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From the National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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All cell lines used in this study (BxPC-3, Capan-1, SU.86.86, Hs 766T, AsPC-1, Capan-2, MIA-PaCa-2, PANC-1, CFPAC-1) were purchased from American Type Culture Collection (Rockville, MD).
Comparative Genomic Hybridization
CGH was performed on normal, sex-matched metaphase chromosomes prepared according to standard procedures. Control DNA was labeled with digoxigenin-12-dUTP (Boehringer Mannheim, Indianapolis, IN) by nick translation. Tumor DNA was extracted from pancreatic carcinoma cell lines using proteinase K digestion and phenol extraction. Labeling of genomic tumor DNA was performed by nick translation substituting dTTP by biotin-16-dUTP (Boehringer Mannheim). Differentially labeled genome (300 ng of each) was precipitated together with an excess (30 µg) of the Cot-1 fraction of human DNA (Gibco BRL, Gaithersburg, MD). The probe DNA was resuspended in 10 µl of hybridization solution (50% formamide, 2x SSC, 10% dextran sulfate), denatured (5 minutes, 75°C), and preannealed for 1 hour at 37°C. The normal metaphase chromosomes were denatured separately (70% formamide, 2x SSC) for 2 minutes at 75°C, and dehydrated through an ethanol series. Hybridization took place under a coverslip for 2 to 4 days at 37°C. Posthybridization washes and immunocytochemical detection was performed as described.19 Biotin-labeled tumor sequences were detected with avidin conjugated to fluorescein isothiocyanate (Vector Laboratories, Burlingame, CA), and the digoxigenin-labeled reference DNA was developed using a mouse anti-digoxin antibody, followed by a tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-mouse antibody (Sigma, St. Louis, MO). The slides were counterstained with DAPI 4;6-diamino-2-phenylindole and embedded in an antifade solution containing para-phenylene-diamine (Sigma).
Gray level images were acquired for each fluorochrome with a cooled
charge-coupled device camera (Sensys, Photometrics, Tucson, AZ) coupled
to a Leica DMRXA epifluorescence microscope using sequential exposure
through fluorochrome specific filters (TR1, TR2, TR3, Cy5; Chroma
Technology, Brattleboro, VT) using the Leica Q-FISH ACAPS imaging
system. Chromosomes were identified using DAPI-banding and
co-hybridization with centromere-specific DNA probes. Fluorescence
ratio images were calculated with the Cytovision CGH software (Applied
Imaging, Ltd., Tyne & Wear, UK). Average ratio profiles were calculated
from at least eight ratio images. An example of the results is shown in
Figure 1
for the cell line AsPC-1. The
vertical lines on the right side of the chromosome ideogram represent
different values of the fluorescence ratios between the tumor and the
normal DNA (Figure 1)
. The center line reflects a ratio of 1, the line
left to the central line indicates the threshold for chromosome losses
(ratio of 0.75), and the line on the right of the central line marks
the threshold for gains (ratio of 1.25, see du Manoir et
al20
for details of the image analysis). The
curves show the ratio profiles that were computed as mean values of at
least 8 metaphase spreads. Average ratio profiles were the basis for
the evaluation of copy number changes in all cases.
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Metaphase chromosomes for SKY were prepared from pancreatic cell lines according to standard procedures. SKY was performed as described.14,21 Briefly, flow-sorted human metaphase chromosomes were amplified using degenerate oligonucleotide polymerase chain reaction (PCR).22 Chromosome-specific painting probes were labeled by PCR with rhodamine-11-dUTP, spectrum green-dUTP, texas red-dUTP, biotin-16-dUTP, digoxigenin-11-dUTP, and combinations thereof. Two hundred ng each of the differentially labeled chromosome painting probes were precipitated in the presence of 50 µg of the Cot-1 fraction of human DNA (Gibco BRL). Hybridization took place over 2 days at 37°C. The biotinylated probe sequences were visualized using avidin Cy5 (Amersham Life Sciences, Buckinghamshire, UK), and the digoxigenin-labeled probe sequences by incubation with an anti-mouse digoxin antibody (Sigma) followed by a goat anti-mouse-antibody conjugated to Cy5.5 (Amersham Life Sciences). Chromosomes were counterstained with DAPI and mounted and coverslipped in paraphenylene-diamine (Sigma).
Image acquisition was performed using a SD200 Spectracube (Applied
Spectral Imaging, Ltd., Migdal Haemek, Israel) mounted on a Leica DMRXA
microscope through a custom designed optical filter (SKY-1, Chroma
Technology, Brattleboro, VT). Using a Sagnac interferometer in the
optical head, an interferogram was generated at all image points that
is deduced from the optical path difference of the light, which in turn
depends on the wavelength of the emitted fluorescence. The spectrum was
recovered by Fourier transformation. The spectral information was
displayed by assigning red, green, or blue colors to certain ranges of
the spectrum using SkyView software (Applied Spectral Imaging). This
red, green, blue-display renders chromosomes, which were labeled with
spectrally overlapping fluorochromes or fluorochrome combinations,
a similar color (see Figure 3B
). Based on the measurement of the
spectrum for each chromosome, a spectral classification algorithm was
applied to assign a pseudocolor to all points in the image that have
the same spectrum.23
This algorithm forms the
basis for chromosome identification by spectral karyotyping (see Figure 3C
). DAPI images were acquired from all metaphases using a
DAPI-specific optical filter, inverted, and contrast enhanced using
SkyView (Applied Spectral Imaging, Ltd.).
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Region-specific FISH probes for the following tumor suppressor
genes and proto-oncogenes were performed using standard procedures.
DNA-probes for the following loci were applied: MDR
(7q21–22), p16 (9p21), p53 (17p13),
DCC (18q21), AIB1 (20q12), and a
microdissection-derived painting probe for chromosome band
18q12.24
Chromosome specific centromere probes
were prepared by alphoid PCR of monochromosomal cell hybrids.
Hybridization and detection followed routine FISH procedures. Images
were acquired using a Leica DMRXA microscope, a cooled CCD camera
(Sensys, Photometrics, Tucson, AZ), and Leica Q-FISH software.
Amplification of the AIB1 gene was scored as moderate
when more than 70% of the cells revealed a ratio of higher than 2
compared with the centromere of chromosome 20 (+). Ratios that exceeded
5 were scored as high level amplifications (++). A gain of
AIB1 was defined as a simultaneous increase of
AIB1 and centromer 20 signal numbers. Signals were
enumerated in 50 to 100 metaphase and interphase cells. The results
were summarized in Table 1
.
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Total RNA was isolated from cell lines using Rneasy Midi Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Electrophoresis, northern transfer, and hybridization were performed by standard methods. In brief, 15 µg of RNA was size fractionated on a 1% agarose and 2.2 mol/L formaldehyde gel and transfered by alkaline method to a charged nylon membrane. The Northern blot was hybridized sequentially with a 2.2-kb fragment from the 3' untranslated region of AIB1 and a ß-actin loading control probe (Clontech, Palo Alto, CA). Probes were labeled with 32P by random priming.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table 1
), and
Northern-blot analysis indicated increased expression of this gene (see
Figure 5
).
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DNA from nine pancreatic carcinoma cell lines (BxPC-3, Capan-1,
SU.86.86, Hs 766T, AsPC-1, Capan-2, MIA-PaCa-2, PANC-1, CFPAC-1) was
analyzed using comparative genomic hybridization. All cell lines
revealed copy number changes, and all chromosomes were involved in
gains and losses. An example of a CGH analysis is shown in Figure 1
for
the cell line AsPC-1. Chromosomal gains were identified on chromosomes
and chromosomal arms and bands 1q, 2, 5, 7, 8, 10, 11, 12q, 16p,
18q11–12, and 20; chromosomal losses were mapped to 9p, 18q, and Xp.
The summary of all aberrations identified in all cell lines is
presented as a karyogram of chromosomal gains and losses in Figure 2
. The number of chromosomal copy number
changes ranged from 14 per case (AsPC-1) to 27 (Capan-2). The average
number of copy alterations (ANCA) is 21.6 per case. Despite this
exceedingly high number of chromosomal aberrations a clear pattern of
nonrandom DNA gains and losses could be identified. Recurring gains
were mapped to chromosomes and chromosome arms 5p (eight of nine
cell lines), 3q (seven of nine), 7p (seven of nine), 8q (six of
nine), 11q (six of nine), 20q (six of nine), 12p (five of nine), and
14q (five of nine). Consistent chromosomal losses were identified on
chromosomes and chromosome arms 9p (eight of nine), 18q (eight of
nine), 8p (six of nine), 17p (five of nine), 19p (five of nine), 21q
(five of nine), Xp (five of nine), and Y (five of nine). The Y
chromosome was lost in all cases of male patients. High-level copy
number increases (amplifications) were mapped to chromosome arms and
chromosome bands 5p, 5p13, 7q21–22, 8q22–24, 12p, 18q23, 19q13.1–2,
20q, and Xq24–28.
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CGH identifies and maps chromosomal copy number changes; however,
aberrations such as balanced chromosomal translocations cannot be
detected by this technique. Conventional chromosome banding analysis
can be difficult in solid tumors, in particular when highly rearranged
genomes confound a complete karyotype analyses. We have therefore
applied SKY to metaphase chromosomes of six of the cell lines that were
also analyzed by CGH (BxPC-3, Capan-1, SU.86.86, Hs 766T, AsPC-1,
MIA-PaCa-2, PANC-1). SKY allows one to simultaneously identify all
human chromosomes in different colors, therefore greatly facilitating
the identification of complex chromosomal aberrations. SKY is
particularly useful when highly shuffled cancer chromosomes produce a
banding pattern in which the origin of rearranged chromosomal material
is impossible to identify with certainty. An example of the SKY
analysis of a metaphase cell from cell line AsPC-1 is shown in Figure 3
. In a single hybridization, all
chromosomes could be identified unambiguously. Figure 3A
shows a
representative metaphase spread after DAPI-banding. The 24-color
hybridization is displayed after visualization of the fluorescent
signals by assigning a red-green-blue look-up table to certain spectral
ranges (Figure 3B
, see Materials and Methods). Based on the pixel by
pixel spectral measurement, a pseudocolor could be assigned to all
pixels that have similar spectra. This assignment is the basis for
spectral karyotyping, the result of which is shown together with the
banded chromosomes in Figure 3C
. Based on the combination
of DAPI banding and SKY, the karyotype of the diploid cell line AsPC-1
was interpreted as follows:
54,X,der(X)t(X;20)(q10;q10),+2,+5,del(7)(q22)x2,+8,der(9)t(7;9)(q31;p13)del(7)(q22),+10,+11,der(12)t(1,12,14)(q12;p12q23;q11),del(16)(q12),der(19)t(1;19)(?;q13.3),der(19)t(8;19)(q22;13.3),+20.
Table 2
presents the karyotypes for all
cell lines analyzed by SKY in accordance with conventional cytogenetic
nomenclature.29
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. Accordingly, the majority of the chromosomal
aberrations detected by SKY is reflected by abnormal CGH profiles of
the involved chromosomes. For instance, the complex translocation
involving chromosomes 14, 12, and 1 would predict gains on chromosomes
1q and 12; this was confirmed by CGH (Figure 1)
. The gain of parts
of chromosome 12 coincides with translocated material from
chromosome 12 that extends from 12p12 to 12q23. SKY revealed two normal
copies of chromosome 7, two copies of a deleted chromosome 7 (q22), and
a translocation between chromosomes 7 and 9. Again, this pattern is
consistent with the CGH results that indicate an overrepresentation of
the short arm of chromosome 7. The amplification on band 7q21–22,
however, was only visible by CGH. FISH with a probe for the
MDR gene gave no evidence of an amplification of this
gene (not shown). Tetrasomy for part of 8q is caused by the 8;19
translocation. Loss of 9p was identified both by CGH and SKY, and so
was the loss of the short arm of the X-chromosome. CGH also revealed
additional information about chromosome 18; it indicated a loss of
18q21 to 18qter, accompanied by copy number increases at chromosome
bands 18q11 and 18q12. SKY and chromosome banding analyses revealed
two normal chromosomes 18. We therefore used FISH with a
microdissection derived painting probe for chromosome band 18q12 that
explained that the apparent discrepancy was because of a duplication of
chromosome band 18q12 (Figure 4, E and F)
. All of the aberrations detected in the cell line AsPC-1 by SKY were
also visible by CGH, therefore all chromosomal aberrations in this cell
line resulted in copy number changes. Balanced structural chromosomal
aberrations are not present in the cell line AsPC-1. In fact, comparison of the CGH and SKY data in all of the cell lines has demonstrated that chromosomal aberrations result predominantly in copy number changes: of 144 chromosomal aberrations only 6 were balanced ones. No recurring balanced chromosomal aberration was identified. Recurring breakpoints were mapped to chromosome bands 7q21 (Capan-1 and MIA-PaCA-2) and 7q31 (BxPC-3, AsPC-1, and Su.86.86).
The cell line Su.86.86 showed a particularly interesting pattern of chromosomal aberrations. In contrast to all other cell lines analyzed, 12 of 13 translocation events involved whole arm chromosomal translocations including isochromosomes. With the exception of a translocation t(7;15)(q31;?), all translocation events were unbalanced.
FISH and Northern Blot Analysis
Based on the results obtained by CGH and SKY, we performed FISH
with DNA probes for commonly involved chromosome regions on tumor
metaphase chromosomes and interphase nuclei (Figure 4)
. For example,
FISH analysis of the cell line AsPC-1 with a DNA probe for the tumor
suppressor gene p16, which maps to chromosome band 9p21,
revealed only one signal in the majority of the tumor cells (Figure 4A)
. The loss of the DCC gene, which was suggested by CGH,
was confirmed as well. Thirty percent of the cell revealed homozyguous
deletion of this loci (Figure 4E)
. This finding was confirmed by FISH
analysis of islets of 10 to 20 cells of clonally derived single cells
(data not shown). The potential involvement of the gene for the
MDR gene in the amplicon on chromosome 7q21–22 could
not be verified by FISH; four signals were observed in the majority of
metaphase cells and interphase nuclei (data not shown).
Six of nine cell lines revealed copy number increases on chromosome arm
20q by CGH. One of the genes in this amplicon that was recently shown
to be amplified in breast carcinomas is the AIB1 nuclear
receptor coactivator gene.28
To explore the
involvement of this receptor in pancreatic carcinomas we performed dual
color FISH with a DNA probe for this gene and a chromosome 20
centromere probe as an internal control. Copy number increases beyond
the one expected from CGH analyses were identified in the cell lines
Capan-1, SU.86.86, Hs 766T, Capan-2, MIA-PaCA-2, and CFPAC-1. Examples
of representative FISH experiments are displayed along with the
respective CGH ratio profiles in Figure 4, B
-D. The quantifications of
the FISH results using a probe for the AIB1 nuclear
receptor coactivator gene are summarized in Table 1
.
Based on these observations, the expression status of this recently
identified gene was evaluated by Northern blot analysis. The results
indicate an overexpression in seven cell lines (Figure 5)
, which with the exception of cell line
Capan-2, correlated with the genomic amplification status.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The frequent amplification of
the nuclear receptor coactivator gene AIB1 may indicate that
estrogen receptor mediated transcriptional activation confers a growth
advantage even to cells that are not primarily controlled by endocrine
stimuli, such as breast and ovary. Alternatively, one could hypothesize
that estrogen receptors are not the only target of AIB1. Its
interaction with CBP/p300 could also influence different signal
transduction pathways,34
which could contribute
to the rapid growth kinetics of pancreatic cancers. The frequent amplification of loci on chromosome 5p further corroborates the important involvement of genes on this chromosome arm in tumor progression. Amplifications of 5p were frequently found in advanced stage cervical cancers35 and in lung carcinomas.36,31 In one case, we detected an amplification that mapped to the terminal band of chromosome 18q, whereas the majority of this chromosome arm was underrepresented. The same chromosomal band was also involved in amplification events in colorectal carcinomas.30
The comparison of our data with data published using CGH on primary pancreatic carcinomas indicates a striking similarity of chromosomal gains and losses.11-13 Whereas the overall number of chromosomal aberrations in our series of cell lines is higher than in primary carcinomas, the distribution of chromosomal gains and losses is virtually identical. This similarity clearly validates the use of established cell lines to identify chromosomal loci that are recurrently involved in pancreatic carcinomas. The use of cell line DNA also omits one of the thorny problems in the analysis of these cancers. Pancreatic carcinomas show histologically a high amount of connective tissue and contaminating non-neoplastic cells. Therefore, the use of whole tissue blocks is problematic because low copy number changes may escape detection. This might explain the relatively few copy number changes that were identified by Solinas-Toldo and colleagues11 in which 25% of the carcinomas did not show any copy number changes. Such a low number of chromosomal copy number changes would not be expected from highly aggressive and advanced pancreatic carcinomas.
The analysis of a large series of solid tumors by CGH over the past
years9,10
has led to the identification of a
highly tumor specific pattern of chromosomal copy number changes. The
question whether balanced chromosomal translocations are present in
similar frequencies can obviously not be addressed using CGH.
Conventional chromosome banding analyses of pancreatic carcinomas are
difficult because the interpretation of extremely rearranged
chromosomes is oftentimes impossible. However, SKY allows one to
reconstruct even complex chromosomal aberrations with a high degree of
accuracy because the origin of rearranged chromosomal fragments can be
identified unambiguously. In an attempt to evaluate the relevance of
reciprocal, balanced chromosomal aberrations in pancreatic carcinomas
we have applied SKY to six of the cell lines. In general, SKY and CGH
results matched closely. In the cell line AsPC-1, for instance, whole
gains of chromosomes identified by CGH were because of trisomies for
chromosomes 2, 5, 10, 11, and 20. Structural unbalanced chromosome
translocations resulted in copy number increases, such as a marker
chromosomes containing sequences of chromosome 1q and a translocation
8;19 resulted in extra copies for chromosome 8q. None of the
chromosomal aberrations identified by SKY was balanced. Surprisingly,
two apparently normal chromosomes 18 were identified by SKY, whereas
CGH suggested the loss of the terminal bands of this chromosome along
with copy number increases on band 18q11–12. We hypothesized that an
explanation for this apparent discrepancy was because of a duplication
of chromosome band 18q12 along with a deletion of the terminal region
of the long arm. Such an aberrations would be cytogenetically
indistinguishable from a normal chromosome 18. Indeed, FISH with a
chromosome band-specific probe for 18q12 revealed a duplication of this
region (Figure 4, E and F)
. The cytogenetic pattern was further
clarified using a DNA probe for the DCC tumor suppressor
gene on chromosome band 18q21 which indicated a heterozygous loss of
DCC in 70% and homozygous loss in 30% of the cells. This
finding is in agreement with previous studies regarding deletions of
18q21 in pancreatic cancers.37
To exclude that
detection artifacts contributed to the interpretation of null signal in
30% of the cells as homozygous deletions, we repeated the FISH
analysis on clusters of interphase cells after seeding the cells in low
density on microscopic slides. Indeed, approximately 75% of the
clonally derived clusters showed one signal (with two signals for the
centromer of chromosome 18), whereas in approximately one quarter of
the clusters, no signal was present (data not shown).
Based on the extent of the deletion, it is likely that the DPC4 tumor suppressor gene is lost as well.26
We compared the number of numerical and unbalanced structural
aberrations with the number of balanced, reciprocal translocations in
the six cell lines analyzed by SKY. Among the 144 chromosomal
aberrations detected, only 6 were balanced, all of which were
different. Only chromosome bands 7q21 and 7q31 were involved twice and
three times in translocation events, respectively. Previous cytogenetic
analyses suggest the involvement of these chromosome bands in
translocation events.38
Interestingly, Achille
and colleagues39
have mapped a region of frequent
allelic loss in pancreatic carcinomas to this chromosomal band. This
could indicate that chromosome bands that are recurrently involved in
chromosomal breakpoints point to chromosomal regions that are
frequently lost and, therefore, to sites of potential tumor suppressor
genes. However, the sites of frequent LOH in pancreatic carcinomas,
such as chromosome 9p do not coincide with chromosomal translocation
events. We can therefore not conclude that the elimination of the
second allele of a tumor supressor gene is caused by chromosomal
translocations. However, the prevalence of unbalanced chromosomal
aberrations and numerical chromosomal aberrations in pancreatic cancers
indicates an aberration pattern clearly different from the one observed
in hematological malignancies, in which the reciprocal exchange of
chromatin seems to play a more important role.40
Biologically, this would mean that the translocation-induced activation
of oncogenes or the translocation-induced generation of a chimeric
protein is less important in pancreatic tumors (and perhaps in other
carcinomas as well) as compared with hematological malignancies. Dosage
variations of a specific gene or a set of specific genes via copy
number changes as a consequence of numerical and unbalanced structural
chromosome aberrations seems to be the predominant genetic mechanism.
Loss or gain of entire chromosomes, isochromosomes, insertions,
deletions, and unbalanced translocations contribute to these copy
number changes. In striking contrast to the variety of chromosomal
aberrations observed in the pancreatic carcinomas cell lines, SKY
analysis of metaphase chromosomes of Su.86.86 revealed a unique pattern
of chromosomal rearrangements. The vast majority of translocation
events involved whole chromosome arms, including the frequent formation
of isochromosomes (Table 2)
. This pattern might indicate a
mechanistically different generation of chromosomal aberrations in this
particular cell line such as a frequent transverse division of the
centromere.41,42
The molecular and cytogenetic analysis of tissue prepared from primary pancreatic carcinomas has proven difficult because of a strong host desmoplastic reaction and contaminating non-neoplastic cells.43 The identification of premalignant lesions in pancreatic carcinomas remains a diagnostic challenge and the sequence of genetic events is poorly understood compared with, eg, colorectal tumorigenesis.44 We have previously shown that early chromosomal aberrations as determined by CGH are maintained at later stages of tumor progression.19,30,32,45 The striking similarity of our data with data from previous studies using CGH on primary pancreatic tumors may suggest that the identification of early chromosomal aberrations in dysplastic lesions could be attempted using interphase cytogenetics with DNA probes for recurrently involved chromosomal regions directly on cytological preparations or tissue sections from bioptic material or surgically removed masses. The advantage of such an interphase approach would be that pertinent genetic markers could be directly correlated to the cellular phenotype.46 A defined set of DNA probes for commonly involved chromosomal loci in pancreatic carcinomas could also become an important diagnostic tool which could be applied directly to fine needle aspirations and cytological preparations from endoscopic sampling.
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Acknowledgements |
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Footnotes |
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Accepted for publication October 29, 1998.
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M. Heidenblad, E. F. P. M. Schoenmakers, T. Jonson, L. Gorunova, J. A. Veltman, A. G. van Kessel, and M. Hoglund Genome-Wide Array-Based Comparative Genomic Hybridization Reveals Multiple Amplification Targets and Novel Homozygous Deletions in Pancreatic Carcinoma Cell Lines Cancer Res., May 1, 2004; 64(9): 3052 - 3059. [Abstract] [Full Text] [PDF]
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 A. Goel and R. Janknecht Concerted Activation of ETS Protein ER81 by p160 Coactivators, the Acetyltransferase p300 and the Receptor Tyrosine Kinase HER2/Neu J. Biol. Chem., April 9, 2004; 279(15): 14909 - 14916. [Abstract] [Full Text] [PDF]
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B. M. Ghadimi, M. Grade, T. Liersch, C. Langer, A. Siemer, L. Fuzesi, and H. Becker Gain of Chromosome 8q23-24 Is a Predictive Marker for Lymph Node Positivity in Colorectal Cancer Clin. Cancer Res., May 1, 2003; 9(5): 1808 - 1814. [Abstract] [Full Text] [PDF]
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 E. P. Tamm, P. M. Silverman, C. Charnsangavej, and D. B. Evans Diagnosis, Staging, and Surveillance of Pancreatic Cancer Am. J. Roentgenol., May 1, 2003; 180(5): 1311 - 1323. [Full Text] [PDF]
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