Clinical Variability of Fanconi Anemia (Type C ...

Clinical Variability of Fanconi Anemia (Type C) Results From Expression of an Amino Terminal Truncated Fanconi Anemia Complementation Group C Polypeptide With Partial Activity By Takayuki Yamashita, Nan Wu, Gary Kupfer, Cristin Corless, Hans Joenje, Markus Grompe, and Alan D. D'Andrea Fanconi anemia (FA) is an autosomal recessive disease characterized by congenital anomalies, aplastic anemia, and cancer susceptibility.Mutations within the FA complementation group C (FAC) gene account for approximately 14% of diagnosed FA cases. Two mutations, one in exon 1 (delG322) and one in exon 4 (IVS4 + 4 A to TI, account for 90% of known FAC mutations. The delG322 mutation results in a mild FA phenotype, while the IVS4 + 4 A to T mutation results in a severe FA phenotype. To determine the molecular basis for this clinical variability, we analyzed patient-derived cell lines for the expression of characteristic mutant

FAC polypeptides. All cell lines with the delG322 mutation expressed a 50-kD FAC polypeptide, FRP-LO (FAC-related protein), shown to be an amino terminal truncated isoform of FAC reinitiated at methionine 55. All cell fines with the IVS4 + 4 A to T mutation lacked FRPQO. Overexpression of a cDNA encoding FRP-LO in an FAW cell line resulted in partial correction of mitomycin C sensitivity. In conclusion, expression of an amino terminal truncated FAC protein accounts, at least in part, for the clinical heterogeneity among FAK) patients. 0 1996 by The American Society of Hematology.

F

ANCON1 ANEMIA (FA) is an autosomal recessive disease characterized by multiple congenital abnormalities, progressive pancytopenia, and cancer susceptibility. Cells from FA patients have genomic instability and are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB).3Cell fusion studies have identified at least five complementation p u p s for FA:.' and a cDNA for FA complementation group C (FAC) was recently cloned.' The primary amino acid sequence of the predicted FAC polypeptide has no homology with known proteins and the function of FAC remains unknown. FA is characterized by a wide variation in clinical severity.' Some FA patients have a relatively mild phenotype, with normal skeletal development, subclinical hematopoietic abnormalities, and survival into their third and fourth decades." Other patients have a more severe phenotype, with skeletal abnormalities and early onset of aplastic anemia and cancer. A clinical scale provides a quantitative assessment of mild versus severe FA phenotype.' ','* While phenotypic variation makes the diagnosis of FA difficult,I3 the DEB sensitivity assay now provides a sensitive and specific test for FA.^ Within complementation group C, clinical severity corre-

lates, at least in part, with specific FAC mutations." The human FAC gene is composed of 14 exons that encode its protein s e q ~ e n c e .In ' ~general, mild disease appears to result from mutations in exon 1, and severe disease results from mutations in exons 4, 6, or 14. The molecular basis for this clinical variability among group C FA patients is unknown. We reasoned that the mild form of FA(C) (exon 1 mutation) could be accounted for by the expression of specific isoforms of the FAC polypeptide that retain partial function. To test this hypothesis, we analyzed FAC polypeptides expressed in cells derived from patients with either mild or severe disease. Cells derived from group C patients with mild disease, but not severe disease, expressed a truncated FAC polypeptide (FRP-50), resulting from loss of the normal amino terminus of FAC. FRP-50 was also expressed in cells derived from normal controls. Moreover, the ectopic expression of FRP-50 in an MMC-sensitive FA(C) cell line conferred partial MMC resistance. These findings demonstrate that the carboxy portion of the FAC polypeptide is required for its cellular function and that internal translation initiation of the FAC polypeptide can account, at least in part, for the clinical heterogeneity among some patients with group C FA.

From the Division of Pediatric Oncology and Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; Department of Human Genetics, Free University, Amsterdam, The Netherlands; and Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, OR. Submitted September 26, 1995; accepted December 21, 1995. Supported by a grant from the National Institutes of Health, Grant No. (ROl-HL5725).A.D.D. is a scholar of the Leukemia Society of America. This work was also supported in part by a grant from the Lucille P. Markey Charitable Trust. Address reprint requests to Alan D. D'Andrea, MD, Division of Pediatric Oncology and Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115. 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. 0 1996 by The American Society of Hematology. 0006-4971/96/871O-OOl5$3.O0/0

Cell lines and culture conditions. Epstein-Barr virus (EBV)transformed normal and mutant FA lymphoblast lines have been previously described.'.'' Cells were maintained as suspension cultures in RPMI media supplemented with 15% heat-inactivated fetal calf serum (FCS). They were grown at 37°C in a humidified, 5% C0,-containing atmosphere. Development of specific anti-FAC antiserum. A polyclonal antiserum against the carboxy terminus of FAC (amino acid 281-558) has previously been described." For affinity-purification, the 3' end of the FAC cDNA (amino acids 502-558) was subcloned into the pGEX2T vector (Pharmacia), maintaining the glutathione-s-transferase (GST) protein and FAC in frame. This GST fusion protein (GST-FAC-C 1) was synthesized and coupled to CNBr-activated Sepharose 4B via its primary amino group. The anti-FAC antiserum was first depleted of antibodies against GST by passage over a GSTSepharose 4B column. The flow-through was then applied to the GST-FAC-C1-Sepharose 4B column. After extensive washing with phosphate-buffered saline (PBS) (pH 7.4), the antibody against FACC1 (C1 antibody) was eluted from the GST-FAC-C1-Sepharose 4B column with 0.1 m o m glycine and 1 m o m acetic acid (pH 2.4). dialyzed against a large volume of PBS (pH 7.4), and concentrated

MATERIALS AND METHODS

4424

Blood, Vol 87, No 10 (May 15). 1996: pp 4424-4432

4425

CLINICAL VARIABILITY OF FANCONI ANEMIA

( ( 0

"*

q4*

I

exon 4 deletion (IV-4 A to T)

C

Fig 1. Mutations of the FAC polypeptide found in patients with FA (Group C). The wild type FAC polypeptide (558 amino acids) is shown schematically at the top. The indicatedFAC alleles (shown at the right) are prediied to encode the FAC mutant polypeptides shown. The NS4+4 A to T allele encodes a FAC mutant with an in frame delation of the 37 amino acids encoded by exon 4. The de10322 mutation encodes a truncated FAC polypeptide with a frame shift and a premature STOP codon at amino acid 44. A hatchedbar shows the frameshifted region of the protein.

by Centricon 30 (Amicon, Danvers, MA). The flow-through of the GST-FAC-Cl column was next incubated with GST-FAC (281-558) immobilized on a nitrocellulose membrane. The bound antibody fraction (C3 antibody) was eluted with 0.1 m o m glycine, 1 m o m acetic acid (pH 2.4). dialyzed against a large volume of PBS (pH 7.4), and concentrated by Centricon 30. The C1 antibody and C3 antibody recognize amino acids 502-558 and 281-502, respectively. Metabolic labeling and immunoprecipitation. Metabolic labeling of EBV-transformed lymphoblasts with "S-methionine (2 hours) and immunoprecipitation of labeled proteins were performed as described previously." Briefly, cell extracts in TBS (50 mmovL Trisbuffered isotonic saline, pH = 7.5) containing 5 mmom EDTA and 1% (voVvol) Triton X-100, 0.2% sodium dodecyl sulfate (SDS)

were precleared with preimmune serum and mixed with C1 and C3 antibody (4 pg/mL). Protein A-sepharose (Pharmacia) immune complexes were washed three times with TBS containing 1% Triton X-100 and 0.1% SDS, and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Statistical analysis. Statistical correlations were calculated using the Fischer's exact test in a two by two contingency table." Transfection of FA lymphoblasts. FAC cDNAs encoding mutant forms of the FAC polypeptide were generated by polymerase chain reaction (PCR) and cloned into the KpnI and BamHI sites of the pREP4 expression vector (Invitrogen, San Diego, CA). All cDNAs were confirmed by sequencing. Plasmid DNA (10 pg) was transfected by lipofection into HSC536N cells (2.5 X 106 cells), as previously described.I6 Selection with hygromycin (0.1 mg/mL) in RPMI medium 1640 with 15% FCS was initiated 72 hours after transfection. Selected cells were analyzed for the presence of wild type or mutant polypeptides by immunoprecipitation. MMC sensitivity of the HSC536N transfectants was performed as previously described.I6 Assay for correction of cell cycle arrest. Transfected HSC536N cells (2.5 X lo6 cells) were grown in the absence or presence of mitomycin C (MMC) at 0.1 pmovL for 48 hours. Cells were washed once with PBS, resuspended in 0.5 mL of a solution containing 0.1% citrate, 0.03% NP-40, and 0.7 p m o m propidium iodide, and incubated at room temperature for 30 minutes. RNaseA (20 pg/mL) was added, and the incubation continued for another 30 minutes at 37°C. Approximately 10,OOO cells were analyzed for fluorescence intensity at an event rate of 2,500 by FACScan (Becton Dickinson). The percentage of cells in each phase of the cell cycle was determined by analysis with the computer program CellFIT (Becton Dickinson). Data shown is representative of at least three separate transfections. RESULTS

Mild group C FA correlates with the expression of an FAC-related Protein, FRP-50. Several specific mutations

Table 1. Genotype of FAG) Lymphoblast Lines FACC Genotype Cell Line

Allele 1

NSK VU168 PD7 VU003 PD4 vu001 vu002 VU158 VU 166 VU239 PD25 PD153 4510C PD77 GM449 PD149 BD0215 PD182 PD391 PD162

wt wt delG(322) delG(322) delG(322) delG(322) delG(322) delG(322) delG(322) delG(322) delG(322) IVS4 + 4 A t o T IVS4 + 4 A t o T IVS4 + 4 A to T IVS4+4AtoT IVS4+4AtoT C to T (808) IVS4 + 4 A t o T IVS4 + 4 A t o T IVS4 + 4 A to T

Allele 2

Phenotype*

wt C to T (808) delG(322) delG(322) delG(322) delG(322) IVS4 + 4 A to T delG(322) IVS4 4 A to T IVS4 + 4 A to T IVS4 4 A t o T IVS4 4 A t o T IVS4 4 A to T C to T (808) IVS4 + 4 A to T IVS4 + 4 A to T IVS4+4AtoT

Normal Normal Normal Normal Mild FA (1) Mild FA (1) Mild FA (2) Mild FA (1) Mild FA (1) Mild FA (3) Mild FA ( 2 ) Severe FA (5) Severe FAt Severe FAt Severe FAt Severe FA (4) Severe FA (4) Severe FA (4) Severe FA (4) Severe FA (5)

wt wt

wt

+ + + +

FRP-50

Full-Length FACC

+ + + + + + + + + +

+ + +

+

Truncated FACC -

-

-

-

-

-

-

-

-

-

-

-

ND

ND

ND

-

-

-

-

-

-

-

-

-

-

-

ND ND ND

ND ND ND

ND ND ND

IVS4 + 4 A t o T indicates a single base change in the fourth intronic base, changing the sequence from A to T. Abbreviation: ND, immunoprecipitation results not available. * Simplified severity score (Auerbach et all'). A score of less than 3 is considered a mild phenotype. t Multiple skeletal abnormalities (exact clinical data not available).

+ + + + +

YAMASHITA ET AL

4426

B

A PD7

n I

P

HSC536N

4

PD153

IO

PD

nnnn

-200

I

FRP-160 4

I

P

@*"c.ll +

kD

-

-9 7

U -6 8

FACC

FACC 4 FRP-50

97 68

4- F R P - 5 0

-

4

1

2

3

4

5

6

7

43

8

C VU168

VU166

VU001

vu002

PD17

VU158

nnnnnn P

I

P

I

P

I

P

I

P

I

P

I

FRP-50*

- 43

C-FRP-50

b 1

2

3

4

5

6

7

0

9

have been identified within the FAC The most frequent mutations are the IVS4 + 4 A to T mutation, which results in the loss of exon 4" and the delG322 mutation, which results in a frameshift and a premature STOP codon in exon Together, these two mutations account for approximately 90% of known FA(C) mutations (Fig I). Initially, we compared the clinical severity of several FA patients by using the clinical score of Auerbach et al" (Table I). This score was originally described for distinguishing FA patients from non-FA patients with aplastic anemia and, more recently, was shown to be proportional to the severity of the FA phenotype." The score is calculated by adding one point each for growth retardation, birthmarks, kidney and urinary tract abnormalities, microophthalmia, low plate-

10

11 12

Fig 2. Expression of the FACrelated protein, FRP-BO, correlates with the mild FA phenotype. The indicated cell lines, derived from normal controls or patients with FA, were metabolically labeled with "S-methionine. Radiolabeled proteins were immunoprecipitated with preimmune serum (PI or antiFAC (C11antiserum (1). Molecular weight markers are shown in kD. Each cell line is described in table l.

lets, or thumb and radial abnormalities and by subtracting one point each for leaming disabilities and other skeletal abnormalities. The score thus ranges from -2 to +6; a score below 3 is defined as a mild phenotype. There is no evidence that the score is a useful predictor of bone marrow failure or cancer susceptibility. Based on this clinical score, we compared the severity of patients with either the IVS4 + 4 A to T mutation or the delG322 mutation (Table I). A mild FA clinical score (3) correlated with the homozygous IVS4 + 4 A to T genotype ( P = .0008). We next analyzed FAC polypeptides expressed in EBVtransformed lymphoblast lines derived from normal adult


4427

-

--

.

kD

FRP-150+

Fig 3. FRP-50 is an amino-terminal truncated isoform of the FAC polypeptide. NSK (non-FA) lymphoblastswere metabolically labeled, and radiolabeled proteins were immunoprecipitated with either preimmune serum (lane 1). C1 antibody (lanes 2 and 31, or C3 antibody (lanes4,5, and 6). Immunoprecipitation was performed in the presence of excess GST-FAC(C1) (502-558) (lanes 3 and 61, or GST-FAC(281-558) (lane 5). The band labeled as "C3specific" in lanes 4 and 6 is only immunoreactive with the C3 antibody.

- 68

FACC 4 FRP-50

+F R P - 5 0

4

controls and FA patients (Fig 2). A polyclonal antiserum that recognizes the carboxy terminus of FAC (CI antibody) immunoprecipitated the 60-kD FAC polypeptide from a normal control cell line (Fig 2A, lane I), as previously described.16.20.21 In addition, the antiserum specifically immunoprecipitated a SO-kD (FAC-related protein, or FRP-SO) and a I SO-kD (FRP-150) polypeptide. Using immunoprecipitation, we next screened multiple EBV immortalized lymphoblast lines, derived from patients with either exon 1 or exon 4 mutations in the FAC gene (Fig 2B and 2C). Cell lines from patients with delG322 did not express full length FAC, but did express FRP-SO (Fig 2B, lane 7 and Fig 2C, lanes 4, 6, 8, and IO). Cell lines from patients with IVS4 + 4 A to T did not express full length FAC, but did express a 55-kD truncated protein (Fig 2B, lanes 3, S and Fig 2C, lane 12), consistent with the in frame deletion of 37 amino acids from the FAC polypeptide." Table 1 summarizes the immunoprecipitation results and corresponding clinical phenotype for multiple cell lines from normal controls and patients. Cell lines derived from four normal controls and six patients with the delG322 mutation expressed FRP-SO. In contrast, all five cell lines from patients with homozygous exon 4 mutations lacked the FRP50 polypeptide. Among cell lines tested, the presence of FRP-50 correlated with the homozygous delG322 genotype, while the absence of FRP-SO correlated with the homozygous IVS4 + 4 A to T genotype ( P = .008). Most cell lines from mild FA patients ( 5 of 7) were homozygous for delG322. Two additional patients were compound heterozygotes. For PD4, one allele was delG322, and the second allele was (C to T [808]).For VU239, one allele was delG322, and the second allele was IVS4 + 4 A to T. Patients who are homozygous for (C to T [808])'9.2"or IVS4 + 4 A to TIs have severe disease. Interestingly, the two compound heterozygotes had mild FA and endogenous FRP-SO expression. Taken together, these results suggest that the delG322 allele is dominant over the IVS4 + 4 A to T allele or the (C to T [808]) allele and confers a mild phenotype. The FRP-50 pol?peptide is encoded by the FAC gene.

-

+~ 3 - r p e c i f t c

1

2

3

4

5

43

6

By several criteria, FRP-SO seems to be derived directly from the FAC gene, either as a splice variant or as an intemal translation initiation product.''.'J First, FRP-SO is directly immunoprecipitated with the anti-FAC antiserum, even in the absence of full-length FAC polypeptide (Fig 2B. lane 7, and Fig 2C. lanes 4. 6, 8. and IO). Therefore, FRP-SO is not an associated protein of FAC or a degradation product of FAC but rather, shares an epitope with FAC. Second, FRPSO was not present in cells where both FAC alleles contained mutations in exon 4 (Fig 2B, lanes 3 and S and Fig 2C, lane 12). To confirm that FRP-SO is derived from the FAC gene, we prepared two discrete antisera against FAC, directed against different linear epitopes (Fig 3). Both C1 and C3 antisera immunoprecipitated FAC and FRP-SO (Fig 3, lanes 2 and 4, respectively), suggesting that these two proteins are highly related isoforms. In contrast, CI, but not C3, recognized an epitope of FRP-IS0 (lane 2 v lane 4). suggesting that FRP1 SO and FAC are only related in the CI epitope. Immunoprecipitation of FAC and FRP-SO by CI antibody was competed by an excess of GST-FAC-CI (lane 3). but not by GST (data not shown). Immunoprecipitation of FAC and FRP-SO by C3 antibody was competed by GST-FAC, containing amino acids 281-558 of FAC (lane 5). but not by GST-FAC-CI (lane 6), confirming that CI and C3 antisera recognize nonoverlapping epitopes. FRP-SO from PD4 cells (Table 1 ) also is recognized by the Cl and C3 antibody (data not shown). Taken together, these results demonstrate that FAC and FRPSO share two discrete epitopes and that FRP-SO is probably encoded by the FAC gene. FRP-50 is an isojbrm of the FAC polvpeptide, initiated inteninlls at methionine 55. Because FAC and FRPJO have related epitopes at the carboxy terminus, we reasoned that FRP-SO is an isoform of FAC that lacks the normal amino terminus encoded by exon I . To test this hypothesis, we generated mutant FAC cDNAs by in vitro mutagenesis (Fig 4). These cDNAs encode FAC polypeptides with amino terminal truncations. The TI, T2, and T3 cDNAs encode FAC polypeptides that initiate at methionine 48 (M48), me-

YAMASHITA ET AL

4420

wlld-type

11 ~

I

1

12

013

B

Fig 4. Expression of wild-type and mutant FAC polypeptides in transfected HSC536N FA(C1 cells. (AI Schematic representation of wild-type and truncated FAC polypeptides. (Bl HSC536N cells were transfected with either pREP4 (lanes 1 and 21, pREP4-FAC(WTl (lanes 3 and 4). or pREP4-FAC(T31(lanes 5 and 61. Labeled proteins were immunoprecipitated with either preimmune antiserum (lanes 1, 3, and 5) or t h e C1 antiserum (lanes 2,4 and 61. Proteins were analyzed o n a 6% t o 12% gradient polyacrylamide gel. Protein sizes are indicated in kD. (C) HSC536N cells were transfected with pREP4 (lanes 1 and 41, pREP4-FAC(T2) (lane 21, or pREPCFAC(T11(lane 31. Immunoprecipitation was with preimmune (lane 11or anti-FAC (lanes 2 t o 41. Proteins were analyzed o n a 7.5% polyacrylamide gel.

C .

kD

m200

C

FRP-150

FRP-1504

-97

-97

-68 FACC 4 FRP-504

-68

-0-

FACC

-43 1 1 4 T2-(

1

2

3

4

5

6

thionine 55 (M55), and methionine 350 (M350), respectively (Fig 4A). An MMC-sensitive, type C FA cell line, HSC536N, was transfected with the cDNAs encoding either full-length or truncated FAC polypeptides. Transfected cells were selected in hygromycin and assayed for the presence of the mutant polypeptides (Fig 4B and C). Untransfected HSC536N cells expressed a mutant FAC polypeptide .(60 kD) and mutant FRP-50, (Fig 4B, lane 2). as previously described.’“ Both FAC and FRP-50 in these cells have an L554P missense mutation and have no functional activity.” Cells transfected with full-length FAC cDNA had increased expression of both FAC and FRPJO (Fig 4B, lane 4), indicating that both FAC and FRPJO are derived from the fulllength cDNA. In contrast, the expression of FRP-150 remains unchanged following transfection (compare lanes 2 and 4). Cells transfected with PREP-FAC(TI ), PREPFAC(T2), and PREP-FAC (T3). expressed proteins of 52

C FRP-SO

1

2

3

4

kD,50 kD, and 25 kD,respectively (Fig 4B and C). The T2 FAC polypeptide had the same electrophoretic mobility as FRPJO, suggesting that these polypeptides are identical. Consistent with these results, the in vitro transcription and translation of the full-length FAC cDNA resulted in synthesis of both FAC and FRP-50 (data not shown). Heterologous expression of FRP-50 partially complements the M M C sensitivify of an FA(C)cell line. Because FRPJO expression correlates with the mild FA phenotype, we reasoned that FRPJO might have functional activity in vivo. To assess the functional activity of FRPJO and other mutant FAC polypeptides, we analyzed their ability to complement the MMC-sensitivity of a severe type C FA cell line, as previously described.16 HSC536N cells, transfected with wild-type FAC, demonstrated enhanced cellular resistance to MMC, similar to normal lymphoblasts (Fig 5 ) . HSC536N cells, transfected with the T3 mutant, an

4429


severity. Cell lines derived from patients with delG322 and mild disease expressed a 50-kD protein ( F R P J O ) . Cell lines derived from patients with IVS4 +4 A to T and severe disease did not express FRP-50.We have demonstrated that F R P J O is an amino terminal truncated FAC isoform, encoded by the FAC gene. Moreover, we have demonstrated that ectopic overexpression of FRP-50partially corrects the MMC sensitivity of severe FA(C) cells. Taken together, these data suggest that the expression of FRP-50 accounts, at least in part, for the mild phenotype of FA patients with exon 1 mutations in the FAC gene. The FAC gene directs the synthesis of the FRP-50 polypeptide by one of two possible mechanisms. First, an alternative splicing mechanism could generate an FAC mRNA that lacks exon 1 sequences. Second, a ribosomal skip mecha10-3 nism could generate a truncated protein (FRP-50) that initiates at an internal methionine, downstream of methionine 1 MMC (M) (Ml) (Fig 4). In either mechanism, the region of exon 1 that contains an in-frame STOP codon would be omitted, and a Fig 5. Complementation of the MMC-sensitive phenotype by FAC continuous reading frame would be generated that encodes and FAC mutants. HSC536N cells were transfectedwith either pREP4mock (Wl, T1 (AI, T2 (01,T3(0), FAC(L554P) (A),or wild-type FAC (0). FRP-50. MMC concentrationsranged from 2 nmol/L to 300 mmol/L. Optical While we cannot rigorously distinguish between these density (OD) at 450 nm. possibilities, our data strongly support the mechanism of internal translation i n i t i a t i ~ nFirst, . ~ ~ FRP-50 has the same FAC(L554P) mutant, or mock-transfected remained sensimolecular weight as the T2(M55) polypeptide that is extive to Mh4C. Interestingly, overexpression of both the T1 pressed in transfected cells (Fig 4C) or synthesized by in and T2(FRP-50) mutant polypeptides resulted in partial corvitro translation (data not shown). Second, increased levrection of the MMC sensitivity of the FA(C) cell line. els of both FAC and FRP-50 are found in cells transfected To verify the partial functional activity of T2(FRP-50), with a full length FAC cDNA that contains no intron sewe designed a second in vitro complementation assay (Fig quences (Fig 4B, lane 4). Third, the DNA sequence sur6). This assay exploits the fact that FA cells, unlike normal rounding codon 55 (AAGAGATGG) - resembles a Kozak cells, accumulate in the G2 phase of the cell cycle following - particularly at consensus sequence (CC~GCCATGG), low-dose exposure to MMC.1326,27 Control cells (HSC536Nbases -3 and +1, and is, therefore, a plausible site for mock) and cells transfected with wild-type FAC (HSC536Ntranslation reinitiati~n.'~.'~ Finally, the delG322 mutation wt FAC) showed a similar cell cycle distribution in the abin codon 22 (Fig 1)is found upstream of M55, and translasence of MMC (Fig 6A and C). After treatment with a low tion of FRP-50 is not affected by this m~tation.~' In conconcentration of MMC (0.1 pmol/L) for 48 hours, trast, the IVS4 +4 A to T mutation is downstream of HSC536N-mock cells accumulated in the G2 phase of the M55 and translation initiation at M55 would not yield a cell cycle (Fig 6B). In contrast, HSC536N-wt FAC cells did functional FAC isoform. Several mammalian genes have not accumulate in G2 to the same extent (Fig 6D). The been described that generate protein isoforms by alternareduction of MMC-induced G2 accumulation by wild-type tive initiation of translation. For instance, internal translaFAC expression was defined as a 100%correction. Transfection initiation can account for a comparatively mild varition with the mock pREP4 vector resulted in a 0%correction. ant of Duchenne Muscular Dy~trophy.~' In these cases, Transfected HSC536N cells expressing either wild-type translation of carboxy terminal polypeptides from downor amino terminal truncated FAC polypeptides were next stream start codons account for the milder phenotype. analyzed by this functional assay (Table 2 and Fig 7). In the Primary cells and immortalized cell lines, derived from absence of MMC, all transfected cell lines had similar cell patients with either delG322 or IVS4 + 4 A to T mutacycle distributions (Table 2). Following treatment with tions, exhibit similar sensitivity to MMC in vitro (A. MMC for 24 hours, HSC536N-T1 and HSC536N-T2 cells D' Andrea, unpublished results, June 1995). The endogecorrected MMC-induced G2 accumulation by 37% and 49%, nous low-level expression of FRP-50 in cells with the respectively (Fig 7). Cells transfected with vector alone, the delG322 mutation, therefore, does not improve their celluT3 mutant, or a full-length FAC(L554P) mutant accumulated lar sensitivity to MMC. The partial correction of MMC in G2 phase similar to parental untransfected cells. Taken sensitivity by the T2 mutant observed in the cytoxicity together, these data are concordant with the cytotoxicity data assay (Fig 5) and in the G2 accumulation assay (Fig 7), (Fig 5) and demonstrate that the amino terminus of FAC can may, therefore, result from overexpression of FRP-50 in be truncated with only partial loss of FAC function. transfected cell lines. Still, overexpression of other mutant DISCUSSION FAC polypeptides such as FAC (T3) (Fig 4) and In the current work, we have analyzed FAC polypeptides FAC(L554P)'6.25fails to even partially correct the MMC expressed in cell lines from FA patients with variable clinical sensitivity of the HSC536N cells. Taken together, these

1

YAMASHITA ET AL

4430

-MMC

+MMC

A

HSC536N pREP4-mock

.-

B-...

-

I

a W

iI

m

I

I

I

N

4N

D

HSC536N pREP4-FACC(Wt) !

i

2N

4N

PROPlDlUM FLUORESCENCE Fig 6. Correction of MMC-induced cell cycle arrest by wild-type FAC cDNA. HSC536N cells were transfected with either pREP4 (mock) IA and E) or pREP4(FAC-wt) (C and D). Cells were untreated (A and C) or treated with 0.1 pmol/L MMC for 48 hours (6 and D), stained with propidium iodide, and analyzed by FACS as described in Materials and Methods. The percentage of cells in the 62 M phase of the cell cycle was determined by analyzing data with the computer program CELLFIT (Becton Dickinson) and is shown in Table 2. Correction with FAC(wt) is defined as a 100% correction

+

results suggest that the endogenous FRPJO levels in cells with delG322 has no effect on cellular MMC sensitivity in vitro, but a measurable protective effect against severe phenotypic features of FA.

Table 2. Effect of Mitomycin C on G2+M Percentage of Flow Histograms From Transfected HSC536N Cells ~

cDNA Transfected

-MMC

+MMC (0.1 umolR)

Mock FACC T1 T2 T3 FACC (L554P)

23.3 19.3 22.7 19.7 22.5 18.4

40.2 27.1 35.3 33.8 39.2 39.8

HSC536N cells were transfected with the indicated cDNA. The percentage of cells in G2+M was determined by the computer program CELLFIT. Transfection with the indicated cDNAs was performed three times, with similar results obtained each time.

Other mutations in the FAC gene may account for mild or even subclinical cases of FA. As discussed above, patients with the delG322 mutation frequently have no skeletal abnormalities and develop hematologic disease later. It is possible that even more subtle mutations in the FAC gene could account for the later onset of aplastic anemia or leukemia. The actual incidence of FA, therefore, may be higher than previously calculated.’ Also, because the DEB test is a highly sensitive and specific test,3 the test should be employed for more patients with sporadic onset of aplastic anemia or acute myeloid leukemia.3’ even in the absence of congenital abnormalities. Finally, the cellular function of the FAC polypeptide remains unknown. FA genes have been proposed to play a role in DNA repair,””.” cell cycle regulation,'"^*' cellular response to oxidative stress,”‘ and prevention of apoptoskMAs FRPJO has partial functional activity and lacks the amino terminal 54 amino acids, a critical functional domain must exist in the more carboxy portion of the protein.

4431


C

.-0 c 0

e

5

0

n

s + cy

s T1

T2

FACC (wl)

T9

FACC

LSMP

Yock

Fig 7. Heterologous overexpression of FRP-50 partially complements the MMC sensitivity of a FA cell line. Data from Table 1, representing the percentage of cells in 6 2 M following MMC treatment, was normalized to demonstrate the percent correction of 6 2 phase accumulation. By definition, transfection with the cDNA encoding FAC (wt) corrected the MMC sensitivity of HSC536N cells by 100%. Vector alone (mock) did not correct the MMC sensitivity of HSC536N cells (0%). Mutant FAC polypeptides T1, T2, T3, and FAC (L554P) corrected the MMC sensitivity to 37%. 49%. 8%. and 3% of FAC (wt), respectively. Percent correction was calculated as [%G2(mock) %G2(test)l + [%G2(mock) - %GZ(FAC)I.

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ACKNOWLEDGMENT

We thank C. Mathew for the BD0215 cell line and P. Jakobs for helpful discussions. We thank M. Buchwald for the human FAC cDNA. We thank Drs H. Caron, G. Kardos, K.J. Roozendaal, and E.J.G.M. Wichgers for providing the clinical severity scores for their patients. We thank Barbara Keane for preparation of this manuscript. REFERENCES

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