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.

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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

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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).

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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.



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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.

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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.



359



AcknowledgmentsWe thank Dr. Maree Overall and Prof. Patrick Tam for critical comments andsuggestions during the preparation of this manuscript.

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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.


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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-

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