Download erbA-related Sequence Coding for DNA-binding

[CANCER RESEARCH 48, 682-685, February 1, 1988]
erbA-related Sequence Coding for DNA-binding Hormone Receptor Localized to
Chromosome 3p21-3p25 and Deleted in Small Cell Lung Carcinoma1
Alexander Dobrovic,2 Benoit Houle, Abdelmajid Belouchi, and W. E. C. Bradley3
Institut du Cancer de Montréal,Montreal, Quebec, Canada H2L 4M!
ABSTRACT
Small-cell lung carcinoma (SCLC) is characterized by a consensus
deletion in the short arm of chromosome 3. Using a panel of cell lines
and somatic cell hybrids containing various rearrangements involving
chromosome 3, we have localized the erbAß
sequence (which codes for a
thyroid hormone receptor) to the region 3p21 —»
3p25 which overlaps
the consensus deletion in SCLC. Moreover, we have shown by Southern
blot analysis that at least one copy of the erbAßsequence is deleted in
all six SCLCs so far studied. Normalized ratios of hybridization intensi
ties of the erbAßprobe to intensities of probes for somatostatin (3q28)
and ra/(3p24-25) ranged from 0.28 to 0.56 and 0.32 to 0.71, respectively,
in the six tumors and tumor lines. In view of the importance of the role
these genes are known or suspected to play in biological regulation, our
results suggest that the erbAßsequence is a candidate for a recessive
oncogene involved in the genesis of SCLC.
Recently, several sequences coding for receptor molecules
have been isolated and mapped to chromosome 3, without
regional localization. One of these is the cellular retinol binding
protein (8), and the others are erbA-re\ntea sequences which are
members of a superfamily of regulatory genes coding for hor
mone receptors (9,10). This latter group is of particular interest
as they include a sequence coding for a DNA binding region of
the receptor and as such are probably directly involved in
specific control of gene expression. One of the genes has been
shown to code for a thyroid hormone receptor (10).
In this communication, we regionally assign the erbA-related
sequences which code for the thyroid hormone receptor to
3p21-3p25 and show that in six of six small cell lung tumors
studied, the sequences are present in reduced copy number
relative to other markers on chromosome 3.
INTRODUCTION
MATERIALS
Chromosomal rearrangements which are nonrandomly as
sociated with certain types of malignant proliferations have
been reported for a wide variety of tumors (1). These abnor
malities probably reflect important genetic changes which con
tribute to the progression of the tumors. In the case of intersti
tial deletions, such as are found in chromosome band 13ql4 in
many retinoblastomas (2), the abnormality is thought to allow
expression of a recessive tumor phenotype (3). It is of consid
erable interest to isolate the sequences for which loss may be
responsible for a given tumor phenotype, but a major problem
is that microscopically visible chromosomal deletions are very
large on the molecular scale. In principle, isolation of the critical
sequences can be facilitated by molecular mapping of the region
in question. Thus, a candidate retinoblastoma gene has recently
been cloned with the aid of a marker DNA fragment which was
known to map within the 13ql4 deletion (4).
Another tumor which is characterized by a particular inter
stitial deletion is small cell lung carcinoma SCLC,4 in which
DNA Probes. The plasmids pgHS7-2.7 (11), pMSl-37 (12), and
p628 (13) were maintained in Escherichia coli HB101 and carry, re
spectively, human somatostatin cDNA (SST), the D3S3 sequence, and
the human raf-l oncogene. The plasm id pheA4 (10) was also main
tained in HB101 and carries a cDNA sequence which in blotting
experiments detects two erbA-re\ated genes collectively called hc-erbAß.
Cells and Cell Culture. Human fibroblast cultures GM2808,
GM1533, and GM0366 were obtained from the Human Genetic Mu
tant Repository (Camden, NJ) and were maintained in RPMI Medium
1640 with 10% fetal calf serum and antibiotics. These cells carry,
respectively, the following abnormalities: translocation
t(3;17)
(p21:pl3); translocation t(X;3) (q26:pl2); and monosomy for 3p25pter plus trisomy for 3q21-qter. The cloned SCLC lines NCI-H69 and
NCI-HI28 (14) were obtained from American Type Culture Collection
and carry deletions in 3p21-3p25 and 3pl4-pter, respectively. The
SCLC line RG1 was established in this laboratory from a pericardial
effusion of a patient (R. G.) with metastatic SCLC, as has been
described by Gazdar et al. (14). The cells were plated in dishes with
RPMI 1640 containing 20% fetal calf serum plus antibiotics and grew
as characteristic floating masses of cells (14). Confirmation of SCLC
phenotype was obtained by cytological examination.
SCLC tumors SCT4 and SCT13 were obtained from two other
patients at autopsy and SCT2 was a biopsy sample from a fourth
patient. All tumor samples were essentially uncontaminated by normal
tissue. Normal tissue was obtained at the same time as the tumors. All
samples were frozen immediately at —70°C
until DNA extraction.
Autopsies were performed 4-20 h after death, and occasionally some
DNA degradation had occurred by this time.
Somatic Cell Hybrids. T317L clones were obtained from the fusion
of GM2808 cells with the thymidine kinase-deficient mouse cell line
LTK~. TX3R clones were obtained from the fusion of GM1533 with
the hypoxanthine phosphoribosyl transferase-deficient mouse cell line
RAG. Fusion was carried out by plating the parental cell lines together
and treating a subconfluent monolayer with 50% polyethyleneglycol
1000 (BDH) in Dulbecco's phosphate-buffered saline (calcium- and
magnesium-free). After recovery overnight in nonselective medium, the
cells were subcultured into Petri dishes at low density and hybrid clones
were selected by growth in HAT medium supplemented with 10~6 M
deletions frequently occur on the short arm of chromosome 3,
most commonly between bands pl4 and p23 (5). This is a
particularly long stretch of DNA (perhaps 30-40 megabases),
and so far only a few cloned sequences have been mapped to
within or near the region. These include D3S3 (pi4; Ref. 6),
D3S2 (pl4-p21), and raf-1 (p24-p25) (7). To better understand
the nature of the change responsible for the SCLC phenotype,
there is clearly a need for further mapping information. It
would be of particular interest to identify any potential regula
tory genes which may lie within this region, as these may be
candidates for the SCLC oncogene(s).
Received 7/14/87; revised 10/23/87; accepted 10/29/87.
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.
1Supported by the National Cancer Institute of Canada, The Medical Research
Council of Canada, and the Fonds de Recherche en Santédu Québec.
2 Present address: Department of Hematology, Flinders University of South
Australia, Adelaide, Australia.
3To whom requests for reprints should be addressed, at Institut du Cancer de
Montreal, 1560 est, rue Sherbrooke, Montréal,Québec,Canada H2L 4ML
4 The abbreviations used are: SCLC, small cell lung carcinoma; cDNA, com
plementary DNA; hprt, X-linked hypoxanthine phosphoribosyl transferase.
AND METHODS
ouabain. Hybrid clones were apparent between 7 and 21 days. Inde
pendent clones were picked using glass cloning cylinders and main
tained in HAT medium. Clones of interest identified by DNA analysis
using chromosomes 3 markers were subcloned and reanalyzed.
682
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£rò/l-RELATEDGENE DELETED IN SCLC
DNA Extraction. Cultured cells were harvested and their DNA was
extracted by a standard procedure (15). Tissue material (tumor and
normal) was ground in a mortar and pestle, maintaining the material
constantly submerged in liquid N2. DNA was extracted from the re
sulting powder in the standard way.
DNA Analysis. Fifteen-/¿gsamples of DNA were digested with re
striction enzymes EcoRI, BamHl, Bglll, or Hindlll (from BoehringerMannheim or BRL) according to the manufacturer's instructions. Sam
ples were size fractionated by electrophoresis on 0.7% agarose gels
(IBI) and transferred to a nylon membrane (Gene-Screen Plus; NEN)
according to instructions furnished by NEN. Blots were dried, incubated
for 6 h at 65°Cin prehybridization mixture (Sx SSC, 5x Denhardt's
solution, 20 mM Tris, pH 7.5, 10% dextran sulfate 1% sodium dodecyl
sulfate, and 250 Mg/ml salmon sperm DNA; Ix SSC is 0.15 M NaCl,
0.015 M Na citrate, and Ix Denhardt's is 0.02% polyvinyl pyrolidone,
0.02% Ficoll, and 0.02% bovine serum albumin). The blot was then
hybridized in the same mixture at 65°Covernight to 100 ng DNA
probe which had been 32Plabeled by nick translation to >3 x 10" cpm/
ng (15), washed as suggested by NEN, and exposed to X-ray film.
RESULTS
digested DNA of human cells and each of the somatic cell
hybrids described in Table 1 were probed with both the £coRl
pheA4 fragment and the SST sequences (Fig. 1). Human DNA
(lanes 2 and 7) gives an SST band at 4.4 kilobases and two
erbAßbands of higher and two of lower molecular weight. All
four erbA human bands were identifiable in each of the hybrids
which had already been identified as raf positive (Fig. 1, lanes
3, 4, and 6). The complementary hybrid t317LC8 did not carry
human er¿>/Ã--specific
bands. Mouse and human SST bands
comigrate at 4.4 kilobases under these conditions and the
distinction between them is therefore not clear.
The DNA from the culture GM0366 was also probed with
erbA and SST sequences (Fig. 1, lane 7), and hybridization
intensities of the various bands were quantitated by densitometry. Comparison with intensities of a normal DNA sample
(lane 2) yielded normalized ratios of erbAß:SST of about 0.7 in
two separate experiments (Table 2), very close to the expected
ratio of 0.67 if all of the erbAßsequences were disomic and
clearly different from the 0.33 expected if the sequence was
Mapping of hc-erbAßGenes. The strategy for determining
the regional localization of the erbAß
sequences was to construct
somatic cell hybrids between mouse cells and human cells
carrying translocations involving chromosome 3, either at 3pl2
or 3p21 ("Materials and Methods"). DNA probes specific for
the two termini of human chromosome 3 could then be used to
determine which hybrids would be useful. The series of hybrids
identified as 1317L had, as the human parent, a line carrying
the 3p21 —>ter segment translocated to chromosome 17,
whereas the series tX3R had a human X carrying a translocation
from 3pl2 —»
ter. About 10 clones were picked from each of
the two hybridizations performed. DNA from each clone was
digested with £coRIor Hindlll in order to generate SST (3q28),
D3S3 (3pl4), and ra/(3p24-25) fragments which differed be
tween human and mouse. Blots were probed with SST, D3S3,
and raf. Since thymidine kinase is located on 17q and the
chromosome 3p fragment was translocated onto 17p, about
one-half of the HAT-selected hybrids were expected to be
human raf positive, human SST negative. It was not known
where the X-linked hprt mapped relative to the Xq26 translo
cation point in the human parent GM2808 prior to fusion.
Analysis with the three probes (pertinent results summarized
in Table 1) showed that ra/and hprt cosegregated, indicating
that hprt is proximal to the translocation breakpoint. Five
hybrid clones (Table 1) were chosen for mapping studies, two
carrying 3p21-ter, two carrying 3pl2-ter, and one, t317Lc8,
which carried 3p21-3qter.
The 1.5-kilobase £c0RIfragment from pheA4 which carries
the erbAßcDNA sequence hybridizes at high stringency with
four bands of fig/II-digested human DNA which are derived
from two different, but homologous, genes (10). A blot of BglllTable
hybridsDNA
1 Segregation analyses of SST, D3S3, and raf among somatic cell
from each hybrid was digested with EcoRI (D3S3 and SST) or Hindlll
(raf), blotted, and probed with the three cloned sequences shown. Raf bands were
at 18 and 8 kilobases (mouse and human, respectively), SST bands were at 1
2and
6 kilobases, respectively, and
only).Presence
the D3S3 band was at 8 kilobases (human
toraf
SSTHybrid
Humanline
Humant317Lc2 Mouse
+t317Lc4
+
+t317Lc8
+
+tX3Rc2
+
-tX3Rc4
+
+
Human
+
+
+
+
of bands corresponding
D3S3
+
+
+
Mouse
+
+
+
CM
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9.46-6 4.4-
2-3Fig. 1. Southern blot of DNA samples from mouse, human, and various
mouse-human hybrid lines. DNA (15 ng) was digested with Bgltt, size fractionated
by gel electrophoresis, blotted, and probed with nick translated sequences of erbAß
(EcoRI fragment of pheA4) and SST (Hhal fragment of pgHS7-2.7). The SST
bands of both mouse and human comigrate at 4.4 kilobases. Lane 1, mouse LTK~;
lane 2, normal human; lanes 3-6, human-mouse hybrids (Table 1); lane 7, human
CM 0366. Left, \ Hindlll size markers (in kilobases).
Table 2 Relative band intensities of erbAß,
SST, and raf
DNA samples were digested with Bglll, blotted, and probed with erbAßand
SST sequences, erbAß
and raf sequences, or all three together. After exposure to
X-ray film, densitometer tracings were made and areas under the peaks were
measured. Values shown are the ratio of the erbAß
band at 4.2 kilobases to the
4.4-kilobase SST band (some samples shown in Fig. 2, left) and the ratio of the
same erbAß
band to the 2-kilobase ra/band, in all cases normalized to values for
tissue Normal 1. When more than one blot was probed, the range of ratios is
given. Results of probing some samples with raf are shown in Fig. 2, right.
Normalized ratios of inten
sity
n.iiu|in.:iNormalI3SUC
ui
vin
uni.
Normal 1
2Normal
Normal
4GM
ter;tnsomy
0366 (monosomy 3p25 —
»
ter)SCLC
3q21 —
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H69H128SCT2SCT4SCT13RG11
f i/.'l11.00
i J..1.1
0.89 ±0.09
010.920.73
1
0.88
251.321.090.46
1
0.040.43
±
0.260.380.39
±
0.010.370.470.320.710.54
±
0.020.28
±
0.080.560.561.00
±
-i
683
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&M-RELATED
GENE DELETED IN SCLC
within the monosomic region p25-ter in this line. Thus the hcerbAßgenes have been assigned to 3p21-p25. As indicated
below, blot analysis of the tumor line H69 gave results consist
ent with this assignment.
hc-erbAßGenes Deleted in SCLC. DNA from a variety of
SCLC tumors and cell lines was digested with Bglll, size
fractionated by gel electrophoresis, blotted, and probed with
hc-erbAß,SST, and raf sequences. Visual inspection of Fig. 2
shows that by comparison with SST, eré/i/î-specifïc
bands hy
bridize much less strongly in the lanes with tumor DNA than
those with normal DNA. Densitometer tracings of each of
several normal DNA samples and six SCLCs were made and
ratios of band intensities were calculated. For many samples,
at least two separate blots were probed. Table 2 shows normal
ized ratios (±range where appropriate) of the 4.2-kilobase
erbAßBglll band to the 4.4-kilobase SST band (see Fig. 2).
(This erbAß
band was chosen for this analysis since it was close
in size to the SST band and hence minimized any intensity
variations due to slightly degraded DNA, such as occurred with
sample SCT4. In all samples relative intensities of all erbAß
bands were uniform.) Table 2 also shows intensity ratios of the
same erbAßband to the raf band at 2 kilobases. (Fig. 2, right;
the blot was hybridized with raf, SST, and an erbAßfragment
which detects only two of the four Bglll bands).
Reproducibility among independent estimates of the same
sample was good (Table 2), with the sole exception that in one
of the four experiments in which H69 was probed with erbAß
and SST, a somewhat uneven background raised the apparent
intensity of the erbAß
band. In general, however, the data clearly
show that in the tumors, there are fewer copies of the erbAß
genes than of SST and raf, which are genes near the termini of
the long and short arms, respectively. We conclude that chro
mosome 3 sequences, including erbAß,are deleted in these
tumors. This is particularly plausible in light of the known
karyotypic change in these cells. Other formal possibilities exist
such as specific degradation of erbAßsequences during extrac
tion and these could be tested if restriction fragment length
polymorphisms could be detected by the erbAß
sequence. How
ever, a preliminary search for such polymorphisms has been
unsuccessful, so at this point we must depend on the intensity
differences shown in the blots.5
A further noteworthy observation is that in most tumors the
erbAß:SST ratio was about the same as the erbAß:rafratio.
Even in those cases where ratios were not very close (e.g.,
SCT13) hybridization intensity of the raf probe was greater
than that of erbAß.This suggests that the deletion event(s)
5A. Dobrovic, B. Houle, A. Belouchi, and W. E. C. Bradley, unpublished
results.
T-
CM
c\j
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CO
Fig. 2. Left, southern blot of DNA samples
from normal (lanes 1 and 2) and SCLC tumor
tissue (lanes 3 and 4) and the SCLC cell line
H69 (lane 5). DNA (15 Mg)was digested with
Bglll, electrophoresed, blotted, and probed
with SST and erbAßsequences as in Fig. 1. \
//mi/Ill size markers (in kilobases). Right, the
same blot was stripped of probe and rehybridized with SST, ra/(the 3' Bgttl-EcoRl frag
ment of p628), and the 350 bp Sacl-Sacl frag
ment of pheA4. The latter detects only two of
the erbAßbands, including the 4.2-kilobase
band.
tended to be interstitial, leaving the ra/gene at p24-p25 intact.
The results of the wide karyotype survey of 16 SCLCs by
Whang-Peng et al. (5), however, suggest that the majority of
the visible karyotypic deletions on 3p (including these on H128
which we analyzed) were terminal. Such a spectrum of kary
otypic changes should yield erbAßirafratios of close to unity,
since raf would also be in the deletion. The fact that this was
not so in our six tumors, including H128, suggests that either
a proportion of the deletions may be submicroscopic or that raf
is frequently amplified in SCLC. It is also quite possible that
the terminal karyotypic deletions in fact are the result of a
translocation coupled with loss of the region carrying erbAß
but
not raf.
DISCUSSION
We have shown that the two erbA-related genes hc-erbAßare
localized to 3p21-3p25 and that they are deleted in SCLC
tumors. These conclusions are consistent with each other, since
it is known that most SCLC tumors carry interstitial deletions
in chromosomes 3p and that the region most consistently
deleted is around 3p23 (5). In particular, cells of the line H69
carry two normal chromosomes 3 and (in virtually all cells)
another with an interstitial p21-p25 deletion. This coincides
exactly with the assignment region deduced from results pre
sented in Fig. 1. In most cases, our results indicated that the
ra/gene, at p24-p25, was not included in the deletion events
detected.
An important consideration in interpreting and evaluating
results based on hybridization intensities of Southern blots is
that cells of a given tumor are karyotypically heterogeneous, so
we do not necessarily expect monosomy of a given alÃ-eleto be
manifested by intensity ratios of exactly 1 to 2 relative to an
undeleted alÃ-ele.Thus SCT4, with an erbAß:SSTratio of about
0.3, may be a triploid line with erbAßdeletions on two homo
logues, and these deletions may be identical (endoreduplication
of the original abnormal chromosome) or may result from
sequential events. The tumor may also be a mixture of cells
hemizygous and homozygous for the deletion. In any event the
net result is underrepresentation of c-erbAßsequences relative
to the rest of chromosome 3.
Does the relative loss of erbAßsequences play a key role in
the evolution of SCLC? The consensus deletion in 3p is very
large (pl4-p23), and it is therefore certainly possible that the
important SCLC oncogene(s) lies elsewhere in this region.
However, two lines of evidence suggest that submicroscopic
deletions may also contribute to the loss of the erbAßgenes.
One of these is the simple fact that not all SCLCs have a visible
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684
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frfi^-RELATED GENE DELETED IN SCLC
3p deletion (16). The second (see "Results") lies in the discord
ance between erbA:raf ratios (Table 2) and the observed fre
quency of terminal deletions in 3p (5). We cannot yet eliminate
the alternative explanation for the discordance, namely, ampli
fication of raf sequences, but it seems an unlikely coincidence
that raf would be amplified in each of the tumors to the precise
extent that the number of raf copies always corresponded to
the number of SST copies (Table 2). We therefore conclude
that some of the deletions are probably relatively small and this
decreases the chance that erbAßdeletions have arisen merely
because of proximity to the real SCLC oncogene.
A further reason to suspect that the erbAßgenes are impor
tant in SCLC is that they play a key role in genetic regulation.
The genes are members of a family coding for DNA-binding
proteins which have a hormone receptor function; one of the
two hc-erbAßgenes has been shown to have thyroid hormone
binding capacity. The other is not yet characterized but has
sufficient homology that it almost certainly codes for a protein
with similar activity.
The mechanism of action of this family of regulatory proteins
is known in some detail. For example activation of glucocorticoid-sensitive genes such as tyrosine aminotransferase requires
the presence of the hormone, and interaction in vivo between
the tyrosine aminotransferase-enhancer
sequences and the re
ceptor protein also occurs only in the presence of glucocorticoid
(17). Other members of this receptor family also require the
presence of the appropriate hormone to activate their target
genes and this activation is associated with translocation of the
hormone-receptor complex to the nucleus. In such a positive
regulatory context a recessive tumor phenotype would be the
result of silencing of the genes which are targets of the product
of the deleted regulatory gene. However, the target genes in
question are presumably silenced at least occasionally in many
normal cell types, with no apparent ill effects, so it is not
immediately evident how erbAßdeletion may contribute to
tumorigenicity in SCLC. Moreover, SCLC tumors are charac
terized by inappropriate activation (rather than inactivation) of
a battery of genes, such as gastrin-releasing peptide and its
receptor ( 18).
These conceptual difficulties would be resolved if the erbAß
product(s) had a negative regulatory function as well, and some
recent observations have raised this possibility, (a) In the spe
cific example of glucocorticoid regulation of expression of the
proopiomelanocortin gene, a represser effect has been very well
documented (19). (b) Several studies have established the prin
ciple that other regulatory proteins which activate transcription
of certain genes can in fact repress expression of other genes.
This has best been shown with adenovirus E1A gene and an asyet-uncharacterized transacting factor in embryonal carcinoma
cells (20). In this light, one could imagine the hc-erbAßgene
products acting as a constitutive represser of some key gene(s),
and deletion of one copy would represent a step toward malig
nancy.
Identification of the hc-erbAßgene(s) as a recessive oncogene
clearly awaits direct proof of the association between deletion
of these sequences and expression of the SCLC (or other tumor)
phenotype. Even if this is shown, loss of other genes in addition
to erbAß
on the short arm of chromosome 3 may contribute to
SCLC progression, since the deletion which is frequently seen
is very large. Simple inactivation of hc-erbAßgenes could be
accomplished by a variety of mutational events most of which,
in normal cells in vivo, have been shown to be either point
mutations or relatively small (<40-kilobase) deletions (21). We
therefore anticipate that the complete documentation of the 3p
sequences which play a role in SCLC will require much further
investigation.
ACKNOWLEDGMENTS
We thank France Laviolette for excellent technical assistance, Dr.
R. Evans for furnishing the plasmici pheA4, and Drs. U. Kapp, G. I.
Bell, T. Dryja, and R. White for providing other cloned sequences. We
particularly thank Drs. C. Hajj and J. Ayoub for close collaboration in
obtaining clinical samples.
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erbA-related Sequence Coding for DNA-binding Hormone
Receptor Localized to Chromosome 3p21−3p25 and Deleted in
Small Cell Lung Carcinoma
Alexander Dobrovic, Benoit Houle, Abdelmajid Belouchi, et al.
Cancer Res 1988;48:682-685.
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