-environmental factors, and survivorship-related outcomes with the race-based
differences observed in our study.
Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/cancers16101807/s1, Figure S1: Alkaline and neutral CometChip
assay optimization for DNA damage, Bleomycin dose, and repair kinetics; Figure S2: Effect of race on
basal, and damage susceptibility and repair of BLM-induced double-strand break in cancer-free NHB
and NHW leukocytes; Table S1: Demographics of the Breast Cancer survivors whose samples were
utilized in this study at GLCCC.
Cancers 2024,16, 1807 12 of 14
Author Contributions: Conceptualization, R.R. and C.D.; methodology, S.D., R.K., J.H., M.G.M. and
R.R.; software, S.D., R.K., H.S., K.T., K.M., C.D. and R.R.; validation, S.D., R.R. and C.D.; formal
analysis, S.D., R.K., H.S., K.T., K.M., C.D. and R.R.; investigation, S.D., R.K. and R.R.; resources,
R.R. and C.D.; data curation, S.D., R.R. and C.D.; writing—original draft preparation, S.D., R.R. and
C.D.; writing—review and editing, S.D., R.R. and C.D.; visualization, S.D., H.S., K.M., C.D. and R.R.;
supervision, R.R. and C.D.; project administration, R.R. and C.D.; funding acquisition, R.R. and C.D.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Institute of Health/National Cancer Institute R21
CA264489. This study was also partially supported by development funds from the National Institute
of Health/National Cancer Institute grant P30 CA051008. We acknowledge Survey, Recruitment,
and Biospecimen Collection Shared Resource (SRBSR) for biospecimen procurement and clinical
data abstraction; Tissue Culture and Biobanking Shared Resource (TCBSR) for sample processing
and storage; and Biostatistics Shared Resource for statistical analysis. SRBSR, Biostatistics Shared
Resource, and TCBSR are partially supported by the National Institute of Health/National Cancer
Institute grant P30 CA051008.
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and approved by the Institutional Review Board of Georgetown MedStar (protocol code
#STUDY00003904 and date of approval 24 June 2021).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study.
Data Availability Statement: The data presented in this study are available upon request from
the contact corresponding author. We used no accession codes, unique identifiers, or web links for
publicly available datasets used in this study.
Acknowledgments: We thank Mehek Thapar at Georgetown University for some image captur-
ing and analysis. We also thank Bevin Engelward and Simran Kaushal at MIT for providing the
polydimethylsiloxane (PDMS) stamp and helping us set up the CometChip assay in the lab.
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
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