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Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 275-287
By
From the Division of Hematology and Medical Oncology, Department of
Medicine, the Department of Molecular and Medical Genetics, and the
Department of Pediatrics, Oregon Health Sciences University; and
Portland Veterans Affairs Medical Center, Portland, OR.
Cells from individuals with Fanconi anemia (FA) arrest excessively
in the G2/M cell cycle compartment after exposure to low doses of DNA
cross-linking agents. The relationship of this abnormality to the
fundamental genetic defect in such cells is unknown, but many
investigators have speculated that the various FA genes directly
regulate cell cycle checkpoints. We tested the hypothesis that the
protein encoded by the FA group C complementing gene (FAC)
functions to control a cell cycle checkpoint and that cells from group
C patients (FA[C]) have abnormalities of cell cycle regulation
directly related to the genetic mutation. We found that retroviral
transduction of FA(C) lymphoblasts with wild-type FAC cDNA
resulted in normalization of the cell cycle response to low-dose
mitomycin C (MMC). However, when DNA damage was quantified in terms of
cytogenetic damage or cellular cytotoxicity, we found similar degrees
of G2/M arrest in response to equitoxic amounts of MMC in FA(C) cells
as well as in normal lymphoblasts. Similar results were obtained using
isogenic pairs of uncorrected, FAC- or mock-corrected (neo
only) FA(C) cell lines. To test the function of other checkpoints we
examined the effects of hydroxyurea (HU) and ionizing radiation on cell
cycle kinetics of FA(C) and normal lymphoblasts as well as with
isogenic pairs of uncorrected, FAC-corrected, or mock-corrected FA(C)
cell lines. In all cases the cell cycle response of FA(C) and normal
lymphoblasts to these two agents were identical. Based on these studies
we conclude that the aberrant G2/M arrest that typifies the response of
FA(C) cells to low doses of cross-linking agents does not represent an
abnormal cell cycle response but instead represents a normal cellular
response to the excessive DNA damage that results in FA(C) cells
following exposure to low doses of cross-linking agents.
FANCONI ANEMIA (FA) is an autosomal
recessive disorder characterized by progressive pancytopenia; a high
risk of malignancies, especially acute myelogenous leukemia
(AML)1; and in some cases by congenital malformations
including skin pigmentation abnormalities, skeletal deformities, and
renal anomalies.2-5 Most patients are diagnosed during the
first 10 years of life and die as young adults of bone marrow failure
or AML. Standard medical therapy is directed at supporting bone marrow
function and may include the use of androgens, hematopoietic growth
factors, antibiotics, and transfusion of blood products.6
Although the National Heart Lung and Blood Institute has recently
instituted a limited trial of gene therapy for individuals with
FA(C),7 currently the only known definitive
therapy for bone marrow failure or AML in FA patients is allogeneic
bone marrow transplantation from a histocompatible
donor.6,8
The cellular hallmark of FA is a unique hypersensitivity to DNA
cross-linking agents. Treatment of FA cells with agents such as
mitomycin C (MMC) or diepoxybutane (DEB) at doses that have little
impact on normal cells results in chromosomal instability and cellular
death.6,8-10 Using cell-cell fusion techniques, it has been
established that there are at least five FA complementation groups
(FA[A] to FA[E]).11
The gene encoding the defective or missing protein in patients with
FA(C) has been cloned by Strathdee et al using a functional
complementation strategy.12 The FA group C complementing
(FAC) gene encodes a protein of 558 amino acids with a
predicted molecular mass of ~63 kD and has been localized to
chromosome 9q22.3.12 The FAC protein
contains numerous hydrophobic residues, consensus sites for Ser/Thr
phosphorylation, and binding sites for the molecular chaperone
immunoglobulin heavy-chain binding protein.13 However, the
sequence contains no significant homologies with other known proteins
in genetic data banks, and the exact biochemical function of the
FAC protein remains unknown. Using polyclonal rabbit
antiserum, a number of groups including ours have found the
FAC protein localized primarily in the
cytoplasm.13,14 Using immunoprecipitation or affinity
purification techniques, a family of cytosolic proteins that bind ex
vivo to FAC protein have been identified, but their function
and in vivo significance are unknown.14,15
FAC mRNA has been found in every normal tissue tested to date,
and several different types of mutations have been described in FA(C)
patients.12,16,17 FAC gene expression appears to be
constitutive, and we have recently found that neither FAC mRNA
or protein is induced in hematopoietic cells after cellular exposure to
MMC, DEB, hydrogen peroxide, Because of the universal occurrence of bone marrow failure in patients
with FA, we and others postulated that the FAC protein may play
some direct role in the regulation of hematopoietic cell proliferation.
Gain- and loss-of-function strategies have confirmed this hypothesis.
We have shown that suppression of FAC gene expression reduced
clonal growth of normal erythroid and granulocyte-macrophage progenitor
cells and that FA(C) progenitor cells are hypersensitive to the mitotic
inhibitory effects of interferon- Abnormalities in the cell cycle kinetics of cells derived from
individuals with FA were first reported by Sasaki in 1975. In these
studies it was noted that FA cells passed more slowly than did normal
cells through the G2/M phase of the cell cycle. Treatment of FA cells
with MMC was noted to further increase the delay in cell cycle
transit.24 An increased percentage of cells in the G2/M
compartment has been noted in untreated cells but is most marked in
cells that have been exposed to DNA cross-linking agents such as MMC or
DEB.25-27
Recent advances in the understanding of cellular proliferation has lead
to the realization that there are certain restriction points for
controlling cell cycle progression. Cell cycle checkpoints order events
in the cell cycle so that initiation of later events is dependent on
the completion of earlier events. For example, the initiation of
mitosis is dependent on the completion of DNA synthesis.28
By ordering the cell cycle in this way, checkpoints ensure that cells
maintain genome integrity (remain euploid) and withstand episodic DNA
damage and delays in DNA replication.29 Studies using yeast
mutants have defined multiple checkpoints for the G1, S, and G2/M
phases of the cell cycle, and homologues of these checkpoints have now
been defined in mammalian cells.29-34 Each checkpoint may
serve multiple functions. For example, the G2/M checkpoint serves (1)
to delay mitosis or arrest cells in response to DNA damage; (2) to
maintain the dependence of mitosis on chromosome replication; and (3)
to make the exit from mitosis dependent on completion of spindle
assembly and proper chromosome segregation.33
Many investigators have speculated that the observed G2/M accumulation
of FA cells after low-dose MMC treatment is caused by defective cell
cycle checkpoint control and that the FA genes may directly regulate
one or more checkpoints.2,3,35-46 Such speculation has been
fueled by the report that the protein product of a novel cyclin-related
gene is able to correct the repression of DNA synthesis seen in FA(A)
fibroblasts after treatment with psoralen and ultraviolet-A
light.39 Others suggest that the observed cell
cycle abnormalities are only secondary to DNA damage and that FA genes
serve to prevent and/or repair DNA damage.6 To
distinguish between these alternatives, we sought to determine whether
the aberrant G2/M arrest in FA(C) cells is a manifestation of primary
cell cycle checkpoint dysfunction. For the studies described here we
have used lymphoblast cell lines derived from patients with various
mutations of the FAC gene, retrovirally transduced isogenic
cells, and cells from normal individuals. Based on these studies we
argue that the aberrant G2/M arrest that characterizes the response of
FA(C) cells to low doses of cross-linking agents is a secondary result
of excessive DNA damage in these cells.
Plasmids
pLXSN.
This retroviral vector was the generous gift of Dr A.D. Miller (Fred
Hutchinson Cancer Research Center, Seattle, WA) and has been previously
described.47
pLFACSN.
A 314-bp polymerase chain reaction (PCR) product (FAC ATG start
to EcoRI site [bases 256-569 Genbank sequence X66893]) was
cloned into the PCR Script SK(+) vector (Stratagene, LaJolla, CA) to
form PCR-Script FAC ATG-RI. The sense primer was designed to add an
Xho I site just 5 Retroviral Vector Amplification
Cell Lines
MMC Cytotoxicity Assay Cellular sensitivity to MMC was assayed by plating cells at a density of 2 × 105/mL in 96-well plates. Increasing doses of MMC were added to the cells, which were then incubated in the dark for 120 hours. Following treatment, cellular viability was determined using an XTT-based assay as described previously.45 Each sample was tested in replicates of 12.Cytogenetic Studies Lymphoblast cultures were treated with doses of MMC from 0 to 400 ng/mL and incubated in the dark for 48 hours. Cultures were obtained after a 1-hour exposure to 0.25 µg/mL colcemid. After a 10-minute treatment with 0.075 mol/L KCl, the cells were fixed with a 3:1 mixture of methanol:acetic acid. Wet-mount slides were prepared, air-dried, and stained with Wright's stain. Fifty metaphase figures from each culture were scored for DNA breaks (gaps greater than one chromatid width) and radial formation. A radial was defined as a chromatid interchange in which chromatids remained paired in mitosis giving rise to a structure with multiple arms.22Cell Cycle Analysis Cells were obtained and resuspended in 100 µL of Dulbecco's phosphate buffered saline and transferred to a polystyrene tube. Following transfer, 250 µL of pH 7.2 propidium iodide (PI) stain (50 µg/mL PI, 30 mg/mL polyethylene glycol 8000, 2 µg/mL RNase A, 0.1% Triton X-100, 36 mmol/L sodium citrate) was added to each tube, and the cells were incubated at 37°C for 20 minutes. Next, 250 µL of PI salt solution (50 µg/mL PI, 30 mg/mL polyethylene glycol 8000, 0.1% Triton X-100, 0.38 mol/L NaCl) was added, and the samples were incubated at 4°C for 10 minutes. Following staining, the samples were analyzed for DNA content using a Becton Dickinson FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Data were analyzed by the Multicycle software program, which uses the polynomial S-phase algorithm (Phoenix Flow Systems, San Diego, CA).53
Retroviral Transduction of FAC cDNA Normalizes FA(C) Lymphoblast Cell Cycle Response to MMC We inserted the human FAC cDNA into the retroviral vector pLXSN to create pLFACSN. Amphotropic virus was generated by transfection of a coculture of ecotropic and amphotropic retroviral packaging cells (ping-pong amplification) with pLXSN or pLFACSN. FA(C) lymphoblasts were exposed to cell-free supernatants from a ping-pong culture, and stably transduced cells were selected in G418 (250 µg/mL). To confirm that retroviral transduction of FAC cDNA resulted in phenotypic correction of cell lines derived from FA patients, we measured the effect of MMC on cell viability. FA(C) cells transduced with pLFACSN amphotropic retrovirus had normal sensitivity to MMC, whereas FA(C) cells transduced with pLXSN amphotropic retrovirus remained as abnormally sensitive to MMC as the original nontransduced cell lines (data not shown). Expression of retrovirally derived FAC mRNA and protein was verified by Northern and Western blot analysis, respectively (data not shown).
Normal and FA(C) Lymphoblasts Have Similar G2/M Compartment
Accumulations in Response to Equivalent Amounts of MMC-Induced
Cytogenetic Damage
FA(C) Lymphoblasts Treated With High-Dose MMC Develop Both S Phase
and G2/M Compartment Accumulation
Normal and FA(C) Lymphoblasts Have Similar Cell Cycle Responses to MMC Doses That Have Equivalent Cytotoxicity We treated FAC-corrected and uncorrected FA(C) cell lines with a range of doses of MMC. Cell cycle parameters were measured after 24 and 48 hours of continuous exposure, and cytotoxicity was measured after 120 hours of continuous MMC exposure (the duration of our standard MMC cytotoxicity assay). Cell cycle responses of uncorrected, mock corrected, or FAC corrected HSC536N cells were similar when comparing doses of MMC that induced similar amounts of cytotoxicity (Table 3). Of note, G2/M accumulation occurred in both FA(C) and corrected FA(C) cells treated with doses of MMC that induced equivalent cytotoxicity. However, the G2/M accumulation was accompanied by either no significant change or a decrease in the percent of cells in S phase. The decrease in percent of cells in S phase was accompanied by a proportional decrease in the percent of cells in the G1 compartment so that the G1/S ratio did not change (data not shown). Thus, no abnormalities of G1/S or S-phase cell cycle checkpoints in response to these equitoxic doses of MMC was found in either mock- or FAC-corrected FA(C) lymphoblasts.
The S-Phase Checkpoint Response to Hydroxyurea (HU) Is Intact in FA(C) Lymphoblasts S-phase checkpoints prevent cell cycle progression into the G2 compartment until DNA replication/repair is complete. To further test the function of these checkpoints in FA(C) cells, we treated cells with HU, which is known to inhibit the enzyme ribonucleotide reductase resulting in depletion of intracellular dNTP precursors and cell cycle arrest at the G1/S boundary.57,58 Yeast mutants have been described that lack normal S-phase checkpoint function and have abnormally high levels of cellular death after exposure to HU.29,32,34 We treated normal or FA(C) lymphoblasts with control media or media containing 1 or 5 mmol/L HU for 24 hours. At the end of the treatment period these cells were harvested for cell cycle analysis. Both normal and FA(C) lymphoblasts had similar degrees of cell synchronization at the G1/S boundary after treatment with HU (Table 4, Fig 4).
Radiation-Induced G1 and G2/M Checkpoints Function Normally in FA(C) Lymphoblasts The above data strongly suggest that the G2/M accumulation seen in FA(C) lymphoblasts is secondary to appropriate cellular recognition of DNA damage and normal functioning of the G2 checkpoint. However, an alternative explanation is that FA cells have a defective checkpoint function in G1 or S phase such that cells with DNA damage abnormally progress through G1 and/or S phase and then appropriately arrest in G2/M. Therefore, we decided to test G1/S and G2/M checkpoint function in response to ionizing radiation, a genotoxic stressor known to induce both G1- and G2/M-compartment accumulation in normal cells.59-61 We treated normal and FA(C) lymphoblasts with 5 Gy of -irradiation and measured cell cycle parameters of control and
irradiated cells after 6 and 24 hours (Fig
5, Table 5).Both normal and FA(C) cells had similar cell cycle responses to 5 Gy of
-irradiation.
The excessive G2/M accumulation seen in FA cells, especially after DNA cross-linker exposure, is caused not only by a prolongation of the G2/M in each cell cycle but also by complete arrest of some cells in the G2/M phase.36,37,56 Hoehn et al noted that the minimum duration of the G2 phase during the first cell cycle of lymphocytes after phytohemagglutinin stimulation was 10.7 hours in FA patient samples and 5.2 hours in normal cells. Additionally, 19.5% of the cycling FA cells arrested after entering the first G2/M compartment as opposed to 2.7% of cycling lymphocytes isolated from normal subjects.56 FA lymphocytes also have delayed G2/M transit and increased arrest in the G2/M compartment of the second cell cycle compared with normal lymphocytes.36,37 Although initial reports suggested that FA cells had slowed transit through G1- and/or S-phase compartments, more recent analyses of FA cells using sensitive flow cytometry techniques have not found any evidence of such delays. Indeed, some investigators have noted that the G1 phase of the second cell cycle is abnormally short (3.7 hours v 6.1 hours in normal cells).37
Submitted March 17, 1997;
accepted August 21, 1997.
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T. Taniguchi, I. Garcia-Higuera, P. R. Andreassen, R. C. Gregory, M. Grompe, and A. D. D'Andrea S-phase-specific interaction of the Fanconi anemia protein, FANCD2, with BRCA1 and RAD51 Blood, September 18, 2002; 100(7): 2414 - 2420. [Abstract] [Full Text] [PDF]
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L. S. Haneline, H. E. Broxmeyer, S. Cooper, G. Hangoc, M. Carreau, M. Buchwald, and D. W. Clapp Multiple Inhibitory Cytokines Induce Deregulated Progenitor Growth and Apoptosis in Hematopoietic Cells From Fac-/- Mice Blood, June 1, 1998; 91(11): 4092 - 4098. [Abstract] [Full Text] [PDF]
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| Copyright © 1998 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Abstract
Introduction
Abstract
1, or interferon-
of the FAC start codon. A 1,452-bp
HindIII to Xba I (stop codon) fragment of FAC
cDNA derived from pFAC3 was cloned into the HindIII and
Xba I sites of pUC18 to create pUC FAC-D1. The pFAC3 plasmid
contains the entire human FAC cDNA sequence and was the
generous gift of Dr Manuel Buchwald (The Hospital for Sick Children,
Toronto, Ontario, Canada).
-2
and PA12 cells.
T mutation on both alleles.
100 ng/mL
continuous exposure for 48 hours) FA(C) cells develop both S-phase and
G2/M-compartment accumulation. In normal or FAC-corrected FA(C)
cells the S-phase accumulation always accompanied the G2/M-compartment
accumulation and was only seen with high-dose MMC. In contrast, in
uncorrected FA(C) cells the G2/M accumulation was seen with both low-
and high-dose MMC, and the S-phase accumulation was seen only with
high-dose MMC treatment.
