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(Received for publication, June 21, 1995; and in revised form, August 9,
1995) From the
In human cervical carcinomas papillomavirus DNA is frequently
integrated in the cell genome. We have cloned the integration site of
human papillomavirus-18 DNA in human chromosome region 12q13-15
present in the SW756 cervical carcinoma cell line. Viral DNA is broken
from nucleotides 2643 to 3418 in the E1 and E2 open reading frames,
resulting in a deletion of 775 bases of viral DNA. Cloning and sequence
analysis of the rearranged and germline alleles shows that there is no
homology between the target cellular and viral DNA, suggesting it is a
nonhomologous recombination. The target cellular region is called
papillomavirus associated locus 2 (PAL2). The 5`- and
3`-flanking probes derived from the hybrid viral-cellular clone detect
completely different germline restriction fragments in DNA from cells
with normal chromosome 12. There is no overlap between the restriction
maps of the target germline clones obtained with 5`- and 3`-flanking
probes. Probes from these germline clones beyond the breakpoint
position do not detect any DNA rearrangement in SW756 cells DNA. These
data prove that there is a deletion of cellular DNA as consequence of
the integration, with an estimated minimum size of 14 kilobases. Both
cellular flanking probes are outside the amplicon of this chromosome
region identified in the OSA and RMS13 sarcoma cell lines, comprising SAS-CHOP-CDK4-MDM2 genes and where translocation breakpoints
are located in liposarcomas. The integration at 12q13-15 might
have been selected by its contribution to the tumor phenotype.
Recurrent chromosomal alterations are a hallmark of many types
of tumors, and their cloning and characterization in lymphoid tumors
has been very instrumental in the identification of oncogenic
loci(1, 2, 3) . In solid tumors, the
characterization of chromosome aberrations lags further behind.
However, specific translocations associated to specific types of solid
tumors are often identified (4, 5, 6, 7) and are also likely to
be related to some aspect of tumor phenotype(8) . These
chromosomal alterations are usually manifested as translocations,
amplifications, deletions, or losses of heterozygosity. In
virus-associated tumors, such as cervical carcinoma linked to
papillomaviruses and hepatocellular carcinoma linked to hepatitis B
virus, the integration of viral DNA represents an additional type of
DNA damage. This viral DNA integration can be considered an alternative
to translocations but with similar biological
consequences(9, 10, 11) . Furthermore the
viral DNA in these integration sites will provide a tag for their
identification, cloning, and characterization. Human
papillomaviruses have been associated with tumors of genital and skin
origin(12) . There are more than 70 viral DNA types, but two of
them, HPV16 ( Integration of HPV DNA does not form
part of the viral life cycle. The integration mechanism appears to be
the result of illegitimate recombination near DNase I hypersensitive
sites(17, 18, 19) . The integrated viral DNA
confers growth advantage to the cell due to viral gene
deregulation(20) . Cytogenetic analysis of cervical carcinomas (21) shows that integrations are clonal and that the viral DNA
is located in some chromosomal regions that have already been
associated with other tumor phenotypes(9, 10) . The
observation of clonal population is likely to be the result of a strong
selection for the biological consequences of some integration events,
thus their detection in regions linked to the tumor
phenotype(9, 10) . These findings are very similar to
those already described for the role of nontransforming retroviruses in
oncogenesis(21) , where the most characteristic observation is
the detection of common sites of provirus insertion, an event known as
insertional mutagenesis, which are linked to a tumor
phenotype(22) . Two human chromosomal regions, 8q24 and
12q13-15, are common integration regions for papillomavirus DNA
in genital cancers. Both HPV-16 and HPV-18 DNA have been found in the
two
regions(21, 22, 23, 24, 25, 26, 27) .
The cervical carcinoma SW756 cell line has HPV type 18 DNA integrated
in 12q13-15(26, 28) , and this locus, called papillomavirus-associated locus 2 (PAL2), is the
object of the present report. Human chromosomal region
12q13-15 undergoes several types of genetic alterations.
Translocations and amplifications have been found in
melanoma(7) , lipoma(29) ,
liposarcomas(6, 30, 31, 32) ,
gliomas(33) ,
leiomyosarcomas(29, 34, 35, 36) ,
pleomorphic adenomas(29) , and lymphomas (37, 38) . Integration of hepatitis B virus DNA in
this region has been reported in one case of hepatocellular
carcinoma(39) , and integration of HPV-16 DNA has been found in
another cervical carcinoma cell line, SK-v(27, 28) . The detection of such a variety of gross 12q13-15 chromosomal
alterations suggests that this is a region likely to be rich in genes
related to the tumor phenotype(40) , most of them related to
cell cycle control(41) . Among them there are membrane
receptors and proteins like WNT1 and two members of the
transmembrane four superfamily of proteins, SAS and ME491, which are implicated in the control of cell
proliferation(42) ; the antigen ME491/CD63 has been
correlated with the prognosis of melanoma(43) . There are
several genes coding for proteins implicated in cell cycle regulation
like CDK2 and CDK4(44) and for oncogenes
like MDM2, which interacts with p53(45) . Genes coding
for signal transduction molecules like RAP1A and RAB5b and for transcription factors like GLI1, CHOP,
and ATF1. However, in most cases these genes have not been
linked to any specific tumor with the exception of translocations
affecting CHOP in liposarcomas resulting in a gene
fusion(6, 32, 47) . Furthermore the
12q13-15 region is also likely to contain a tumor suppressor gene
based on data from the detection of loss of heterozygosity in
seminomas(48, 49) , gliomas(50) , and prostate
cancer, where the presence or absence of a fragment derived from region
12q13-15 determined its oncogenic potential(51) . We
report the cloning and characterization of PAL2, the
integration site of HPV-18 DNA in chromosome region 12q13-15
present in the cervical carcinoma cell line SW756 (26) as well
as of the corresponding germline target sequences. This viral-cellular
DNA recombination is nonhomologous and causes the deletion of cellular
DNA and a small region of viral DNA. The integration of HPV18 is not
located in the area of DNA amplification described in 12q13-15 in
some sarcoma cell lines. This is a genomic region likely to be
implicated in the tumor phenotype.
Hybridization of DNA to different
probes was carried out in 5
To obtain
human germline clones, we used a commercially available human genomic
library made by partial Sau3A digestion of DNA from a
3-year-old Caucasian male and addition of EcoRI linkers,
followed by cloning in
Figure 1:
Structure of the phage clone containing
the integrated human papillomavirus type 18 DNA in the genome of SW756
cells surrounded by cellular sequences. The
Figure 2:
Detection of different genomic cellular
sequences (PAL2) with flanking probes derived from the two
ends of the integrated viral DNA. Southern blots of cellular DNA from
the Ramos cell line with normal chromosome 12 digested with several
restriction enzymes and hybridized to cellular probe pS3 derived
flanking the 5` breakpoint (A) or to cellular probe p500
flanking the 3` breakpoint (B).
Figure 3:
Structure of germline clones corresponding
to flanks of the integrated viral DNA. A, germinal clone,
From the 3`-end, two clones were isolated with probe
p500, The comparison of restriction maps
from clones
Figure 4:
Deletion of internal genomic probes in
SW756 cells DNA. 10 µg of DNA from OSA and RMS13 cell lines as
reference for not rearranged alleles and from SW756 cells were digested
to completion with EcoRI and fractionated in an 0.8% agarose
gel. The DNA was transferred to Hybond-N
Figure 5:
Relationship of integration site to two
amplicons of 12q13 region. Both ends of the integrated viral DNA are
outside the amplicon of 12q13. EcoRI-digested DNA from five
cell lines, Colo320, Daudi, OSA, RMS13, and SW756 was hybridized to
several probes from genes located in 12q13, some of which, SAS and CDK4, are included in both amplicons, and one, MDM2, is amplified only in the OSA cell line. From the PAL2A locus, probe S3 was used. The probe used in each case is
indicated to the right side of the corresponding blot. In some
blots, the detection of several bands is due to the use of cDNA probes
that contain several exons.
Figure 6:
Nucleotide sequence of germline target
sequence at both ends of integration site of HPV18 DNA in SW756 cells. A, nucleotide sequence of the PAL2A germline spanning
the 5` cellular-viral recombination breakpoint in the hybrid clone
described in Fig. 1. B, nucleotide sequence of the
germline PAL2B region spanning the viral-cellular
recombination breakpoint corresponding to the 3` in the integration
site described in Fig. 1. The location of each of the
breakpoints is indicated by an arrow. The deleted cellular DNA
that is replaced by viral DNA in the recombinant clone is indicated in boldface characters. The accession numbers are X88931 and
X88932 for PAL2A and PAL2B respectively.
The germline sequence spanning the
3` breakpoint was obtained from clone p500 and is shown in Fig. 6B. The sequence comprising the 3` breakpoint was
determined from the viral HincII site (nucleotides 2643) to
the most proximal cellular HincII site in the hybrid clone (Fig. 1). The breakpoint of viral DNA is located at nucleotide
2643 within the E1 ORF. There was no significant homology between
the viral DNA and the sequence of the target alleles, PAL2A and PAL2B. A search did not reveal any homology
of the target sequences to any known sequence in the GenBank data base. The retained viral DNA has several point mutations in the region
proximal to the breakpoints with respect to the reference HPV18 DNA. In
the 5` breakpoint 190 viral nucleotides were sequenced, and we detected
viral mutations at positions 3462 (A The detection of integrated HPV DNA in the host genome is a
common characteristic in cases of advanced cervical carcinoma. This
observation can therefore be considered a recurrent type of genetic
damage that could be selected for and thus contribute to the tumor
phenotype. This integration has two different aspects. First, it can be
looked at from the point of view of the viral DNA. There might be a
selection for or against certain viral genes that will determine which
viral genes are retained and the position where the recombination will
occur in the viral genome. Thus, it is important to retain the genes
that would favor cell proliferation, like E6 and E7, and delete or
damage its repressor, E2, and that is what actually is observed in
SW756 (Fig. 1) and in other cervical carcinoma cell
lines(17, 28, 53) . Most of the work on the
role of HPV integration has been focused from this
perspective(11, 28, 56) . Furthermore, the
integrated viral genes might have an altered regulation as a
consequence of the cellular flanking sequences. Thus in SW756 cells,
the regulation by glucocorticoids of E6 and E7 gene expression is
affected by the integration site(57) . The expression of the
viral oncogenes might favor additional chromosome instability that
could be the cause of other genetic defects not directly linked to the
integration itself (58) . Secondly, the integration chromosomal
location might also be selected for the effects on the host genome
either by gene damage or by a cis effect of viral sequences on
nearby cellular genes that might lead to a deregulated expression.
Therefore, the cellular genetic alteration resulting from viral DNA
integration is a potentially oncogenic event. In most of the cases,
where the chromosomal location has been mapped, like 8q24 or
12q13-15(9) , it is a genomic region already associated
with the tumor phenotype suggesting a possible role that has not yet
been demonstrated in cancer cases associated to DNA tumor viruses. The
limited evidence available is consistent with this
view(9, 10) , although that is a common observation in
oncogenesis by nontransforming retroviruses(22) . In this
report, we have shown in the SW756 cervical carcinoma cell line that
the viral integration has resulted in a deletion of viral and cellular
DNA, at chromosome region 12q13-14. Three types of evidence
support this interpretation. There are completely different restriction
patterns in germline PAL2A and PAL2B DNA hybridized
to cellular probes derived from both flanks on the integrated viral DNA (Fig. 2); there is no overlap between PAL2A and PAL2B genomic clones derived from both ends (Fig. 3),
and there is lack of detection of rearranged allele by cellular probes
from the cellular region replaced by viral DNA (Fig. 4). This
integration is a likely consequence of nonhomologous recombination
because of the lack of any significant homology between viral and
cellular sequences. The location of the break within the viral genome
and, probably, the chromosomal location are likely to be the result of
strong biological selection for them. It is interesting to note the
large differences in deleted DNA, 0.775 kb for viral DNA and more than
14 kb of cellular DNA. Although the deleted cellular DNA is likely to
be much larger based on cytogenetic data, partial loss of a band occurs
in the 12q13-15 region(25) . The discrepancy between the
sizes of cellular and viral deleted DNA might be explained as the
result of illegitimate recombination between viral DNA and a loop of
chromosomal DNA. The cellular DNA, due to its highly organized
superstructure, might have in close proximity two distant regions; in
this DNA, the viral DNA recombines by forming a bridge between the two
distal ends of the loop and results in deletion of the intervening
cellular DNA. The DNA sequence at the viral-cellular junctions (Fig. 6) shows no significant homology, suggesting that it is a
nonhomologous recombination (59) in agreement with other cases
of recombination between HPV and cellular DNA(60) . The lack
of any significant homology between viral DNA and the two target, PAL2A and PAL2B, sequences at the integration site
and the subsequent observation of its localization in chromosome
regions associated to the tumor phenotype, like the case of PAL1 in 8q24 (55) strongly suggest that there is a strong
biological selection for some chromosomal locations. This selection is
more likely to be related to the properties of this chromosomal region
than to the retention of some viral genes, like E6 and E7. The
expression of E6 and E7 could be achieved from almost any position
where they might integrate in so far as they are structurally intact.
If that was the case, there would be no reason to expect the
integrations to be located in oncogenic regions. The location of the
integration in the SW756 cell line maps to a genomic region implicated
in several oncologic phenotypes. The type of alteration includes both
translocations and amplifications. Most recurrent chromosomal
alterations contribute to the tumor phenotype by altering a gene. This
gene modification can be very heterogeneous, ranging from its
overexpression to changes in regulation, mutations, or deletions. The
type of modification depends on the biological function of the gene.
Thus the so called oncogenes are deregulated or overexpressed, or their
products are altered while tumor suppressor genes undergo inactivating
alterations, like mutations or deletions. Deletion of cellular
sequences is a characteristic observation in genetic alterations of
tumor suppressor genes. There are three lines of evidence pointing to
the existence of a tumor suppressor gene in the 12q13-15 region.
The first is loss of heterozygosity in 40% of male germ cell tumors (48) and in glioblastomas(50) . Furthermore, in a
prostate cancer cell line containing a deletion in this region, the
malignant phenotype could be reversed after introduction of a normal
chromosome 12, and the malignant phenotype reappeared following the
loss of the introduced chromosome(51) . The deletion detected
by HPV-18 integration might be an indicator of the location of this
putative suppressor gene, which is also known to be outside the OSA and
RMS13 amplicons in the 12q13-15(49) . This human
region is syntenic with regions of mouse chromosomes 10 and
15(40) . The amplified genes in OSA and RMS13 cell line are all
located in the region that is syntenic with mouse chromosome
10(40) . The data on the amplicons in different cell lines
combined with data from translocation breakpoints in different types of
tumors suggests the following gene order in this region: COL2A1-WNT1-ATF1-GPDH-SP1-RAR HPV
integration was postulated to be a late event in the process of
oncogenesis by human papillomaviruses(61) . During the early
steps of the oncogenic process by HPV, the extrachromosomal location of
the E6 and E7 viral genes is enough to generate an expanding
population, because of cell cycle deregulation, where additional
genetic alterations might occur. In cervical carcinoma, there are
frequent rearrangements of region 11q13 not linked to HPV(5) .
Some of these additional genetic alterations might be the consequence
of viral DNA integration. If the integration happens in an oncogenic
region, it is likely to be selected for its contribution to some
aspects of the tumor phenotype, which are unlikely to be related to the
initial steps of transformation. In this case, the viral E6 and E7
contribute to expand the cells carrying important oncogenic
mutation(5) . The detection of PAL1 in 8q24 (55) and PAL2 in 12q13-15 is consistent with
this view. But their role in the tumor phenotype will only be known
when the implicated genes are identified.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s) X88931 [GenBank]and X88932[GenBank].
Volume 270,
Number 41,
Issue of October 13, 1995 pp. 24321-24326
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
15 by Integration of Human
Papillomavirus DNA in a Cervical Carcinoma Cell Line (*)
)and HPV18, are linked to over 70% of the
cervical cancers(12) . The mechanism by which these viruses are
implicated in oncogenesis is very complex and has two main components.
First of all, the oncogenic early viral genes E7 and E6 are capable of
interacting with the products of RB(13) and P53(14, 15) genes respectively, thus achieving a
functional inactivation of two cellular tumor suppressor genes.
However, E6 and E7 viral genes can also be expressed from viral DNA in
an episomal form (16) . Second, most malignant tumors of the
genital tract have viral DNA integrated in the cellular genome causing
a genomic rearrangement(11, 12) . Nevertheless, these
integrations are usually studied from the consequences on viral genes (16) rather than on the cellular genome; this latter aspect is
the object of the present work.
Cell Culture
The SW756 cell line was obtained
from Dr. J. A. DiPaolo, National Cancer Institute, Bethesda, MD. The
cervical carcinoma SW756, the colon carcinoma Colo320, and the
rabdomyosarcoma RMS13 cell lines were grown in Dulbecco's
modified Eagle's medium with 10% fetal calf serum. The MOLT4
T-cell lymphoma, the OSA osteosarcoma (from O. Mykeblost, Oslo,
Norway), and the Burkitt lymphoma cell lines (from G. Klein, Stockholm)
were grown in RPMI 1640 supplemented with 10% fetal calf serum. All the
other cell lines were obtained from ATCC (Rockville, MD).Probes
HPV18 genomic clone was provided by H. zur
Hausen and E. M. De Villiers (Heidelberg, Germany). CHOP probe, plasmid
WT10, was obtained from P. Aman (Lund University, Sweden). CDK2 and
CDK4 probes, plasmids pCDK2 and pCDK4, were from D. Beach (Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY). MDM2 probe, plasmid
MDM2-FL5, was from B. Vogelstein (John Hopkins University, Baltimore,
MD). SAS probe, plasmid pSJP2, was from P. Meltzer (National Center for
Human Genome Research, National Institutes of Health, Bethesda, MD).
SP1 probe, plasmid pPacSP1, was from R. Tjian (University of
California, Berkeley, CA). RAR probe was from P. Chambon (INSERM
U184, Faculté de Medicine, Strasbourg, France).
WNT1 probe was obtained from the ATCC (Rockville, MD). The DNA insert
to be used as probe was isolated by restriction digestion and purified
with a Geneclean II kit (Bio 101, Inc., La Jolla, CA). The probes were
labeled using a commercial random priming kit from Amersham Corp.
DNA and RNA Preparation and Analysis
DNA was
prepared following standard procedures based on cell lysis and
treatment with proteinase K and ribonuclease A(52) . Afterward,
the DNA was cleaned by repeated phenol and phenol-chloroform
extractions(53) . Digestions with restriction enzymes were
performed following manufacturer's instructions (Promega or
Boehringer Manheim)(52, 53) . The digested DNA was
fractionated in an 0.8% agarose gel in Tris/sodium acetate/EDTA buffer (52) with phage DNA digested with HindIII or BstE2 as size markers. The DNA was transferred to
Hybond-N
nylon membranes (Amersham Corp.) and fixed by
alkali treatment. RNA was prepared by cell treatment like guanidinum
thyocianate followed by centrifugation in cesium chloride according to
a standard protocol(54) .
SSC (SSC is 0.15 M NaCl,
0.015 M sodium citrate), 0.1% SDS, at 65 °C for 18 h. The
filters were washed in 2 SSC, 0.1% SDS at 62 °C for 15 min
followed by three washes in 0.2 SSC, 0.1% SDS at 62 °C for
20 min. Northern blots were hybridized under the same conditions,
except that the high stringency washes were done at 55
°C(55) . Quantitation of the blots were performed using a
densitometer from Bio-Rad.Genomic Cloning
To clone the rearranged allele
from SW756 cells with integrated HPV18 DNA, 75 µg of cellular DNA
were digested to completion with EcoRI and fractionated in a
sucrose gradient by centrifugation(52) . DNA fractions
comprising the 10-15-kilobase pair range were pooled and ligated
to EcoRI-digested DASH (Stratagene, La Jolla, CA) arms
phage vector purified by sucrose gradient centrifugation.
FIXII vector (Stratagene, La Jolla, CA).
This library was screened with cellular DNA sequences flanking the
integrated viral DNA. Positive phage clones were partially digested
with different restriction enzymes and mapped by hybridization to
end-labeled T7 and T3 primers. DNA fragments free of repetitive
sequences were subcloned in pBluescript SKII(-) plasmid and used
for different studies as probes.
DNA Sequencing
The different clones in pBluescript
SKII(-) vectors were sequenced by the chain termination method using
dideoxynucleotides with T7 DNA polymerase (Sequenase) from Amersham
Corp. The sequences have the following GenBank accession numbers:
X88931 for PAL2A and X88932 for PAL2B.
Cloning of HPV-18 DNA Integrated in the Genome of SW756
Cells
The SW756 cell line is derived from a cervical carcinoma
and contains HPV18 DNA integrated in the chromosome region
12q13-15 (26, 28) . The integrated HPV18 viral
DNA can be identified as bands hybridizing to viral DNA generated by
enzymes that do not cut within the viral genome, like SalI, XhoI, EcoRI, and HindIII. By restriction
enzyme analysis and hybridization to subgenomic viral probes, we first
determined that viral DNA breakpoint must be located between the HincII sites at positions 2472 and 3605 (not shown). To clone
the integrated viral DNA, we made a library of EcoRI-digested
DNA from SW756 cells screened with a complete HPV18 DNA probe. We
obtained a 12.5-kb clone, 12QHP, that was mapped with several
restriction enzymes (Fig. 1) and hybridized to subgenomic viral
probes, confirming that the breakpoint of the circular and
extrachromosomal viral DNA before integration is between the HincII restriction sites at the end of the E1 and beginning of
E2 ORFs. This clone lacks the internal EcoRI site at
nucleotide 2440 within the E1 ORF. The phage clone,
12QHP, has
cellular DNA in both flanks. From the
12QHP clone, we prepared
three cellular flanking probes. Two probes were from the 5`-flank; the
pS3 probe is a 1.6-kb EcoRI-SpeI fragment, and pHH
probe is a 0.7-kb HindIII-HincII fragment with some
viral nucleotides near the HincII site. From the 3`-end we
prepared probe p500, which is an 0.5-kb HincII-HincII
fragment (Fig. 1) also with some viral nucleotides at one end.
12QHP clone was
isolated from a genomic library of SW756 cells and screened with HPV-18
DNA. The thickline represents the region where DNA
of viral origin is located within the clone. The arrow indicates the start and direction of transcription of retain viral
early genes. Under the map, the locations of several probes used in
this work are indicated, pS3, pHH from the 5`-flank,
and p500 from the 3`-flank. R, EcoRI; Sp, SpeI; X, XbaI; H, HindIII; Hc, HincII; B, BamHI; K, KpnI; A, ApaI.
The restriction sites were determined by partial digestion of the clone
followed by hybridization to labeled T3 and T7 oligonucleotide primers.
The clone was hybridized to viral subgenomic probes and to total human
DNA to detect fragments with repetitive sequences. The retained viral
open reading frames are indicated as well as the relative position of
viral genes. The breakpoints affect the E2 and E1 ORFs. The lcr is the viral long control region where the origin of replication
and several transcription control sequences are
located.
Flanking Cellular Probes Detect Different Genomic Regions
Suggesting a Deletion at the Integration Site
As a consequence
of the integration, there might be complex cellular DNA rearrangements
or deletions. To ascertain if both flanking probes detected the same
cellular genomic region, we determined the germline restriction pattern
with both probes in DNA from several cell lines. We used probe pS3 from
the 5`-flank and probe p500 from the 3`-flank. To develop this pattern,
we used a cell line, Ramos, that does not have any alteration on
chromosome 12. The pS3 (5`) probe detects the following bands, EcoRI (3.2 kb), HindIII (9.6 kb), XbaI (5.8
kb), SacI (15 kb), ApaI (24 kb), BamHI (19
kb), KpnI (15 kb), and SpeI (3 kb). The probe derived
from the 3`-flank, p500, detects the following fragments: EcoRI (4.5 kb), HindIII (2.8 kb), XbaI (6.9
kb), SacI (6.7 kb), ApaI (1.2 kb), BamHI (14
kb), KpnI (35 kb), and SpeI (30 kb). Probes pS3 (Fig. 2A) and p500 (Fig. 2B) detected a
completely different genomic region in all restriction digestions
performed. This observation suggests that the region detected by each
probe is not overlapping and therefore there is a deletion of cellular
DNA as a consequence of the integration of viral DNA in SW756 cells.
The size of the deletion is likely to be at least larger than 14 kb
based on the size of the observed restriction fragments.
Cloning the Target Germline Alleles
The genomic
target region corresponding to both flanks of the integration site were
obtained by screening a genomic library made from a healthy individual.
One clone, h12/8HH, was obtained with probe S3 from the 5`-flank.
The clone is 18 kilobase pairs long and spans the 5` breakpoint, of
which approximately 6 kb were replaced as result of HPV 18 integration (Fig. 3A). This part of the target region is called PAL2A.
h12/8HH, corresponding to the 5` breakpoint and isolated with
probe pS3. B, germinal clone,
h12/1-500, derived
from the 3` breakpoint obtained with probe p500. The location of
different probes used in this work are indicated. The restriction map
of the two overlapping clones is shown, with the relative position of
the probe used for their isolation, the cellular flanking probe from
the rearranged allele. In these clones, we have also determined the
presence of repetitive DNA sequences. The arrow represents the
location where the recombination between cellular and viral DNA took
place. R, EcoRI; X, XbaI; S, SacI; K, KpnI; A, ApaI; Sp, SpeI; H, HindIII. The arrow indicate the location of the
breakpoint and the separation between retained and lost
DNA.
h12/32 and
h12/1-500, of 13 (not shown) and 17
kb, respectively (Fig. 3B). These two clones are
overlapping and cover a total genomic region of 23 kb, of which 8 kb
were lost as a result of the integration. This side of the target
region is called PAL2B.
h12/8HH,
h12/32, and
h12/1-500 shows
no overlap between PAL2A (5`) or PAL2B (3`) regions.
These data support the conclusion of a deletion of genomic DNA at the
integration site, which has a minimum size of 14 kb, and the cloned
genomic region, which has been replaced by viral DNA in the rearranged
allele.
Internal Genomic Probes Detect Germline But Not
Rearranged Alleles
The use of cellular probes derived from the
germline clones corresponding to the region that has been replaced by
viral DNA should detect only the germline allele, but not the
rearranged one, if there is a deletion as a result of HPV18 DNA
integration. To confirm this interpretation, we hybridized DNA from
three cell lines, RMS13, OSA, and SW 756, with probes derived from the
two phage germline clones, h12/8HH and
h12/1-500, in
such a way that they are to both sides of the breakpoints. The SW756
cell line has a 17-kb EcoRI band that is detected with an
HPV18 probe (Fig. 4A). The 5` breakpoint was examined
with the pS3 probe and with probe pXR, a 0.25-kb XbaI-EcoRI fragment (Fig. 3A) from
the region replaced by the viral DNA. The results for PAL2A, containing the 5` breakpoint, are shown in Fig. 4, B and C. The 3` breakpoint was studied with probes
p500 and pRH, a 0.5-kb EcoRI-HindIII fragment, from
the region replaced by viral DNA (Fig. 3B). The results
for the PAL2B region, containing the 3` breakpoint, are shown
in Fig. 4, D and E. The two probes retained in
the hybrid clone, pS3 and p500, detect germline and rearranged alleles (Fig. 4, B and D). The internal probes, from
replaced cellular DNA, pXR and pRH, only detect germline alleles (Fig. 4, C and E), ruling out their retention
as part of a more complex rearrangement. These observations confirm the
interpretation of a cellular DNA deletion at the integration site of
HPV18 DNA in 12q13 between the regions defined by the PAL2A and PAL2B loci.
membranes and
hybridized to different probes. A, detection of rearranged
allele with viral DNA detected by hybridization to HPV-18 DNA. B, detection of rearranged and germline alleles from the 5`
breakpoint with flanking probe pS3. C, lack of detection of 5`
rearrangement of genomic DNA from the 5` genomic clone with probe pXR,
derived from the region replaced by viral DNA. D, detection of
rearranged and germline alleles with a probe from the 3`-flank, p500. E, lack of detection of rearrangement of genomic DNA replaced
by viral DNA with probe, pRH, derived from the 3` genomic
clone.
HPV-18 DNA Integration Site Is Outside Known Amplicons of
Human 12q13-15 Region
There are two cell lines, OSA and
RMS13 with an amplicon in the 12q13-15 region(45) . This
amplicon contains the region where most translocations
occur(29, 30, 31, 32, 33, 46) .
We determined if the cellular flanking probes are located within these
amplicons. For this purpose, we digested DNA from five cell lines, Colo
320, MOLT4, OSA, RMS13, and SW756 and hybridized them to the breakpoint
flanking probes; to probes from genes within the amplicon like CDK4 and
SAS; to probes from genes outside the amplicon like SP1, WNT1,
RAR, and CDK2; and to MDM2, which is within the OSA amplicon but
outside the RMS13 amplicon. Densitometric analysis of these Southern
blots detected a 5-fold amplification of the genes included within the
amplicon. The results with these cellular gene probes and with flanking
probe pS3 are shown in Fig. 5. The results with the other three PAL2 probes are shown in Fig. 4, but only data for
three cell lines is included in this figure. These data demonstrate
that both ends of the integration site in SW756 cell line, the PAL2A and PAL2B loci, are outside the genomic region
defined by the amplicons of both cell lines, OSA and RMS13.
Sequence of the Cellular-Viral Junctions and Target
Cellular Sequences
The sequence of the viral cellular junctions
in the rearranged allele were determined and compared with the cellular
and viral sequences. We sequenced 761 nucleotides from the germline PAL2A region (Fig. 6A). The cellular sequence
spanning the 5` breakpoint does not contain repetitive elements and is
AT-rich. There is no DNA homology between the viral and cellular target
sequences. The 5` recombinant breakpoint was sequenced from the
cellular HindIII to the most proximal viral HincII
(probe pHH in Fig. 1) and occurs within the early region E2 gene
at nucleotides 3418 (not shown).
T), 3533 (T
C), 3557
(C
A), 3577 (C
T), 3584 (A
C), and 3951 (T
G). In
the 3` breakpoint, the small region sequenced has mutations at
positions 2482 (T
C), 2509 (G
A), and 2551 (G
T) and
also in the EcoRI site within E1 ORF that is lost, but this
mutation was only identified by the loss of the restriction site and
not by sequencing.
-A2MR-GLI(40) . The
region amplified in OSA and RMS13 comprises A2MR-GLI-MDM2-ERB3-GST3 and CHOP, but it does not include RAR
, SP1, or WNT1(33, 45) . Thus, the lack of
amplification of PAL2 places it in a region outside this gene
cluster(25) . The precise location of PAL2 will be
determined when the yeast artificial chromosome containing it is
isolated and placed within the existing map of the region.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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