of 9
Supporting Information
Cellular Target of a Rhodium Metalloinsertor is the DNA
Base Pair Mismatch
Kelsey M. Boyle, Adela Nano, Catherine Day, and Jacqueline K. Barton*
[*] K. M. Boyle, Dr. A. Nano, C. Day, Prof. J. K. Barton
Division of Chemistr
y and Chemical Engineering
California Institute of Technology
1200 East California Boulevard, MC 127
-
72,
Pasadena, California 91125
(USA)
Email: jkbarton@caltech.edu
S
2
Table S1:
Growth c
onditions for
cell lines used
.
Cell Line
Obtained
from
Typ
e of
Media
%
FBS
Media Supplements
CaCo2
a,b
AMGEN
DMEM
20
100
U/mL PenStrep
Colo205
a,b
AMGEN
RPMI 1640
10
100
U/mL PenStrep
Colo320DM
a
AMGEN
RPMI 1640
10
100
U/mL PenStrep
Colo678
a,b
AMGEN
DMEM
10
100
U/mL PenStrep
CW2
a,b
AMGEN
DMEM
10
100
U/mL
PenStrep
DLD
-
1
a,b,c,d
AMGEN
RPMI 1640
10
100
U/mL PenStrep
HCC2998
a,b
AMGEN
RPMI 1640
10
100
U/mL PenStrep
HCT116
a,b,c
AMGEN
McCoy's 5A
10
100
U/mL PenStrep
HCT15
a,b,d
AMGEN
RPMI 1640
10
100
U/mL PenStrep
HT29
a,b,d
AMGEN
McCoy's 5A
10
100
U/mL P
enStrep
KM12
a
AMGEN
RPMI 1640
10
100
U/mL PenStrep
LoVo
a
AMGEN
Ham's F
-
12K
10
100
U/mL PenStrep
Ls1034
a,b
AMGEN
RPMI 1640
10
100
U/mL PenStrep
Ls123
a,b
AMGEN
DMEM
10
100
U/mL PenStrep
Ls174T
a,b
AMGEN
DMEM
10
100
U/mL PenStrep
NCI
-
H716
a,b
AMGEN
RPMI 1640
10
100
U/mL PenStrep
NCI
-
H508
a
AMGEN
RPMI 1640
10
100
U/mL PenStrep, 2
mM GlutaMAX
RKO
a,b
AMGEN
DMEM
10
100
U/mL PenStrep
SW1116
a,b
AMGEN
RPMI 1640
10
100
U/mL PenStrep
SW1463
a
AMGEN
RPMI 1640
10
100
U/mL PenStrep
SW403
a,b
AMGEN
RPMI 16
40
10
100
U/mL PenStrep, 2
mM GlutaMAX
SW48
a,b
AMGEN
RPMI 1640
10
100
U/mL PenStrep
SW480
a,d
AMGEN
RPMI 1640
10
100
U/mL PenStrep
SW620
a,b,d
AMGEN
RPMI 1640
10
100
U/mL PenStrep
SW837
a
AMGEN
RPMI 1640
10
100
U/mL PenStrep
SW948
a,b
ATCC
RPMI 1640
10
100
U/mL PenStrep, 2
mM GlutaMAX
WiDr
a,b,d
AMGEN
DMEM
10
100
U/mL PenStrep
AN3
-
CA
c
AMGEN
DMEM
10
100
U/mL PenStrep
DU
-
145
c
ATCC
DMEM
10
100
U/mL PenStrep
HCT
-
116N
c
--
RPMI 1640
10
100
U/mL PenStrep, 2
mM L
-
glutamine, 0.1
mM
non
-
essential amino a
cids, 1 mM sodium
pyruvate, 400
ug/mL Geneticin (G418)
HCT
-
116O
c
--
RPMI 1640
10
100
U/mL PenStrep, 2
mM L
-
glutamine, 0.1
mM
non
-
essential amino acids, 1 mM sodium
pyruvate, 400
ug/mL Geneticin (G418)
HEC
-
1
-
A
c
ATCC
McCoy's 5A
10
100
U/mL PenStrep
a
C
e
ll lines used in cytotoxicity assay
b
Cell lines used in whole cell uptake assay
c
Cell lines used in
RhCy3
fluorescence assay
d
DLD
-
1/HCT15, HT29/WiDr, SW480/SW620 pairings are derived from a common patient.
S
3
Table S2:
IC50 values of
RhPPO
and cisplatin
in all tested cell lines
.
Cell Line
IC50
RhPPO
(
μ
M)
IC50 Cisplatin
(
μ
M)
IC50 RhPPO
/IC50 Cisplatin
Cancer
Type
MMR Status,
(Mutated Protein)
CaCo
-
2
1.5 ± 0.8
9.5 ± 3.7
6.2
Colorectal
MMR+
Colo 205
0.063 ± 0.03
36.4 ± 2.8
580
Colorectal
MMR+
Colo 320DM
18.0 ± 2.9
9.5 ± 2.0
0.5
Colorectal
MMR+
Colo 678
0.81 ± 0.15
18.2 ± 0.7
22.4
Colorectal
MMR+
CW
-
2
9.2 ± 1.3
9.0 ± 2.1
1.0
Colorectal
MMR
, (
MLH1)
DLD
-
1
c
3.6 ± 0.3
10.7 ± 2.2
3.0
Colorectal
MMR
, (
MSH6)
HCC2998
a
1.6 ± 0.6
20.4 ± 1.4
12.6
Colorectal
MMR+, (
POLE)
HCT
-
116
c
0.25 ± 0.01
18.5 ± 0.9
73.3
Colorectal
MMR
, (
MLH1)
HCT
-
15
9.5 ± 2.5
16.1 ± 0.3
1.7
Colorectal
MMR
, (
MSH6)
HT
-
29
0.21 ± 0.01
22.1 ± 1.1
106
Colorectal
MMR+
KM
-
12
0.83 ± 0.07
13.9 ± 0.9
16.7
Colorectal
MMR
, (
MLH1)
LoVo
1.
7 ± 0.2
7.0 ± 1.2
4.0
Colorectal
MMR
, (
MSH2)
Ls1034
5.6 ± 0.3
14.4 ± 1.9
2.6
Colorectal
MMR+
Ls123
0.23 ± 0.03
9.0 ± 7.0
39.7
Colorectal
MMR+
Ls174T
2.0 ± 0.2
5.4 ± 0.6
2.8
Colorectal
MMR
, (
MLH1)
NCI
-
H716
1.8 ± 0.6
13.9 ± 2.9
7.7
Colorectal
MMR+
N
CI
-
H508
1.5 ± 0.4
8.8 ± 1.4
5.9
Colorectal
MMR+
RKO
0.12 ± 0.01
11.6 ± 0.7
97.5
Colorectal
MMR
, (
MLH1)
SW
-
1116
4.4 ± 1.2
9.1 ± 1.4
2.1
Colorectal
MMR+
SW
-
1463
1.6 ± 0.2
9.5 ± 1.1
6.0
Colorectal
MMR+
SW
-
403
0.34 ± 0.04
9.1 ± 1.5
27.1
Colorectal
MMR+
SW
-
48
0.34 ± 0.02
2.2 ± 0.2
6.4
Colorectal
MMR
, (
MLH1)
SW
-
480
c
0.44 ± 0.13
8.3 ± 0.6
12.0
Colorectal
MMR+
SW
-
620
0.33 ± 0.04
4.8 ± 0.8
14.7
Colorectal
MMR+
SW
-
837
1.8 ± 0.7
11.4 ± 1.3
6.2
Colorectal
MMR+
SW
-
948
9.7 ± 1.9
22.1 ± 1.7
2.3
Colorectal
M
MR+
WiDr
0.13 ± 0.01
25.5 ± 14.1
198.7
Colorectal
MMR+
AN3
-
CA
c
0.086 ± 0.003
--
--
Endometrial
MMR
, (
MLH1)
DU
-
145
c
0.67 ± 0.04
--
--
Prostate
MMR
, (
MLH1,
PMS2)
HEC
-
1
-
A
c
0.39 ± 0.02
--
--
Endometrial
MMR
, (
PMS2)
HCT
-
116N
b
,
c
1.12 ± 0.27
--
--
Colorectal
MMR+
HCT
-
116O
b
,
c
0.15 ± 0.06
--
--
Colorectal
MMR
, (
MLH1)
a
HCC2998 is mutated in the POLE gene, leading to an increase in polymerase errors such as
mismatches and indels. While it is not technically MMR
, it will have higher mismatch a
nd
indel occurrences than other MMR+ cell lines.
b
IC50 values from reference
[5]
c
Cell lines used as the test set for the RhCy3 assay. Note that since the colorectal cancer cell
lines examined in the cytotoxicity studies were primaily deficient in the ML
H1 gene, this test set
includes several cell lines of non
-
colorectal origin that span deficienicies in different MMR
genes as well.
S
4
Table S3:
Purity of genomic DNA extracted from the cancer cell lines used in this study as
determined using a NanoDrop sp
ectrophotometer.
Phenotype
a
A260/A280
b
A260/A230
c
SW480
-
i
1.94
2.05
SW480
-
ii
1.93
2.06
SW480
-
iii
1.94
2.06
HCT116N
-
i
2.02
2.02
HCT116N
-
ii
2.00
2.04
HCT116N
-
iii
2.01
2.02
HCT116O
-
i
1.95
1.97
HCT116O
-
ii
1.97
2.01
HCT116O
-
iii
1.99
2.00
H
CT116
-
i
1.97
2.06
HCT116
-
ii
1.98
2.05
HCT116
-
iii
1.94
2.03
AN3
-
CA
-
i
1.91
1.99
AN3
-
CA
-
ii
1.92
1.99
AN3
-
CA
-
iii
1.91
1.98
DU
-
145
-
i
2.00
1.97
DU
-
145
-
ii
1.95
1.90
DU
-
145
-
iii
1.96
1.91
HEC
-
1A
-
i
1.95
2.03
HEC
-
1A
-
ii
1.97
2.06
HEC
-
1A
-
iii
1.93
2.04
DLD
-
1
-
i
2.00
2.04
DLD
-
1
-
ii
1.94
1.98
DLD
-
1
-
iii
1.99
2.06
a
For each phenotype there are three biological replicates (i, ii, iii).
b
The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA. A ratio of
~1.8 is consider
ed as “pure” for DNA; If the ratio is appreciably lower it may indicate the
presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.
c
This ratio is used as a secondary measure of nucleic acid purity. The 260/230 values fo
r “pure”
nucleic acid are commonly in the range of 2.0
-
2.2. If the ratio is appreciably lower than
expected, it may indicate the presence of contaminants which absorb at 230 nm.
S
5
Figure S1
.
Cytotoxicity of a
therapeutic
in 27 colorectal cancer cell
lines. A) Dose response
curves of cisplatin in MMR
and MMR+ CRC cell lines. B)
Comparison of the IC50 values of
RhPPO
in MMR+ (MSS) and MMR
(MSI
)
cell lines shown as boxplots. The average IC50 of
RhPPO
in MMR+ cells is 3.22
μ
M and the median is 1.37
μ
M.
The average IC50 of
RhPPO
in
MMR
cells is 2.62
μ
M and the median is 1.28
μ
M. C) Comparison of the IC50 values of
cisplatin in MMR+ and MMR
cell lines shown as boxplots. The average IC50 of cisplatin in
MMR+ cells is 15.65
μ
M and the median is 12.09
μ
M. T
he average IC50 of cisplatin in MMR
cells is 10.38
μ
M and the median is 10.30
μ
M.
D) Comparison of the IC50 values of
RhPPO
broken down based on mutated MMR
(or POLE)
protein (MMR+ = NA). For MLH1 deficient
cells,
the IC50 of
Rh
PPO
is
2.06
μ
M and the medi
an is
0.59
μ
M
.
0
0.2
0.4
0.6
0.8
1
1.2
0.1
1
10
100
Viability
[Cispla-n] (μM)
MMR+
MMR−
A
C
B
D
S
6
Figure S2
:
A correlation between whole cell uptake and IC50 for
RhPPO
. MMR
cell lines are
shown in red and MMR+ are shown in blue, with all cell lines labeled.
S
7
Figure S3.
Scatterplots comparing fluorescence output of
RhCy3
with g
DNA versus the
Log(IC50) of
RhPPO
in various cell lines. A) shows all data and has a Pearson’s r =
-
0.52,
making it significant at the p < 0.2 level. B) By removing the possible outlier, DU145, the
Pearson’s r increases to
-
0.81, which is significant at th
e p < 0.05 level. C) When comparing only
MMR+ ce
ll lines and MMR
cell lines with mutations in the MLH1 gene, r =
-
0.47 with
significance at the p < 0.35 level. D) When comparing only MMR+ cell lines and MMR
cell
lines that are not mutated in PMS2, r increases to
-
0.87 with significance at the p < 0.05
level.
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
Log(IC50)
I/
I
0
at 600
μM
gDNA
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
Log(IC50)
I/
I
0
at 600
μM
gDNA
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
Log(IC50)
I/
I
0
at 600
μM
gDNA
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
Log(IC50)
I/
I
0
at 600
μM
gDNA
0
1
2
3
4
5
6
7
0
100
200
300
400
500
600
I/I
0
[gDNA]
μ
M
DU-145
AN3-CA
HCT116O
HCT116
DLD-1
HEC-1A
SW480
HCT116N
r = -0.52
p < 0.20
r = -0.81
p < 0.05
r = -0.47
p < 0.35
r = -0.87
p < 0.05
A
C
B
D
S
8
Supplemental Discussion on RhCy3 as a reporter on gDNA lesions
There is a clear relationship between the identity of the deficient MMR protein and
RhCy3
fluorescence output. Titrations of
RhCy3
with genomic DNA extracted from cell lines
with a
deficiency in the MLH1 protein (i.e. HCT116O, AN3
-
CA, DU
-
145, and HCT116)
resulted in the highest fluorescence intensities, indicating there are an abundance of mismatches,
indels, and/or abasic sites present in these cell lines. The final two MMR
cell li
nes, DLD
-
1 and
HEC
-
1
-
A, display low fluorescence intensity that is comparable to the intensity observed for the
MMR+ cell lines, HCT116N and SW480. It may be possible to explain the low intensity
observed with DLD
-
1 and HEC
-
1
-
A by considering their specifi
c MMR
-
deficiencies: MSH6 and
PMS2, respectively. Functioning MMR generally involves two heterodimers, MutS
α
(MSH2 +
MSH6) and MutL
α
(MLH1 + PMS2), however other homologues to these heterodimers also
exist: MutS
β
(MSH2 + MSH3), MutL
β
(MLH1 + MLH2), and MutL
γ
(MLH1 + MLH3).
[6]
These
different MutS and MutL homologues have different roles in the cell, with MutS
α
and MutL
α
correcting mismatches and some indels, and MutS
β
, MutL
β
, and MutL
γ
contributing to the
correction of long and short indels but not mismatche
s. Therefore, a cell line deficient in MSH2
or MLH1 cannot correct any mismatches or indels, but a cell line deficient in MSH6 or PMS2
may still be able to correct some indels via MutS
β
, MutL
β
, and MutL
γ
homologues. Considering
this, the fluorescence of DL
D
-
1 and HEC
-
1
-
A may be relatively low because these cell lines
have MMR machinery that can correct some indels, meaning they will have fewer total lesions
than MLH1
-
deficient
cells that correct neither mismatches nor indels. It is also of note that the
bas
eline fluorescence in the presence of DNA from MMR+ cells and the variations between
DNA from different MMR
cells may be due to
RhCy3
binding abasic sites or other
thermodynamically destabilized lesions that are not associated with MMR pathways.
Overall
R
h
Cy3
shows great promise as a direct reporter on the number of thermodynamically
destabilized lesions in gDNA, and it may also have the potential to be developed into an early
diagnostic reporter of MMR
-
deficiencies in cells.
S
9
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