|
|
Next Article 
Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 1-8
Loss of FancC Function Results in Decreased Hematopoietic Stem
Cell Repopulating Ability
By
Laura S. Haneline,
Troy A. Gobbett,
Rema Ramani,
Madeleine Carreau,
Manuel Buchwald,
Mervin C. Yoder, and
D. Wade Clapp
From the Department of Pediatrics, Herman B Wells Center for
Pediatric Research and the Departments of Microbiology/Immunology and
Biochemistry and Molecular Biology, Indiana University School of
Medicine, Indianapolis, IN; and the Department of Genetics, The
Hospital for Sick Children, Toronto; and the Department of Molecular
and Medical Genetics, University of Toronto, Toronto, Ontario, Canada.
 |
ABSTRACT |
Fanconi anemia (FA) is a complex genetic disorder characterized by
progressive bone marrow (BM) aplasia, chromosomal instability, and
acquisition of malignancies, particularly myeloid leukemia. We used a
murine model containing a disruption of the murine homologue of
FANCC (FancC) to evaluate short- and long-term
multilineage repopulating ability of FancC  / cells in
vivo. Competitive repopulation assays were conducted where "test"
FancC / or FancC +/+ BM cells (expressing
CD45.2) were cotransplanted with congenic competitor cells (expressing
CD45.1) into irradiated mice. In two independent experiments, we
determined that FancC / BM cells have a profound decrease
in short-term, as well as long-term, multilineage repopulating ability.
To determine quantitatively the relative production of progeny cells by
each test cell population, we calculated test cell contribution to
chimerism as compared with 1 × 105 competitor cells. We
determined that FancC / cells have a 7-fold to 12-fold
decrease in repopulating ability compared with FancC +/+
cells. These data indicate that loss of FancC function results in reduced in vivo repopulating ability of pluripotential hematopoietic stem cells, which may play a role in the development of the BM failure
in FA patients. This model system provides a powerful tool for
evaluation of experimental therapeutics on hematopoietic stem cell function.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
FANCONI ANEMIA (FA) is a complex,
heterogeneous genetic disorder characterized clinically by a
predisposition to congenital anomalies, a progressive bone marrow (BM)
aplasia, and the development of malignancies.1-3 The
diagnosis of FA is established by a characteristic hypersensitivity of
FA cells to bifunctional alkylating agents such as mitomycin C (MMC) or
diepoxybutane (DEB).3,4 This cellular hypersensitivity is
manifested by induction of chromosome breaks, a G2/M cell
cycle arrest, and cell death (reviewed in D'Andrea and
Grompe5 and Auerbach and Verlander6). The
hypersensitivity of FA cells to MMC has enabled the use of somatic cell
fusion studies to identify eight complementation groups (A to H),
inferring the existence of eight genes.7 Four of the eight
genes have been mapped to different chromosomal loci (FANCA,
FANCC, FANCD, FANCG), and the FANCA, FANCC,
and FANCG genes have been cloned.8-12
Many patients with FA have non-life-threatening congenital
abnormalities particularly of the skeletal, genitourinary, and central
nervous systems.13,14 However, the major causes of morbidity and mortality of FA relate to dysfunction of the
hematopoietic system. Most patients die of BM failure (80%) or develop
malignancies, particularly myeloid leukemia. The mean age of onset of
BM failure occurs at 8 years, and the mean survival is 19 years.2 Data from many laboratories indicate that there is
a reduction in the frequency of lineage restricted erythroid
(burst-forming unit-erythroid [BFU-E]) and granulocyte macrophage
(colony-forming unit-granulocyte-macrophage [CFU-GM]) progenitors
from BM aspirates of asymptomatic,15 as well as
pancytopenic FA patients.16,17 These studies have also demonstrated a decrease in colony size, possibly reflecting a reduction
in proliferation of lineage restricted progenitors. Together these data
infer that loss of function of an FA protein results in a qualitative
or quantitative alteration in the progenitor compartment. In addition,
the development of progressive BM failure in most FA patients suggests
that loss of FA gene function results in injury to the stem cell, as
well as the progenitor cell compartment.
Recently, two murine models containing a disruption of the murine
homologue of FANCC (FancC) were developed to facilitate functional studies of primary cells in vitro and in vivo. Chen et
al18 created a disruption in exon 8 of FancC, while
Whitney et al19 used homologous recombination to create a
disruption in exon 9. In both lines of mice, spontaneous chromosomal
breaks in splenic lymphocytes were observed, as well as an increase in chromosomal breaks in response to bifunctional alkylating agents. In
addition, a number of abnormalities in the progenitor cell compartment
were identified in both murine models, including the hyperresponsiveness of FancC / progenitors to
MMC20,21 and a predisposition of FancC
/ progenitors to undergo apoptosis in response to
inhibitory cytokines.20,22 The murine line developed by
Whitney et al19 develops an age-dependent decrease in the number of hematopoietic progenitors cultured from the BM, while FancC / mice developed by Chen et
al18 do not acquire these functional abnormalities,
suggesting a somewhat milder phenotype. Cumulatively, the similarities
between in vitro BM assays in FA patients and those in FancC
/ mice suggest that these mice will be a good model
system to evaluate the role of FancC on short- and long-term in
vivo stem cell repopulating ability.
A major advantage of using murine models to study genetic diseases
affecting the hematopoietic system is the ability to evaluate hematopoietic stem cell function by repopulation in vivo, the only
universally accepted assay for these cells. These transplantation studies can be accomplished using well-established, competitive repopulation assays, which allow highly accurate, quantitative determinations of stem cell activity.23-30 Congenic mouse
strains that differ only in the expression of genetically
distinguishable isoenzymes, such as the CD45.1/CD45.2 antigens, allow
identification of progeny cells from two different stem cell
populations with high sensitivity. Coinjection of "competitor"
cells of one isotype and "test" cells with a distinct isotype
into irradiated recipients provides a means to identify stem cells that
have different capacities to compete for repopulation.
We used FancC / mice to address two questions
related to the role of FancC in regulating hematopoietic stem
cell control. First, we used the competitive repopulation assay to
determine whether FancC is required for normal repopulating
ability of short- and long-term reconstituting hematopoietic stem
cells. Second, because the hematopoietic system is hierarchical in
nature and has multiple compartments, we evaluated whether
FancC is required to regulate the growth of specific
populations of cells. We demonstrate that loss of FancC results
in a profound impairment in short- and long-term repopulating stem
cells. These data have significance for understanding the basic
pathogenesis of the disease and in providing an approach to test
experimental therapies.
| |
MATERIALS AND METHODS |
Mice.
FancC / and FancC +/+ mice (C57Bl/6 × SV129) were backcrossed into a C57Bl/6 strain
(CD45.2+). Congenic C57Bl/6 (CD45.2+) and
B6.SJL-PtrcaPep3b/BoyJ (B6.BoyJ) mice (CD45.1+) were
purchased from Jackson Laboratories (Bar Harbor, ME) and maintained in
our animal facility.
Harvesting BM samples.
BM cells were flushed from the tibias and femurs of 6- to 8-week-old
FancC / and FancC +/+ littermates and
B6.BoyJ mice using Iscove's Modified Dulbecco's Media (IMDM)
(GIBCO-BRL, Gaithersburg, MD) containing 5% fetal calf serum (FCS)
(Hyclone Laboratories, Logan, UT). Low-density mononuclear cells
(LDMNC) were prepared by centrifugation on ficoll-hypaque (density,
1.119; Sigma, St Louis, MO). B6.BoyJ LDMNC were used as competitor
cells for both competitive repopulation experiments. FancC
/ and FancC +/+ LDMNC were further purified
by fluorescence cytometry for Sca1+
lin /dim cells to be used as test cells.
Purification of Sca1+ lin/dim
cells.
BM LDMNC from littermates of each genotype were pooled before cell
sorting. The cells were resuspended in cold phosphate-buffered saline
(PBS) 0.1% bovine serum albumin (BSA) at a concentration of 1 to 2 × 108 cells/mL. The cells were stained for 20 minutes
at 4°C with the following antibodies: Sca1-phycoerythrin (PE),
CD4-fluorescein isothiocyanate (FITC), CD8-FITC, B220-FITC, Mac1-FITC,
Gr1-FITC, and Ter119-FITC. All antibodies were purchased from
PharMingen (San Diego, CA). Cell samples were washed twice and
resuspended at a concentration of 5 to 10 × 106
cells/mL cold PBS 0.1% BSA. The cells were sorted for
Sca1+ lin/dim cells using a Becton
Dickinson (San Jose, CA) FACSTAR sorter using identical sorting
gates. This selection enriches for immature hematopoietic
progenitor and stem cells and excludes all differentiated cells.
Competitive repopulation experiments.
Ninety 8- to 10-week-old female C57Bl/6 mice (experiment 1) or B6.BoyJ
mice (experiment 2) were lethally irradiated (1,100 cGy split dose)
before transplantation as previously described.31,32 Limiting dilutions of Sca1+ lin/dim
cells (0 to 5,000 experiment 1 and 0 to 10,000 experiment 2) from
FancC / and FancC +/+ mice were mixed
with a constant number of B6.BoyJ low-density competitor cells (5 × 105). Each cell mixture was resuspended in 0.5 mL
of IMDM 2% FCS and injected into the tail vein of six lethally
irradiated recipients. Some mice (n = 4) were irradiated, but did not
receive exogenous cells to be certain that the irradiation dose was
lethal and to control for any residual contribution of the host's
hematopoietic system. Finally, to control for any potential late
contribution of endogenous hematopoiesis from the irradiated recipient
animals, some mice (n = 6) were transplanted with competitor cells only.
Chimerism analysis by fluorescence cytometry.
Tail-vein blood samples (100 µL) were obtained monthly
posttransplantation for analysis of chimerism and every other month for
multilineage analysis. Peripheral blood cells were incubated in red
blood cell (RBC) lysis buffer (0.16 mol/L
NH4Cl, 0.1 mol/L KHCO3, 0.1 mmol/L EDTA) for 5 minutes at 4°C. The cells were washed twice, resuspended in PBS
0.1% BSA, and aliquoted into seven individual tubes for antibody
staining. Each sample was stained with CD45.1-FITC (B6.BoyJ strain) and
CD45.2-FITC (C57Bl/6 strain) plus four individual lineage markers
conjugated to PE at 4°C for 20 minutes. Samples were washed twice
and resuspended in PBS 0.1% BSA before analysis by fluorescence
cytometry. Three mixtures of C57Bl/6 and B6.BoyJ cells were stained
with CD45.1-FITC and CD45.2-FITC individually as controls to assist
with appropriate gate settings and to assure that no errors occurred
during antibody staining. A total of 5,000 events were collected from
each sample. All data were analyzed using CELLQuest software (Becton
Dickinson). Instrument settings and gates used to analyze data were
identical from month to month and between genotypes. An unpaired
Student's t-test was used to determine whether significant
differences existed in chimerism between genotypes.
Repopulating unit calculation.
Relative repopulating ability of the donor cells compared with the
competitor cell population was determined using a repopulating unit
(RU) calculation described previously by Harrison et
al.27,28 This calculation allows quantitative comparison of
repopulating ability between different donor cell populations, ie,
FancC / and FancC +/+. We calculated RU
for mice transplanted with the highest dose of Sca1+
lin/dim cells for both experiments. Because a
constant number of competitors (5 × 105) were used
for all experiments, the following equation was used to compute RU: RU = 5 × Measured Donor Chimerism/(100 Measured Donor Chimerism). Differences in RU between groups were compared using
an unpaired Student's t-test.
| |
RESULTS |
Chimerism detection using fluorescence cytometry.
Preliminary studies were conducted to evaluate the reliability of
fluorescence cytometry to detect CD45.1 and CD45.2 antigen positive
cells over a range of C57Bl/6 and B6.BoyJ cell mixtures. Defined
numbers of nucleated peripheral blood cells of each respective strain
were mixed and stained with antibodies to either CD45.1-FITC or
CD45.2-FITC. Addition of the measured CD45.1 and CD45.2 percentages was
used as an internal control to verify that the sum of the two
percentages approximated 100%. The results from these studies are
shown in Fig 1. The experimental result
obtained from measuring CD45.1 or CD45.2 was consistently within 3% of
the predicted value. These results support the use of fluorescence
cytometry as a sensitive and accurate methodology to analyze donor cell
contribution in animals transplanted with cells expressing these two
isoantigens over a wide range of chimerism.
View larger version (28K):
[in this window]
[in a new window]
| Fig 1.
Detection of test cell chimerism using fluorescence
cytometry. Venous peripheral blood cells were isolated from the tail
veins of C57Bl/6 (CD45.2+) and B6.BoyJ
(CD45.1+) mice. Defined mixtures of nucleated blood cells
from each mouse were stained with antibodies to CD45.1 ( ), CD45.2
( ), or a nonspecific isotype control ( ) and analyzed using
fluorescence cytometry. Each histogram represents the staining profile
of a single cell mixture. The white peak is the isotype control. The
gray and black peaks represent CD45.1 and CD45.2 positive cells,
respectively. The numbers in the upper right corner of each histogram
show the expected percentages of CD45.1/CD45.2 for each histogram.
CD45.1 and CD45.2 chimerism measured using fluorescence cytometry were
consistently within 3% of the predicted value over a wide range of
cell mixtures.
|
|
FancC / hematopoietic stem cells have decreased short-
and long-term repopulating ability.
A schematic of the competitive repopulation assay is illustrated in
Fig 2. BM cells from 6- to 8-week-old
FancC / and FancC +/+ littermates were
isolated, fractionated using ficoll-hypaque separation, and sorted by
fluorescence-activated cell sorting (FACS) to obtain Sca1+
lin/dim cells. The percentage of nucleated BM cells
from both FancC / and FancC +/+ mice that
were Sca1+ lin/dim ranged from 0.4% to
0.5%. The absolute number of nucleated and Sca1+
lin/dim cells per femur were similar in both
FancC +/+ and FancC / cells, consistent
with previously published studies20 (data not shown). These
cells were mixed with 5 × 105 competitor
cells and injected into irradiated recipient mice.
View larger version (20K):
[in this window]
[in a new window]
| Fig 2.
Schematic of competitive repopulation assay. Test cells
from the BM of FancC / and FancC +/+
littermates were isolated and enriched for
Sca1+lin/dim cells. Each respective test
cell population was cotransplanted into irradiated recipient mice with
competitor cells from B6.BoyJ mice that were genetically identical to
C57Bl/6 mice with the exception of expression of a different CD45
isoantigen.
|
|
Peripheral blood samples were collected from recipient mice monthly
after transplantation, and aliquots of cells from each specimen were
stained with CD45.1-FITC and CD45.2-FITC, respectively. Representative
histograms of chimeric peripheral blood cell specimens from mice
transplanted with 5,000 Sca1+ lin/dim
FancC +/+ or FancC / (express CD45.2)
and 500,000 low-density mononuclear competitor cells (express
CD45.1) 6 months after transplantation are shown in
Fig 3. The CD45.2 chimerism measured from
the mouse transplanted with FancC / cells was
only 18%, while the CD45.2 chimerism in the recipient transplanted
with FancC +/+ cells was 71%. As expected, in each case the
sum of CD45.1 chimerism (representing competitor cell proliferation)
and CD45.2 chimerism (representing test cell proliferation)
approximated 100% ± 3%.
View larger version (17K):
[in this window]
[in a new window]
| Fig 3.
Chimerism of representative irradiated recipient mice
transplanted with FancC / or FancC +/+ BM
cells 6 months after transplantation. Representative histograms of
peripheral blood cells obtained from recipient mice transplanted with
500,000 low-density competitor cells expressing the CD45.1 antigen and
5,000 Sca1+lin/dim FancC /
cells (left panel) or 5,000 Sca1+lin/dim
FancC +/+ cells (right panel), which express the CD45.2
antigen. Nucleated cells from aliquots of each specimen were stained
with antibodies to CD45.1 (), CD45.2 (), and the isotype control
(). The contribution of FancC / and FancC +/+
cells to the chimerism is indicated in the upper righthand corner
of each histogram.
|
|
Short-term repopulating ability is characteristically measured 1 month
after transplantation and long-term repopulating ability is established
by 4 to 6 months after transplantation in the murine system.26-28 The CD45.2 chimerism of all transplanted mice
from a representative experiment at 1, 3, and 6 months after
transplantation is shown in Fig 4. As
expected, all animals irradiated, but not transplanted with exogenous
cells, died in 14 days, and recipient animals (CD45.2+)
transplanted with competitor cells (CD45.1+) only had low
levels of residual endogenous CD45.2 chimerism (<5%, data not
shown). The mean chimerism of mice transplanted with FancC +/+
cells 1 month after transplantation ranged from 15% when 100 Sca1+ lin/dim cells were transplanted to
55% chimerism when 5,000 Sca1+ lin/dim
cells were transplanted. In contrast, the mean chimerism of FancC / cells over these same Sca1+
lin/dim concentrations ranged from 3% to a maximum
of 12%.
View larger version (13K):
[in this window]
[in a new window]
| Fig 4.
Summary of chimerism in recipient mice transplanted with
FancC / or FancC +/+ test cells. Chimerism of
peripheral blood cells was determined by fluorescence cytometry for
mice transplanted with FancC / () or FancC
+/+ ( ) Sca1+lin/dim cells and
500,000 competitor cells. The mean chimerism for each
Sca1+lin/dim cell number transplanted was
calculated for each genotype, and the data generated at 1, 3, and 6 months after transplantation are summarized in the graphs as indicated.
The error bars represent standard error of the means. Twelve mice (six
per genotype) were transplanted at each test cell concentration.
*P < .05 FancC / cells exhibit a significant
decrease in repopulating ability as compared with FancC +/+
cells over several Sca1+lin/dim cell doses
transplanted at every time point evaluated.
|
|
Long-term repopulating ability of the two test cell populations was
determined by evaluating CD45.2 chimerism in peripheral blood cells of
recipient mice 6 months after transplantation. A dose-dependent
increase in donor chimerism of mice transplanted with FancC +/+
and FancC / cells was observed. A mean chimerism of 59% was detected in recipient mice transplanted with the highest cell dose of FancC +/+ cells. While there was an increase in
chimerism in the recipients of FancC / donor
cells, only a 10% peripheral blood chimerism was achieved in the
recipients transplanted with the highest number of Sca1+
lin/dim cells. Together, the data indicate that the
short- and long-term repopulating ability is markedly reduced in
FancC / hematopoietic stem cells over a wide
range of input test cells.
FancC / Sca1+
lin/dim cells have decreased RU activity.
To compare the repopulating ability between FancC
/ and FancC +/+ hematopoietic cells in a
quantitative fashion, RU activity was calculated as
described.27,28 Table 1
contains the RU calculations for two independent experiments. In both
experiments, the FancC / hematopoietic cells had
a lower repopulating ability as compared with FancC +/+
hematopoietic cells at each monthly analysis. By 4 months after
transplantation, there was a 9-fold to 12-fold difference in RU
activity of BM cells from the FancC +/+ and FancC
/ mice.
Multilineage analysis of FancC / and FancC
+/+ hematopoietic cells in reconstituted mice.
To determine whether mice transplanted with FancC
/ donor cells exhibited any evidence of a
preferential repopulation defect in myeloid cells, we evaluated the
peripheral blood samples for test cell chimerism using a series of
antibodies that are specific to myeloid or lymphoid cells. This was
accomplished by dual staining cells for simultaneous expression of
CD45.2 (test cell population) and specific lineage markers. The
lineages examined by two-color fluorescence cytometry included T cell
(CD3), B cell (B220), granulocyte (Gr1), and monocyte/macrophage
(Mac1). The summary of multilineage analysis for a representative
experiment 5 months posttransplantation is shown in
Table 2. Although there were significant
differences in the repopulating ability of FancC
/ and FancC +/+ test cells, the data
demonstrate that FancC / cells contribute equally
to lymphoid and myeloid lineages. We conclude that these data are consistent with a defect in the pluripotential hematopoietic stem cell
compartment.
| |
DISCUSSION |
FA is the most common genetic cause of BM failure.2
Currently, the only cure for the progressive aplasia found in the high majority of FA patients is allogeneic BM transplantation. Many investigators have demonstrated that there is a defect in the hematopoietic progenitor compartment in FA patients.15-17
However, progenitor assays, which grow in culture over a few weeks, do not correlate with in vivo stem cell function. Therefore, while the
observation that FA patients acquire BM failure suggests a defect in
the stem cell compartment, a formal experimental proof of this
hypothesis in human stem cells has not been conducted. Further, the
evaluation of human hematopoietic stem cell function experimentally is
difficult due to the lack of a quantitative in vitro assay and inherent
problems in transplanting human cells into xenograft systems.
Murine models provide powerful tools for basic research and answering
clinical questions that cannot be practically or ethically addressed in
human systems. We used a murine model of FancC to determine
whether FancC / hematopoietic stem cells have
reduced repopulating ability in vivo as compared with FancC
+/+ cells using a competitive repopulation assay. A key feature of
this assay is that the result measures a functional ability of the hematopoietic stem cell and does not rely on phenotypic determinants of
output cells. The assay is extremely sensitive to even small differences in test cell populations because evaluation of the entire
differentiation pathway is analyzed. In addition, the use of a wide
series of limiting dilutions of test cells further strengthens the
sensitivity of this assay. Our data in two independent experiments indicate that FancC / hematopoietic stem cells
have a marked early, as well as late, repopulation defect over a
50-fold range of test cells.
To quantitatively assess the relative difference in repopulating
ability between FancC / and FancC +/+
cells, we calculated a repopulating unit activity from recipients who
received 5 to 10,000 Sca1+ lin/dim test
cells in two independent experiments. These cell doses were chosen
because the low chimerism detected in mice transplanted with fewer
FancC / cells would introduce significant error
in the calculation. Calculated RU activity values generated from donor
FancC +/+ cells and transplanted into C57Bl/6 recipients were
comparable to previously published data for normal C57Bl/6 marrow
cells.27 A 7-fold to 9-fold reduction in early repopulating ability and a 9-fold to 12-fold reduction in late repopulating ability
was detected in recipient mice transplanted with FancC / cells.
The test cell chimerism detected in experiment 2 was lower than the
chimerism noted in experiment 1 (Table 1). Potential explanations for
the lower levels of chimerism detected in experiment 2 may relate to
variability between the two experiments resulting from differences in
test or competitor cell populations (ie, ficoll-hypaque separation,
Sca1+ lin/dim cell purification) or
differences in the response of the hosts to the conditioning regimen.
Two experimental controls were used to evaluate this latter
possibility. First, we showed that a lethal irradiation dosage was
administered to both CD45.1 and CD45.2 recipients. All irradiated mice
that did not receive exogenous cells died 10 to 14 days after
irradiation. Second, we included a set of control animals (CD45.2) that
were transplanted with only competitor cells (CD45.1) to examine
whether residual endogenous hematopoiesis contributed significantly to
the measured chimerism. Another plausible explanation for the
differences between experiments 1 and 2 is that, although CD45.1 and
CD45.2 are isoenzymes, these antigens could potentially induce a weak
immune response when transplanted into hosts with disparate CD45
antigen expression. Other investigators have also suggested this
possibility using milder conditioning regimens of the recipient
animals.33 However, despite the difference in absolute test
cell chimerism between the two experiments, the relative difference in
repopulating ability between FancC / and
FancC +/+ test cells was similar in both experiments.
The hematopoietic system is characterized by a hierarchy of multiple
compartments (ie, stem, progenitor, and differentiated cell
compartments), as well as cell lineages (ie, myeloid, lymphoid, and
erythroid). Loss of specific gene products may affect some lineages and
compartments, but not others. In FA patients, for instance, there is a
selective attrition of myeloid cells in vivo, while lymphocyte
populations remain normal, and patients with FA are highly predisposed
to myeloid, but not lymphoid leukemias. We used the competitive
repopulation assay to determine whether FancC is required for
normal lymphoid, as well as myeloid cell repopulation. While we were
able to detect profound differences between the repopulating ability of
FancC / and FancC +/+ test cells, no
differential requirement for FancC was detected between myeloid
or lymphoid lineages. Our observation that recipient mice transplanted
with FancC / cells have an equivalent
repopulation defect in multiple lineages is consistent with this gene
being important in hematopoietic stem cell function in FancC
/ mice.
Murine models of FancC have recently been used to begin to
delineate the consequences of loss of FancC function in
differentiated hematopoietic cells, as well as in hematopoietic
progenitor cells. Differentiated splenic lymphocytes from FancC
/ mice were shown to exhibit increased spontaneous,
as well as induced chromosomal aberrations, similar to lymphocytes from
FA patients.18 Further, FancC /
hematopoietic progenitors are hypersensitive to MMC20,21 and inhibitory cytokines,19,20 including interferon-
(IFN-), tumor necrosis factor- (TNF-), and macrophage
inflammatory protein-1. In addition, loss of
FancC predisposes progenitors to inhibitory cytokine-mediated
apoptosis.20,22 Other studies evaluating the hematologic
consequences of in vivo administration of MMC demonstrated that
FancC / mice develop pancytopenia, BM aplasia, and death after serial weekly intraperitoneal injections of MMC for 3 to 8 weeks.21 These studies show that hematopoietic cells from FancC / mice are hypersensitive in vivo, as
well as in vitro, to bifunctional alkylating agents, analogous to human
FA blood and BM cells. Together, the previous studies and our present results demonstrate that loss of FancC results in a profound
alteration in hematopoietic cell function in multiple hematopoietic compartments.
Recent discoveries have now resulted in the positional identification
of four FA complementation types and the cloning of three of the
cDNAs.8-10,12 These genes account for the genetic abnormalities detected in approximately 65% to 70% of known FA patients. The isolation of the cDNAs, continued improvements in somatic
gene transfer technology, and an improved understanding of the role of
these proteins in normal cellular homeostasis22,34-38 may
allow new pharmacologic and gene transfer approaches to ameliorate or
cure the hematopoietic disease. Our studies demonstrating a clear
repopulating defect in FancC / hematopoietic stem
cells will allow use of the competitive repopulation assay to determine whether FancC gene transfer will restore normal proliferation to FancC / cells in vivo. Such studies will have
direct application regarding the ability to correct the proliferation
of human hematopoietic stem cells in FA patients.
If gene transfer corrects the biochemical defect and restores normal
proliferation to FancC / hematopoietic stem
cells, two questions regarding the in vivo consequences of transduced stem cells will need to be addressed. The first question is: what are
the conditions in which the genetically corrected cells will engraft
and proliferate? The potential of transduced FA hematopoietic stem
cells to engraft and proliferate in the absence of myeloablation is
particularly important, as FA patients are hypersensitive to many
chemotherapeutic agents and may be at an increased risk for secondary
malignancies. It has been previously determined in the murine system
that wild-type cells will engraft in the absence of myeloablation when
high numbers of donor cells are used.39,40 In addition,
other investigators have determined that significantly higher chimerism
of donor cells is obtained in nonmyeloablated normal recipients when
mobilized peripheral blood or BM cells are used.26,41 It
will be important to evaluate the engraftment and in vivo proliferation
potential of transduced FancC /, BM, and
mobilized peripheral blood stem cells after transplantation into
nonmyeloablated FancC / mice.
Second, gene transfer, using current strategies, will result in
transduction of only a small subpopulation of the total number of
hematopoietic stem cells. It remains to be determined what the fates of
the untransduced stem cells, as well as the endogenous stem cells, will
be after transplantation. The noncorrected cells may remain quiescent,
while the genetically corrected cells maintain hematopoiesis. There is
now precedence for this pattern of clonal proliferation occurring in an
FA patient where a somatic reversion resulted in a single
stem/progenitor cell restoring normal hematopoietic cell function for
at least 5 years.42 Alternatively, the population of
untransduced stem cells may undergo further damage leading to apoptosis
or leukemic transformation. The FancC / mice will provide a valuable model system to evaluate these basic questions.
| |
ACKNOWLEDGMENT |
We thank our colleagues at Indiana University and Dr Kevin Shannon
(University of California, San Francisco, CA) for reading the
manuscript. We also thank Patricia Fox for secretarial support.
| |
FOOTNOTES |
Submitted December 2, 1998; accepted February 26, 1999.
Supported by US Public Health Services Grants No. P01 HL53586, P50
DK49218, R29 CA74177-01, and IF32 HL09851-01.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to D. Wade Clapp, MD, Cancer Research
Institute, 1044 W Walnut St, Room 408, Indianapolis, IN 46202-5254.
| |
REFERENCES |
1.
Fanconi G:
Familial constitutional panmyelocytopathy, Fanconi anemia (F.A.).
Semin Hematol
4:233, 1967[Medline]
[Order article via Infotrieve]
2.
Alter B, Young N:
The Bone Marrow Failure Syndromes (ed 4). Philadelphia, PA, Saunders, 1998.
3.
Auerbach A, Rogatko A, Schroeder-Kurth T:
International Fanconi Anemia Registry; Relation of clinical symptoms to diepoxybutane sensitivity.
Blood
73:391, 1989[Abstract/Free Full Text]
4.
Auerbach A, Zhang M, Ghosh R, Perhament E, Verlinsky Y, Nocolas G, Boue J:
Clastogen-induced chromosomal breakage as a marker for first trimester prenatal diagnosis of Fanconi anemia.
Hum Genet
73:86, 1986[Medline]
[Order article via Infotrieve]
5.
D'Andrea A, Grompe M:
Molecular biology of Fanconi anemia: Implications for diagnosis and therapy.
Blood
90:1725, 1997[Free Full Text]
6.
Auerbach A, Verlander P:
Disorders of DNA replication and repair.
Curr Opin Pediatr
9:600, 1997[Medline]
[Order article via Infotrieve]
7.
Joenje H, Oostra A, Wijker M, di Summa F, van Berkel C, Rooimans M, Ebell W, van Weel M, Pronk J, Buchwald M, Arwert F:
Evidence for at least eight Fanconi anemia genes.
Am J Hum Genet
61:940, 1997[Medline]
[Order article via Infotrieve]
8.
Strathdee C, Gavish H, Shannon W, Buchwald M:
Cloning of cDNAs for Fanconi's anaemia by functional complementation.
Nature
356:763, 1992[Medline]
[Order article via Infotrieve]
9.
The Fanconi Anaemia/Breast Cancer Consortium:
Positional cloning of the Fanconi anaemia group A gene.
Nat Genet
14:324, 1996[Medline]
[Order article via Infotrieve]
10.
Lo Ten Foe J, Rooimans M, Bosnoyan-Collins L, Alon N, Wijker M, Parker L, Lightfoot J, Carreau M, Callen D, Savoia A, Cheng N, van Berkel C, Strunk M, Gille J, Pals G, Kruyt F, Pronk J, Arwert F, Buchwald M, Joenje H:
Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA.
Nat Genet
14:320, 1996[Medline]
[Order article via Infotrieve]
11.
Whitney M, Thayer M, Reifsteck C, Olson S, Smith L, Jakobs P, Leach R, Naylor S, Joenje H, Grompe M:
Microcell mediated chromosome transfer maps the Fanconi anaemia group D gene to chromosome 3p.
Nat Genet
11:341, 1995[Medline]
[Order article via Infotrieve]
12.
de Winter J, Waisfisz Q, Rooimans M, van Berkel C, Bosnoyan-Collins L, Alon N, Carreau M, Bender O, Demuth I, Schindler D, Pronk J, Arwert F, Hoehn H, Digweed M, Buchwald M, Joenje H:
The Fanconi anaemia group G gene FANCG is identical with XRCC9.
Nat Genet
20:281, 1998[Medline]
[Order article via Infotrieve]
13.
Giampietro P, Adler-Brecher B, Verlander P, Pavlakis S, Davis J, Auerbach A:
The need for more accurate and timely diagnosis in Fanconi anemia: A report for the International Fanconi Anemia Registry.
Pediatrics
91:1116, 1993[Abstract/Free Full Text]
14.
Alter B:
Fanconi's anaemia and its variability.
Br J Haematol
85:9, 1993[Medline]
[Order article via Infotrieve]
15.
Doneshbod-Skibba G, Martin J, Shahidi N:
Myeloid and erythroid colony growth in non-anemic patients with Fanconi's anemia.
Br J Haematol
44:33, 1980[Medline]
[Order article via Infotrieve]
16.
Broxmeyer H, Douglas G, Hangoc G, Cooper S, Bard J, English D, Arny M, Thomas L, Boyse E:
Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells.
Proc Natl Acad Sci USA
86:3828, 1989[Abstract/Free Full Text]
17.
Gluckman E, Broxmeyer H, Auerbach A, Friedman H, Douglas G, Devergie A, Esperou H, Thierry D, Socie G, Lehn P, Cooper S, English D, Kurtzberg J, Bard J, Boyse E:
Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from an HLA-identical sibling.
N Engl J Med
321:1174, 1989[Medline]
[Order article via Infotrieve]
18.
Chen M, Tomkins D, Auerbach W, McKerlie C, Youssoufian H, Liu L, Gan O, Carreau M, Auerbach A, Groves T, Guidos C, Freedman M, Cross J, Percy D, Dick J, Joyner A, Buchwald M:
Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia.
Nat Genet
12:448, 1996[Medline]
[Order article via Infotrieve]
19.
Whitney M, Royle G, Low M, Kelly M, Axthelm M, Reifsteck C, Olson S, Braun R, Heinrich M, Rathbun R, Bagby G, Grompe M:
Germ cell defects and hematopoietic hypersensitivity to -interferon in mice with a targeted disruption of the Fanconi anemia C gene.
Blood
88:49, 1996[Abstract/Free Full Text]
20.
Haneline L, Broxmeyer H, Cooper S, Hangoc G, Carreau M, Buchwald M, Clapp D:
Multiple inhibitory cytokines induce deregulated progenitor growth and apoptosis in hematopoietic cells from Fac / mice.
Blood
91:4092, 1998[Abstract/Free Full Text]
21.
Carreau M, Gan O, Liu L, Doedens M, McKerlie C, Dick J, Buchwald M:
Bone marrow failure in the Fanconi anemia group C mouse model after DNA damage.
Blood
91:1, 1998[Free Full Text]
22.
Rathbun R, Faulkner G, Ostroski M, Christianson T, Hughes G, Jones G, Cahn R, Maziarz R, Royle G, Keeble W, Heinrich M, Grompe M, Tower P, Bagby G:
Inactivation of the Fanconi anemia group C gene augments interferon-gamma-induced apoptotic responses in hematopoietic cells.
Blood
90:974, 1997[Abstract/Free Full Text]
23.
Harrison D:
Competitive repopulation: A new assay for long-term stem cell functional capacity.
Blood
55:77, 1980[Abstract/Free Full Text]
24.
Szilvassy S, Humphries R, Lansdorp P, Eaves A, Eaves C:
Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy.
Proc Natl Acad Sci USA
87:8736, 1990[Abstract/Free Full Text]
25.
Yoder MC, Hiatt K, Dutt P, Mukherjee P, Bodine DM, Orlic D:
Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac.
Immunity
7:335, 1997[Medline]
[Order article via Infotrieve]
26.
Bodine D, Seidel N, Orlic D:
Bone marrow collected 14 days after in vivo administration of granulocyte colony-stimulating factor and stem cell factor to mice has 10-fold more repopulation ability than untreated bone marrow.
Blood
88:89, 1996[Abstract/Free Full Text]
27.
Harrison D, Astle C:
Short- and long-term multilineage repopulating hematopoietic stem cells in late fetal and newborn mice: Models for human umbilical cord blood.
Blood
90:174, 1997[Abstract/Free Full Text]
28.
Zhong R, Astle C, Harrison D:
Distinct developmental patterns of short-term and long-term functioning lymphoid and myeloid precursors defined by competitive limiting dilution analysis in vivo.
J Immunol
157:138, 1996[Abstract]
29.
Yoder M, Hiatt K, Mukherjee P:
In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus.
Proc Natl Acad Sci USA
94:6776, 1997[Abstract/Free Full Text]
30.
Rebel V, Miller C, Eaves C, Lansdorp P:
The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their adult bone marrow counterparts.
Blood
87:3500, 1996[Abstract/Free Full Text]
31.
Clapp D, Freie B, Srour E, Yoder M, Fortney K, Gerson S:
Myeloproliferative sarcoma virus directed expression of  -galactosidase following retroviral transduction of murine hematopoietic cells.
Exp Hematol
23:630, 1995[Medline]
[Order article via Infotrieve]
32.
Zhang Y, Vik T, Ryder JW, Srour E, Jacks T, Shannon K, Clapp D:
Nf1 regulates hematopoietic progenitor cell growth and ras signaling in response to multiple cytokines.
J Exp Med
187:1893, 1998[Abstract/Free Full Text]
33.
Sheridan TM, Robinson S, Mauch PM, van Os R:
Potential immunogenicity of LY5 antigens in bone marrow transplantation.
Exp Hematol
26:756, 1998
34.
Kruyt F, Youssoufian H:
The Fanconi anemia proteins FAA and FAC function in different cellular compartments to protect against cross-linking agent cytotoxicity.
Blood
92:2229, 1998[Abstract/Free Full Text]
35.
Youssoufian H:
Cytoplasmic localization of FAC is essential for the correction of a prerepair defect in Fanconi anemia group C cells.
J Clin Invest
97:2003, 1996[Medline]
[Order article via Infotrieve]
36.
Kruy |
TOP
ABSTRACT
INTRODUCTION