Download A novel 4 cM minimal deletion unit on chromosome 6q25.1

Oncogene (1998) 16, 555 ± 559
 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00
SHORT REPORT
A novel 4 cM minimal deletion unit on chromosome 6q25.1-q25.2 associated
with high grade invasive epithelial ovarian carcinomas
Cristiano V Colitti1, Kerry J Rodabaugh1, William R Welch2, Ross S Berkowitz1,
and Samuel C Mok1
1
Laboratory of Gynecologic Oncology, Department of Obstetrics, Gynecology and Reproductive Biology; 2Department of
Pathology, Brigham and Women's Hospital; Harvard Medical School, Boston, Massachusetts 02115, USA
Detailed deletion mapping of chromosome 6q has shown
that the highest percentage of loss of heterozygosity
(LOH) is located at 6q25-q27 and suggested that an
ovarian cancer associated tumor suppressor gene may
reside in this region. To further de®ne the smallest region
of common loss, we used 12 tandem repeat markers
spanning a region no more than 18 cM, located between
6q25.1 and 6q26, to examine allelic loss in 54 fresh and
paran embedded invasive ovarian epithelial tumor
tissues. Loss of heterozygosity was observed more
frequently at the loci de®ned by marker D6S473 (14 of
32 informative cases, 44%) and marker D6S448 (17 of
40 informative cases, 43%). Detailed mapping of
chromosome 6q25-q26 in these tumor samples identi®ed
a 4 cM minimal region of LOH between markers
D6S473 and D6S448 (6q25.1-q25.2). Loss of heterozygosity at D6S473 correlated signi®cantly both with
serous versus non-serous ovarian tumors (P=0.040) and
with high grade versus low grade specimens (P=0.023).
The results suggest that a 4 cM deletion unit located at
6q25.1-q25.2 may contain the putative tumor suppressor
gene which may play a role in the development and
progression of human invasive epithelial ovarian carcinomas (IEOC).
Keywords: ovary; cancer; loss of heterozygosity;
chromosome 6
Ovarian carcinoma persists to be the leading cause of
mortality among gynecological malignancies in the
Western world. The high mortality rate is due to the
late diagnosis of the disease, usually occurring during
stages III and IV. Even though several oncogenes and
tumor suppressor genes have been identi®ed in recent
years, the genetic mechanisms involved in the initiation
and progression of ovarian carcinoma remain largely
unknown.
Growing evidence suggests that a broad number of
tumor suppressor genes might be involved in the
development of di€erent tumor types. Functional
inactivation of these genes would deprive a constraint
for cell proliferation. Although frequent LOH has been
detected on chromosomes 1p, 3p, 4p, 5q, 6q, 7p, 8p, 9,
11p, 12, 13q, 16, 17p, 19p, 21q in IEOC (Lee et al.,
1996; Ehlen Dubeau, 1990; Leary et al., 1993; Merlo et
Correspondence: SC Mok
Received 29 April 1997; revised 27 August 1997; accepted 27 August
1997
al., 1994; Zheng et al., 1991; Sato et al., 1991; Cliby et
al., 1993; Cooke et al., 1996b; Lee et al., 1990; Foulkes
et al., 1993; Orphanos et al., 1995b; Saito et al., 1992;
Rodabaugh et al., 1995a; Wertheim et al., 1996), no
tumor suppressor genes has yet been identi®ed. In
recent years, chromosome 6q has been under a lot of
scrutiny in ovarian cancer studies because of earlier
evidence of high LOH at the estrogen receptor (ESR)
site (Cooke et al., 1996b; Lee et al., 1990).
Furthermore, frequent LOH on chromosome 6 has
also been observed in gastric carcinoma (Queimado et
al., 1995), breast cancer (Devilee et al., 1991; Iwase et
al., 1995; Orphanos et al., 1995a; Sheng et al., 1996),
small cell lung cancer (Merlo et al., 1994), and
malignant melanoma (Millikin et al., 1991).
Although frequent allelic deletion on chromosome
6q has been reported in the region 6q21-23.3, with a
43% allelic imbalance at the D6S287 locus (Orphanos
et al., 1995b), more studies have been focused on the
terminal region 6q24-27. (Lee et al., 1990) reported
high frequencies (64%) of LOH at the ESR locus at
6q24-27. (Saito et al., 1992), using Southern blot
hybridization, located a 1.9 cM commonly deleted
region between loci D6S195 and D6S149, at 6q27.
Furthermore, using Southern blot analysis and
microsatellite markers, (Rodabaugh et al., 1995b)
observed the highest rate of LOH at the 6q25-27
region, with locus D6S255 having the highest frequency
of allelic loss of 43%. Recently, (Cooke et al., 1996b)
has used 20 polymorphic microsatellite repeat markers
to perform a more detailed investigation of LOH on
6q, particularly in the 6q27 region. Thirteen of thirty
informative tumor cases (56%) showed allele loss at the
estrogen receptor locus, located on 6q25, and twentythree of thirty-eight (62%) revealed LOH at locus
D6S193, which falls on the 6q27 region.
In this report, a set of twelve microsatellite markers,
on chromosome 6q25-q26, were used to perform LOH
studies on 54 invasive ovarian epithelial cancers. All
tumors were informative (heterozygous) for at least one
marker. The map positions and genetic distance
between primers are based on previously published
human genetic maps of Chromosome 6 (Cooke et al.,
1996a; Gyapay et al., 1994; Volz et al., 1994), and they
are shown in Figure 1. The frequency of LOH for each
locus is also summarized in Figure 1. Twenty-seven of
the 54 (50%) informative ovarian tumors exhibited
LOH in at least one locus. Figure 2a shows the allelic
deletion map for the selected cases that displayed LOH
in at least one locus. Five out of 28 samples (cases 341,
353, 357, 530, ES3) showed loss at all informative loci
studied, suggesting loss of the entire region. Analysis of
Deletion mapping of chromosome 6q25 ± q26 in ovarian tumors
CV Colitti et al
556
the pattern of LOH for the other samples illustrated a
novel minimal deletion region of 4 cM ¯anked by, but
not including, markers D6S441 and D6S442. This unit
is de®ned by LOH at D6S448 and/or D6S473 with
retention of heterozygosity at D6S441 and/or D6S442
in tumors 332, 403, 443, 498, 508, B17. Tumors 351
and 516 also show LOH for loci D6S415 and D6S437,
with retention of heterozygosity at the ¯anking
markers. This may suggest a second region of allelic
loss, but it was not considered signi®cant because of
the low LOH percentage at the two loci.
Examples of LOH at four di€erent markers on
chromosome 6q25.1-q25.2 are shown in Figure 2b. The
highest frequency of allelic loss occurred on loci
D6S448 (43%) and D6S473 (44%) at 6q25.1-q25.2.
Other loci that revealed high frequencies were D6S442
(36%) and D6S1007 (35%), also located at 6q25.1q25.2. Our data supports earlier studies showing a high
frequency of allelic loss on the terminal portion of
chromosome 6q. Several of these studies have described
high frequencies of LOH at 6q27. Saito et al. (Saito et
al., 1992) reported a 1.9 cM region of common allelic
loss between loci D6S193 (11 of 23 informative serous
cases, 48%), and D6S149 (10 of 22 informative serous
cases, 45%). A 3 cM minimal region of allelic loss
located between D6S264 and D6S297 at 6q27 was also
documented by Cooke et al. (Cooke et al., 1996b).
Other published data also point to chromosome 6q27
for the site of a possible tumor suppressor gene (Ehlen
Dubeau, 1990; Zheng et al., 1991; Foulkes et al., 1993).
However we identi®ed another area in the terminal
region of 6q other than 6q27 which is distal to the ESR
locus, at 6q25. The smallest common region of allelic
loss was narrowed to a novel 4 cM segment between,
but not including, loci D6S441 and D6S442, corresponding to the map position 6q25.1-q25.2. This
®nding will facilitate the identi®cation of a tumor
suppressor gene(s) in the region by the positional
cloning approach.
Among all the markers used, loci ESR and D6S437
both showed the lowest percentage of LOH rate at
17%. Our results for ESR are in accordance with the
®ndings of both (Gallion et al., 1992) and (Leary et al.,
1993), which reported LOH frequencies of 15% and
Figure 1 Ideogram of chromosome 6q summarizing the 12 loci studied, map position and physical distance for each locus (Cooke
et al., 1996a; Gyapay et al., 1994; Volz et al., 1994), and frequency of loss of heterozygosity in ovarian invasive tumors for each
locus studied. LOH, number of tumors that showed allelic loss at each marker. Inf, number of informative samples (heterozygous).
Surgical specimens of human ovarian tissue were obtained from 54 patients following a protocol approved by the Human Subjects
Committee of Brigham and Women's Hospital. Archival material consisted of 11 sets of paran blocks. Control tissues consisted of
segments of normal fallopian tube, uninvolved round ligament, or peripheral blood lymphocytes. All histopathological diagnoses of
the invasive epithelial tumors were con®rmed and graded by a gynecological pathologist, and all cases were surgically staged
according to the FIGO criteria. The invasive tumors consisted of 41 serous adenocarcinomas, ®ve mucinous cystadenocarcinomas,
four endometroid adenocarcinomas, and four mixed epithelial carcinomas of the ovary. DNA was extracted from fresh specimens of
43 invasive cancers. In 11 cases DNA was extracted from archival material which had previously been ®xed in formalin and
embedded in paran. Prior to the extraction, blocks containing predominantly tumor tissue were marked and selectively trimmed
for tumor concentration. Paran sections were deparanized with xylene followed by absolute ethanol. All selected samples
contained at least 80% of tumor cells. DNA extraction was performed using previously published methods (Edelson et al., 1997)
Deletion mapping of chromosome 6q25 ± q26 in ovarian tumors
CV Colitti et al
557
498
528
T N
T N
T N
T N
T N
B17
T N
508
T N T N
498
443
B17
B27
T N
T N
D6S442
d
T N T N
403
443
T N
351
T N
508
443
403
T N T N
498
D6S448
b
351
T N T N
332
403
T N
508
332
T N
498
B17
T N
D6S473
c
D6S441
321
a
T N
T N
Figure 2 (a) Schematic representation of allelic deletion patterns of selected tumor cases that displayed LOH in at least one locus.
The pattern of allelic loss de®ned a 4 cM minimal deletion unit ¯anked by, but not including, markers D6S441 and D6S442. (b)
Autoradiographs of LOH results obtained at four of the markers used. For each panel, the marker name is shown above the patient
identi®cation numbers. Tumor (T) and normal (N) tissues are matched for each patient. (a) At D6S441, patient B17 showed LOH at
the tumor sample. Patients 332, 403, 498, and 508 all showed retention of heterozygosity. (b) At D6S448, allelic loss is observed in
patients 403, 443, 498, 508, B17, and B27. (c) At D6S473, depicting LOH is shown in patients 332, 351, 443, 498, and 528. Tumor
403 showed retention of heterozygosity. (d) At D6S442, patients 321, 351, and 443 showed LOH at the tumor sample. Patients 498
and B17 are informative cases with no LOH. Patient 508 is an uninformative case. LOH studies were carried out by performing PCR
ampli®cations of tandem repeats on chromosome 6q25-q26. Twelve oligonucleotide primer pairs were used (Research Genetics,
Huntsville, AL). The di€erent primers and their corresponding map positions are summarized in Figure 1. The forward primer of
each set was end radio-labeled with Polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN) and gamma [32P]ATP (ICN,
Irvine, CA). After a 30 min incubation period, the reaction mix was diluted to a ®nal volume of 320 ml of primer-PCR mixture
containing 40 ml of 10X PCR bu€er (0.1 M Tris-HCl, 0.5 M KCl, pH 8.3), 20-50 mM MgCl2, 20 ml of 1.25 mM deoxynucleotide
triphosphate mixture, and 2 ml (10 units) of Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT). Ampli®cation was carried out with
50 ng of genomic DNA using 30-35 cycles of PCR with denaturation at 958C for 1 min, annealing at 458 ± 658C for 1 min, and
elongation at 728C for 1 min. 45 ml of loading bu€er consisting of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and
0.05% xylene cyanol FF (Sigma, St. Louis, MO) was added to 5ml of the PCR product, and 3 ml of this mixture was loaded onto a
6% polyacrylamide gel. The electrophoresis gel was then run at a constant 1700 V, transferred onto 3 MM chromatography paper,
dried, and exposed to X-ray ®lm with an intensifying screen, for a duration determined by the level of radioactivity count. The
autoradiographs were then developed and analysed for indication of LOH. Cases were considered informative when heterozygosity
was detected in the control tissue sample. LOH was de®ned as a visible reduction of 50% or more in the band intensity of one of the
two alleles of the tumor tissue sample when compared to the normal tissue. Microsatellite instability (MSI) was de®ned as an
electrophoretic mobility shift of equal intensity in tumor compared to normal tissue
Deletion mapping of chromosome 6q25 ± q26 in ovarian tumors
CV Colitti et al
558
Table 1 Correlation between histology, di€erentiation, stage, and LOH in ovarian tumors for each locus
Locus
Histology
Serous
Mucinous
Endometrioid
Mixed
Di€erentiation
Poor
Moderate
Well
Stage
I
II
III
IV
LOH/Inf (%)
D6S442 D6S1007 D6S255
ESR
D6S420
D6S441
D6S448
D6S473
4/19
(21)
1/4
(25)
0/3
(0)
0/3
(0)
4/20
(20)
0/2
(0)
1/2
(50)
0/2
(0)
8/28
(29)
0/2
(0)
1/4
(25)
0/3
(0)
15/29
(52)
1/4
(25)
1/4
(25)
0/3
(0)
13/24
(54)
1/4
(25)
0/2
(0)
0/2
(0)
12/28
(43)
0/2
(0)
1/3
(33)
0/3
(0)
10/22
(45)
1/4
(25)
0/1
(0)
0/4
(0)
2/14
(14)
2/10
(20)
1/5
(20)
5/18
(28)
0/6
(0)
0/2
(0)
8/24
(33)
1/8
(12)
0/5
(0)
13/23
(57)
2/11
(18)
2/6
(33)
11/17
(65)
2/10
(20)
1/5
(20)
10/24
(42)
2/10
(20)
1/2
(50)
0/1
(0)
0/2
(0)
4/24
(17)
1/2
(50)
0/3
(0)
0/1
(0)
4/19
(21)
1/3
(33)
0/3
(0)
0/4
(0)
7/27
(26)
2/3
(67)
2/4
(50)
0/5
(0)
14/28
(50)
1/3
(33)
0/3
(0)
0/2
(0)
14/26
(54)
0/1
(0)
1/4
(25)
0/3
(0)
11/26
(42)
1/3
(33)
23% respectively. We found a LOH occurrence of only
17% for the ESR locus. In contrast to our ®ndings,
Cooke et al. demonstrated that LOH occurred at the
ESR locus (6q25.1) at a high frequency of 56% (Cooke
et al., 1996b). Another study that showed a high
frequency of LOH at ESR was conducted by Lee et al.,
which showed that nine out of 14 informative cases
(64%) exhibited allelic loss (Lee et al., 1990). The
contrasting results may be due to the di€erent number
of informative cases and the number of cases of
di€erent histologic grades. While the ESR locus has
been explored for allelic loss, with di€ering results, the
region in the proximity of the ESR locus has not been
examined. A greater quantity of closely-spaced markers
in this region has recently become available, allowing
us to further de®ne the minimally deleted segment. In
this study, we mapped a region about 5 cM distal from
the ESR locus, suggesting that a tumor suppressor
gene may reside in the vicinity of the estrogen receptor
gene but not involve it directly.
The correlation between LOH and histology,
di€erentiation, and stage for all the ovarian tumors
studied at each locus is shown in Table 1. Twenty-six
of the 28 tumors showing LOH on chromosome 6q are
serous adenocarcinomas, with only one endometrioid
adenocarcinoma and one mucinous cystadenocarcinoma showing allelic loss in the same region. For each
locus studied, chi-square P values were calculated for
di€erent clinicopathological parameters. P50.050 was
considered signi®cant; P values between 0.050 and
0.100 were considered to suggest a di€erence. The
frequency of LOH at locus D6S473 (P=0.040) was
signi®cantly higher for the serous cancers than of the
other cancer types (Table 2). LOH was also
signi®cantly higher at loci D6S255 (P=0.028) and
D6S415 (P=0.033), while loci D6S448 (P=0.056) and
D6S1007 (P=0.070) showed LOH to be suggestively
more frequent in serous tumors than in non-serous
D6S415
D6S437
D6S363
IGF2R
10/28
(36)
0/3
(0)
0/3
(0)
0/4
(0)
8/25
(32)
0/4
(0)
0/4
(0)
0/3
(0)
4/17
(24)
0/3
(0)
0/2
(0)
0/1
(0)
7/25
(28)
0/3
(0)
0/0
(0)
0/3
(0)
4/13
(31)
0/1
(0)
0/1
(0)
0/0
(0)
7/19
(37)
2/7
(29)
2/5
(40)
7/20
(35)
2/10
(20)
1/8
(13)
5/20
(25)
2/8
(25)
1/8
(13)
4/18
(22)
0/4
(0)
0/1
(0)
6/21
(29)
1/7
(14)
0/3
(0)
4/11
(36)
0/2
(0)
0/2
(0)
1/2
(50)
1/2
(50)
9/25
(36)
0/2
(0)
1/3
(33)
0/3
(0)
8/28
(29)
1/4
(25)
1/4
(25)
0/4
(0)
5/25
(20)
2/3
(67)
0/2
(0)
0/2
(0)
3/17
(18)
1/2
(50)
0/2
(0)
0/2
(0)
6/24
(25)
1/3
(33)
0/1
(0)
0/0
(0)
3/12
(25)
1/2
(50)
Table 2 Relationship with clinicopathological parameters for locus
D6S473
Clinicopathological
characteristics
Histology
Serous
Non-serousb
Graded
High
Low
D6S473
Total
LOH (%) No LOH (%)
Pa
24
8
13 (54)
1 (12)
11 (46)
7 (88)
0.040c
17
13
11 (65)
3 (23)
6 (35)
10 (77)
0.023c
a
LOH vs. No LOH. bNon-serous types include mucinous cystadenocarcinomas, endometrioid adenocarcinomas, and mixed epithelial
carcinomas. cChi-square test. dHigh grade specimens include poorly
di€erentiated tumors. Low grade specimens include well and
moderately di€erentiated tumors
ones. This ®nding seems to be in accordance with other
reports that suggested that LOH in serous adenocarcinomas occurs more frequently than in other histological types (Sato et al., 1991; Foulkes et al., 1993). This
suggests that losses of speci®c chromosomal regions on
6q may be involved with the development or
progression of serous ovarian adenocarcinomas (Sato
et al., 1991). Furthermore, a signi®cant correlation was
observed between allele loss and low grade and high
grade tumors (P=0.023) at locus D6S473. Loci
D6S441 (P=0.083) and D6S448 (P=0.051) also
showed LOH to be suggestively more frequent in high
grade tumors. No other signi®cant correlation between
LOH and other clinicopathological parameters was
observed by Chi-Square analysis.
In recent studies, allelic deletions on the terminal
end of chromosome 6q have also been reported for
di€erent tumors. In small cell lung cancer, Merlo et al.
used markers D6S255 and D6S264 on 6q25-q27 to
report a 47% LOH frequency (Merlo et al., 1994). In
Deletion mapping of chromosome 6q25 ± q26 in ovarian tumors
CV Colitti et al
breast cancer, Devilee et al. reported chromosome 6q
being involved in allelic imbalance in more than 50%
of informative cases. They also suggested that a
potential tumor suppressor gene is thought to reside
on 6q25.1 (Devilee et al., 1991). Furthermore, in
malignant melanomas, Millikin et al. demonstrated
that one of the markers de®ning the chromosomal
region bearing the highest frequency of 6q allelic loss
was ESR with 50% (Millikin et al., 1991). All these
results may suggest the presence of a tumor suppressor
gene in the 6q25 region that is common to ovarian and
other carcinomas.
On chromosome 6q25, only the superoxide dismutase
2 (SOD2) gene, which encodes the antioxidant enzyme
manganese superoxide dismutase (MnSOD), has been
suggested as a tumor suppressor in malignant melanoma
(Volz et al., 1994; Bravard et al., 1995). Using expression
vectors containing MnSOD cDNAs, Church et al. have
shown that increased SOD2 gene expression modi®es
the transformed phenotype of melanoma cells (Church
et al., 1993). In ovarian cancer, although the SOD2 gene
locus has previously been shown to have high
frequencies of LOH (Cliby et al., 1993; Foulkes et al.,
1993), there is no evidence that the gene has any
involvement in ovarian carcinogenesis. Furthermore, the
SOD2 gene locus has been located about 5 cM telomeric
to D6S437 which is positioned outside our minimally
deleted region (Gyapay et al., 1994; Volz et al., 1994).
This suggests that there may be another novel tumor
suppressor gene located in our candidate region which is
involved in ovarian carcinogenesis.
In conclusion, we report the identi®cation of a novel
4 cM minimal deletion unit on chromosome 6q25.1q25.2. The small size of our region may allow for the
construction of a physical map of the minimally
deleted region by yeast arti®cial chromosomes (YAC)
cloning strategy, giving us an opportunity for direct
cloning and identi®cation of a potential tumor
suppressor gene associated with invasive epithelial
ovarian cancer.
Acknowledgements
This work was supported by Public Health Service Grants
R01CA63381, R01CA69453 and R01CA69291 from the
National Institute of Health, Department of Health and
Human Services.
References
Bravard A, Sabatier L, Ho€schir F, Ricoul M, Luccioni C
and Dutrillaux B. (1995). Int. J. Cancer, 51, 476 ± 480.
Church SL, Grant JW, Ridnour LA, Oberley LW, Swanson
PE, Meltzer PS and Trent JM. (1993). Proc. Natl. Acad.
Sci. USA, 90, 3113 ± 3117.
Cliby W, Ritland S, Hartmann L, Dodson M, Halling KC,
Keeney G and Jenkins RB. (1993). Cancer Res., 53, 2393 ±
2398.
Cooke IE, Cox SA, Shelling AN, Le Meuth VG, Spurr NK
and Ganesan TS. (1996a). Mammal. Genome, 7, 157 ± 159.
Cooke IE, Shelling AN, Le Meuth VG, Charnock ML and
Ganesan TS. (1996b). Genes Chromosome & Cancer, 15,
223 ± 233.
Devilee P, van Vliet M, van Sloun P, Kuipers Dijkshoorn N,
Hermans J, Pearson PL and Cornelisse CJ. (1991).
Oncogene, 6, 1705 ± 1711.
Edelson MI, Scherer SW, Tsui LC, Welch WR, Bell DA,
Berkowitz RS and Mok SC. (1997). Oncogene, 14, 2979 ±
2984.
Ehlen T and Dubeau L. (1990). Oncogene, 5, 219 ± 223.
Foulkes WD, Ragoussis J, Stamp GW, Allan GJ and
Trowsdale J. (1993). Br. J. Cancer, 67, 551 ± 559.
Gallion HH, Powell DE, Morrow JK, Pieretti M, Case E,
Turker MS, Hunter JE and van Nagell JR, Jr. (1992).
Gynecol. Oncol., 47, 137 ± 142.
Gyapay G, Morissette J, Vignal A, Dib C, Fizames C,
Millasseau P, Bernardi G, Lathrop M and Weissenbach J.
(1994). Nat. Genet., 7, 246 ± 339.
Iwase H, Greenman JM, Barnes DM, Bobrow L, Hodgson S
and Mathew CG. (1995). Br. J. Cancer, 71, 448 ± 450.
Leary JA, Doris CP, Boltz EM, Houghton CRS, Ke€ord RF
and Friedlander ML. (1993). Int. J. Gynecol. Cancer, 3,
293 ± 298.
Lee JH, Kavanagh JJ, Wildrick DM, Wharton JT and Blick
M. (1990). Cancer Res., 50, 2724 ± 2728.
Lee WC, Balsara B, Liu Z, Jhanwar SC and Testa JR. (1996).
Cancer Research, 56, 4297 ± 4301.
Merlo A, Gabrielson E, Mabry M, Vollmer R, Baylin SB and
Sidransky D. (1994). Cancer Res., 54, 2322 ± 2326.
Millikin D, Meese E, Vogelstein B, Witkowski C and Trent J.
(1991). Cancer Res., 51, 5449 ± 5453.
Orphanos V, McGown G, Hey Y, Boyle JM and SantibanezKoref M. (1995a). Br. J. Cancer, 71, 290 ± 293.
Orphanos V, McGown G, Hey Y, Thorncroft M, Santibanez-Koref M, Hickey I, Atkinson RJ and Boyle JM.
(1995b). Br. J. Cancer, 71, 666 ± 669.
Queimado L, Seruca R, Costa-Pereira A and Castedo S.
(1995). Genes Chromosome & Cancer, 14, 28 ± 34.
Rodabaugh KJ, Biggs RB, Qureshi JA, Barrett AJ, Welch
WR, Bell DA, Berkowitz RS and Mok SC. (1995a).
Oncogene, 11, 1249 ± 1254.
Rodabaugh KJ, Blanchard G, Welch WR, Bell DA,
Berkowitz RS and Mok SC. (1995b). Cancer Res., 55,
2169 ± 2172.
Saito S, Saito H, Koi S, Sagae S, Kudo R, Saito J, Noda K
and Nakamura Y. (1992). Cancer Res., 52, 5815 ± 5817.
Sato T, Saito H, Morita R, Koi S, Lee JH and Nakamura Y.
(1991). Cancer Res., 51, 5118 ± 5122.
Sheng ZM, Marchetti A, Buttitta F, Champeme MH,
Campani D, Bistocchi M, Lidereau R and Callahan R.
(1996). Br. J. Cancer, 73, 144 ± 147.
Volz A, Boyle JM, Cann HM, Cottingham RW, Orr HT and
Ziegler A. (1994). Genomics, 21, 464 ± 472.
Wertheim I, Tangir J, Muto MG, Welch WR, Berkowitz RS,
Chen WY and Mok SC. (1996). Oncogene, 12, 2147 ± 2153.
Zheng JP, Robinson WR, Ehlen T, Yu MC and Dubeau L.
(1991). Cancer Res., 51, 4045 ± 4051.
559