c-myb Heterozygous Mice Are Hypersensitive to 5 ... · c-myb Heterozygous Mice Are Hypersensitive...
Transcript of c-myb Heterozygous Mice Are Hypersensitive to 5 ... · c-myb Heterozygous Mice Are Hypersensitive...
c-myb Heterozygous Mice Are Hypersensitive to5-Fluorouracil and Ionizing Radiation
Robert G. Ramsay,1 Suzanne Micallef,2 Sally Lightowler,1 Michael L. Mucenski,3
Theo Mantamadiotis,1 and Ivan Bertoncello1
1Peter MacCallum Cancer Centre, East Melbourne, Australia; 2Institute for Reproduction and Development,Monash Medical Centre, Clayton, Australia; and 3Division of Pulmonary Biology, Children’s Hospital Medical Centre,Cincinnati, Ohio
AbstractHypersensitivity to chemo- and radiotherapy
employed during cancer treatment complicates patient
management. Identifying mutations in genes that
compromise tissue recovery would rationalize treatment
and may spare hypersensitive patients undue tissue
damage. Genes that govern stem cell homeostasis,
survival, and progenitor cell maintenance are of
particular interest in this regard. We used wild-type
and c-myb knock-out mice as model systems to explore
stem and progenitor cell numbers and sensitivity to
cytotoxic damage in two radiosensitive tissue
compartments, the bone marrow and colon. Because
c-myb null mice are not viable, we used c-myb
heterozygous mice to test for defects in stem-progenitor
cell pool recovery following ;-radiation and
5-fluorouracil treatment, showing that c-myb+/� mice
are hypersensitive to both agents. While apoptosis is
comparable in mutant and wild-type mice following
radiation exposure, the crypt beds of c-myb+/� mice
are markedly depleted of proliferating cells.
Extrapolating from these data, we speculate that acute
responses to cytotoxic damage in some patients may
also be attributed to compromised c-myb function.
(Mol Cancer Res 2004;2(6):354–61)
IntroductionBone marrow (BM) and the gastrointestinal tract are
continuously renewing tissues containing highly ordered stem
and progenitor cell compartments with tightly controlled pro-
liferation, differentiation, and apoptosis throughout life. Despite
insights gained from analysis of mutant mice with specific
genetic lesions, the molecular control of homeostasis in BM,
and the gastrointestinal tract in particular, is poorly understood.
Analysis of c-myb heterozygous mutant (c-myb�/�) mice
has confirmed that this gene is essential for establishing
definitive hematopoiesis (1) and colonic crypt formation (2).
Whether c-myb contributes to the maintenance of these two
compartments once established in adult animals is unclear.
c-myb has been studied extensively in the hematopoietic
system and blood cell malignancies. It is highly expressed in
immature cells of most lineages and declines as cells differenti-
ate (3). However, the role of c-myb in regulating hematopoiesis
is complex because it seems to be employed and required
at multiple differentiation steps (4-6). The overexpression of
c-myb , or expression of activated forms of Myb that transform
hematopoietic cells, implicates c-myb in hematopoietic malig-
nancies (7-9). Conversely, very fewmature adult-like blood cells
are generated by the c-myb�/� fetal liver (1), and c-myb�/�mice
die at the critical developmental checkpoint when fetal liver
erythropoiesis is established (1, 6).
c-myb is also essential for colon development and its over-
expression modifies normal colon biology. Basic expression
data show that rat, mouse, and human colons have levels of
c-myb mRNA similar to, or exceeding, that observed in thymus
and BM (10-13). Colon cancer cells from humans (14-16) and
rodents (11, 17, 18) frequently overexpress c-myb compared
with normal colon mucosa and differentiation of colon cancer
cells results in down-regulation of c-myb in a fashion com-
parable to that observed with c-myb in differentiating hema-
topoietic cells (10). Importantly, c-Myb increases during colon
(19) and esophageal adenocarcinoma progression (20). In colon
cancer, this increase is important because the level of c-myb
expression has prognostic significance for patient survival (21).
Colonic crypts are exquisite structures that generate a vast
and continuous epithelial surface allowing efficient water and
ion reabsorption. As with other rapidly renewing tissues like the
skin and hematopoietic system, the architectural features and
function of each colonic crypt is maintained by a fine balance of
high proliferation through the provision of progenitor cells that
arise from a single steady-state stem cell at the crypt base; cell
differentiation into columnar, goblet, and endocrine cells as
they migrate away from the base; and apoptosis into the crypt
lumen near or at the top of the crypt. c-Myb expression is
strongest at the crypt base and decreases toward the middle
of the crypt where it partitions to be expressed weakly in
differentiated columnar cells (10, 12). Understanding the regu-
lation of colonic crypt homeostasis is key to unraveling colon
hyperproliferative disorders, initiation of colon cancer, and
recovery of colon integrity following tissue damage. We have
reported that one functional allele of c-myb is required for fetal
crypt formation in the mouse.
Received 1/6/04; revised 5/10/04; accepted 5/19/04.Grant support: National Health and Medical Research Council of Australia.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Requests for reprints: Robert Ramsay, Differentiation and TranscriptionLaboratory, Trescowthick Research Laboratories, Peter MacCallum CancerCentre, Locked Bag #1 A’ Beckett Street, Melbourne 8006, Australia. Phone:61-3-9656-1863; Fax: 61-3-9656-3738. E-mail: [email protected] D 2004 American Association for Cancer Research.
Mol Cancer Res 2004;2(6). June 2004354on July 3, 2020. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Although c-myb haploinsufficiency has little or no demon-
strable effect under steady-state conditions, we reasoned that
cytotoxic challenge might reveal a stress response role for this
gene. Both colonic crypts and BM undergo rapid renewal and
their stem and progenitor cell populations are extraordinarily
sensitive to cytotoxic insult. In the case of BM, we find c-myb
expression in the most immature stem cell population, and
c-myb expression is at its strongest in the bottom third of
the colonic crypt. Thus, such immature cells in these tissues
were the focus of our study. We employed ionizing radiation
and 5-fluorouracil (5-FU) to stress these two highly prolifer-
ative compartments. Ionizing radiation kills both stem cells and
progenitor cells, whereas a single 5-FU treatment preferentially
kills progenitor cells. Such cytotoxicity necessitates the
recruitment of new cells to allow tissue regeneration. Under
these conditions, c-myb+/� mice show significant impairment
in BM and colonic crypt recovery compared with wild-type
controls. Crypt beds are depleted of cells in the c-myb hete-
rozygous mice leading to their progressive disintegration. These
observations suggest that c-myb is required for stem and/or
progenitor cell renewal in BM and colonic crypts.
ResultsCrypt Length and Induced Apoptosis Are Identicalin c-myb+/� and Wild-Type Mice Following IonizingRadiation Treatment
c-myb�/� fetal colon fails to form normal crypts (2) and the
distribution of apoptosis is markedly different in these mutant
transplants (see Discussion). Assessment of the specific
consequences of c-myb loss in adult colon has not been
investigated. Thus, although the hematopoietic (1, 6) and
gastrointestinal tract (data presented below) compartments of
adult c-myb+/� mice seem essentially normal, we speculated
that differences in the regenerative ability of c-myb+/�
hematopoietic stem cells and colonic crypt cells might be
revealed when these mice were challenged by irradiation or
chemotherapeutic drugs.
Wild-type and c-myb+/� mice at 4 hours, 2, 3, and 4 days
post-lethal irradiation were examined. Irradiation resulted
in a precipitous decline in BM cellularity from 21.0 F 1.2
(c-myb+/+) and 19.8F 2.0 (c-myb+/�) nucleated cells per femur,
respectively, at 4 hours to 1.7 F 0.3 (c-myb+/+ ) and 1.4 F 0.2
(c-myb+/�) nucleated cells per femur at day 4 post-irradiation
(data not shown). Although abnormal morphologic changes
were evident by day 3 in c-myb+/� colons, no significant
differences in cell number or apoptosis (Fig. 1A–G) were
observed until day 4.
c-myb Heterozygous Crypts Disintegrate at Day 4Post-Irradiation
Sections from c-myb+/� mice at day 4 were not amenable
to morphometric analysis because the crypt architecture was
disrupted and frequently hypocellular (Fig. 2A). By day 4, the
cycle of radiation-induced apoptosis was minimal as judged by
the absence of pyknotic bodies (see Fig. 1E and G) indicative of
apoptosis (22-24). The disintegration of the normal architecture
(Fig. 2A) that is essentially restored in the wild-type mice
(Fig. 2B) is shown for four heterozygous mice. Higher
magnification of an additional two heterozygous colon sections
(Fig. 2C) reveals either empty crypt beds depleted of cells or
poorly defined crypt structures compared with wild-type colons
(Fig. 2D). This is the location in which stem and progenitor
cells normally reside, suggesting that these populations of cells
have been selectively deleted in the c-myb+/� crypts.
Similar Apoptosis but Markedly Different Proliferation inWild-Type and c-myb+/� Colons
Detecting apoptotic bodies in normal crypts is problematic
because most apoptosis occur at the luminal surface such that
FIGURE 1. Steady-state crypt length and radiation-induced apoptosisis identical in wild-type and c-myb+/� distal colon for the first 3 days. Wild-type (A) and c-myb+/� (B) distal colon. Crypt length reduces markedlyfollowing ionizing radiation treatment shown at day 2 for wild-type (C) andc-myb+/� (D) colonic crypts in part due to the elevated apoptosis at thecrypt base (E) identified by pyknotic densely staining subnuclear bodies(indicated by arrows ; wild-type crypt section shown). F. Average numbersof crypt cells per 50 longitudinal sections for wild-type and c-myb+/� micemeasured at steady state (0), days 2 and 3 post-irradiation are depictedgraphically. Average numbers of apoptosis per crypt were quantified,finding that at 4 hours, post-irradiation induces the maximum frequency ofapoptosis that is comparable in both wildtype and heterozygous colon. G.Columns , mean for five mice at each time point; bars , SE.
c-myb Mice Are Hypersensitive to Cytotoxic Damage
Mol Cancer Res 2004;2(6). June 2004
355
on July 3, 2020. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
dead cells are transported away by peristalsis or engulfed by
macrophages. However, cells in the early stages of apoptosis
can be identified using antibodies directed against activated
caspase-3. Figure 3A and B demonstrate that apoptosis occurs
in both c-myb heterozygous and wild-type crypts in a similar
fashion. Intense staining for pro-apoptotic Bak was also equally
evident in both wild-type and mutant colons (not shown).
Therefore, it seems that enhanced apoptosis at either the crypt
base or luminal surface is not primarily responsible for the
radiosensitivity of the c-myb+/� colons.
Attempts to identify mitotic figures in the recovering crypts
of c-myb+/� mice at day 4 suggested that cell division was not
restored compared with the wild-type colons. To verify this, we
examined a marker of cell proliferation frequently used to
examine gastrointestinal tract cell turn over. PCNA expression
serves as an informative indicator of proliferation in colonic
crypts where its location is normally restricted to the bottom
third of the crypt (12). c-Myb expression is also most intense in
this region, persisting in differentiated columnar cells in mice
and humans (10, 12). In c-myb+/� colon 4 days post-irradiation,
some crypts showed essentially no PCNA staining at the crypt
base, perhaps due to crypt base hypocellularity. PCNA
expression was mostly evident distal to the crypt base
(Fig. 3C and D), suggesting that in c-myb+/� colon, the stem
cell compartment was not replacing progenitor cells as they
differentiate and eventually apoptose.
To characterize the apparent differences in proliferation,
colonic crypts from five wild-type and five heterozygous mice
at day 4 post-irradiation were examined in terms of the crypt
position(s) where PCNA staining occurred. Conceptually, adult
colonic crypts have one steady-state stem cell at the base
located at position 0. With division, daughter cells occupy
positions moving up from the base starting at position 1. The
actual number of functional stem cells is thought to increase
following radiation (25). Figure 4A shows that PCNA staining
in heterozygous crypts is very heterogeneous unlike wild-type
crypts (Fig. 4B). PCNA-positive nuclei in 20 individual crypts
per mouse were scored using the central cell at the crypt base as
the starting position 0. Figure 4B shows the highly significant
difference (P > 0.001, ANOVA) for the frequency of PNCA-
positive nuclei in heterozygous mice at positions 0, 1, 2, and 3.
On average, wild-type crypts showed 2 times or greater number
of positive nuclei at the crypt base. This is most evident at
position 0 where 75% of cells in this position (15 of 20) show
PCNA staining compared with 30% (6 of 20) in heterozygote
crypts. It was also of interest that cells in position 1 showed
significantly less PCNA staining compared with positions 0
(P > 0.02) and 2 (P > 0.002). Distinct differences at position 1
compared with positions 0 and 2 have been observed in other
instances. For example, in mid-colonic crypts at 24 hours post-
irradiation, apoptosis is less at position 1 compared with
position 0 or 2 (26). This cell position may represent the first
and most primitive ‘‘progenitor’’ cell. Collectively, these data
suggest that c-Myb is required to maintain proliferation at the
crypt base and, because stem and progenitor cells reside in this
region, we further suggest that c-Myb is required for progenitor
cell expansion.
5-FU Treatment Reveals a Defect in c-myb HeterozygousBM Stem Cells
Ionizing radiation ablates the vast majority of stem and
progenitor cells at the doses employed in this study, irreversibly
compromising the BM stem cell reserve (17, 27). In com-
parison, 5-FU preferentially ablates proliferating progenitor
cells and spares highly quiescent stem cells with long-term
repopulating ability (LTRA; ref. 28). We have employed the
high proliferation potential-colony forming cell (HPP-CFC)
assay as a surrogate measure of the status of the hematopoietic
stem cell compartment and the low proliferation potential-
colony forming cell (LPP-CFC) assay as a measure of the
committed progenitor cell pool (29). Comparison of the
regenerating BM of 5-FU–treated mice revealed significant
differences in the pattern of recovery for the incidence (Fig. 5C)
and content (Fig. 5D) of high proliferation potential-colony
forming cells (P = 0.005 and P = 0.001, respectively), but not
low proliferation potential-colony forming cells, in c-myb+/�
mice compared with wild-type controls. The incidence and
femoral content of low proliferation potential-colony forming
cell were essentially the same in both wild-type and c-myb
FIGURE 2. Heterozygous mice show colonic crypt disintegration at day4 post-irradiation. A. Low-power fields of four c-myb heterozygous distalcolons at day 4 post-irradiation show aberrant morphology with abnormallevels of cellular debris within the lumen compared with similarly treatedwild-type colons (B). C. High-power fields of additional two mice show thatc-myb+/� crypts were disrupted and hypocellular (indicated by arrows )compared with crypts from two independent wild-type colons (D).
Ramsay et al.
Mol Cancer Res 2004;2(6). June 2004
356
on July 3, 2020. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
heterozygous BM, consistent with the view that c-myb
haploinsufficiency blocks the early recovery or recruitment of
stem cells under such stress conditions (Fig. 5A and B).
Notably by day 5, the c-myb+/� BM high proliferation
potential-colony forming cell numbers return to near normal
levels.
5-FU Treatment Reveals a Defect in c-myb HeterozygousColonic Crypt Recovery
Colonic crypt recovery was also significantly delayed in
5-FU–treated c-myb+/� mice compared with wild-type mice at
days 1 and 5 posttreatment (Fig. 6A) (P = 0.02 and P = 0.033,
respectively). Similarly, there were less apoptotic cells in the
c-myb+/� crypts at day 1 (Fig. 6B) (P = 0.0005), essentially
excluding a primary defect in the degree of apoptosis induction.
As expected, mitosis was severely retarded in crypts of both
genotypes until day 5 (Fig. 6C), suggesting that the difference
in crypt length recovery is not due to a cell division defect
per se in the heterozygous mice but a delay in replenishing
progenitor cells. Indeed both wild-type and heterozygous crypt
length exceeded the steady-state levels at day 5 although the
heterozygotes recovery was delayed.
Collectively, these data suggest that c-myb haploinsuffi-
ciency results in a stem cell defect that impairs mouse BM and
colonic crypt recovery following cytotoxic damage. Ionizing
radiation leads to crypt disintegration, preventing the longer-
term recovery (day 4) of c-myb+/� crypts. Moreover, analysis of
BM and colonic crypt recovery following 5-FU suggests that
the loss of one c-myb+/� allele impedes the replacement of
transit amplifying progenitor cells that by definition are stem
cell derived.
DiscussionThe requirement for c-myb in the stem and progenitor cell
populations of the fetal liver and BM, respectively, is most
evident when c-myb knock-out and knockdown mutant mice
FIGURE 3. Proliferation is defective inirradiated c-myb+/� colonic crypts but apopto-sis is essentially normal. c-myb+/� (A) andwild-type (B) colon sections from day 4 post-irradiated mice were stained for activatedcaspase-3, indicating that the level and distri-bution of residual apoptosis (arrows ) is equiv-alent at this time point. In contrast, proliferationmarker proliferating cell nuclear antigen(PCNA) staining reveals marked differencesbetween the c-myb+/� and c-myb+/+ colons.The characteristic position for PCNA-positivenuclei is at the base of c-myb+/+ crypts (C;located by arrows ), whereas in c-myb+/� cryptsPCNA, staining was scarce in this region orpresent mostly at the middle to top of the crypt(D). Infiltration of macrophage-like cells withcytosol and membrane staining of residualperoxidase is also evident.
c-myb Mice Are Hypersensitive to Cytotoxic Damage
Mol Cancer Res 2004;2(6). June 2004
357
on July 3, 2020. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
are examined (1, 6). Because c-myb knock-out mice are not
viable as adults, we employed wild-type and apparently normal
c-myb heterozygous mice treated with agents that kill stem and
progenitor cells to probe steady-state hematopoiesis is these
animals. Under these conditions, adult c-myb+/� mice show a
defect in stem cell recovery following 5-FU treatment.
At present, there are no ex vivo assays for colonic crypt stem
and progenitor cell activity equivalent to the predictive colony
forming assays used as a surrogate measure of hematopoietic
stem cell activity in BM and fetal liver cells (29). However,
documenting crypt cell numbers, mitosis, and apoptosis by
morphometric assessment of crypts in vivo can be very
informative in cases in which genetic defects influence these
parameters. Both ionizing radiation and 5-FU treatment
revealed significant differences in the recovery and survival
of stem and progenitor cells in the colon of c-myb+/� mice
compared with wild-type controls. This was most evident when
crypts were stained for proliferation antigen, PCNA, in which
irradiated heterozygous crypts showed a substantial deficit in
proliferation.
Therefore, the combined examination of colonic crypt and
BM cells using in vivo and ex vivo assays in c-myb+/� and
wild-type mice has advanced our understanding of the role of
c-myb in stem and progenitor cells, suggesting that diploid
levels of c-myb are required for tissue recovery.
The defect in c-myb+/� mouse colon and BM recovery is
unlikely to be due solely to the absence of the c-Myb target
gene bcl-2 or an increase in induced apoptosis. One report
suggested that a Bcl-2 deficiency led to colon and small
intestinal disorganization (30), whereas others reported a
normal gut phenotype in bcl-2�/� mice with elevated
spontaneous apoptosis that was further increased following
cytotoxic damage (24). In contrast, c-myb+/� colons had normal
steady-state crypt apoptosis and cell death that was either the
same or lower following cytotoxic damage compared with
wild-type colon. This was puzzling because previously we
reported that apoptotic cells were more frequent in colon grafts
from c-myb�/� fetuses (2). We now suggest that this difference
between the knock-out and wild-type colon transplants is due
to the inability of the knock-out grafts to extrude dead cells
because of the lack of defined crypts with internal mucinous
channels that exit into the lumen. In wild-type grafts, apoptotic
bodies are not trapped and thus accumulate at the top of crypts
and are evident because peristalsis does not operate in the graft
tissue.
Some insights into the regulation of stem and progenitor
cell homeostasis have been gained in which c-myb expression
has been reduced. Blocking the characteristic c-myb mRNA
induction observed when resting T cells are stimulated to exit
G0-G1 (31-33) using antisense oligonucleotides suggests that
a threshold level of c-myb is required for this process. This
requirement may also be paralleled for quiescent BM and
colonic crypt stem cells to reenter the cell cycle to give rise to
progenitor cells.
A single 5-FU treatment did not seem to deplete prolifer-
ating progenitor cells to a greater extent in the c-myb+/� mice as
measured directly by BM colony forming assays. However, the
high proliferation potential assay revealed a significant delay
in recovery at day 1 but not the characteristic rebound of
measurable stem cell activity at days 3 and 5 in BM of the
heterozygous mice. These data suggest that the c-myb+/� bone
stem cell population is not inherently more sensitive to the
antimetabolic effects of 5-FU, because this defect would be
evident in the low proliferation potential population as well, but
rather stem cells are slower to reenter into cycle, divide, and
generate cell colonies ex vivo. This may be due to slower
recruitment of cells with ‘‘stem cell’’– like properties that
emerge under stressed conditions, such as 5-FU treatment.
An alternative but not mutually exclusive view is that
diploid levels of c-myb are required to maintain normal
radioresistance and the loss of one copy sensitizes stem cells
in crypts and perhaps BM (although this was not examined).
The crypt data suggest that c-myb+/� stem cells do not recover
at the doses of radiation used in these studies. These questions
will need to be addressed in an in vitro setting in which
FIGURE 4. PCNA staining reveals a deficit of dividing cells in thec-myb heterozygote colons at day 4 post-irradiation. A. Distribution ofPCNA staining in heterozygous crypts suggests less proliferation in thestem and progenitor cell compartment of the crypt that is located at thecrypt base (as indicated by arrows ). B. Quantitation of PCNA-stainednuclei in relation to crypt position shows significant (P > 0.001) lessproliferating cells at positions 0 (center of crypt base; equivalent to thestem cell location in untreated colonic crypts), 1, 2, and 3. Position 1 cellsshowed significantly less staining than position 0 or 2. Twenty crypts permouse were evaluated. Columns , mean for five mice of both genotypes;bars , SE. Asterisks indicate significant difference between wild-type andc-myb+/� samples.
Ramsay et al.
Mol Cancer Res 2004;2(6). June 2004
358
on July 3, 2020. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
epithelial and BM stem and progenitor cells are isolated from
c-myb heterozygous mice. This will assist in avoiding the
potential compounding problems that irradiation of tissue
stroma and microvessels brings to in vivo experiments. In the
case of the c-myb null fetal liver, a defect in the stromal
production of stem cell factor (SCF) plus the reduction of c-kit
on the few evident hematopoietic cells (34) is likely to
contribute to the overall deficit in high proliferation potential
and low proliferation potential in the c-myb null embryo (35).
Perhaps these two target genes are also in haploinsufficiency in
the c-myb+/� BM such that this reduces recovery of stem cells
following 5-FU treatment.
Collectively, these findings have implications that were not
predicted. Extrapolating from the mouse c-myb+/� data it is
tempting to suggest that in humans, impaired c-Myb function
need not be evident under non-stressed conditions but may
manifest severe BM and colon cytotoxicity when challenged
during radio- and/or chemotherapy. Because there are groups of
patients that respond in such a manner during cancer treatment,
adjuvant therapy, and BM depletion before transplantation,
investigating c-Myb function in these individuals should be
considered.
Materials and MethodsMice
c-myb+/� mice have been described previously (1, 2) and
were maintained on a C57Bl/6J background and housed in SPF
Thoren racking system.
BM Colony Forming AssaysColony-forming cells of low and high proliferation potential
were assayed in a double layer nutrient agar culture system as
previously described (36).
Radiation and 5-FU TreatmentsA single dose of lethal whole body irradiation (13 Gy) was
delivered at a rate of 67 cGy/min. Mice were sacrificed at
4 hours, 2, 3, and 4 days post-irradiation. 5-FU (David Bull
Laboratories, Mulgrave, Australia) was made up to 15 mg/mL
in saline, administered to mice (150 mg/kg, i.p.), and tissues
harvested from euthanized mice were analyzed at days 1, 3, 5,
and 7 administration.
Morphometric Analysis and ImmunohistochemistryDistal colon sections were fixed in 4% buffered paraformal-
dehyde overnight, embedded, sectioned, and H&E stained. Full
crypts exposing a lumen and identifiable base were scored. Crypt
cells per 40 to 50 longitudinal sections were scored and mitotic
figures and apoptotic bodies enumerated based on established
morphologic criteria (22-24). Sample groups were subjected to
ANOVA using Origin software. Mouse anti-PCNA antibody,
PC-10 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), was
used at a 1:100 dilution to identify PCNA followed by goat anti-
mouse horseradish peroxidase at 1:250 (Bio-Rad, Hercules,
CA). Anti-activated caspase-3 (Promega, Madison, WI) was
used at 1:500 and detected with goat anti-rabbit horseradish
peroxidase at 1:250 (Bio-Rad). Each was developed with Pierce
Metal enhanced detection reagent (Pierce, Rockford, IL).
FIGURE 5. Bone marrow from c-myb+/� mice shows hypersensitivity to 5-FU. BM cells were plated under conditions that allow colony formation of cellswith low proliferative potential (LPP) and high proliferative potential (HPP ) per 25,000 plated cells (A and C) or per femur (B and D), in the c-myb+/� micecompared with wild-type controls at days 1 to 5. The c-myb+/� recovery of stem cells was significantly impaired compared with the wild-type bone marrowbased on absolute numbers or per femur.
c-myb Mice Are Hypersensitive to Cytotoxic Damage
Mol Cancer Res 2004;2(6). June 2004
359
on July 3, 2020. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
AcknowledgmentsWe thank Dr. Maree Overall and Prof. Patrick Tam for critical comments andsuggestions during the preparation of this manuscript.
References1. Mucenski ML, McLain K, Kier AB, et al. A functional c-myb gene is requiredfor normal murine fetal hepatic hematopoiesis. Cell 1991;65:677-89.
2. Zorbas M, Sicurella C, Bertoncello I, et al. c-Myb is critical for murine colondevelopment. Oncogene 1999;18:5821-30.
3. Weston K. Myb proteins in life, death and differentiation. Curr Opin GenetDev 1998;8:76-81.
4. Allen RD 3rd, Bender TP, Siu G. c-Myb is essential for early T celldevelopment. Genes Dev 1999;13:1073-8.
5. Bender TP, Thompson CB, Kuehl WM. Differential expression of c-mybmRNA in murine B lymphomas by a block to transcription elongation. Science1987;237:1473-6.
6. Emambokus N, Vegiopoulos A, Harman B, Jenkinson E, Anderson G,Frampton J. Progression through key stages of haemopoiesis is dependent ondistinct threshold levels of c-Myb. EMBO J 2003;22:4478-88.
7. Fu SL, Lipsick JS. Constitutive expression of full-length c-Myb transformsavian cells characteristic of both the monocytic and granulocytic lineages. CellGrowth & Differ 1997;8:35-45.
8. Gonda TJ, Ramsay RG, Johnson GR. Murine myeloid cell lines derived byin vitro infection with recombinant c-myb retroviruses express myb fromrearranged vector proviruses. EMBO J 1989;8:1767-75.
9. Gonda TJ, Buckmaster C, Ramsay RG. Activation of c-myb by carboxy-terminal truncation: relationship to transformation of murine haemopoietic cellsin vitro . EMBO J 1989;8:1777-83.
10. Thompson MA, Rosenthal MA, Ellis SL, et al. c-Myb down-regulation isassociated with human colon cell differentiation, apoptosis, and decreased Bcl-2expression. Cancer Res 1998;58:5168-75.
11. Alexander RJ, Buxbaum JN, Raicht RF. Oncogene alterations in rat colontumors induced by N -methyl-N -nitrosourea. Am J Med Sci 1992;303:16-24.
12. Rosenthal MA, Thompson MA, Ellis S, Whitehead RH, Ramsay RG.Colonic expression of c-myb is initiated in utero and continues throughout adultlife. Cell Growth & Differ 1996;7:961-7.
13. Thompson MA, Ramsay RG. Myb: an old oncoprotein with new roles.BioEssays 1995;17:341-50.
14. Torelli G, Venturelli D, Colo A, et al. Expression of c-myb protooncogeneand other cell cycle-related genes in normal and neoplastic human colonicmucosa. Cancer Res 1987;47:5266-9.
15. Alitalo K, Winqvist R, Lin CC, de la Chapelle A, Schwab M, Bishop JM.Aberrant expression of an amplified c-myb oncogene in two cell lines from acolon carcinoma. Proc Natl Acad Sci USA 1984;81:4534-8.
16. Kanei-Ishii C, Yasukawa T, Morimoto RI, Ishii S. c-Myb-induced trans-activation mediated by heat shock elements without sequence-specific DNAbinding of c-Myb. J Biol Chem 1994;269:15768-75.
17. Baird MC, Hendry JH, Dexter MT, Testa NG. The radiosensitivity ofpopulations of murine hemopoietic colony-forming cells that respond tocombinations of growth factors. Exp Hematol 1991;19:282-7.
18. Alexander RJ, Buxbaum JN, Raicht RF. Enhanced expression of oncogene-encoded mRNA in a rat model of colon cancer. Am J Med Sci 1991;301:238-45.
19. Ramsay RG, Thompson MA, Hayman JA, Reid G, Gonda TJ, WhiteheadRH. Myb expression is higher in malignant human colonic carcinoma andpremalignant adenomatous polyps than in normal mucosa. Cell Growth & Differ1992;3:723-30.
20. Brabender J, Lord RV, Danenberg KD, et al. Increased c-myb mRNAexpression in Barrett’s esophagus and Barrett’s-associated adenocarcinoma.J Surg Res 2001;99:301-6.
21. Biroccio A, Benassi B, D’Agnano I, et al. c-Myb and Bcl-x overexpressionpredicts poor prognosis in colorectal cancer: clinical and experimental findings.Am J Pathol 2001;158:1289-99.
22. Potten CS, Grant HK. The relationship between ionizing radiation-induced
FIGURE 6. Colons harvested from c-myb+/�
mice following exposure to 5-FU show signifi-cantly delayed crypt length restoration com-pared with c-myb+/+ mice. Three parameters ofcrypt response and recovery were assessed:cell number per longitudinal section (A), apo-ptosis (B), and mitoses (C) per crypt. In additionto the delayed crypt length recovery, signifi-cantly fewer cells per crypt and apoptoses wereobserved in the c-myb+/� mice compared withwild-type controls at day 1 excluding the roleof enhanced apoptosis in the reduced cryptrecovery process in c-myb+/� colons. Columns ,mean for five mice at each time point; bars , SE.Asterisks , significant difference between wild-type and c-myb+/� samples.
Ramsay et al.
Mol Cancer Res 2004;2(6). June 2004
360
on July 3, 2020. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
apoptosis and stem cells in the small and large intestine. Br J Cancer 1998;78:993-1003.
23. Pritchard DM, Print C, O’Reilly L, Adams JM, Potten CS, Hickman JA.Bcl-w is an important determinant of damage-induced apoptosis in epithelia ofsmall and large intestine. Oncogene 2000;19:3955-9.
24. Merritt AJ, Potten CS, Watson AJ, Loh DY, Nakayama K, Hickman JA.Differential expression of bcl-2 in intestinal epithelia. Correlation with attenuationof apoptosis in colonic crypts and the incidence of colonic neoplasia. J Cell Sci1995;108:2261-71.
25. Potten CS. Stem cells in gastrointestinal epithelium: numbers, characteristicsand death. Philos Trans R Soc Lond B Biol Sci 1998;353:821-30.
26. Pritchard DM, Potten CS, Korsmeyer SJ, Roberts S, Hickman JA. Damage-induced apoptosis in intestinal epithelia from bcl -2-null and bax -null mice:investigations of the mechanistic determinants of epithelial apoptosis in vivo .Oncogene 1999;18:7287-93.
27. Neben S, Redfearn WJ, Parra M, Brecher G, Pallavicini MG. Short- andlong-term repopulation of lethally irradiated mice by bone marrow stem cellsenriched on the basis of light scatter and Hoechst 33342 fluorescence. ExpHematol 1991;19:958-67.
28. Hodgson GS, Bradley TR. Properties of haematopoietic stem cells sur-viving 5-fluorouracil treatment: evidence for a pre-CFU-S cell? Nature 1979;281:381-2.
29. Bertoncello I, Bradford GB. Surrogate assays for hematopoietic stem cell
activity. In: Garand J, Quesenberry P, Hilton D, editors. Colony-stimulatingfactors. 2nd ed. New York: Marcel Dekker, Inc.; 1997. p. 35-47.
30. Kamada S, Shimono A, Shinto Y, et al. bcl -2 deficiency in mice leads topleiotropic abnormalities: accelerated lymphoid cell death in thymus and spleen,polycystic kidney, hair hypopigmentation, and distorted small intestine. CancerRes 1995;55:354-9.
31. Churilla AM, Braciale TJ, Braciale VL. Regulation of T lymphocyteproliferation. Interleukin 2-mediated induction of c-myb gene expression isdependent on T lymphocyte activation state. J Exp Med 1989;170:105-21.
32. Gewirtz AM, Anfossi G, Venturelli D, Valpreda S, Sims R, Calabretta B.G1/S transition in normal human T-lymphocytes requires the nuclear proteinencoded by c-myb . Science 1989;245:180-3.
33. Stern JB, Smith KA. Interleukin-2 induction of T-cell G1 progression andc-myb expression. Science 1986;233:203-6.
34. Sumner R, Crawford A, Mucenski M, Frampton J. Initiation of adultmyelopoiesis can occur in the absence of c-Myb whereas subsequent developmentis strictly dependent on the transcription factor. Oncogene 2000;19:3335-42.
35. Sicurella C, Freeman R, Micallef S, Mucenski ML, Bertoncello I, RamsayRG. Defective stem cell factor expression in c-myb null fetal liver stroma. BloodCells Mol Dis 2001;27:470-8.
36. Bradford GB, Williams B, Rossi R, Bertoncello I. Quiescence, cycling,and turnover in the primitive hematopoietic stem cell compartment. Exp Hematol1997;25:445-53.
c-myb Mice Are Hypersensitive to Cytotoxic Damage
Mol Cancer Res 2004;2(6). June 2004
361
on July 3, 2020. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
2004;2:354-361. Mol Cancer Res Robert G. Ramsay, Suzanne Micallef, Sally Lightowler, et al. Medical Research Council of Australia.
National Health and115-Fluorouracil and Ionizing Radiation Heterozygous Mice Are Hypersensitive tomybc-
Updated version
http://mcr.aacrjournals.org/content/2/6/354
Access the most recent version of this article at:
Cited articles
http://mcr.aacrjournals.org/content/2/6/354.full#ref-list-1
This article cites 35 articles, 15 of which you can access for free at:
Citing articles
http://mcr.aacrjournals.org/content/2/6/354.full#related-urls
This article has been cited by 2 HighWire-hosted articles. Access the articles at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's
.http://mcr.aacrjournals.org/content/2/6/354To request permission to re-use all or part of this article, use this link
on July 3, 2020. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from