(CANCERRESEARCH57. 1166-1 170, March15. 1997)
Positions of Chromosome 3pl'L2 Fragile Sites (FRA3B) within the FHIT Gene'
Drazen B. Zimonjic,
and Kay Huebner4
Laboratory
Teresa
of Experinwntal
Druck,
Carcinogenesis.
Masataka
Ohta,2
Kumar
Kastury,3
Carlo
M. Croce,
Nicholas
C. Popescu,
Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892 (D. B. 1, N. C. P.!; Kimmel Cancer institute,
JeffersonMedicalCollege.ThomasJeffersonUniversity.Philadelphia,Pennsylvania19107fT. D..M. 0.. K.K., C.M. C., K.H.J
ABSTRACT
The FHIT gene spans approximately 1 Mb of DNA at chromosome
band 3p14.2, which includes the famffial renal ceHcarcinoma chromosome
translocation
breakpoint
(between
FHIT
exons 3 and 4), the most Ire
quently expressed human constitutive chromosomal fragile site (FRA3B,
telomeric to the t(3;8) translocation), and numerous homozygous deletions
in various human cancers, frequently involving FHIT exon 5. The FRA3B
has previously been shown to represent more than one specific site, and
some specific representatives
of FRA3B breaks have been shown to fall in
two regions, which we know to be in FHIT introns 4 and intron 5. Because
breakage
and integration
frequent
in aphidicolin-treated
of exogenous DNA in this chromosome
somatic
cell hybrids,
cancer
region is
cells,
and,
presumably, aphidicolin-treated normal lymphocytes that exhibit gaps or
breaks, we determined by one- and two color fluorescence in situ hybrid
ization, using cosmids covering specific regions of the FHIT gene, that
most of the aphidicolin-induced
gaps at FRA3B fall within the FHIT gene,
with the higjtest frequency ofgaps falling in intron 5 ofthe FHIT gene, less
than 30 kb telomeric to FHIT exon 5. Gaps also occur in intron 4, where
a human papillomavirus 16 integration site has been localized, and in
intron 3, where the t(3;8) break point is located. These results suggest that
the cancer-specific deletions, which frequently involve introns 4 and 5,
originated through breaks in fragile sites.
INTRODUCTION
Chromosomal fragile sites are regions susceptible to breakage
under specific experimental conditions. Antifolates, fluorodeoxyuri
dine plus methotrexate, and ape,5 a DNA polymerase inhibitor (1),
induce the expression of fragile sites during metaphase as nonstaining
gaps or breaks, usually involving both chromatids (for review, see
Ref. 2). The latest compendium comprises 103 fragile sites classified
as common and rare, constitutive and inherited, that have been local
ized in all human chromosomes except chromosomes 21 and Y (3).
Although fragile sites are dispersed throughout the genome, they tend
to cluster at G-light chromosome bands (4) that are known to contain
GC-nch Alu repeats and are constitutionally more relaxed and un
folded during transcription (5). The location of fragile sites has been
correlated cytogenetically with locations of recurrent translocation
and deletion breakpoints in cancer cells, as well as the integration sites
of oncogenic viruses (6—9).Moreover, structural rearrangements in
duced by mutagens or carcinogens through fragile sites can be cyto
genetically indistinguishable from those in cancer cells (10), and the
constitutive familial clear-cell renal carcinoma-associated transloca
tion t(3;8)(pl4.2;q24) (1 1, 12) has been found to be indistinguishable
from the most frequent of the common fragile sites, FRA3B (13).
Nevertheless, evidence implicating fragile sites in the process of
neoplastic development has been circumstantial. It has indeed been
argued that it is very unlikely that breaks in a fragile site should be
associated with cancer (14).
The constitutive ape-inducible fragile site at chromosome band
3pl4.2 (FRA3B) is of special interest, because it is the most com
monly expressed in humans (15), and the region 3pi4.2 is involved in
deletions in a variety of histologically different cancers (16—18),the
t(3;8) translocation in familial RCC, and Kaposi's sarcomas (19). In
addition, consistent with previous cytogenetic observations linking
viral integration sites and fragile sites (8, 20), the first molecular
evidence for HPV integration in FRA3B was provided recently (21).
The positions of some ape-induced gaps and breaks in FRA3B have
been determined by isolation of YAC clones encompassing the breaks
(22—25)and mapping ape-induced hybrid breaks (21, 26) or plasmid
integration sites (27, 28) to regions of the YAC map.
We have recently isolated the FHJT gene, which encompasses
perhaps 1 Mb, straddling the t(3;8) break, the FRA3B, and cancer
cell-specific homozygous deletions (29), and have suggested that the
FHIT gene may be the target of FRA3B-induced deletions in cancer
cells. The FlIT gene is ex@pressedin all tissues thus far examined
(29), including normal lymphocytes (30) and ape-induced lympho
cytes, in which the normal-sized FHJT transcript was observed (data
not shown).
To determine the relationship of the FHJT gene to cancer cell
deletions and FRA3B breaks and gaps, we have determined the posi
tions of FHlTexons relative to the translocation, viral integration site,
cancer cell deletions, and FRA3B breaks described previously, and
have then used FHIT cosmid probes to determine the positions of
specific
MATERIALS
Received I 1126/96;accepted 1/13/97.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
work
was
supported
partially
by
United
Public
Health
Service
Grants
CA51083, CA39860, and CA21 124, and a gift from R. R. M. Carpenter Ill and Mary K.
Carpenter.
2 Present
address:
Banyu
Tsukuba
Research
Institute,
Okubo
3, Tsukuba
300-33
address:
Department
of
Endocrinology.
R.
Drew
University
of
requests
for reprints
should
be addressed,
at Kimmel
Cancer
abbreviations
used
are: ape.
aphidicolin;
of the sequences.
HPV,
human
papillomavirus;
YAC,
yeast artificial chromosome; STS, sequence-tagged site; BAC, bacterial artificial chro
mosome; RCC, renal cell carcinoma; FISH, fluorescence in situ hybridization.
U39799
and U39804
primer
pairs
are
(5'-CGUGTAGTFCTGA'ITFGCA-3')
and
U06l l8R
(5'-
GGATA1T1'GTCACTGCC1TF-3') at nucleotides 20 and 330, respectively;
U60203F (5'-GTGCTCAGAGATFACATGC-3'
and U60203R (5'-GTATGT
and
c5B 1AR
(5'-AlTFCCAA
Each primer pair was used in a PCR reaction with template DNA containing
Institute,
Room 1008, Jefferson Medical College, Thomas Jefferson University, 233 South 10th
Street, Philadelphia, PA 19107. Phone: (215) 503-4656; Fax: (215) 923-4498.
5 The
numbers
CAAGCFGCCA-3'), which amplify a —2l8-bpproduct containing a TA
dinucleotide repeat (locus name D3S4490; GenBank accession no. U76285).
Medical Science, Room 3069, 162 1 E. I20th Street, MPO2, Los Angeles, CA 90059.
Phone:(213)563-5959.
4 To whom
in
AND METHODS
(5'-CAAAGCAGAAATAAAACTGC-3')
Charles
gaps and breaks
CAGCCAAACACC-3') at positions 786 and 1069, respectively; and cSB1AF
Japan.
Phone: 81-298-77-2000; Fax: 81-298-77-2024.
L1 Present
to the ape-induced
listed in Druck et a!. (30). Other newly designed primer pairs are: U39793F
(5'-TGGATTGAmCACACC-3')
and U39793R (5'-GCCAGAGG
GAGA ri-i-i- iC-3') at bp 104 and 265 of the U39793 sequence, respectively;
U061 l8F
States
relative
Localization of STSs by PCR. Primer pair sequences for amplification of
STSs were obtained from the Genome Data Base or were designed from
published sequences in GenBank; the numbers beginning with U are GenBank
accession
I This
FHJT exons
human lymphocyte metaphase chromosomes.
some portion of the FHIT genomic locus. PCR amplifications were carried out
in 12.5-pJ
reaction
volumes
with 10 ng of cloned
genomic
DNA or 100 ng of
total genomic DNA, 20 ng of each primer, 10 mMTris-HCI (pH 8.3), 50 mr@i
KC1,0.1 mg/rn)gelatin, 1.5 mMMgCl2,0.2 mMeach deoxynucleotidetriphos
1166
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POS@ONS OF FRA3B SITES WITHIN THE FH!T GENE
Telomere
pSVZn.o
4-
Centromere
HPV16
-@
Q)0)Q)
5'PTPRG
@
F:::I
@
I
@
+
I
118k;
II@I@
@+J!@
I
I
C,)
I
U
63
E4
@8E8@ll14—@—14-c06#7c05#9c05#1
.I—c84
@
El.-4j..cX3—O-'cX2
E3E2
.—ll-cX4
—c76
—c36
‘I,
LI)
II
—II-cXl
c19-20 —
BAC
@!Af@ O@AAb
) 00 kb
Fig. 1. Map of the Fllfflocus.
The chromosome 3pl4.2 region (—1Mb) is illustrated with approximate placement of the cosmid and BAC clones relative to each other. Positions
of relevant STSs were determined by PCR amplification with cosmid and BAC DNA templates. Localization of the telomeric side of the lung carcinoma deletion (U60203), the pSV2neo
plasmid integrations, the c13break, the HPVI6 integration site, the t(3;8) break, as well as 5'-PTPRG are shown (arrows). D3S4260 and IA6G/H primers are derived from the cosmid
1A6, to which 10 fragile site breaks in hybrid cells have been mapped (37).
phate, and 0.5 units of Taq polymerase. Amplifications were for 30 cycles of
94°Cfor 30 s, 57°Cfor 30 s, and 72°Cfor 30 s. Products were analyzed by
electrophoresis in ethidium bromide-stained 1.5% agarose gels. Presence of an
amplification
product
determined
localization
of a STS to a particular
genomic
clone.
Isolation of Genomic Clones. New cosmid clones were isolated as de
scribed previously (29), and BAC clones were identified by PCR amplification
of specific STS fragments from pooled BAC library template DNAs (Research
Genetics, Huntsville, AL); individual BAC clones were then ordered from
Research Genetics, single colonies for specific STSs were identified and
grown, and end sequences were determined, as for cosmids.
FISH Analysis.
Normal peripheral lymphocytes and lymphoblastoid
cells
derived from a member of the RCC family with the t(3;8) translocation were
cultivated
in RPM!
lin-streptomycin
1640 supplemented
(50 mg/mI),
with 15% fetal bovine
and 0.2 m@i glutamine.
serum,
The expression
penicil
of FRA3B
was induced by ape (0.2 p.M;Sigma Chemical Co., St. Louis, MO) dissolved
in DMSO
(31), or by combined
ape and ethanol
(0.2%)
during
the last 26 h of
culture (32). Metaphase chromosomes were obtained after a 1-h colcemid (50
ILg/ml) treatment
by standard
KC1 hypotonic
incubation
and acetic acid
methanol fixation. Six previously described cosmid probes (25, 29) [19-20
(representing the PTPRG 5' end, as shown in Fig. 1), cX3 (including FHJT
exon 3), cX4 (exon 4), c76 (exon 5), c36, and c63 (intron 5)] labeled with
biotin or digoxigenin, as well as chromosome 3 and 8 painting probes (Oncor,
Gaithersburg,
MD)
were
used
for in situ hybridization.
The conditions
for
cosmid.
The hybrid cl3 break, representing
our 3pl4.2
hybrid de novo
break (35, 36), had previously been placed telomeric to FHJT exon 5
based on Southern blot data (29), but we now know the break falls in
intron 4, as shown here. The U60203 marker falls in cosmid cP4 in
intron 5, based on amplification of a fragment from this template. This
marker represents the telomeric side of a homozygous deletion ob
served in a lung carcinoma.6 Human sequences U40597 and U40401,
representing portions of plasmid integration flanking sites (27, 28)
were mapped within 4 kb telomenc of FHIT exon 5 and in cosmids
c36 and c76, respectively, by sequence identity; sequence U061l8,
another plasmid flanking sequence, was mapped to cosmid c36; see
Fig. 1 for positions of each of these sequences in intron 5. Addition
ally, numerous tumor-specific deletion end points fall within introns 4
and 5 (30), and seven hybrid breakpoints identified by Paradee et a!.
(37) are in intron 5. The relationship of these various chromosome
landmarks to each other and to the FH!T gene is summarized in
Fig.1.
FISH, detection ofthe signal, and digital imaging were carried out as described
previously (33, 34).
Position of Exons Relative to FRA3B Gaps. Metaphase spreads
prepared from ape-treated peripheral lymphocytes derived from two
normal donors were hybridized with the six cosmid probes; 19-20,
cX3, cX4, c76, c36, and c63 probes that span the t(3;8) and FRA3B
sites (25, 29); see Fig. 1 for a map of the region. To obtain a sufficient
number of metaphases with informative signals at FRA3B, two or
three slides were hybridized with each cosmid probe. Only symmet
rical signals were included in statistical analysis, although, due to the
chromatid displacement at expressed fragile sites, single fluorescent
spots were observed frequently. The location of the hybridization
signal was assigned as medial (crossing the gap or break), proximal
RESULTS
The FHIT/FRA3B Map. We have previously described a YAC
and partial cosmid contig for the FlIT locus, which extends from
more than 300 kb centromeric to the t(3;8) break to an estimated 700
kb telomeric to the t(3;8) (25, 29). A map of the FHITlocus is shown
in Fig. 1, on which we have placed a number of relevant markers and
the cosmid probes and the position of the FH!T exons; the relative
distances of the FHIT exons 1, 2, and 3 relative to each other and the
5' end of the PTPRG gene is not known, nor is the position of c5BlA
relative to exon 1 known. The positions of markers IA6G/H and
6 T. 0. Ong, K. Fong,
D3S4260
shownbetween
exons4 and5 (Fig.1,E4andES,respec
tively) were deduced by PCR amplification from our YAC, BAC, and
cosmid templates, with pairs of primers derived from published se
quences. The positions of the U39793, U39804, and U39799 markers,
which represent the HPV16 integration site (21), were deduced by
amplification of appropriate fragments from the BAC clone (shown in
Fig. 1) and from knowledge of their positions relative to the nearby
Miul site and other markers that we mapped within the exon 4
S. Bader,
J. Minus,
M. Le Beau, T. McKeithan,
and F. Rassool.
Precise localization of the common fragile site at 3pl4.2 to the putative tumor suppressor
gene FHJT and characterization of homozygous deletions in tumor cell lines within
FRA3B affecting FHIT transcription, unpublished,
obtained from GenBank (accession
number U6O203).
1167
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posmoNs OFFRA3B
SITESWITHIN
ThE FHITGENE
a
b
C
the fragile region and were retained on chromosome 3 in metaphases
derived from lymphocytes with the t(3;8) translocation. The remain
ing probes were found translocated to chromosome 8 in the t(3;8)
lymphocytes. Among these, the signals produced with the probes c36
and c63 (both in FHJT intron 5) were located distal to the gaps,
whereas probes cX4 (exon 4) and c76 (exon 5) hybridized proximal to
the gaps in some metaphases but distal to the gaps in other met
aphases, with different frequencies, as summarized in Table 1; exam
pies are shown in Fig. 2, a, b, d, and e. Experiments with two-color
detection of digoxigenin and biotin label were also carried out to
distinguish the placement of pairs of the probes relative to the FRA3B
gaps. The combination of two probes hybridized to individual met
aphase slides revealed distinct separation of the doublets to sites
proximal and distal to the gaps with paired probes cl9-20 and c63 (see
Fig. 2c for an example) or cX3 and c36. Probe cX4 and c76 hybrid
ization signals were not separable in cohybridized metaphase or
prometaphase spreads. Signal at the median site (overlapping the gap)
was observed with all cosmids, but the incidence varied significantly
among the probes, ranging from 7.14% for probe 19-20 to 33.34% for
probe c76 (see Table 1 for summary).
The data presented in Table 1 are represented graphically in Fig. 3.
The difference between the percentages at proximal and distal sites
was plotted as an absolute value for each probe (Fig. 3, line), together
with the values representing percentages of signals at the median site
(Fig. 3, columns). Thus, probes 19-20 and cX3, and probes c36 and
c63 flank, proximally and distally, respectively, the ape-induced gaps
or breaks at FRA3B. On the other hand, probe cX4 and particularly
c76 contain sequences crossing the fragile sites, and the signal gen
crated with these probes could fall on either side of the breakpoint at
FRA3B. This analysis delineates the FRA3B to a region of a few
hundred kb between FHIT exon 3 (cosmid cX3) and FHJT intron 5
(c36), with the majority of gaps occurring in introns 4 and 5, flanking
the first FHIT protein-coding exon.
DISCUSSION
By FISH analysis of chromosomes derived from normal lympho
cytes treated with ape, we localized the majority of chromosome gaps,
collectively known as FRA3B, to a region telomeric to the breakpoint
of the t(3;8) translocation in RCC, encompassing the region involved
in deletions in tumor cell lines and bordered distally by a portion of
intron 5 of the recently identified FH!T gene (29). In fact, the majority
of gaps map just to either side of FHJT exon 5, the exon that is most
frequently lost in cancer cells with homozygous deletions in the FHJT
gene (30).
Rare folate-sensitive fragile sites that have been cloned (FRAXA,
FRAXF, FRAJ6A, and FR@411B)involve unstable expansion of CCG
trinucleotide repeats (38—42),and their expression is mediated by an
excessive copy number of CCG repeats. Regions of 3pl4.2 thus far
tested do not contain CCG repeats (21, 28), suggesting that the
distinction between constitutive and heritable fragile sites is not only
d
e in inducing agent and frequency of expression within the population,
Fig. 2. FISH localization of cosmid probes at FRA3B on metaphase chromosomes
but also in molecular structure and the mechanisms mediating their
derived from ape-treated normal peripheral lymphocytes. Partial and complete metaphase
expression.
spreads with fluorescent hybridization signal located proximal (a; probe cX3), distal (d;
Several groups of investigators have made important contributions
probe c36), and on both sides (c; probes c19-20 red, rhodamine, and c63 green; FITC
detection) on the FRA3B. 4',6'-diamidino-2-phenylindole-generated chromosome band
to our current understanding of the fragile region at 3pl4.2. A group
ing (b and e) of the labeled spreads permits an accurate localization of the fluorescent
of investigators studying FRA3B-associated break points induced in
signal relative to the gaps and breaks at FRA3B.
hamster-human hybrids (21, 22, 37) has most recently shown that two
clusters of aphidicolin induced hybrid break points, both telomeric to
(centromeric), and distal (telomenc) to the gaps or constrictions
the t(3;8) 3pl4.2 break, are separated by more than 200 kb and flank
marking the fragile sites, in a minimum of 60 metaphases with
an Miul site estimated to be —185kb from t(3;8). We have mapped
4',6'-diamidino-2-phenylindole
enhanced G-like banding (Fig. 2; Ta
this M!ul site within intron 4, perhaps —100kb centromeric to exon
ble 1). Probes 19-20 and cX3 hybridized predominantly proximal to
5 (see Fig. 1); thus, the two hybrid break point clusters flank FH!T
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POS@ONS
OFFRA3B
SITESWITHIN
ThE FHJTGENE
Table 1Distribution (percentage) offluorescent
FRA3BProbeNaN
signals generated by cosmid probes spanning the
D)19-208478
(%) P
N (%) D
N (%) M(P
(7.14)92.86cX36248
(19.35)73.59cX48860
(92.86)
(77.42)
0(0.00)
2 (3.83)
6
12
(31.82)68.18c766614(21.21)
(68.18)
0 (0.00)
28
22(33.34)24.24c36760
(15.79)84.21c63
a Number
of metaphase72
30(45.45)
64 (84.21)
60 (83.33)
(0.00)
(0.00)
spreads examined0; P.
proximal;
D, distal; M, medial;
P —D, percentage
exon 5. In fact, the cluster of fragile sites represented by 10 hybrid
breaks in the proximal cluster map very close to FHJT exon 4, as
shown in Fig. 1 by the position of markers D3S4260 and IA6GIH. We
were also able to determine that the HPV16 integration site (21, 37)
falls in FHJT intron 4 as shown in Fig. 1; integration of HPV16 in this
site resulted in a large deletion (21) in the cervical carcinoma FlIT
locus, which included exon 5. Interestingly, our own cl3 human
hamster hybrid with a de novo break in 3pl4.2 (35, 36) also falls in
intron 4, probably not far telomeric to the HPV16 integration site and
between the two hybrid breakpoint clusters defined by Paradee et a!.
(37).
A second group of investigators has been isolating DNA sequences
from FRA3B by cloning plasmid integration sites (27) and sequencing
and mapping the flanking regions (28). This group had shown previ
ously that, under conditions of fragile site induction, the pSV2neo
plasmid preferentially integrates into chromosomal regions harboring
fragile sites (27). These investigators reported isolation and sequenc
ing of two plasmid integration sites that they mapped to the telomeric
end ofthe 850A6 YAC, which carries the t(3;8) break and the FRA3B.
We have mapped one of the sequences flanking the centromeric side
of a plasmid integration, U40597, to 4 kb telomenc to FHIT exon 5.
Additionally, a sequence cloned by this group (GenBank accession
no. U60203), encompassing one side of a tumor cell deletion, maps in
intron 5, —100kb from exon 5, as shown in Fig. 1. Thus, the cloned
fragile sites probably fall within a region ofless than 200 kb, flanking
FHIT exon 5, the protein coding exon most frequently homozygously
deleted in cancer cell lines. It also follows that the cosmids c36 and
c63 in intron 5 are telomeric to many of the identified fragile sites, in
agreement with our finding that these probes are almost always found
by FISH to be distal to the gaps induced in 3pl4.2. Wilke et a!. (21)
FRA3B
Probe
proximal
-
12
12 (16.66)
minus percentage
distal in absolute
numbers.83.33
and Rassool et a!. (28) have also performed FISH analyses using
probes from regions near their identified fragile sites, and their results
are largely in agreement with ours. Interestingly, c76 and cX4 cos
mids, containing exons 5 and 4, respectively, can be proximal or distal
to the gaps or can overlap the gaps, as summarized in Fig. 3. Most
gaps occur within cosmid c76, the exon 5-containing cosmid, mirror
ing the findings in tumor cells (30), but a number of gaps fall within
the exon 4 cosmid and even in the exon 3 cosmid, two cosmids that
flank the t(3;8) break, suggesting that the t(3;8) translocation, as well
as the loss and rearrangement of FHIT exons 3 and 4 observed in
cancer cells (30), could have derived from breaks in sequences that are
“fragile,―
although the actual fragile sequences, the bases for breaks
and gaps in this region, have not yet been identified.
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Positions of Chromosome 3p14.2 Fragile Sites (FRA3B) within
the FHIT Gene
Drazen B. Zimonjic, Teresa Druck, Masataka Ohta, et al.
Cancer Res 1997;57:1166-1170.
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