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Oncogene (2000) 19, 1392 ± 1399
2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00
www.nature.com/onc
Combined LOH/CGH analysis proves the existence of interstitial 3p
deletions in renal cell carcinoma
Andrei Alimov1,2, Maria Kost-Alimova1,2, Jian Liu1, Chunde Li3, Ulf Bergerheim3, Stefan Imreh1,
George Klein1 and Eugene R Zabarovsky*1,2,4
1
Microbiology and Tumor Biology Center, Karolinska Institute, 171 77, Stockholm, Sweden; 2Engelhardt Institute of Molecular
Biology, Russian Academy of Sciences, 117 984, Moscow, Russia; 3Department of Urology, Karolinska Hospital, 171 76
Stockholm, Sweden; 4Center for Genomics Research, Karolinska Institute, 171 77, Stockholm, Sweden
We have recently developed an allele titration assay (ATA)
to assess the sensitivity and in¯uence of normal cell
admixture in loss of heterozygosity (LOH) studies based
on CA-repeat. The assay showed that these studies are
biased by the size-dependent di€erential sensitivity of allele
detection. Based on these data, we have set up new criteria
for evaluation of LOH. By combining these new rules with
comparative genome hybridization (CGH) we have shown
the presence of interstitial deletions in renal cell carcinoma
(RCC) biopsies and cell lines. At least three out of 11
analysed RCC cell lines and three out of 37 biopsies
contain interstitial deletions on chromosome 3. Our study
suggests the presence of several regions on human
chromosome 3 that might contribute to tumor development
by their loss: (i) 3p25-p26, around the VHL gene
(D3S1317); (ii) 3p21.3-p22 (between D3S1260 and
D3S1611); (iii) 3p21.2 (around D3S1235 and D3S1289);
(iv) 3p13-p14 (around D3S1312 and D3S1285). For the
®rst time, AP20 region (3p21.3-p22) was carefully tested
for LOH in RCC. It was found that the AP20 region is the
most frequently a€ected area. Our data also suggest that
another tumor suppressor gene is located near the VHL
gene in 3p25-p26. Oncogene (2000) 19, 1392 ± 1399.
Keywords: tumor suppressor genes; allele titration
assay; deletion mapping; human chromosome 3
Introduction
Cytogenetic data showed that deletions within the
short arm of human chromosome 3 occur frequently in
a variety of solid tumors (Kok et al., 1997). LOH in 3p
could be detected by restriction fragment length
polymorphism (RFLP) and microsatellite markers in
practically all cases of clear cell type nonpapillary renal
cell carcinoma (RCC, Presti et al., 1993; Van der Hout
et al., 1993). LOH-analysis of a number of RCC
biopsies resulted in the identi®cation of several
frequently a€ected regions (FAR) in 3p. It has been
suggested that these FARs carry one or several tumor
suppressor genes (TSG) that contribute to the genesis
of RCC when deleted (Latif et al., 1993; Van der Hout
et al., 1991; Yamakawa et al., 1991; Bergerheim et al.,
1989; Kovacs et al., 1988).
*Correspondence: ER Zabaraovsky, Microbiology and Tumor
Biology Center, Karolinska Institute, S-171 77, Stockholm, Sweden
Received 4 February 1999; revised 10 January 2000; accepted 14
January 2000
Di€erent studies de®ned di€erent FARs in the short
arm of chromosome 3, in 3p12 (Lubinski et al., 1994),
in 3p13-p14 (Yamakawa et al., 1991; Lubinski et al.,
1994), in 3p21 (Van der Hout et al., 1991; Yamakawa
et al., 1991), and in 3p25-pter (Foster et al., 1994),
respectively. A recent analysis of sporadic RCCs has
shown that 3p deletions most frequently (in 33 out of
44 analysed cases) included the 3p21 region between
D3S643 and D3S1235 (Van den Berg et al., 1996a), i.e.
they were very close to, if not overlapping the 3p21.3
region that is often deleted in lung cancer (Daly et al.,
1993; Van den Berg et al., 1996b).
Van den Berg and Buys (1997), analysing data from
several laboratories, concluded that sporadic renal
tumors indicate an association of allelic losses of the
VHL and 3p12-p14 regions with adenomas and suggest
that losses of the 3p21 region are necessary for
malignant development to clear cell nonpapillary RCC.
The most above mentioned publications describe
partial 3p losses, some of which are regarded as
interstitial deletions. In contrast, Wilhelm et al. (1995)
reported terminal deletions a€ecting the major part of
the 3p arm in all 41 analysed tumors. They used shortterm cultures that had a low level of contaminating
normal cells. They have suggested that the interstitial
deletions found in other studies were artefacts due to
the contamination of tumors with normal cells. Later,
Chudek et al. (1997) reported that in a small
percentage (six cases from a total of 104), these
`terminal' deletions retain some material from the most
telomeric 3p region.
The admixture of stroma, lymphocytes and other
normal cells in a tumor is an unavoidable parameter in
LOH studies of solid tumors. If di€erent markers are
a€ected to varying degrees by this admixture, arti®cial
LOH patterns may be obtained. This may hamper
localization of tumor suppressor genes. To test the
potential impact of this problem, we have developed an
allele titration assay (ATA) using ®ve available mousehuman microcell hybrid lines (MCH) from di€erent
patients (Liu et al., 1999). In this assay LOH analysis
was carried out on DNA samples from two MCH lines
containing di€erent alleles. DNAs were mixed in
di€erent proportions to mimic the contamination of
tumor with normal cells in biopsies. These experiments
did not reveal signi®cant di€erences in the sensitivity of
detection for di€erent markers, but there was a
systematic di€erence between the low (L) and high
(H) molecular weight alleles of the same marker. The
absence of H was detected with higher sensitivity than
the absence of L allele.
Interstitial deletions in renal cell carcinoma
A Alimov et al
This asymmetry introduces a potential source of
error. In the presence of contaminating normal cells
the same marker in the same tumor may be considered
as deleted or retained, depending on which allele (H or
L) is deleted. Random screening of 100 papers
published between 1994 and 1999 revealed that the
loss of L allele is detected only at 52% frequency of the
loss of H allele. Statistical analysis of these results
based on the z approximation (z=8.95) of a binomial
test (two-tailed P550.00001) showed that the observed di€erence in detection of allele loss is highly
signi®cant. These results suggest that about 50% of the
L allele deletions in tumor samples may go undetected.
We have developed new rules for the evaluation of
LOH experiments to avoid this bias (Liu et al., 1999;
see Materials and methods).
In the current study we used these ATA rules
together with the comparative genome hybridization
(CGH) to con®rm the presence of interstitital deletions
revealed by LOH. As others (Muleris et al., 1994), we
noticed the importance of visual analysis of chromosome images in high resolution CGH. We have shown
that visual analysis is superior in resolution compared
to CGH pro®les (Kost-Alimova et al. submitted).
Using visual analysis, the small homozygous deletions
(around 1 Mb) and hemizygous deletion (less than
7 Mb) were successfully identi®ed and localized at the
corresponding sites on chromosome 3. Thus, in our
experiments we have decided to use the combination of
visual (for detection of small deletions) and pro®le (for
detection of large deletions) CGH.
The aim of this work was to reexamine the
occurrence of interstitial deletions in 3p in cases of
RCC, combining new approaches.
Results and discussion
LOH and CGH analysis of 3p in human RCC cell lines
Eleven RCC (clear cell type) cell lines (see Materials
and methods) were studied. Twenty-one microsatellite
Table 1
and RFLP markers distributed over the whole short
arm of chromosome 3 were used for LOH analysis
(Table 1). Five lines (A498, KH39, TK164, HN51,
KRC/Y) contained only one allele for all markers
tested, indicating either terminal deletion of 3p or loss
of the whole chromosome 3. The other six cell lines
(CAKI 1, CAKI 2, TK10, UOK115, UOK117-5,
UOK123) had two alleles for at least some of the
markers studied.
Analysis of cell lines for LOH is not in¯uenced by
contaminating normal cells but the absence of normal
control cells creates a problem. A single allele can be
originally homozygous and is thus non-informative.
However, the exclusive presence of only a single allele
of several contiguous highly polymorphic markers in a
cell line is suggestive of hemizygous deletion in the
region concerned. For example, the presence of only
one allele for three neighboring markers (D3S1611,
heterozygous in 66%; D3S1298 ± 87% and D3S1260 ±
64%) by chance is about 1.661072 (see Table 1). The
probability that these three markers have one allele in
three cell lines (CAKI 1, CAKI 2 and TK10) by chance
is 461076. In many cases (e.g. TK10, CAKI 2) we
found more than eight contiguous markers represented
by a single allele only suggesting major deletion.
Seven RCC cell lines (A498, TK164, KRC/Y, CAKI
1, CAKI 2, TK10, UOK123) were selected for CGH
(Figure 1A,B). Four of them (CAKI 1, CAKI 2, TK10,
UOK123) were suggested to contain interstitial deletions by LOH. Figure 1B shows the combined results
of visual and pro®le CGH. We consider the existence
of the small interstitial deletion if it is identi®ed as a
gap or decrease of intensity in tumor hybridization
signal compared to normal at the speci®c chromosome
region (identi®ed by DAPI banding) (Figure 1B, a).
The deletion pattern was estimated, based on visual
analysis of more than 20 chromosome 3 images (Figure
1B, b,c). The CGH pro®les averaged for more than ten
chromosomes 3 have con®rmed only the existence of
large deletions, identi®ed as a shift of the ¯uorescence
intensity ratio (FIR) pro®le to the left with the
threshold level 0.2 (Figure 1B, d). The interstitial
Microsatellite and RLFP analysis of RCC cell lines
Marker
Localization
on chr.3
Heterozygosity A498 KH39 TK164 HN51 KRC/Y CAKI1
D3S1297
D3S1620
D3S1317
D3S1560
D3S1304
D3S1539
D3S1283
D3S1611
D3S1298
D3S1260
NL2-007
D3S966
D3S32
D3F15S2
D3S1235
D3S1289
D3S1312
D3S1285
D3S1217
D3S6
D3S30
3p26
3p25
3p25
3p25
3p25
3p25
3p24
3p22
3p22
3p22
3p21.3
3p21.33
3p21.3
3p21.3
3p21.2
3p21.1
3p14.2
3p14.2
3p14.2
3p14.1
3p13
82
61
68
81
79
83
70
66
87
64
77
51
80
75
72
77
1
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1393
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CAKI2
TK10
1
1 and 2
1
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2 1 and 2
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UOK115 UOK117-5 UOK123
1
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1 and 2 ± Both alleles are present; 1 ± One allele is present; nd ± not done
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Interstitial deletions in renal cell carcinoma
A Alimov et al
1394
deletions suggested by LOH were con®rmed in three
out of the four RCC lines (CAKI 1, TK10 and
UOK123, see Figure 1B). CGH analysis suggested the
occurrence of interstitial deletions in the A498 cell line,
that contained only one allele of all markers tested (see
Table 1, Figure 1B).
LOH and CGH examination of 3p in RCC biopsies
Thirty-seven tumors and normal controls were tested
for LOH using seven markers located in frequently
deleted regions (D3S1297-D3S1304-D3S1611-D3S1260NL2007-D3S1289-D3S1217). In view of the considerations presented at the end of the Introduction, 12
tumors that have lost an L allele were selected for
further LOH analysis. The results are presented in
Figure 2. In six of these cases (T1, T5, T8, T16, T20
and T51) LOH was detected for all markers tested,
implying terminal deletion of 3p or loss of the whole
short arm. In the other six cases (T2, T4, T6, T7, T9
and T18) some loci retained heterozygosity, suggesting
interstitial deletions. Five of these biopsies (T2, T4, T6,
T7 and T9) were selected for the same kind of CGH
analysis as was described in the previous section
(Figure 3). Interstitial deletions were con®rmed by
CGH in three of them (T2, T4 and T6).
Comparison of LOH and CGH analysis of 3p in RCC cell
lines and biopsies
Hemizygous interstitial deletion (HID) is de®ned in this
study as the retention of only one of two original
alleles in cases where surrounding markers are
represented with two alleles in the same tumor sample.
We did not analyse in detail which kinds of
rearrangements in RCC samples led to the HID but
cytogenetic and CGH analysis suggested that in some
cases (e.g. CAKI 1, CAKI 2, TK10) these could be true
cytogenetic interstitial deletions.
A limitation of CGH is that it can identify losses or
gains in comparatively large chromosome regions but an
advantage is that it permits the analysis of the whole
chromosome in one experiment. This allows detection of
deletion(s) and/or gain(s) not revealed by microsatellite
analysis due to the unavailability of appropriate markers
in the region concerned. The technology is limited mainly
by relative imprecision, and particularly the fuzziness of
the deletion borderlines. Its sensitivity is fairly low and
deletions smaller than 1 ± 2 Mb cannot usually be
detected. On the other hand, such deletions can be easily
detected by the appropriate microsatellite marker. A
combination of CGH and LOH is therefore mutually
complementary. Figure 4 shows a comparison between
LOH and CGH data.
In two cell lines (TK164 and KRC/Y) both LOH
and CGH showed terminal deletion of 3p. In three
cases (CAKI 2, T7 and T9) CGH data argued for
terminal deletions as well, but LOH showed retention
of some markers in T7 (D3S1317, D3S1289 and
D3S1217), in T9 (D3S1611) and in CAKI 2
(D3S1304). The retention of some markers in T7 and
T9 is probably not an artefact, because markers close
to the retained alleles showed loss of the L allele. Since
the presence of two alleles in CAKI 2 can not be
explained by admixture of normal cells, these data
indicate that a part of 3p is not deleted but this could
not be detected by CGH due to low resolution. In the
case of A498 the situation is the reverse. CGH suggests
the presence of some non-deleted regions in 3p but
LOH analysis failed to reveal it.
It should be noted here that since one of our most
important aims was to prove the existence of
interstitital deletions, we considered only such cases
when both methods con®rmed HID. For instance, it
does not mean that A498 does not contain HID. Since
the CGH results clearly reveal retention in 3p, this most
probably means that LOH analysis failed to detect
deletion due solely to the low density of microsatellite/
RFLP markers in the region concerned. Anyway, in
three RCC cell lines (UOK123, CAKI 1 and TK10) and
three biopsies (T2, T4 and T6) interstitial deletions were
suggested by both methods. In CAKI 1, TK10, T2 and
T6 the agreement between the two methods was fairly
good. It was less good in UOK123 and T4. Thus, the
presence of at least one retained region in 3p was
con®rmed in six cases by both methods.
A
b
Oncogene
Interstitial deletions in renal cell carcinoma
A Alimov et al
Most frequently affected 3p regions in RCC biopsies and
lines
Our results suggest that only a fraction of clear cell
RCC has terminal deletions. Interstitial deletions occur
in about 25 ± 50% of the cases described here. Using
the rules stated in the Materials and methods we have
excluded the L allele bias in evaluating the data. As a
result, we have detected 45 H and 44 L allele deletions.
The discrepancy between our ®ndings and those of
Wilhelm et al. (1995) and Chudek et al. (1997) may be
due to the fact that only a limited number of markers
in 3p21-pter, which di€ered from ours, were analysed
in the latter studies. Neither reverse painting nor CGH
was used to check the results. We have analysed all
biopsies and cell lines for frequently retained and
commonly deleted markers. No marker was deleted or
retained in all cases.
1395
B
Figure 1 CGH analysis of RCC cell lines. (A) CGH analysis of TK164 (a) versus DAPI banded chromosomes (b). Tumor DNA ±
green hybridization signal, normal kidney DNA ± red signal (redder ± chromosome's losses; greener ± chromosome's gain).
Chromosomes 3 are marked by arrow. (B) Summary of CGH results. (a) example of visual analysis of tumor hybridization signal
along the chromosome 3. (b) The deletion pattern estimated on the basis of more than 20 chromosomes; visual analysis (black ±
loss of region). (c) Chromosome 3 ideogram. (d) CGH tumor/normal signal ratio average pro®le (n=number of analysed
chromosomes)
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Interstitial deletions in renal cell carcinoma
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1396
Seven loci (D3S1539, D3S1260, NL2-007, D3S966,
D3S1235, D3S1312 and D3S1285) showed LOH or
Figure 2 Microsatellite analysis of RCC biopsies. RCC cases
were divided into two subgroups: containing terminal deletions
and retaining some markers in 3p
were non-informative in all 12 biopsies (Figure 2).
However, only D3S1539 showed LOH in all six cases
that had interstitital deletions (T2, T4, T6, T7, T9
and T18), while D3S1260 and D3S1235 showed LOH
in only two of the six cases. In all cases with
interstitial deletions, two loci (NL2-007 and D3S966)
showed neither detectable deletions nor detectable
retentions. Only D3S1539 can thus be considered as a
commonly eliminated marker on the basis of LOH
analysis on biopsies. The other six loci can be
considered candidate regions. The same marker
(D3S1539) was, however, represented with two alleles
in the RCC cell line CAKI 1. This speaks against
hemizygous deletion.
In contrast, D3S1260 and D3S1611 were presented
only with a single allele in all lines. Other markers
(D3S1620, D3S1298, D3S6 and D3S30) were also
represented by only one allele, but they were not
included in the biopsy analysis due to the limited
amount of material available. The right part of Figure
4 indicates for comparison 3p regions that have been
suggested to contain TSGs (Latif et al., 1993; Murata
et al., 1994; Roche et al., 1996; Szeles et al., 1997; Wei
et al., 1996; Ohta et al., 1996).
Figure 3 CGH analysis of RCC biopsies. (a) Example of visual analysis of tumor hybridization signal along the chromosome 3.
(b) The deletion pattern estimated on the basis of more than 20 chromosomes; visual analysis (black ± loss of region). (c)
Chromosome 3 ideogram. (d) CGH tumor/normal signal ratio average pro®le (n=number of analysed chromosomes)
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Interstitial deletions in renal cell carcinoma
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1397
Figure 4 Combined LOH and CGH analysis of RCC cell lines and tumor biopsies. Bars on the right side show location of VHL
region, two regions in 3p21 homozygously deleted in SCLC, common eliminated region in SCID mice (CER and CER1) and FRA3
region
It may be concluded from the results that 3p carries
at least four FARs: (1) 3p25-26, around the VHL
gene (Latif et al., 1993). It is interesting that D3S1317
assigned to the gene was retained in three out of four
informative tumor biopsies with interstitial deletions
(Figure 2). In six RCC lines with interstitial deletions
D3S1317 was represented with 2 alleles in two cases
(Table 1). This raised the question whether the genetic
map of this region was suciently precise or if
another TSG is present in this region. The latter
possibility was supported by the ®nding of a
homozygous deletion near D3S1297 in nasopharyngeal
carcinoma (Hu et al., 1996). This deletion is at least
7 cM but does not include the VHL gene. (2) 3p21.322 (AP20 region, Kashuba et al., 1995; between
D3S1611 and D3S1260). This region is also involved
in the common eliminated region (CER) in SCID
mouse tumors derived from human chromosome 3containing hybrid cell lines (Imreh et al., 1994;
Kholodnyuk et al., 1996). The more restricted CER1
described in later studies is centromeric to AP20,
however (Szeles et al., 1997). The present work is not
informative with regard to the frequency of LOH in
the CER1 region. D3S32 which is located in the
CER1 region is a low frequency polymorphic marker
and was not used for the analysis of the RCC biopsies
because of the limited amount of available DNA.
Homozygous deletions in the AP20 region have been
reported in ®ve SCLC cell lines and three tumor
biopsies (Murata et al., 1994; Roche et al., 1996). The
present study identi®es this region as the most
consistently deleted region in RCC (Table 1, Figures
2 and 4). (3) 3p21.2 (around D3S1235 and D3S1289).
Several groups reported homozygous deletions in this
region for SCLC (Daly et al., 1993; Wei et al., 1996).
The above mentioned CER1 (Szeles et al., 1997) is
located close but more telomeric to this region. (4)
3p13-14 (around markers D3S1312 and D3S1285).
This region contains homozygous deletions in the
SCLC cell line U2020 and is frequently a€ected by
LOH in RCC (Rabbitts et al., 1990; Van den Berg et
al., 1996a; Ohta et al., 1996).
To sum up, using new stringent criteria for
evaluation of LOH, we have proved that RCC
samples contain interstitial deletions in the 3p21
region. By analysing more samples and by using more
markers, this opens a way to narrow down the region
where TSGs may be localized. In general, our data are
dicult to accurately compare with previous studies
like Yamakawa et al., 1991 or Van den Berg et al.,
1996a since di€erent markers were used in these
studies but we have con®rmed roughly that FARs are
located in 3p13-p14, 3p21 and 3p25-p26 regions. It is
important, that we have found that the most
frequently a€ected region is located between
D3S1260 and D3S1611 (the AP20 region). This region
has not been intensively characterized in the previous
studies. The distance between these markers is less
than 2.5 Mb (Kashuba et al., 1995). We have
generated new probes in the region and are continuing
®ne deletion mapping of the area. This region is also
frequently deleted in SCLC (see Kok et al., 1997) and
can thus represent the area where a general TSG
involved in di€erent cancers is located. The most
straightforward approach to localize TSG will be
®nding and ®ne mapping of homozygous deletions. In
this respect it is worthwhile to mention that our
preliminary data using quantitative real-time PCR
using ABI PRISM 7700 Sequence Detection System
revealed in AP20 region 21% (six of 28 cases) of
homozygous deletions in RCC biopsies. These homozygous deletions allowed us to narrow down the
suspected region to 1.5 Mb. It is interesting that using
the same quantitative real-time PCR approach we
found that 7% of RCC samples (two of 28 cases)
have homozygous deletions in the GNAI2-3PK region
(Liu et al. manuscript in preparation). Exactly in the
same region homozygous deletions were detected in
SCLC and breast cancer (Wei et al., 1996; Sekido et
al., 1998). This ®nding narrows down the third FAR
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Interstitial deletions in renal cell carcinoma
A Alimov et al
1398
to less than 200 kb and proves that this FAR also
contains a general TSG. The results of this study also
suggest that in addition to VHL another TSG can be
located in 3p25-p26.
Materials and methods
DNA samples and Southern blotting
Paired normal and tumor tissue samples were obtained
immediately after resection and stored at 7808C before
DNA extraction. Each tumor piece was examined histopathologically. Only clear cell type tumors were included.
DNAs for ATA were obtained from mouse-human microcell
hybrid cell lines (Wang et al., 1994). RCC lines used in this
study: A498, UOK-115, UOK-117-5, UOK-123 (Gnarra et
al., 1994), CAKI 1 and CAKI 2 (ATCC catalog No. HTB46
and No. HTB47), HN51 (Bergerheim et al., 1996), KH39
(Tomita et al., 1996), KRC/Y (Yano et al., 1988), TK10 and
TK164 (Bear et al., 1987).
High molecular weight DNA was isolated from tumor and
normal cells by the phenol extraction method, described by
Sambrook et al. (1989). Southern blotting analysis of
D3F15S2, D3S6, D3S32 and D3S30 RFLP markers was
done as described previously (Kovacs et al., 1988; Bergerheim
et al., 1989). Genomic DNA digested with HindIII was
examined for LOH of D3F15S2, with MspI for LOH of
D3S6 and D3S30, and DNA digestion with RsaI was used for
testing of polymorphism of D3S32. We used these RFLP
markers in our study because they have been well de®ned in
previous studies (Kovacs et al., 1988; Bergerheim et al.,
1989).
Microsatellite markers and LOH analysis
The most likely order of the markers used in this study is
shown in Table 1. Most of them are listed according to the
Genethon human genetic linkage map (Dib et al., 1996). The
following order has been established for the 3p21 markers,
proceeding from the distal to the proximal markers:
D3S1298 - D3S1260-NL2-007 -D3S966-D3S32-D3F15S2-D3S1235-D3S1289 (Van den Berg et al., 1995 and unpublished
data). The order is based on at least two independent events,
e.g. multiple cross-overs in families from the Centre d'Etude
du Polymorphism Humane (CEPH) DNA collection and
results of FISH analysis, or a combination of FISH-mapping
and deletion-hybrid mapping (Van den Berg et al., 1995).
Marker NL2-007 is not integrated into the CEPH microsatellite map (Dib et al., 1996). Our own FISH mapping and
positioning data showed that NL2-007 is located in 3p21.3
between D3S1260 and D3S32 (data not shown). The
estimated position of these three markers is not in contradiction to our ®nal results.
D3S1317 has been mapped to the VHL gene region located
between D3S587 and D3S18 (Latif et al., 1993). For
chromosome region 3p25-26 an order established using
radiation hybrids and YAC contig map of human chromo-
some 3 (Gemmill et al., 1995) was used. D3S1539 has been
mapped to 3pter-24.2 (http://cedar.genetics.soton.ac.uk/pub/
chrom3/p8).
PCR was performed in a volume of 25 ml according to
standard protocols. To avoid bias in detection of LOH with
H and L alleles we used the following rules (Liu et al., 1999).
(1) More weight was given to LOH found with the L than
with the H allele (rule of allele L). For instance, markers that
appear to be retained were considered as such only in cases
where the suspected allele is close to a locus showing LOH
with respect to allele L. Otherwise, if no independent
supportive data were available we considered such markers
as non-informative. For example, analysis of the ®lms for
contiguous informative markers A-B-C revealed, that H allele
is deleted in A or/and C, and B seemed to be undeleted. In
this case we consider B as uninformative. If L allele would be
deleted in A and C then we would consider B as retained. (2)
The number of deleted H and L alleles was established for
each of the normal/tumor pairs. These numbers should be
(and were) about the same. (3) Comparative genome
hybridization was used in parallel with CA-repeat analysis
to con®rm interstitial deletions.
Comparative genome hybridization (CGH)
CGH was performed according to Kallioniemi et al. (1994)
with some modi®cations. A Bionick kit (Gibco BRL,
Bethesda, MD, USA) was used to label tumor and normal
kidney DNA with digoxygenin-11-dUTP and biotin-14-dATP
respectively. Labeled DNAs were hybridized to normal
human metaphases, prepared from PHA stimulated lymphocytes. Five hundred ngs of each labeled DNA and 20 mg of
human Cot1 DNA were used in one hybridization experiment
(total volume 5 ml). Hybridization was performed for 48 ±
72 h at 378C (26SSC, 50% formamide, 1% Tween 20, 10%
SDS). Slides were washed three times in 26SSC, 50%
formamide for 10 min at 458C. Hybridization signals were
detected using avidin-rhodamine for biotin and anti-digFITC system for digoxygenin labeled DNA. A ¯uorescent
microscope (LEITZ-DMRB, Leica, Heidelberg, Germany)
equipped with Photometrics PXL-KAF-1400 CCD camera
was used for capturing of metaphases images. Image analysis
was performed using PowerGene 710 Karyotyping, FISH and
CGH system (PSI Scienti®c Systems) and Adobe Photoshop.
Fluorescence intensity rates for tumor and normal DNA
hybridizing signals were calculated and CGH pro®les were
produced using CGH PSI software.
Acknowledgments
This work was supported by research grants from Swedish
Cancer Society, Karolinska Institute and AÊke Wiberg
foundation. M Kost-Alimova and J Liu were recipients
of fellowships from the Concern Foundation in Los
Angeles and the Cancer Research Institute in New York.
A Alimov was a recipient of a fellowship from Wenner
Gren foundation.
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