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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3970-3977
RED CELLS
Posttranscriptional cell cycle-dependent regulation of human
FANCC expression
Michael C. Heinrich,
Kirsten V. Silvey,
Stacie Stone,
Amy J. Zigler,
Diana J. Griffith,
Michelle Montalto,
Lin Chai,
Yu Zhi, and
Maureen E. Hoatlin
From the Department of Medicine, Division of Hematology and Medical
Oncology, Oregon Health Sciences University and Portland Veterans
Affairs Medical Center, Portland, OR.
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Abstract |
The Fanconi Anemia (FA) Group C complementation group gene
(FANCC) encodes a protein, FANCC, with a predicted
Mr of 63000 daltons. FANCC is found in both the cytoplasmic
and the nuclear compartments and interacts with certain other FA
complementation group proteins as well as with non-FA proteins. Despite
intensive investigation, the biologic roles of FANCC and of the other
cloned FA gene products (FANCA and FANCG) remain unknown. As an
approach to understanding FANCC function, we have studied the molecular regulation of FANCC expression. We found that although FANCC
mRNA levels are constant throughout the cell cycle, FANCC is
expressed in a cell cycle-dependent manner, with the lowest levels seen in cells synchronized at the G1/S boundary and the highest levels in
the M-phase. Cell cycle-dependent regulation occurred despite deletion
of the 5' and 3' FANCC untranslated regions, indicating that information in the FANCC coding sequence is sufficient to mediate
cell cycle-dependent regulation. Moreover, inhibitors of proteasome
function blocked the observed regulation. We conclude that FANCC
expression is controlled by posttranscriptional mechanisms that are
proteasome dependent. Recent work has demonstrated that the functional
activity of FA proteins requires the physical interaction of at least
FANCA, FANCC, and FANCG, and possibly of other FA and non-FA proteins.
Our observation of dynamic control of FANCC expression by the
proteasome has important implications for understanding the molecular
regulation of the multiprotein complex.
(Blood. 2000;95:3970-3977)
© 2000 by The American Society of Hematology.
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Introduction |
Fanconi anemia (FA) is an autosomal recessive disorder
characterized by progressive pancytopenia, high risk for malignancies (especially acute myelogenous leukemia),1 and, in some
patients, congenital malformations including skin pigmentation
abnormalities, skeletal deformities, and renal
anomalies.2-5 The cellular hallmark of FA is a unique
hypersensitivity to DNA cross-linking agents. Treatment of FA cells
with agents such as mitomycin C or diepoxybutane at doses that have
little impact on normal cells results in chromosomal instability and
cellular death.6-9
Despite clinical and cellular similarities in the FA phenotype,
cell-cell fusion techniques have established that there are at least 8 FA complementation groups, FA[A] to FA[H].10 The genes
inactivated in complementation groups A (FANCA), C
(FANCC), and G (FANCG) have been
cloned.11-13 Additionally, the genes mutated in group D and
group E FA have been localized to chromosomes 3p22-26 and 6p21-22,
respectively.14,15
The FANCC gene was cloned by Strathdee et al11
using a functional complementation strategy, and it encodes a protein
of 558 amino acids with a predicted molecular mass of 63 kd. FANCC is
predominately hydrophobic and has no obvious transmembrane domain,
signal sequence, or other functional motifs. The polypeptide sequence
contains no significant homologies with other known proteins in genetic
databanks, and the biochemical function of FANCC is unknown. A number
of groups have found FANCC localized primarily in the cytoplasm, with
recent reports also describing the existence of nuclear
FANCC.16,17 Low endogenous protein expression has limited
all existing studies of FANCC to immunoblot and overexpression immunofluorescent analyses (IFA). Numerous interacting partners for
FANCC have been described, including the cyclin-dependent kinase cdc2,
FANCA, FANCG, the molecular chaperone GRP94, NADPH cytochrome P450
reductase, and FAZF a novel transcriptional repressor with homology to
the acute promyelocytic zinc finger protein.17-24
We report here further evidence that FANCC expression is regulated
during the cell cycle and that this regulation is controlled by a
posttranscriptional mechanism dependent on proteasome function. The
lowest expression was found in cells synchronized at the G1/S boundary,
and the highest expression was found in cells in the M-phase. We infer
that the regulation of FANCC polypeptide expression occurs after
transcription because we found that levels of FANCC mRNA were constant
throughout the cell cycle. Cell cycle-dependent regulation occurred
despite deletion of the 5' and 3' FANCC untranslated regions, indicating that information in the FANCC coding sequence is
sufficient to mediate cell cycle-dependent regulation. Cell cycle
regulation was abrogated with the use of inhibitors of
proteasome-dependent proteolysis. We conclude that FANCC expression is
controlled by proteasome-dependent processes that regulate FANCC mRNA
translation, protein degradation, or both.
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Materials and methods |
Plasmids
pcFANCC.
The plasmid pcFANCC has been described.25 The FANCC cDNA in
this plasmid contains only the coding sequence with no FANCC-derived 5' or 3' untranslated region sequences.
pGEM-11z FANCC.
The FANCC cDNA was excised from pLFACSN using XhoI and
BamHI and was ligated into a
XhoI/BamHI-digested pGEM-11z vector (Promega, Madison,
WI).26
2HA-L554P.
The construct 2HA-L554P has been described.23 It contains
an HA-epitope-tagged cDNA for the L554P mutant cDNA inserted in the
backbone of the pCEP4 vector (Invitrogen, Carlsbad, CA).
pcLUC.
A BglII/XhoI fragment containing the firefly luciferase
cDNA was excised from pSP-luc+ (Promega) and ligated into
BamHI/XhoI-digested pcDNA3.1(+) vector (Invitrogen).
Cell lines
The parental 293 cell line was obtained from ATCC (Manassas, VA) and
maintained in DMEM (GIBCO-BRL, Grand Island, NY) supplemented with 10%
fetal bovine serum (defined, heat-inactivated, low endotoxin; HyClone,
Logan, UT). As described previously,23,25 293 FANCC, 293 LUC, 293 L544P, and 293 NEO cells were generated. For this study,
subclones of 293 LUC, 293 NEO, and 293 L554P were prepared by
limiting-dilution cloning. The 293 FANCC E2 subclone was prepared by 2 successive rounds of limiting-dilution cloning.
FANCC antibodies
Monoclonal antibodies specific for wild-type 8F3, 3A11, or L554P
FANCC 13F5 polypeptide were generated by immunizing mice with a
KLH-peptide conjugated to a peptide containing wild-type FANCC AA
547-558 or with an overlapping peptide containing the inactivating
leucine-to-proline substitution at position 554.11 These
antibodies will be described in detail elsewhere (Zhi et al, manuscript
in preparation). Immunoblots were performed as described
previously.25
Reagents
Polyclonal rabbit anti-luciferase antiserum, dimethyl sulfoxide
(DMSO), nocodazole, and hydroxyurea were purchased from Sigma (St
Louis, MO). Lactacystin and MG132 were purchased from
Calbiochem-Novabiochem (San Diego, CA) and were resuspended in DMSO.
Cell synchronization experiments
Cells were synchronized at the G1/S boundary by culturing in 1.3 mmol/L hydroxyurea for 24 hours.27 To release cells from synchronization, the cells were washed twice with phosphate-buffered saline (PBS) and placed back in regular growth medium. Cell cycle progression was monitored by flow cytometric determination of DNA
content. At timed intervals after release, cells were processed for
protein, DNA, or RNA analysis. To enrich for cells in M-phase, cultures
were treated with 2 µmol/L nocodazole for 24 hours before harvest.28
RNA analysis
Biotin-labeled anti-sense probes to luciferase or FANCC were
generated by in vitro transcription of HincII linearized
pSP-luc+ or NcoI linearized pGEM-11Z FANCC, respectively. A
biotin-labeled anti-sense probe to human  -actin was generated by in
vitro transcription of the pTRI--actin template (Ambion, Austin,
TX). Non-isotopic ribonuclease protection assays were performed using a
commercially available kit (RPA II and Brightstar Biodetect; Ambion).
Cell cycle analysis
Cells were harvested and stained with propidium iodide as described
previously. After 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).29
Indirect immunofluorescence
Then 293 FANCC E2 and control 293 NEO cells were seeded at a density
of 2.5×104 cells/chamber of 2-well Permanox Lab Tek
chamber slides (Nunc Nalge; Rochester, NY). Cells were cultured in
complete growth media for 48 hours. For cell synchronization studies,
slides were treated with fresh medium (asynchronous control) or medium
containing hydroxyurea (final concentration, 1.3 mmol/L) for 24 hours,
washed twice with PBS, and released into complete media. The cells were fixed and permeabilized for analysis at 0, 4, 6, 8, 10, and 12 hours
after release. To enrich for cells in the M-phase, slides were treated
with 2 µmol/L nocodazole for 24 hours and then processed for
IFA.28
The slides were fixed with 3.7% paraformaldehyde and permeabilized
with 0.2% Triton X-100. Cells were blocked with a solution of 3%
normal goat serum in PBS. Primary antibody was added (1:100 8F3) and
allowed to bind overnight at room temperature with gentle rocking.
After 5 washes with PBS at 5 minutes each, Oregon green-conjugated goat
antimouse secondary antibody was added (1:100 [Molecular Probes,
Eugene, OR]). Primary and secondary antibodies were diluted in PBS
containing 3% normal goat serum. Secondary antibody incubation was
performed in the dark. The stained cells were mounted in SlowFade (Molecular Probes).
Fluorescence was observed by using a Bio-Rad MRC 1024ES laser scanning
confocal imaging system (Bio-Rad, Cambridge, MA) attached to an
inverted Nikon eclipse TE330 microscope (Nikon, Tokyo,
Japan). The acquisition system (LaserSharp) uses a
krypton/argon laser with an excitation line at 488 nm and an 8-bit
photomultiplier tube. Settings were optimized using positively stained
cells and were maintained during scanning of control cells to retain
relative brightness. Images were processed using the LaserSharp
postprocessing software and exported as TIFF files into Adobe Photoshop
4.0 (Adobe, San Jose, CA).
Percentage FANCC expression data were gathered with a
Leitz Orthoplan 2 fluorescence microscope (Leitz,
Stuttgart, Germany) using a green filter (488 nm) and a ×100 oil
lens. Two hundred cells were counted from each slide. Cells were scored
as positive for FANCC staining based on a comparison with control cells
(293 NEO). Standard error was computed from the standard deviation divided by the square root of the count using StatView 5.0 (SAS Institute, Cary, NC).
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Results |
Immunofluorescent analysis of FANCC expression
In a previous study using 293 FANCC cells, we noted cell-to-cell
variation of FANCC expression and variable subcellular
localization.25 Because the expression of endogenous FANCC
is regulated during the cell cycle, we wondered whether exogenous FANCC
was subject to the same type of regulation and whether this phenomenon
might partially or completely explain the cell-to-cell variation we observed.25,30
To investigate FANCC expression and subcellular localization during the
cell cycle, we transfected 293 cells with pcFANCC and then cloned the
resultant cell population twice by limiting dilution to obtain
293/FANCC E2 cells. Control cell lines, 293 NEO and 293 LUC, were
created similarly. Using polyclonal and monoclonal anti-FANCC
antibodies, we confirmed our previous observation that the cellular
expression of exogenous FANCC was variable from cell to cell (Figure
1). In contrast, the expression of
luciferase was similar in all 293 LUC cells examined. As we reported
previously, FANCC was found in the cytoplasm and the nuclei of these
cells.23,25 The subcellular localization was highly
variable: it ranged from equal amounts in the cytoplasm and the nuclei,
to much greater nuclear expression than cytoplasmic expression, to much
greater cytoplasmic expression than nuclear expression.
Additionally, we observed FANCC nuclear foci in approximately 25% of
the cells. Figure 1C depicts a typical range of FANCC expression and
localization seen in our experiments.
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| Fig 1.
Heterogeneous cellular expression of FANCC.
Cells were stained with monoclonal anti-FANCC antibody and a secondary
Oregon green-labeled goat antimouse antibody. Fluorescence was
observed using a confocal imaging system. A single slice through the
plane of cells is displayed. (A) 293 NEO cells. No staining of
endogenous FANCC is detectable. (B) 293 FANCC E2 cells. In this field,
1 cell has a large amount of cytoplasmic FANCC, whereas an adjacent
cell is only weakly positive. (C) 293 FANCE2 cells. Some cells have
only cytoplasmic FANCC, others have predominately nuclear FANCC, and
some have FANCC in both compartments. Nuclear foci are clearly visible
in the top and bottom cells. In addition to the subcellular
localization differences, there is marked cell-to-cell variation in
total cellular FANCC expression.
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Cell cycle-dependent regulation of FANCC expression
To test the hypothesis that the concentration of exogenous FANCC was
regulated in a cell cycle-dependent manner analogous to that of
endogenous protein, we used hydroxyurea to synchronize cells at the
G1/S boundary.31,32 After synchronization, the cells were
washed extensively and allowed to progress through the cell cycle. Cell
cycle position was monitored by flow cytometry, and FANCC expression
was assessed using immunofluorescence.
Results of representative fields are shown in Figure
2A, and summarized data are depicted in
Figures 2B and 2C. Although our impression is that all 293 FANCC E2
cells stain at least weakly positive when using our anti-FANCC mAbs and
are brighter than control cells (293 WT, 293 NEO, or 293 LUC), the
level of staining was very low in some cells. For the purposes of our
experiments, we scored 200 cells as either 0+ (negative/low FANCC
expression compared with control cells ["low"]) or 1+
(moderate-to-high FANCC expression compared with control cells
["high"]). In asynchronous cultures approximately 20% of cells
have high FANCC expression. The percentage of high FANCC-expressing
cells drops to 5% in cells arrested at the G1/S boundary. After
release, FANCC expression does not appreciably increase for 8 hours,
and it reaches a maximum 10 to 12 hours after release, at which time
30% of the cells express high levels of FANCC. The greatest number of
high FANCC-expressing cells (60%) was seen in cultures of
nocodazole-treated cells, implying that FANCC expression is highest in
the M-phase because nocodazole arrests cells by the disruption of
microtubules.28
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| Fig 2.
Cell cycle-dependent regulation of FANCC expression.
293 FANCC E2 cells were synchronized with hydroxyurea for 24 hours and
then washed twice and cultured in complete growth medium. Cells were
fixed and permeabilized at different time points after release (0 hours, 4 hours, 6 hours, 8 hours, 10 hours, and 12 hours). An
asynchronous population of 293 FANCC E2 cells (Asynch.) and 293 Neo
cells were also analyzed (Control). Cells were treated with nocodazole
for 24 hours to induce M-phase arrest. (A) IFA of cells using
monoclonal anti-FANCC antibody. No FANCC staining is detectable in 293 Neo (control cells). FANCC expression is lowest in cells at the time of
or immediately after release from hydroxyurea synchronization. Highest
FANCC expression is seen in cells 10 to 12 hours after HU release or in
cells arrested in the M-phase by nocodazole. (B) Cell cycle
distribution of cells shown in panel A. (C) Summary statistics. 200 cells in each condition were scored as negative or positive for FANCC
expression. Bars depict mean ± SEM.
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Interestingly, cell cycle position did not account for all the observed
cell-to-cell variation in FANCC expression; even in the
nocodazole-treated cultures, a full 40% of the cells were scored
as having negative or low FANCC expression. Nor did we observe any correlation between cell cycle position and subcellular localization.
To confirm our IFA results, we used an immunoblot technique to assess
FANCC expression. Whole-cell lysates were prepared from asynchronous
cells, cells synchronized at the G1/S boundary by hydroxyurea, and
populations of cells released from G1/S synchronization (Figure
3A) and probed with a murine monoclonal
antibody that recognizes a carboxy-terminal FANCC epitope. We detected
only a single protein band corresponding to FANCC in 293 FANCC E2
cells. Under these conditions, only exogenous FANCC can be detected. No
bands are seen using whole-cell lysates from 293, 293 NEO, or 293 LUC
cells (data not shown). Using an immunoblot method, we found excellent
agreement in our immunofluorescent results, with FANCC expression
increasing from low levels at the G1/S boundary to maximum levels at 16 hours after release.
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| Fig 3.
Cell cycle-dependent regulation of exogenous FANCC but
not exogenous luciferase expression.
(A) 293 FANCC E2 cells were treated with 1.3 mmol/L HU for 24 hours to
synchronize cells at the G1/S boundary. Cells were then washed twice
and fed with complete growth medium. Whole-cell lysates were prepared
at the indicated times after release. A whole-cell extract was also
prepared from a culture of asynchronous FANCC E2 cells (Control). The
whole-cell extracts were subjected to Western blotting and probed using
a murine anti-FANCC antibody (3A11). Identical results were obtained
using a second monoclonal antibody (8F3; data not shown). Cell cycle
kinetics in these experiments was identical to that shown in Figure 2B.
(B) Whole-cell extracts of 293 LUC cells were prepared under similar
experimental conditions. Extracts were analyzed for luciferase
expression by Western blotting and probing with a rabbit
anti-luciferase antiserum. The top band is a luciferase-specific band
(LUC) detected only in 293 LUC cells and not in 293 WT, 293 NEO, 293 FANCC, or 293 L554P cells (data not shown). The lower 2 bands are
nonspecific and are seen in 293 WT and derivative cells.
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The expression of exogenous FANCC in 293 FANCC cells is controlled by
the cytomegalovirus (CMV) immediate early promoter.33 To
test whether our results were an artifactual effect of cell cycle
position on CMV promoter activity, we used 293 LUC cells in which the
expression of the exogenous firefly luciferase gene was dependent on
the identical CMV promoter used in our FANCC expression vector. We
found that the expression of luciferase protein was constant throughout
the cell cycle (Figure 3B). The FANCC cDNA used in our experiments
consisted of only the coding sequence with no FANCC-derived 5' or
3' UTR sequences. We concluded that the coding sequence of FANCC
contained information sufficient for its regulated expression.
Cell cycle-dependent regulation of L544P FANCC expression
The carboxyl terminus of FANCC is thought to be critical for the
proper functioning of FANCC.17 An inactive form of FANCC, L554P, results from a carboxy-terminal mutation identified in FA(C)
patients.11 We and others23,25,34 have recently
reported that the L554P polypeptide is unable to enter the nucleus,
possibly accounting for its failure to function. To test whether the
L554P mutation would alter cell cycle-dependent FANCC regulation, we generated an isogenic cell line overexpressing the mutant L554P polypeptide (293 L554P). We have developed and characterized a monoclonal antibody (13F5) that selectively recognizes the epitope present in L554P but absent in wild-type FANCC (Zhi et al, manuscript in preparation). The13F5 monoclonal antibody was used to probe whole-cell lysates prepared from asynchronous or HU-synchronized 293 NEO, 293 FANCC E2, and 293 L554P cells. The 13F5 antibody selectively
recognized the L554P but not the wild-type FANCC isoform (Figure
4). The expression of L554P was markedly
decreased in cells synchronized at the G1/S boundary in a manner
identical to that of wild-type FANCC. Thus, the mechanism by which
FANCC expression is regulated during the cell cycle is not affected by
this particular mutation.
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| Fig 4.
Expression of a patient-derived mutant form of FANCC
(L554P) is regulated identically to the wild-type isoform.
293 NEO, 293 FANCC, or 293 L554P cells were grown in the presence (HU
24H) or absence (Asynch.) of 1.3 mmol/L HU for 24 hours. Whole-cell
extracts were prepared and analyzed by Western blotting using a
monoclonal antibody specific for the L554P FANCC isoform (13F5). The
L554P isoform is recognized in asynchronous 293 L554P cells, but no
endogenous (293 NEO) or exogenous (293 FANCC) wild-type FANCC is
detected using this antibody. Synchronization with HU markedly
decreases expression of the mutant isoform in 293 L554P cells.
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Endogenous and exogenous FANCC mRNA levels are constant throughout
the cell cycle
To aid in determining potential mechanisms for the cell
cycle-dependent regulation of FANCC, we measured FANCC mRNA levels in
synchronized 293 FANCC and 293 NEO cells. Cell cycle position did not
change the concentration of exogenous FANCC (293 FANCC), endogenous
FANCC (293 NEO), or -actin. Results of a representative experiment
are shown in Figure 5. Although slight
fluctuations in endogenous FANCC mRNA concentration are seen in Figure
5, these changes were not consistently observed. In multiple
experiments we found no significant change in either endogenous or
exogenous FANCC mRNA levels during cell cycle progression. These
results are similar to those reported by Kupfer et al.31
Cell cycle position also did not change the concentration of luciferase
mRNA in 293 LUC cells (data not shown). We concluded that FANCC
expression varied during the cell cycle despite constant levels of
FANCC mRNA, suggesting the regulation of FANCC mRNA translation, FANCC protein degradation, or both.
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| Fig 5.
FANCC mRNA expression is constant during cell cycle
progression.
(A) 293 NEO cells were synchronized with HU and released from the G1/S
boundary as described above. Total cytoplasmic RNA was prepared from
synchronized cells or from a culture of asynchronous 293 NEO cells
(Asynch.). Endogenous FANCC and -actin mRNA were analyzed by
ribonuclease protection assay. A representative experiment is shown.
(B) Identical experiments were performed using 293 FANCC E2 cells.
Total FANCC (endogenous plus exogenous) and -actin mRNA were
analyzed by ribonuclease protection assay. A representative experiment
is shown. Exposure times were identical for membranes depicted in A and
B to demonstrate that 293 FANCC cells express more FANCC mRNA than 293 NEO cells.
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Inhibitors of proteasome function block cell cycle-dependent
regulation of FANCC
The ubiquitin-proteasome pathway is the best-described system for
the regulation of protein degradation. Therefore, we used inhibitors of
proteasome function to test whether the regulation of FANCC expression
is dependent on proteasome function. For 24 hours, 293 FANCC E2 cells
were treated with vehicle only (DMSO) or with proteasome inhibitor (20 µmol/L MG132 or 10 µmol/L lactacystin in DMSO) ± HU (Figure
6). Treatment with either inhibitor alone increased FANCC expression. However, the percentage of cells in the
G2/M compartment also dramatically increased in inhibitor-treated cells
(Table 1), probably because of the
inhibition of proteasome-mediated cyclin degradation.35,36
Thus, increased FANCC expression could be a direct result of the
inhibition of proteasome-dependent degradation of FANCC or an indirect
result of the accumulation of cells in the G2/M compartment, where
FANCC expression is increased (Figures 2, 3). To help distinguish
between these possibilities, we treated cells with both HU and
proteasome inhibitor. Combined treatment of cells with HU and MG132
resulted in partial synchronization at the G1/S boundary (70% vs. 97%
DMSO + HU). Combined treatment with HU and lactacystin resulted in
nearly complete synchronization at the G1/S boundary (93%). However,
the synchronization of cells at the G1/S boundary in the presence of
either inhibitor did not result in a decrease in FANCC expression when
compared with treatment with inhibitor alone. Thus, the inhibition of
proteasome function prevented the reduction of FANCC expression
associated with partial or complete synchronization at the G1/S
boundary, suggesting that this phenomenon was proteasome dependent.
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| Fig 6.
Proteasome inhibitors increase FANCC expression.
293 FANCC E2 cells were treated with media only (Control) or media
supplemented with vehicle alone (DMSO), 20 µm MG132 (MG132), or 10 µmol/L lactacystin (LACT.) for 24 hours in the presence or absence of
1.3 mmol/L HU. Whole-cell extracts were analyzed for FANCC expression
by Western blotting and probing with monoclonal anti-FANCC antibody
(8F3). Ten percent of the cells from each experimental condition were
analyzed for DNA content by flow cytometry (Table 1). Arrow indicates
full-length FANCC. The apparent increase in size of full-length FANCC
in inhibitor-treated cells is an artifact of this particular gel (see
also Figure 7).
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To further confirm a direct role of proteasome inhibitor on FANCC
expression independent of any secondary effect on cell cycle distribution, we first synchronized the cells at the G1/S boundary for
24 hours using hydroxyurea, and then we added vehicle alone (DMSO),
MG132, or lactacystin to our cultures. Treatment of cells with MG132 or
lactacystin after prior synchronization at the G1/S boundary did not
change the cell cycle distribution compared with treatment with HU
alone or HU + DMSO (data not shown). As shown in Figure
7, the addition of MG132 or lactacystin to
cells synchronized at the G1/S boundary increased FANCC expression.
Thus, our results suggested that the observed decrease in FANCC
expression at the G1/S boundary was an active process dependent on
proteasome function.
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| Fig 7.
Proteasome inhibitors MG132 and lactacystin increase
FANCC expression in cells synchronized at the G1/S boundary.
293 FANC E2 cells were treated for 24 hours with complete growth medium
(Control) or 1.3 mmol/L HU (HU). HU-synchronized cells were treated
with vehicle only (DMSO), 20 µmol/L MG132 (MG132), or 20 µmol/L
lactacystin (LACT.) for an additional 6 hours. Whole-cell extracts were
analyzed for FANCC expression by Western blotting and probing with a
monoclonal anti-FANCC antibody (8F3).
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Discussion |
In the past decade, genes for 3 of the 8 known FA complementation
groups have been cloned. However, the gene products of the 3 cloned FA
genes have no informative homologies to known sequences, nor do the 3 proteins have significant homology to each other.11-13 Thus, the FA genes likely regulate a novel biologic pathway. Various hypotheses have been advanced to explain the clinical phenotype, including defects in DNA repair, pre-repair defects, abnormalities in
coping with cellular oxidative stress, and abnormal regulation of
apoptosis.2,3,37 Additionally, we recently reported that FANCC interacts with and co-localizes in nuclear foci with the novel
transcriptional repressor FAZF, suggesting a possible role in chromatin
remodeling.23
Understanding the basic features of FA protein fate, including
expression and subcellular localization during the cell cycle, is a
reasonable first step in confirming and extending notions about FA
protein function. In this paper we report that FANCC subcellular
localization was surprisingly variable. Under the conditions of our
experiments, no obvious correlation between cell cycle transit and
subcellular localization was observed. In addition, we report that the
level of total cellular FANCC expression was controlled by a
proteasome-dependent posttranscriptional mechanism.
We previously reported that FANCC expression varied in a clonal
population of cells that had been engineered to overexpress FANCC (293 FANCC).25 The expression vector used in these studies lacked FANCC 5' and 3' UTR sequences and contained only the
coding sequence under the control of a CMV promoter. Thus, the variable expression we observed suggested that sequence information contained in
the FANCC coding region might allow cell cycle-dependent regulation of
FANCC expression. Moreover, endogenous FANCC expression was reported by
other investigators to change during the cell cycle in HeLa cells,
suggesting that exogenous expression of FANCC mirrors the behavior of
the endogenous protein in this respect.31 To explore
further the basis of FANCC regulation, we measured FANCC mRNA and
protein levels in asynchronous and synchronous cell populations of a
doubly cloned subline of 293 FANCC (293 FANCC E2). Despite a striking
variation in FANCC protein expression during the passage from the G1/S
boundary to the G2/M compartment assayed by immunoblot and IFA, we
found no corresponding change in FANCC mRNA levels. Regulated
expression was not observed for unrelated exogenous cDNA expressed
under the same conditions, suggesting that this effect is specific for
the FANCC coding sequence. Consistent with a posttranscriptional
regulation model, we found that endogenous FANCC mRNA levels remained
constant during cellular transition from the G1/S boundary to the G2/M
compartment in matched control cells by using a ribonuclease protection
assay. We emphasize these similarities between endogenous and
overexpressed proteins because a drawback in the FA field is that
endogenous FA proteins cannot be detected by IFA because of low
expression levels. However, IFA is crucial for investigating which
factors regulate subcellular localization. One strength of this work is
that endogenous FANCC expression behavior closely matched that observed
for overexpressed proteins wherever comparison was possible. We
conclude that FANCC expression is regulated at a posttranscriptional
level by altering the translation of FANCC mRNA, controlling
degradation of FANCC, or both.
An inactive mutant form of FANCC, L554P, results from a mutation
identified in FA(C) patients.11 Although wild-type FANCC interacts with FANCA, appears in the nucleus during some parts of the
cell cycle, and can form subnuclear foci, the L554P mutation abrogates
the interaction with FANCA and the nuclear localization observed for
wild-type FANCC.23,25,38 To determine whether this missense
mutation alters the cell cycle-dependent regulation of FANCC, we made
a cell line overexpressing L554P and examined protein levels
during the cell cycle. We found that the expression pattern of L554P
was identical to that seen with wild-type FANCC. Thus, the mechanism by
which FANCC expression is regulated during the cell cycle is not
affected by the L554P mutation. Taken together, this suggests that
conditional nuclear localization or interaction with other FA proteins
is required for FANCC function. In this regard,
Youssoufian39 found that forced nuclear expression of FANCC
by attachment of an SV-40 nuclear localization signal (NLS) interferes
with FANCC function, leading to the notion that cytoplasmic localization, not nuclear localization, is required for FANCC function.
The abundant cytoplasmic expression observed for FANCC and the
discovery of cytoplasmic FANCC binding partners are consistent with
this view.17-19,21-24 However, now that FANCC expression
and subcellular localization seem to be much more complex than
originally appreciated, additional interpretations should be
considered. For example, attachment of an SV-40 nuclear localization
signal may have interfered with appropriate subnuclear localization
(eg, in foci). Moreover, experimental evidence suggests that FANCC and
FANCA physically interact and that this association is required for the
nuclear translocation of FANCA.38,40 Other FA proteins may
contribute to the formation of the nuclear FANCA/FANCC complex because
cells from certain other FA complementation groups are deficient in
this complex.41,42 Our observations suggest that the cell
cycle-dependent regulation of FANCC may be an important control
point for modulating the assembly or activity of a multiprotein complex
of FA and non-FA proteins.
Abundant precedent exists for the posttranscriptional
regulation of proteins involved in the control of critical cellular processes. For example, the translation of proopiomelanocortin mRNA is
regulated by the interaction of RNA-binding proteins and a stem-loop
sequence present in the coding region of proopiomelanocortin mRNA.43 This model is potentially of relevance to our
observations of FANCC regulation because the FANCC cDNA used in these
experiment contained only the FANCC coding sequence without 5' or
3' UTR sequences.
Another possibility for the mechanism of the posttranscriptional
regulation of FANCC expression is cellular control of FANCC polypeptide
degradation. The proteasome pathway is the best-described mechanism for
regulated protein degradation and is involved in the control of many
cell cycle-dependent proteins, including some proteins that directly
regulate cell cycle progression such as cyclins and cyclin-dependent
kinase inhibitors.44,45 Proteasome-dependent degradation of
proteins occurs through ubiquitin-dependent (eg, cyclins) and
ubiquitin-independent (eg, ornithine decarboxylase) pathways.44,46-48 Interestingly, the expression pattern of
FANCC during the cell cycle is similar to that of A- and B-type
cyclins. Both cyclin B1 and FANCC are binding partners of
cdc2.31,49,50 However, the relationship between FANCC
binding to cdc2 and the observed regulation of FANCC expression during
cell cycle progression is unknown.
The peptide aldehyde MG132, used in this study, inhibits the
proteolytic activities of the proteasome but also inhibits the cysteine
proteases calpain and cathepsin B. In contrast, lactacystin is the most
selective proteasome inhibitor known, and it inhibits the proteolytic
activities of the 20S proteasome without inhibiting other known
proteases, including chymotrypsin, trypsin, or papain.51-53 Our results with lactacystin strongly suggest a specific role of the
20S proteasome in regulating FANCC expression during the cell cycle.
We do not yet know whether proteasome-dependent proteolysis regulates
the translation of FANCC mRNA, controls FANCC degradation, or both.
Further studies are needed to discover the precise molecular mechanism(s) by which cell cycle position regulates FANCC expression.
Note added in proof. During the manuscript review process,
the cloning and characterization of the FANCF gene was reported by de
Winter et al.54
| |
Acknowledgments |
The authors thank Michael Moody for his help in preparing the
figures and Dr William Skach for his thoughtful review of the manuscript.
| |
Footnotes |
Submitted September 9, 1999; accepted February 8, 2000.
Supported by grant HL56045 from the National Institutes of Health
(M.E.H.), a grant from the Fanconi Anemia Research Foundation (M.E.H.),
and a Merit Review grant from the Department of Veterans Affairs
(M.C.H.).
Reprints: Michael Heinrich, R&D-19, Portland Veterans Affairs
Medical Center, 3710 SW US Veterans Hospital Road, Portland, OR 97207;
e-mail: heinrich{at}ohsu.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction