The Xpc gene markedly affects cell survival in mouse bone marrow.
ABSTRACT The XPC protein (encoded by the xeroderma pigmentosum Xpc gene) is a key DNA damage recognition factor that is required for global genomic nucleotide excision repair (G-NER). In contrast to transcription-coupled nucleotide excision repair (TC-NER), XPC and G-NER have been reported to contribute only modestly to cell survival after DNA damage. Previous studies were conducted using fibroblasts of human or mouse origin. Since the advent of Xpc-/- mice, no study has focused on the bone marrow of these mice. We used carboplatin to induce DNA damage in Xpc-/- and strain-matched wild-type mice. Using several independent methods, Xpc-/- bone marrow was approximately 10-fold more sensitive to carboplatin than the wild type. Importantly, 12/20 Xpc-/- mice died while 0/20 wild-type mice died. We conclude that G-NER, and XPC specifically, can contribute substantially to cell survival. The data are important in the context of cancer chemotherapy, where Xpc gene status and G-NER may be determinants of response to DNA-damaging agents including carboplatin. Additionally, altered cell cycles and altered DNA damage signalling may contribute to the cell survival end point.
- SourceAvailable from: anticancer-research.org[Show abstract] [Hide abstract]
ABSTRACT: Recent laboratory and clinical studies have utilized selenium in the form of pure seleno-L-methionine (SeMet) in combination with DNA-damaging cancer chemotherapy drugs. In mice, the selenium protected bone marrow and other tissues from dose-limiting toxicity. In fact, because of the protection from dose-limiting toxicity, a doubling or even tripling of the maximum tolerated dose (MTD) was enabled. Previously we showed that SeMet protects bone marrow by a DNA repair mechanism that requires the XPC DNA repair protein. XPC is rate-limiting and is required for repair of cisplatin or carboplatin adducts. Herein we used a mouse strain that carries a lambda phage reporter gene in the genome that serves as a mutagenesis target. SeMet protects from carboplatin mutagenesis in mouse bone marrow. Methylseleninic acid (MSA), a metabolite of SeMet, also protected against mutagenesis in mouse bone marrow.Anticancer research 02/2010; 30(2):291-3. · 1.71 Impact Factor
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ABSTRACT: Organic selenium compounds are known to prevent certain cancers although mechanisms may be complex. A widely-held view is that selenium compounds can induce apoptosis in cancer cells, or more precisely, in aberrant cells that are undergoing clonal evolution somewhere along the carcinogenesis process. There are at least 20 different selenium compounds, inorganic as well as organic, that have been used in various published studies. Extrapolation between studies should therefore be undertaken with caution. Similarly, it will be important to ascertain the physiological relevance of the selenium concentrations used in some studies. While cancer prevention by selenium is well-established, recently, organic selenium in the form of pure seleno-L-methionine (SeMet) has been used in combination with cancer chemotherapy drugs. SeMet can induce a DNA repair response in some cell types including bone marrow. Cancer cells generally lack a SeMet-inducible DNA repair response. Thus, SeMet appears to selectively regulate a DNA repair pathway and thereby potentially alter responses to cancer chemotherapy drugs. The specific pathway implicated, nucleotide excision DNA repair (NER) is required for repair of cisplatin or carboplatin DNA damage relevant to chemotherapy. Moreover, some studies have implicated NER as a factor in carcinogenesis processes. Thus, the capacity of SeMet to selectively regulate NER may prove useful in both therapeutic and preventive contexts.Molecular and Cellular Pharmacology 01/2009; 1(4):218-221.
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ABSTRACT: DNA is subject to various types of damage that can impair cellular function or cause cell death. DNA damage blocks normal cellular processes such as replication and transcription and can have catastrophic consequences for the cell and for the organism. It has long been thought that the G1/S cell cycle checkpoint allows time for DNA repair by delaying S-phase entry. The p53 tumor suppressor pathway regulates the G1/S checkpoint by regulating the cyclin-dependent kinase inhibitor p21Waf1/Cip1, but p53 also regulates the nucleotide excision DNA repair protein XPC. Here, using p53-null cell lines we show that additional mechanisms stabilize XPC protein and promote NER in concert with the G1/S checkpoint. At least one mechanism to stabilize and destabilize XPC involves ubiquitin-mediated degradation of XPC, as the ubiquitin ligase inhibitor MG-132 blocked XPC degradation. The retinoblastoma protein, RB, in its unphosphorylated form actually stabilized XPC and promoted NER as measured by host-cell reactivation experiments. The data suggest that XPC protein and XPC-mediated NER is tightly linked to the G1/S checkpoint even in cells lacking functional p53. Indiana University-Purdue University Indianapolis (IUPUI)
Mutagenesis vol. 24 no. 4 pp. 309–316, 2009
Advance Access Publication 16 April 2009
The Xpc gene markedly affects cell survival in mouse bone marrow
Joshua L. Fischer, M.A. Suresh Kumar, Travis W. Day1,
Tabitha M. Hardy, Shari Hamilton2, Cynthia Besch-
Williford2, Ahmad R. Safa1, Karen E. Pollok3and Martin
Department of Microbiology and Walther Oncology Center, Indiana University
Simon Cancer Center and Walther Cancer Institute,1Department of
Pharmacology, Indiana University Simon Cancer Center, Indiana University
School of Medicine, Indianapolis, IN 46202, USA,2Department of Veterinary
Pathology and Research Animal Diagnostic Laboratory, University of
Missouri-Columbia, Columbia, MO 65211, USA and3Herman B. Wells Center
for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN
The XPC protein (encoded by the xeroderma pigmentosum
Xpc gene) is a key DNA damage recognition factor that is
required for global genomic nucleotide excision repair (G-
NER). In contrast to transcription-coupled nucleotide
excision repair (TC-NER), XPC and G-NER have been
reported to contribute only modestly to cell survival after
DNA damage. Previous studies were conducted using
fibroblasts of human or mouse origin. Since the advent of
Xpc2/2 mice, no study has focused on the bone marrow of
these mice. We used carboplatin to induce DNA damage in
Xpc2/2 and strain-matched wild-type mice. Using several
independent methods, Xpc2/2 bone marrow was ?10-fold
more sensitive to carboplatin than the wild type. Impor-
tantly, 12/20 Xpc2/2 mice died while 0/20 wild-type mice
died. We conclude that G-NER, and XPC specifically, can
contribute substantially to cell survival. The data are
important in the context of cancer chemotherapy, where
Xpc gene status and G-NER may be determinants of
response to DNA-damaging agents including carboplatin.
Additionally, altered cell cycles and altered DNA damage
signalling may contribute to the cell survival end point.
The xeroderma pigmentosum Xpc gene is defective in a subset
of human patients exhibiting the cancer-prone disease xero-
derma pigmentosum which results from defective nucleotide
excision DNA repair (NER). XPC patients are sensitive to
sunlight and ultraviolet (UV) radiation-induced DNA damage
and skin cancers. They also exhibit internal cancers with
advanced age. The gene products encoded by Xpc and other
XP genes A-G have been characterized biochemically (1).
Specifically, the XPC protein (encoded by the xeroderma
pigmentosum Xpc gene) is required and is rate limiting for
global genomic nucleotide excision repair (G-NER). XPC is
neither required nor apparently involved in transcription-
coupled nucleotide excision repair (TC-NER). Thus, studies
of Xpc are good models for G-NER separate from TC-NER (1).
Mice lacking Xpc genes (Xpc?/? mice) were generated
some 10 years ago (2). A number of studies of carcinogenesis
and mutagenesis have been conducted, consistent with the
cancer-prone human genetic disease discussed above (2–4).
This is the first study to examine bone marrow in Xpc?/? mice.
Bone marrow is often dose limiting in response to cancer
chemotherapy drugs including carboplatin. While cell survival
is a complex end point, the goal of chemotherapy is to sensitize
can protect bone marrow are important adjuncts to chemother-
apy. Presently, the cytokine granulocyte macrophage colony
stimulating factor is administered which causes proliferation of
bone marrow myeloid stem/progenitor cells and thus repopu-
lates bone marrow after chemotherapy (5). The idea of G-NER
as a protective mechanism in bone marrow is novel. Xpc?/?
mouse bone marrow was highly sensitive to carboplatin.
Patients carrying mutant XPC genes may exhibit an adverse
bone marrow response to carboplatin.
Materials and methods
Mice originated from Sands et al. (2) and were purchased from Taconic Farms
and bred at Indiana University under licence agreement as B6;129s7-XPCtml/Brd
mice. Female mice were 10 weeks old at the time of initiating the experiments.
Bone marrow was directly harvested and cultured for 24–72 h for indicated in
vitro experiments (Figures 1–3). For in vivo experiments (Figures 4 and 5),
carboplatin (Sigma, St Louis, MI, USA) was dissolved in sterile saline and
administered intra-peritoneally at weekly intervals over 6 weeks as indicated in
the figure (0.5-ml injections, 60 or 100 mg/kg body weight). White blood cell
(WBC) counts were by weekly tail vein blood collection in ethylenediaminete-
traacetic acid-treated haematocrit tubes (6). Counting was done using a Hemavet
950 (Drew Scientific, Dallas, TX). Statistical analysis was conducted using a t-
test using Microsoft Office 2003 or GraphPad Prism software. All mouse
treatments were in accord with protocols approved by the Institutional Animal
Care and Use Committee at Indiana University. Mice were sacrificed after 41
days using approved methods. Mice that appeared moribund before 41 days
were sacrificed by veterinary staff.
DNA repair assay
To confirm the DNA repair defect in Xpc?/? bone marrow, we used an
immunoassay in which genomic DNA from UV-irradiated bone marrow cells,
at 0, 4, 8 or 16 h after UV irradiation, was fixed to 96-well microtiter plates at
10 ng per well. Cells were washed and re-suspended in 2-ml phosphate-
buffered saline and irradiated as a thin monolayer in 60-cm2dishes, then placed
back in culture medium. An antibody to 6-4 photoproducts (Trevigen Inc.,
Gaithersburg, MD) was used to detect removal of lesions. Immunoassays were
developed with peroxidase secondary antibody and 2,2#-azino-bis 3-ethyl-
benzthiazoline-6-sulphonic acid chromogenic substrate and read at 405 nm in
a Tecan Spectra plate reader. Immunoblotting of Xpc was with a rabbit
polyclonal antibody (Santa Cruz Biotech, Santa Curz, CA).
*To whom correspondence should be addressed. Indiana University Simon Cancer Center, 1044 West Walnut Street, Room 155, Indiana University School of
Medicine, Indianapolis, IN 46202, USA. Tel: þ1-317-278-0225; Fax: þ1-317-278-3331; Email: email@example.com
? The Author 2009. Published by Oxford University Press. All rights reserved.
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Cell cycle analysis
Bone marrow of wild-type and Xpc?/? genotypes were cultured in Iscove’s
modified Dulbecco medium (IMDM)-containing cytokines as above for 48 h to
stimulate proliferation and then treated with carboplatin for an additional 15 h.
Untreated cultures served as controls for each genotype. Cells were fixed in 70%
(PI) content. To analyse only proliferating cells, 10-lM bromodeoxyuridine
(BrdU) was added to the cultures during the 48-h growth period. Cycling cells
were first gated for BrdU using a fluorescein isothiocyanate-conjugated mouse
monoclonal antibody to BrdU (Becton-Dickinson, Franklin Lakes, NJ, USA)
and then with PI. The data represent ?15 000 cells per data point.
For lineage marker analysis, antibodies to CD4, CD8, B220, Gr-1, lin, c-kit
and Sca-1 were used in accord with manufacturer protocols (Becton-
Dickinson). Samples were analysed on a Becton-Dickinson FACScalibur
using CellQuest software, scoring at least 10 000 events per sample.
Immunoblotting of cell cycle proteins
Initially whole-cell lysates were used to probe for differences in cell cycle
proteins between wild-type and Xpc?/? bone marrow. Given apparent
differences in Cdt1 and Cul4a, we purified total ubiquitinated proteins on
a ubiquitin-binding resin (Pierce Chemical, Rockford, IL). Cell lysates
corresponding to 107viable cells per sample were prepared in RIPA lysis
buffer to which 50 ll of resin was added and rotated overnight at 4?C. The resin
was collected by centrifugation and then boiled 15 min in sodium dodecyl
sulphate/gel-loading buffer and subject to electrophoresis in 4–20% poly-
acrylamide gels (Invitrogen, Carlsbad, CA). Immunodetection was on
nitrocellulose membranes using rabbit anti-Cul4a (34897; Abcam, Cambridge,
MA) or rabbit anti-Cdt1 (sc-28262; Santa Cruz Biotech).
Human H1299 lung cancer cell line was transfected using a FuGene
(Boehringer-Mannheim, Indianapolis, IN) and a short hairpin RNA (shRNA)
served as a control (Origene). Cells were analysed by immunoblotting after 72 h.
Femurs were fixed in 10% buffered formalin overnight, then paraffin embedded
and thin sectioned for histological examination by a veterinary pathologist.
Slides were stained with haematoxylin and eosin for morphological analysis or
4#,6-diamidino-2-phenylindole to visualize intact nuclear DNA. Slides were
visualized by a Nikon HB-10101AF fluorescence microscope using a ?100
objective. Digital photographs were captured.
Bone marrow was collected from femurs, fibulas and iliac crests. Total bone
marrow cells were then added to complete methylcellulose medium consisting
of IMDM liquid medium (Sigma), 20% foetal bovine serum, interleukin-6 (200
U/ml) and stem-cell factor 100 ng/ml (Stem Cell Technologies, Vancouver,
Canada). Triplicate 35-mm culture dishes were seeded with 105cells and
placed in a humidified incubator at 37?C for 10 days at which time colonies
were counted manually as in ref. (7).
MTS assay of cell viability
Bone marrow of wild-type and Xpc?/? genotypes was cultured in 96-well
plates in IMDM medium with cytokines as above and then treated in vitro with
increasing concentrations of carboplatin for 2 h. Cell yield was determined after
72 h in culture by adding MTS (Promega, Madison, WI) to the wells and
reading the absorbance at 490 nm in a Tecan Spectra plate reader. Cells that did
not receive carboplatin served as controls for each genotype representing 100%
The data show a pronounced effect on mouse bone marrow
owing to the presence or absence of a functional XPC gene
product. The first observation was that the number of viable
cells recovered from untreated Xpc?/? bone marrow was
always 2- to 3-fold lower than wild type. To remedy this
problem, we normalized the number of viable cells for both
genotypes for the cell culture experiments. Cell numbers were
normalized typically to 107cells representing pooled cells of
three mice of each genotype. The normalized cell populations
then were used as a source of cells for tissue culture
experiments. Flow cytometric analysis was conducted both
immediately after harvest and after tissue culture experiments,
to ascertain if any one cell lineage was affected in the Xpc?/?
mice. As shown in Figure 1A, no single lineage was uniquely
affected in Xpc?/? mice. Populations representing lineage
markers Gr-1þ, B220þ, Lin?, CD4þ, CD8þ and the triply
gated Lin?/Sca1þ/c-kitþ population were not significantly
different between wild-type and Xpc?/? mice when compar-
ing equal numbers of cells of both genotypes (Figure 1A). The
relative distribution of the lineage markers did not change over
the course of cell culture experiments (results not shown),
although the cell culture experiments were typically conducted
within 48 h and were therefore short-term cultures.
Mice of wild type and Xpc?/? genotypes were assayed for
XPC expression and G-NER activity. Bone marrow was used
directly for western blotting using an XPC antibody. Bone
Fig. 1. G-NER defect in cultured Xpc?/? bone marrow. (A) For cell culture
experiments (Figures 1–3) bone marrow was harvested from untreated wild-
type or Xpc?/? mice. Cell yields were lower in Xpc?/? compared to wild
type; therefore, cell numbers were normalized to 107viable cells per genotype.
Flow cytometry was conducted using six different lineage markers. No
significant differences were observed in the lineage markers in comparing 107
cells of each genotype. As shown in the pie graph, lineage markers tested were
Gr1þ, B220þ, Lin?, CD4þ, CD8þ and the small subpopulation Lin?/
Sca1þ/c-kitþ. The pie segment labelled ‘neg’ was unreactive for any of the
lineage markers tested. (B) XPC protein was undetectable in Xpc?/? bone
marrow (inset). Removal of DNA lesions was markedly slow in Xpc?/? bone
marrow, consistent with the known rate-limiting role of XPC in G-NER.
Pooled bone marrow of three or more untreated 10-week-old mice of
each genotype was used. Cells were cultured in cytokine-containing
medium for 15 h, irradiated with 20 J/m2254 nm UV radiation in phosphate-
buffered saline and returned to tissue culture. Time points were taken at 0, 4, 8
and 16 h and assayed using an antibody to 6-4 photoproducts (P , 0.02
J. L. Fischer et al.
marrow was additionally cultured 24 h, UV irradiated and then
subjected to an immunoassay for removal of DNA damage.
Removal of DNA lesions was 80% complete in wild-type mice
after 16 h, while Xpc?/? mice were defective in removal of
the lesions (Figure 1B). Thus, the Xpc?/? mice exhibit the
expected defective NER phenotype.
We reasoned that the Xpc?/? DNA repair defect might
affect the cell cycle as a secondary consequence of lack of
lesion removal. A comparison was made between wild type
and Xpc?/? for cell cycle distribution in the presence or
absence of carboplatin. The majority (80%) of bone marrow
cells when freshly harvested were in the G1 phase of the cell
cycle irrespective of genotype (Figure 2A). By PI staining
alone, no cell cycle defect of Xpc?/? bone marrow or
difference from wild type was observed in any cell cycle phase
(Figure 2A). To further analyse the cell cycle, we grew the cells
for 48 h in the presence of BrdU, thereby labelling proliferating
cells. Cells were then treated with 10-lM carboplatin for 15 h,
fixed and stained for PI. Cells were gated for BrdU positivity
using a fluorescein-labelled antibody and then for PI staining.
A modest but significant decrease in the G1 phase was
observed in Xpc?/? compared to wild type (Figure 2B). A G2
arrest by carboplatin was observed in both genotypes, which
was more pronounced in Xpc?/? than in wild type (Figure 2B).
The flow cytometric profiles corresponding to the bar graph in
Figure 2B are shown in Figure 2C. The intent of the cell cycle
experiments was to reveal cell cycle differences between wild-
type and Xpc?/? bone marrow, while minimally causing
apoptosis or other cell death. However, use of a higher
concentration of carboplatin revealed a G2 population of 8% in
the wild type and 26% in the Xpc?/?. Moreover, the sub-G1
apoptotic populations were 21% for the wild type and 38% for
Xpc?/?. Thus, G2 arrest may precede apoptosis, both of
which were more pronounced in the Xpc?/? mutant bone
marrow compared to wild type (Figure 2C).
We examined several key cell cycle regulatory proteins for
differences between Xpc?/? and wild type, including Rb,
cyclin D1 and cyclin E, which were unremarkable (results not
shown). Our analyses by western blotting would reveal not
only protein steady-state levels but also phosphorylation and/or
Fig. 2. Evidence of G1 and G2 cell cycle checkpoint alterations in cultured Xpc?/? bone marrow. (A) Bone marrow harvested from untreated 10-week-old mice
was cultured in cytokine-containing medium for 15 h and then treated with 10-lM carboplatin for an additional 24 h. Cells were fixed in 70% ethanol and analysed
by PI staining. By PI staining alone, cell cycle differences were not significant and 68–80% of cells were in G1. (B) Bone marrow harvested from untreated 10-
week-old mice was stimulated with cytokines and cultured for 48 h in the presence of 10-lM BrdU to label proliferating cells, then treated with 10-lM carboplatin
for 15 h and then fixed and stained with PI. A fluorescein isothiocyanate-conjugated antibody to BrdU was used to gate the BrdU-labelled population, which were
then assayed for PI content. Values for carboplatin-treated bone marrow were divided by values for untreated bone marrow conducted side by side. The data
represent three pooled mice of each genotype. Two separate experiments yielded similar results. A modest but significant decrease in G1 population was observed in
Xpc?/? mice compared to wild-type mice (P , 0.05 by t-test). A significant increase was observed in the G2 population in Xpc?/? mice compared to wild-type
mice (P , 0.02 by t-test). The plot shows relative cell cycle distribution after carboplatin treatment. (C) Raw flow cytometric data corresponding to the bar graph
shown in panel (B). Cell number is plotted on the y-axis, at least 15 000 events per sample; PI staining is shown on the x-axis. Use of a higher concentration of
carboplatin (40 lM) revealed a sub-G1 apoptotic population which was more pronounced in the Xpc?/? mutant bone marrow. The G2 population was also more
pronounced in the mutant at the higher carboplatin concentration.
Xpc and cell survival
Fig. 3. (A) CUL4A and CDT1 cell cycle checkpoint proteins in cultured wild-type and Xpc?/? bone marrow. We wanted to determine if the presence or absence of
XPC would alter DNA damage signalling. Immunoblots were conducted using 50 lg of total cell lysates (lanes 1 and 2, upper and lower panels). Equal amounts of
cellular proteins, 5 mg, were then affinity purified on a ubiquitin-binding resin and immunoblotting of bound proteins was conducted. The higher molecular weight
ubiquitinated forms of CUL4A (upper panel) and CDT1 (lower panel) clearly differ between the two genotypes (lanes 3 and 4 of upper and lower panels). The data
suggest a defect in CUL4A and CDT1 ubiquitin modification in Xpc?/? mice. To help identify ubiquitinated CUL4A and CDT1, we used in vitro ubiquitin-
conjugated proteins. Omission of ubiquitin from the reaction shows that mainly ubiquitinated CUL4A is detected by the CUL4A antibody (lanes 5 and 6, upper
panel). Plasmid-encoded CDT1 protein was also used as a marker (lanes 5 and 6, lower panel). (B) Knocking down XPC in H1299 cells alters the ubiquitin
modification of CDT1. We used transiently transfected H1299 cells expressing an shRNA to XPC to test if the results shown in panel (A) were due directly to loss of
XPC. Although Xpc was not completely silenced, it was clearly decreased by the shRNA. Higher molecular weight ubiquitinated forms of CDT1 were largely
absent where XPC was knocked down, consistent with a role for XPC in DNA damage signalling to the CDT1 cell cycle checkpoint protein.
Fig. 4. Effect of carboplatin administration in vivo in bone marrow of wild-type and Xpc?/? mice. (A) Kaplan–Meier plots of mouse survival. Twenty mice of each
genotype received carboplatin or saline only as controls. Saline-only did not affect mouse survival nor alter WBC counts; saline-only control groups are not shown
in order to simplify the figure. Mice received carboplatin at weekly intervals up until day 27. By day 41, 12/20 Xpc?/? mice died, while 0/10 wild-type mice died.
(B) WBC counts were monitored during the course of the experiments. WBC counts were not significantly different prior to day 27, but were significantly different
after day 27 (P , 0.05, lower panel). Each circle represents an individual mouse, black, wild type; red, Xpc?/?. The horizontal bars represent the mean of each
group. By day 41, the few surviving Xpc?/? mice had significantly lower WBC counts (P , 0.05 by t-test). The carboplatin dosing schedule is shown in black,
wild type; red, Xpc?/?. Note that wild-type mice received an additional dose on day 27 that was not administered to Xpc?/? mice. (C) Histological evaluation of
wild-type and xpc?/? mice in carboplatin-treated and saline-control groups, day 41. Haematoxylin–eosin staining of formalin-fixed femur sections. Marked
hypocellularity was observed in Xpc?/? mice receiving carboplatin (upper panel); 4#,6-diamidino-2-phenylindole staining (lower panel). The data are
representative of at least three mice of each treatment group and genotype.
J. L. Fischer et al.
other modifications that might be affected as a secondary
consequence of lack of lesion removal in the Xpc?/? mutant.
Interesting results were obtained for CUL4A and CDT1 cell
cycle checkpoint proteins, which are involved in DNA damage
signalling. The rationale for studying CUL4A and CDT1 was
that XPC is a key substrate for CUL4A after DNA damage;
thus, in the absence of XPC, other downstream targets
including CDT1 might be either exaggerated or attenuated.
Higher molecular weight forms of CUL4A and CDT1 were
largely absent in Xpc?/? bone marrow suggesting that Xpc
may play a role in the DNA damage signalling mechanism
(Figure 3A). The differences in higher molecular weight
CUL4a and CDT1 were observed in untreated, freshly
harvested bone marrow. Both CUL4A and its downstream
substrate CDT1 are ubiquitinated as evidenced by binding to
a ubiquitin-binding resin (Figure 3A), although we do not
exclude other possible post-translational modifications that
may occur as well. The differences are not merely reflective of
cell cycle differences, as cell cycle differences in untreated
cells are not significant (Figure 2A). The differences are not
merely reflective of bone marrow cell subpopulations, which
could theoretically differ between wild type and Xpc?/?. No
significant differences were observed between wild type and
Xpc?/? for any of the six lineage markers shown in Figure 1A.
Fig. 4. (Continued)
Xpc and cell survival
Thus, the apparent defective ubiquitination of CUL4A and its
downstream substrate CDT1 appears to be a characteristic of
Xpc?/? bone marrow. We used transiently transfected H1299
cells to test if knocking down Xpc would mirror the mouse
bone marrow findings. H1299 cells in which Xpc was knocked
down by an shRNA plasmid showed a decrease in ubiquiti-
nated CDT1, similar to that observed in the bone marrow
(Figure 3B). XPC may therefore affect DNA damage signalling
to the cell cycle checkpoint protein CDT1, although further
studies are needed.
Having conducted cell culture experiments to characterize
Xpc?/? bone marrow (Figures 1–3), we conducted experi-
ments to address carboplatin sensitivity in vivo. Twenty mice of
each genotype were divided into four treatment groups and
administered carboplatin at weekly intervals. Mice were 10
weeks old at the time of initiating the experiment. By day 41 of
carboplatin regimen, 12/20 Xpc?/? mice had died unexpect-
edly. No deaths were observed in wild-type mice receiving
carboplatin (Figure 4A). No deaths were observed in saline-
only control groups of either genotype (results not shown).
During the course of carboplatin treatments, peripheral WBC
counts were measured weekly (6). Peripheral WBC counts in
Xpc?/? mice were significantly lower than in wild-type mice
(Figure 4B). Remaining mice were sacrificed on or after day 41
using approved protocols. Mice were evaluated by a veterinary
pathologist. The most significant pathology was marked
hypocellularity in bone marrow in 10/10 Xpc?/? mice
examined by the pathologist (Figure 4C). Brain lesions were
observed in ?50% of the mice which, however, occurred with
equal frequency in wild type and Xpc?/? (results not shown).
No significant lesions were found in cecum, colon, duodenum,
heart, ileum, jejunum, kidney, liver, lymph nodes, spleen,
stomach or thymus. Perivascular lymphoid infiltrates were
observed in lung in both genotypes (results not shown).
To quantify and further characterize the hypocellularity in
Xpc?/? bone marrow, we conducted colony-forming assays
and cell viability assays using a vital dye. A hallmark of
myeloid stem/progenitor cell populations is their colony-
forming ability when placed in culture and stimulated with
cytokines. Colony-forming assays of bone marrow were
conducted as in ref. (7). The data were plotted as total colonies
per femur. In the absence of carboplatin, Xpc?/? mice
exhibited a 3-fold decrease in colony-forming units compared
to wild type (Figure 5A). With carboplatin, the difference was
10- to 12-fold (Figure 5A). We also used the vital stain
thiazolyl blue to measure cell viability after 4 days in culture.
Using this method, carboplatin concentrations corresponding to
50% cell yield (known as IC50dose) was approximately 10-
fold lower in Xpc?/? compared to wild type (Figure 5B). It is
likely that a higher fraction of apoptosis contributed to
decreased cell yield in Xpc?/? bone marrow, evidenced by
the sub-G1 (apoptotic) fractions shown in Figure 2C. We also
conducted experiments utilizing annexin V, an indicator of
apoptosis that is assayed by flow cytometry. The Xpc?/? bone
marrow showed a higher apoptotic fraction by annexin V
staining (results not shown), although neither of these findings
of apoptosis can exclude other mechanisms such as irreversible
cell cycle arrests that may also contribute to overall cell yield.
Untreated Xpc?/? mice always yielded 2- to 3-fold fewer
cells per femur compared to wild type. We used equal numbers
of viable cells for cell culture experiments shown in Figures
1–3, while Figures 4 and 5 reflect the difference in overall bone
marrow yield between wild type and Xpc?/?. All six lineage
markers were decreased in proportion to total cell yield in
Xpc?/? as compared to wild type, i.e. no single lineage was
uniquely affected. Table I shows raw cell numbers of distinct
lineages, per femur, recovered from bone marrow of wild type
and Xpc?/? genotypes after 41 days receiving either saline
only or carboplatin. The Gr-1þ, B220þ, Lin?, CD4þ and
CD8þ lineages differed significantly between wild type and
Xpc?/?, as indicated by P values shown in Table I.
It has long been thought that, in contrast to TC-NER, G-NER
mediated by XPC contributes only modestly to cell survival
after DNA damage (8). Essentially all studies were conducted
in human or mouse fibroblasts which showed 2- to 3-fold
differences in cell survival after DNA damage (8–12). The
present study is the first to examine bone marrow in Xpc?/?
mice. Xpc?/? bone marrow exhibited 10-fold greater
sensitivity to carboplatin compared to strain-matched wild-
type bone marrow (Figures 4 and 5 and Table I). The data
Fig. 5. Quantification of bone marrow hypocellularity in Xpc?/? mice
compared to wild type. (A) Colony-forming assays of bone marrow harvested
from the respective genotypes and carboplatin treatment groups shown in
Figure 4. Bone marrow was harvested and grown in complete methylcellulose
medium containing interleukin-6 and stem cell factor for 10 days. Total
colonies per femur are shown. Xpc?/? bone marrow in the carboplatin-treated
groupwas decreased10- to 12-fold comparedto wild type(P, 0.008by t-test).
Xpc?/? bone marrow in the untreated group was decreased 3-fold compared to
wild type (P , 0.02 by t-test). Each set of experiments utilized bone marrow
from three or more mice of each genotype. (B) Assay of cell yield in bone
marrow cells treated with carboplatin in vitro. Bone marrow of untreated
10-week-old wild-type and Xpc?/? mice was cultured for 24 h and then treated
with indicated concentrations of carboplatin for 2 h. Cell yield after 72 h in
culture is shown (P , 0.006 by t-test). The data shown were averaged from
three experiments. The dose modification factor for 50% cell survival in the
presence of carboplatin is ?10-fold, consistent with the other data.
J. L. Fischer et al.
indicate that the Xpc gene can contribute substantially (10-fold)
to cell survival in bone marrow. The cell survival end point was
biologically relevant as bone marrow myelosuppression was
dose limiting for carboplatin, consistent with other studies (13)
and the majority of Xpc?/? mice died during the course of the
experiments (Figures 4 and 5). No deaths were observed in
wild-type mice irrespective of the carboplatin regimen and/or
duration (Figure 4 and results not shown).
Carboplatin and cisplatin produce a similar DNA damage
spectrum, mainly 1,2 and 1,3 platinum diadducts that are
substrates for NER in vitro (14,15). However, our data do not
exclude that minor lesions such as platinum monoadducts,
interstrand cross-links and/or base damage by reactive oxygen
species may also contribute to the cell survival end point.
Recent studies point to a role for XPC in repair of oxidative
DNA damage (12). A catalytic role for XPC in base-excision
DNA repair (BER) was suggested by the finding that XPC
enhanced the activity of the DNA glycosylase 8-oxoguanine
DNA glycosylate (OGG1) (12). Colony-forming ability of
Xpc?/? bone marrow was decreased 3-fold compared to wild
type even in the absence of carboplatin (Figure 5A). As endo-
genous base damage is removed by the separate BER pathway,
the decreased cell yield in Xpc?/? mice may be due to the XPC
role in BER (12). Interestingly, Xpc?/? fibroblasts exhibited
sensitivity to a 20% oxygen environment compared to wild-type
fibroblasts consistent with a role in base damage repair (11).
Cell survival though is a complex end point that involves
cell cycle effects. We found that Xpc?/? bone marrow cells
were defective in a G1 checkpoints and showed increased
accumulation in G2 compared to wild type (Figure 2). One
possibility is that Xpc?/? bone marrow cells exit the cell cycle
after a prolonged G2 or may be irreversibly arrested in G2
(Figure 2). We show that CUL4A and CDT1 cell cycle proteins
exhibit defective ubiquitination even in untreated Xpc?/?
mice. The data suggest an alteration in DNA damage signalling
in Xpc?/? bone marrow (Figure 3), although further studies
are warranted. Given that these data were obtained in the
absence of carboplatin, it is possible that XPC signalling of
oxidative base damage involves the CUL4A/CDT1 mechanism
(16–18). Thus, XPC may contribute to DNA damage
sensitivity including base damage by (i) controlling the BER
protein OGG (12), (ii) altering cell cycles and (iii) affecting
DNA damage signalling to downstream effectors including
CUL4A and CDT1. Our data pertain to bone marrow; thus, it is
not known if absence of functional XPC would exhibit these
same features in other cell types.
Human patients with xeroderma pigmentosum XPC mutant
alleles typically develop skin cancers well prior to age 20 and
develop internal cancers with ageing. Similarly, Xpc?/? mice
mice as young as possible for the current study. Perhaps human
XPC patients would exhibit myelosuppression were they to live
for Xpc genes, while the human mutant XPC alleles probably
exhibit variable penetrance. One study did show myelosuppres-
sion in an XPC patient (19). Thus, cancer therapeutic regimens
may require modification in the case of XPC patients.
In summary, the Xpc gene product protects mouse bone
marrow from exogenous and probably endogenous DNA
damage with cell survival as an end point. The biological
relevance is indicated by the fact that 12/20 Xpc?/? mice
receiving carboplatin died while 0/20 wild-type mice died. No
deaths were observed in saline-only controls of either
genotype. The proximal cause of death appeared to be bone
marrow failure, although other tissues may have contributed.
The XPC protein may be linked to the CUL4A/CDT1 cell
cycle checkpoint, probably relevant to DNA damage signalling
and may contribute to the cell death mechanism.
National Institutes of Health (R01 HL086978) to M.L.S.;
American Institute for Cancer Research (04B010) to M.L.S.;
US Department of Defense (BC051172) to J.L.F. T.M.H. was
supported by 1R25 GM079657 training grant to Dr. Hal E.
We thank Mark R. Kelley, Laura Haneline and D. Wade Clapp for comments
on the figures. We thank four anonymous reviewers for their helpful comments.
Conflict of interest statement: None declared.
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Table I. Lineage marker distribution after six weeks carboplatin administration
Genotype WT Xpc?/?
Treatment SalineCarboplatinSaline Carboplatin
aIndicates statistical significance for the comparison of wild type and Xpc?/? for carboplatin treatment groups; values represent the mean and standard deviation of
three mice per group.
P , 0.005a
P , 0.005a
P , 0.008a
P , 0.05a
P , 0.05a
5.7 (?0.3) ? 106
1.5 (?0.2) ? 106
3.0 (?0.5) ? 105
2.1 (?0.4) ? 105
3.3 (?0.5) ? 105
2.0 (?0.4) ? 104
1.6 (?0.1) ? 106
6.0 (?1.5) ? 105
0.8 (?0.2) ? 105
1.6 (?0.2) ? 105
1.3 (?0.1) ? 105
2.1 (?0.5) ? 104
3.5 (?0.5) ? 106
7.4 (?0.3) ? 105
1.8 (?0.3) ? 105
1.4 (?0.2) ? 105
1.9 (?0.2) ? 105
3.0 (?0.5) ? 104
0.3 (?0.1) ? 106
1.5 (?0.1) ? 105
0.3 (?0.1) ? 105
1.2 (?0.7) ? 105
1.0 (?0.1) ? 105
0.3 (?0.1) ? 104
Xpc and cell survival
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Received on January 27, 2009; revised on March 6, 2009;
accepted on March 21, 2009
J. L. Fischer et al.