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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2158-2166
RAPID COMMUNICATION
Role of Amplified Genes in the Production of Autoantibodies
By
Nicole Brass,
Alexander Rácz,
Christine Bauer,
Dirk Heckel,
Gerhard Sybrecht, and
Eckart Meese
From the Department of Human Genetics and the Department of Internal
Medicine V, Medical School, University of Saarland, Homburg/Saar,
Germany.
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ABSTRACT |
A variety of previously published studies have shown the presence of
autoantibodies directed against oncogenic proteins in the sera of
patients with tumors. Generally the underlying genetic aberration
responsible for the induction of an immune response directed against an
abnormal protein is unknown. In our studies we analyzed the role of
gene amplification in the production of autoantibodies in squamous cell
lung carcinoma. We screened a cDNA expression library with autologous
patient serum and characterized the isolated cDNA clones encoding tumor
expressed antigens termed LCEA (lung carcinoma expressed antigens). As
determined by sequence analysis, the 35 identified cDNA clones
represent 19 different genes of both known and unknown function. The
spectrum of different clones were mapped by polymerase chain reaction
(PCR) and fluorescence in-situ hybridization, showing that a majority
are located on chromosome 3, which is frequently affected by
chromosomal abnormalities in lung cancer. Gene amplification of 14 genes was analyzed by comparative PCR. Nine genes (65% of all analyzed
genes) were found to be amplified; furthermore, most of them are also
overrepresented in the pool of cDNA clones, suggesting an
overexpression in the corresponding tumor. These results strongly
suggest that gene amplification is one possible mechanism for the
expression of immunoreactive antigens in squamous cell lung carcinoma.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMORAL IMMUNE RESPONSES to oncogenic
proteins have been reported for different types of cancer and are
continuing to be defined. The oncogenic proteins are expressed by
cancer cells themselves and act as tumor-expressed antigens. Oncogenes become activated by a variety of mechanisms including point mutation, translocation, and overexpression. Each mechanism of activation can
result in the expression of proteins with the potential to serve as
tumor antigens.
Autoantibodies directed against mutated oncogenic proteins have been
reported for many different tumors. The ras oncogenes, for
example, are cancer-related genes that become activated by specific point mutations.1 Studies have shown the existence of antibodies directed against the ras protein containing a single amino acid substitution in patients with colon cancer.2 In sera of patients with various types of cancer, eg, breast cancer, colorectal cancer or lung cancer, antibodies against abnormal p53
protein containing multiple mutational hot spots have been detected.3-5 Besides point mutations, translocations can
result in the generation of fusion genes expressing chimeric proteins, which could be the target of an immune system attack. The SSX2 gene,
for example, which is involved in a translocation in synovial sarcomas,
encodes a tumor antigen.6 Furthermore, overexpressed oncogenic proteins represent potential tumor antigens. C-erbB-2 is one
of the genes overexpressed in human tumors, especially in breast
carcinoma. Antibodies against the overexpressed oncogenic protein could
be detected in several patient sera.7 Autoantibodies against the L-myc oncogene, which is known to be overexpressed in tumors with neuroendocrine characteristics, could be identified in
patient sera with small cell lung cancer.8 The
overexpression of L-myc in human tumors has frequently been
correlated with gene amplification, but an amplified gene itself has
never been identified together with the presence of antibodies in the
same patient. In contrast, we were able to show previously that the
translation initiation factor eIF-4 , which is encoded by an
amplified gene, acts as a tumor expressed antigen in squamous cell lung
carcinoma.9
To further elucidate the role of gene amplification as a genetic
aberration evoking the production of autoantibodies, we selected squamous cell lung cancer as a model system. Squamous cell lung carcinomas constitute 40% to 50% of lung cancers. This is the most
frequent lethal tumor in Western societies, with a median survival time
of approximately 6 months and a 5-year survival rate of less than
10%.10 Because of the high frequency of gene amplifications, lung cancer is an ideal neoplasm to analyze downstream effects of gene amplification, especially with regard to the expression of immunoreactive antigens in cancer patients.11,12
For investigation of these effects, we used an immunoscreening
approach. A cDNA expression library generated from a squamous cell lung
carcinoma was screened with autologous patient serum, and cDNA clones
representing tumor-expressed antigens were isolated. Both new and known
gene sequences were identified and analyzed for gene amplification.
Gene amplification is among the most common genetic changes in human
cancer cells.13 There are only a few examples of programmed
gene amplification in early development of lower eukaryotic animals,
but gene amplification does not occur in normal mammalian
cells.14,15 Over the last decade amplification analysis has
been largely restricted to known oncogenes. Using the immunoscreening
approach it is also possible to detect new immunoreactive tumor
antigens whose genes are amplified in cancer cells.
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MATERIALS AND METHODS |
Tumor tissue.
Informed consent was obtained from the patients for use of their tumors
and sera. Tissue samples of squamous cell lung carcinoma were frozen in
liquid nitrogen immediately after biopsy or surgery and stored at
 70°C. The pathological stage of the tumor L10 was T2, N0, M0,
stage I.
DNA and RNA isolation.
Genomic DNA was isolated by standard methods.16 In brief,
after proteinase K digestion at 55°C, proteins were extracted with
chloroform and DNA was precipitated with isopropanol. The DNA
concentration was determined by optical density measurement at 260 nm.
RNA isolation was according to the manufacturer's instructions
(Stratagene, Heidelberg, Germany). Frozen tissue was
homogenized, proteins were phenol-chloroform extracted, and RNA was
precipitated twice with isopropanol and finally resuspended in
diethylpyrocarbonate (DEPC)-treated H2O. Integrity and
concentration of RNA was evaluated using formaldehyde gels.
cDNA expression library.
Total RNA was applied to oligo(dT) cellulose push columns and poly(A)
mRNA was eluted according to the Poly(A) Quick Kit
(Stratagene). cDNA synthesis was performed with the ZAP Express
cDNA synthesis kit (Stratagene). In brief, 4.5 µg of
poly(A) mRNA was reverse transcribed by Moloney murine leukemia virus
(MMLV) reverse transcriptase using an oligo(dT) primer with a 5'
XhoI restriction site. The cDNA was ligated to
EcoRI adapters, XhoI-digested, size-fractionated with
Sephacryl S-500 columns (Stratagene, Heidelberg, Germany) and
cloned into ZAP Express vector arms. The vector was
packaged using the Gigapack III Gold Packaging Extract
(Stratagene). After one round of amplification of the ZAP Express
library, the bacteriophage titer was determined to be 1.3 × 1010 plaque-forming units (pfu)/mL.
Preabsorption of patient serum.
Serum was prepared from 10-mL blood samples using serum gel monovettes
and stored at 70°C. Serum samples were diluted 1:10 in 1× TBS
(Tris buffered saline), 0.5% (wt/vol) low-fat milk, and 0.01%
thimerosal before use. Mechanical preabsorption columns were prepared
by incubation of sonicated Escherichia coli XL1blue MRF'cells
in 1× TBS with Affinity Absorbent (Glutaraldehyde-activated; Boehringer Mannheim, Mannheim, Germany) overnight in
BioRad Polyprep chromatography columns (BioRad,
München, Germany). Lytic preabsorption columns were prepared in
the same way using instead bacteria lysed by nonrecombinant ZAP express
phages. The patient serum was preabsorbed using each column type five
times. The preabsorbed serum was diluted to a final concentration of
1:100 in 1× TBS, 0.5% (wt/vol) low-fat milk, and 0.01% thimerosal.
Serological screening.
E coli XL1-Blue MRF' cells were transfected with recombinant
bacteriophage containing lung carcinoma cDNA and plated onto NZCYM-Tetracyclin-agar plates (Greiner, Frickenhausen,
Germany). After incubation at 42°C for approximately 4.5 hours,
isopropyl- -D-thiogalactopyranoside (IPTG) (10 mmol/L)
pretreated filters were layered on the bacteriophage plaques to induce
protein expression. After a second incubation at 37°C for 4 to 5 hours, plates were cooled at 4°C overnight. Filters were removed and
washed twice for 30 minutes with TBS/0.05% Tween 20. After blocking
with 5% (wt/vol) low-fat milk, filters were incubated with autologous
patient serum diluted 1:100 for about 4 hours. For the detection of
antigen-antibody complexes, filters were overlayered with a solution
containing a secondary antibody directed against the constant region of
human IgG heavy chain and conjugated with alkaline phosphatase. After
the filters were washed three times in TBS, serum-positive clones were
visualized by the addition of
5-bromo-4-chloro-3-indoyl-phosphate-p-toluidine salt (BCIP) and
nitroblue tetrazolium-chloride (NBT), which are substrates of alkaline
phosphatase, resulting in a blue precipitate. For further
analysis, serum-positive clones were isolated and inserted into the
pBK-CMV phagemid vector by in vivo excision. Some of the clones, for
example clone 9-1, were also isolated using a 1:500 dilution of the
autologous patient serum.
Sequence analysis.
Sequencing was performed using the Perkin Elmer ABIPrism
Cycle sequencing kit (Perkin Elmer, Überlingen,
Germany). Clone inserts were sequenced with an automatic sequencer
(373A DNA Sequencer; Applied Biosystems, Weiterstadt,
Germany). For sequence alignment, clone sequences were
compared with known sequences by BLASTN search (BCM search launcher,
Human Genome Center, Baylor College of Medicine, Houston, TX).
Chromosomal localization.
Chromosomal localization of isolated cDNA clones was performed by
hybrid panel mapping or fluorescence in situ hybridization (FISH). For
FISH, 12 ng of biotinylated clone DNA was hybridized against normal
metaphase chromosome spreads. Before hybridization, slides with
chromosomes were treated with RNase A for 1 hour and pepsin for 10 minutes. After dehydration of chromosomes in ethanol, both chromosomes
and biotinylated probe were denatured in 50% formamide solution at
80°C. Slides were incubated at 37°C overnight. After washes in 50%
formamide solution at 45°C and in 0.1× sodium chloride-sodium
citrate (SSC) at 60°C, biotinylated probes were detected using avidin conjugated to fluorescein isothiocyanate. Three
rounds of amplification were performed by using goat anti-avidin antibodies. Slides were counterstained with 4,6-diamino-2-phenyl-indol (DAPI). Fluorescence signals were visualized in an
Olympus microscope (Olympus, Hamburg, Germany) and
analyzed and documented with the program ISIS3 of Metasystems
(Altussheim, Germany).
Alternatively, panels of multichromosomal somatic cell hybrids (hybrid
mapping panel 1) and monochromosomal somatic cell hybrids (hybrid
mapping panel 2) from the Coriell Institute for Medical Research
(Camden, NJ) were used for chromosomal localization by polymerase chain
reaction (PCR). The human chromosome content of these cell hybrids has
been characterized by Drwinga et al17 and Dubois and
Naylor.18 The PCR conditions for each sequence specific
primer pair are summarized in Table 2.
Comparative PCR.
The concentration of peripheral blood DNA, as control DNA, and tumor
DNA was determined by optical density measurement at 260 nm. After
preparation of a master solution containing 5 ng/µL of each DNA
sample, three different DNA dilutions (2 ng/µL, 1 ng/µL, 0.5 ng/µL) were generated to monitor the linear range of amplification
during PCR.19 Before amplification analysis,
the three dilutions of each DNA sample were calibrated using
sequence-specific primers for the MUC gene. A successful calibration
results in PCR products of equal intensity in tumor and blood DNA.
After successful calibration of template DNA, the amplification status of the LCEA clones was determined using specific primer pairs. Gene
amplification results in increased signal intensities of all three
dilutions of a tumor DNA sample. PCR was performed with 5 minutes of
initial denaturation at 94°C, for 25 cycles, with 1 minute of
denaturation at 94°C, 1 minute of annealing at temperatures given in
Table 2, 1 minute of extension at 72°C followed by 10 minutes of
final extension at 72°C in an M.J. Research Minicycler PTC-150 and PTC-100 (M.J. Research, Watertown, MA). All PCR reactions were performed in a total volume of 50 µL with 0.5 µmol/L of each primer, 200 µmol/L deoxynucleotide triphosphates, and 2.5 U of Taq
polymerase (Pharmacia, Freiburg, Germany) in PCR buffer. The PCR
conditions are summarized in Table 2.
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RESULTS |
Identification of cDNA clones representing tumor-expressed antigens.
A cDNA expression library was generated from a squamous cell lung
carcinoma, which has previously been shown to harbor amplification units on several different chromosomes by reverse chromosome painting and comparative genomic hybridization.16 Therefore,
enriched poly(A)mRNA was reverse transcribed into cDNA, inserted into
the ZAP Express expression vector (Stratagene) in the sense orientation with respect to the lacZ promoter and transfected into E coli cells. After induction of polypeptide synthesis, this expression library was screened with autologous patient serum to identify cDNA
clones representing tumor expressed antigens. Antigen-antibody complexes were detected by a secondary antibody binding to the constant
region of the human IgG heavy chain. By screening a total of 800,000 clones, we identified 35 positive cDNA clones. The positive clones that
encode immunoreactive antigens, termed lung carcinoma expressed
antigens (LCEA), were isolated and subjected to a second round of
screening for verification and enrichment. Figure
1 shows the identification of positive
clones in the primary screening and the enrichment during the second
round of screening.
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| Fig 1.
Screening of a cDNA expression library generated from a
squamous cell lung carcinoma with autologous serum. Recombinant
proteins were screened with preabsorbed patient serum (see Materials
and Methods) and antigen-antibody complexes were detected by a color
reaction. As shown for clones 11-4 and 26-4, the serum-positive clones
were identified in a primary screening (left-hand side). The enrichment
of serum-positive clones by replating and subjection to a second round
of screening is shown on the right-hand side.
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Positive clones were isolated and partially or completely sequenced
from both ends using vector- and sequence-specific primers. Sequences
were analyzed by comparison with each other and known sequences of
different databases.
Grouping of identified cDNA clones.
Sequence analysis showed that the 35 isolated cDNA clones represent 19 different genes including both known and unidentified sequences. As
documented in Table 1, the 35 LCEA encoding
cDNA clones were grouped depending on their sequence homology to known genes or to each other. Group 1 is represented by six clones encoding the gene for human translation initiation factor
eIF-4.20 These clones represent almost 20% of all
isolated cDNA clones, indicating the relevance of this translation
initiation factor as a tumor-expressed antigen in squamous cell lung
carcinoma. Our preliminary expression data indicate an mRNA
overexpression of the eIF-4 gene. The second group is represented by
5 clones with homology to the gene for human DnaJ-protein homologue
heat-shock protein.21 Group number 3 consists of 3 clones
identical with the MGEA5 gene, group number 4 of 2 clones homologous to
the RBPJK gene, group number 5 of 2 clones homologous to the NNP-1
mRNA, and group number 6 of 2 clones identical with the human
transcriptional repressor (GCF2) mRNA.22-25 In group 7 we
summarized both individual cDNA clones representing different genes of
known function, for example the gene for protein kinase
p160ROCK, the gene for human transcription factor NFATc.b
and the gene for a human ring zinc-finger protein.26,27 The
clones 9-1/46-1 and 11-2/54-2 represent homologous cDNA sequences
encoding new genes, whereas the cDNA sequences of the successive clones
show homology to known genes with unknown function.
Chromosomal mapping of identified cDNA clones.
The newly isolated LCEA encoding clones were assigned to human
chromosomes by somatic cell hybrid panel mapping. Using
sequence-specific primer pairs and DNA of hybrid cell lines with one or
several human chromosomes in front of a rodent background, genomic
mapping was possible by PCR (Fig 2). The
PCR conditions are listed in Table 2.
Additionally, the exact chromosomal localization was determined by
FISH. The results of the chromosomal localization are summarized in
Table 3. In total, 11 cDNA clones
representing 4 different genes were mapped on chromosome 3. For 3 clones representing 3 different genes, the exact localization has been
determined by FISH showing in all cases a localization to the long arm
of chromosome 3, namely at chromosomal bands 3q25, 3q26.3-qter, and 3q27. The fact that 11 of 32 clones representing 4 of 16 genes are
located on chromosome 3 and, as shown by FISH, in the terminal region
of the chromosomal q-arm, suggests a correlation between this region on
chromosome 3 and the production of tumor expressed antigens.
Furthermore, two different cDNA clones were mapped to chromosome 14 and
two were mapped to chromosome 10. The chromosomal localization of 8 additional clones was mapped to chromosomes 2, 6, 9, 15, 17, 18, 20, and 21. For the remaining cDNA clones the localization has not yet been
determined because of the difficulty in finding appropriate primer
pairs for hybrid panel mapping.
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| Fig 2.
Chromosomal localization of clone 59-2 by hybrid panel
mapping. Sequence-specific primers were used in a PCR reaction with DNA
of hybrid cell lines. In the first PCR reaction, DNA of
multichromosomal somatic cell hybrids with several human chromosomes in
a rodent chromosome background was amplified in the PCR reaction (top
part of the figure). Finally the exact localization was determined
using DNA of monochromosomal somatic cell hybrids with one or several
human chromosomes in a rodent chromosome background (bottom part of the
figure). Clone 59-2 was mapped to chromosome 6. (Top) VI, DNA marker;
lane 1, NA09927; lane 2, NA09929; lane 3, NA 09931; lane 4, NA09935A;
lane 5, NA00347A (hamster DNA); lane 6, NAIMR91 (human genomic DNA);
lane 7, human blood DNA; lane 8, no DNA. (Bottom) VI, DNA marker; lane
1, NA10253 (chromosome 3); lane 2, NA10115 (chromosome 4); lane 3, NA10629 (chromosome 6); lane 4, NA00347A (hamster DNA); lane 5, NAIMR91
(human genomic DNA); lane 6, human blood DNA; lane 7, no DNA.
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In summary, the localization of the isolated cDNA clones correlates
well with the localization of amplified regions in the tumor sample
used to generate the cDNA library. Previously, we have been able to
identify, in this tumor sample, amplifications on chromosome 3q26,
6p21.1, 1q, 5p, and 14 by reverse chromosome painting and one-color
comparative genomic hybridization.28
Analysis of gene amplification of identified cDNA clones.
To analyze the amplification status of the identified and isolated cDNA
clones, we chose comparative PCR, which is a very sensitive technique
to detect gene amplifications from small amounts of tumor
DNA.19 In the first step of comparative PCR one control DNA
and several tumor DNA samples are calibrated using sequence-specific primers from an unamplified chromosomal region. In the second step,
primers specific for a potentially amplified gene are used in the PCR.
Amplifications are indicated by increased signal intensities in the
tumor DNA compared with the control DNA, as shown in Fig 3. A gene is considered to be amplified if
the signal intensities in all three tumor samples are increased in
comparison to the signal intensity of the corresponding dilutions of
the blood DNA. Although this method is highly suited to detect gene
amplification, it does not allow a quantification of the amplification
level. Of the 14 analyzed LCEA encoding clones, 9 were found to be
amplified in the tumor DNA which had been used for generating the cDNA
library. This translates into an amplification frequency of 65%. Most
of the amplified genes are represented by more than one cDNA clone, for
example the 6 cDNA clones homologous to the gene for human translation
initiation factor eIF-4 (group 1), the 5 cDNA clones of group 2 encoding a human heat shock protein, the two clones of group 4 showing
homology to the gene for Jk-recombination signal binding protein, and
the clones 9-1/46-1 and 11-2/54-2 (group 7) encoding tumor-expressed
genes of unknown function. With one exception, all clones that did not
show gene amplification of tumor DNA are representated by a single cDNA
clone isolated from the expression library. Representative results of
the amplification analysis are shown in Fig 3. The results concerning
the determination of the amplification status of the various clones are
summarized in Table 3. The gene for eIF-4 has been found to be
amplified in independent tumor samples (data not shown).
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| Fig 3.
Amplification analysis of clones 26-4, 96-5, and 129-1 in
squamous cell lung carcinomas L115, L10, and L116 by comparative PCR.
Three different concentrations of tumor and blood DNA were amplified
each by PCR using primers specific for clones 26-4, 96-5, and 129-1 and
the MUC gene. The PCR products were separated by gel electrophoresis
and visualized by SYBR green I. While the signal intensities of tumor
and peripheral blood DNA (PB) were comparable using primers specific
for the MUC gene, the signals of all three clones 26-4, 96-5, and 129-1 were significantly increased in tumor DNA L10 versus normal DNA
indicating amplification of the corresponding genes in tumor L10. This
tumor was used to generate the cDNA expression library. Lower
amplification levels or the absence of amplification are observed for
tumors L115 and L116, respectively. Lane 1, DNA marker; lanes 2 through
4, three dilutions of blood DNA; lanes 5 through 7, three dilutions of
tumor DNA L115; lanes 8 through 10, three dilutions of tumor DNA L10;
lanes 11 through 13, three dilutions of tumor DNA L116; lane 14, no
DNA.
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In summary, we were able to isolate, together with our previously
published results, 35 cDNA clones representing 19 LCEA encoding genes.
These data show that squamous cell lung carcinoma expresses a wide
variety of immunogenic antigens. Many cDNA clones encoding tumor-expressed antigens could be mapped to chromosome 3, a chromosome that is frequently involved in chromosomal aberrations, especially in
lung cancer. Furthermore, a great majority of cDNA clones are amplified
in the tumor sample used to generate the expression library.
Additionally, most of them are overrepresented in the pool of isolated
cDNA clones, suggesting an overexpression at the RNA level.
Unfortunately, an expression analysis was impossible because of a lack
of tumor tissue. These results strongly suggest that gene amplification
may be a general mechanism for overexpression of tumor-expressed
antigens, which induce a humoral immune response resulting in the
production of autoantibodies.
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DISCUSSION |
As an early event during lung cancer development, alterations in
oncogenes and/or tumor suppressor genes may lead to the production of
mutant forms or abnormal levels of proteins within tumor cells or on
tumor cell membranes. Recognition by the immune system is possible
through presentation on the cell surface. Alternatively, there may be a
release of proteins from damaged cancer cells due to spontaneous tumor
necrosis resulting in antigen presentation via complex formation with
other proteins such as heat shock proteins. To detect the humoral
response to oncogenic proteins in lung cancer, we generated a cDNA
library from a squamous cell lung carcinoma, expressed the cDNA clones
as recombinant fusion proteins, and screened them with autologous
patient serum. An important advantage of this screening approach is the
possibility of detecting antigens that are encoded by both known and
unknown genes. Moreover, this technique enabled us to identify multiple
antigens, which correlates with the suggestion that immunogenicity of
human tumors is not caused by a single antigen.
The majority of isolated cDNA clones encoding tumor-expressed antigens
represent genes that are known to be involved in tumor formation,
supporting the idea that oncogenic proteins induce an immune system
response. The translation initiation factor eIF-4, for example, is a
critical link between mRNAs and the 40s ribosomal subunit during the
initial steps of translation.29,30 Overproduction of this
factor has been shown to form transformed foci on a monolayer of cells,
to cause anchorage-independent cell growth, and to form tumors in nude
mice.31 Increased levels of eIF-4 protein, which is the
limiting factor of translation initiation in normal cells, may be
responsible for the production of autoantibodies by B
cells.9 Our preliminary data indicate a correlation between
gene amplification and mRNA overexpression of the eIF-4 gene.
Another interesting antigen we identified is the Rho-associated,
coiled-coil containing protein kinase p160ROCK that
functionally associates with the guanosine triphosphate (GTP)-bound
Rho-protein.32 In addition to organizing specific actin cytoskeletons, recent studies have shown that Rho
is involved in cell-cycle progression and in cell
transformation.33,34 Because of its functional association
with Rho-protein, it can be speculated that p160ROCK is
also involved in tumor formation.
In addition to the above-mentioned antigens, we also isolated a
DnaJ-homologue heat-shock protein that is an inducer of a humoral
immune response. This DnaJ-like protein is involved in the regulation
of HSP70 heat-shock protein which plays a major role in antigen
presentation.35 It has been shown that in antibody-elicting breast tumors, a 70-kD heat-shock proteins forms
complexes with p53, whereas none of the antibody-negative tumors
contained any of these complexes.36 This implies that
heat-shock proteins are involved in the antigenic presentation of p53.
This observation has been supported by further studies which identified
antibodies directed against p53 protein complexed with HSP70 protein in
sera from oral cancer patients.37 Because of the results of
these investigations, we suppose that the DnaJ-homologue heat-shock protein may also be involved in antigen presentation in lung cancer. Additionally, we isolated several cDNA clones encoding unknown proteins. The role of these antigens needs to be determined.
In addition to isolating cDNA clones encoding tumor-expressed antigens
in squamous cell lung carcinoma, we also determined their chromosomal
localization by hybrid panel mapping and FISH. Most of the isolated
cDNA clones could be mapped to the long arm of chromosome 3 (3q25-qter). It is of great significance that this chromosomal region
is most frequently involved in chromosomal imbalances in squamous cell
lung carcinoma. More than one third of these tumors show DNA sequence
amplifications of the distal part of chromosome 3q, which may serve as
a source for genes encoding tumor-expressed antigens through
overexpression or abnormal protein formation. Recently, we reported an
amplicon localized at 3q26 containing the genes BCHE and SCL2A2 which
were amplified in 40% of squamous cell lung
carcinomas.12,28,38,39
In general, DNA amplification is frequent in squamous cell lung
carcinoma and may result in immunoreactive antigens via increased protein levels without additional DNA mutations. In our study, we
showed that the majority of immunogenic antigens expressed in the
analyzed squamous cell lung carcinoma are encoded by amplified genes.
It is likely that at least some of the genes amplified in tumor L10 are
also frequently amplified in other squamous cell lung carcinomas. Our
results strongly support the hypothesis that DNA amplification is a
major mechanism provoking the expression of immunoreactive autoantigens
in this type of lung cancer. This is the first report presenting a
detailed investigation of a correlation between gene amplification,
overrepresentation of cloned tumor antigen genes, and the production of
autoantibodies in human cancer. Additional genetic aberrations may be
responsible for the expression of antigens which are not encoded by
amplified genes.
Newly isolated antigens are likely to be useful as novel prognostic
parameters of squamous cell lung carcinoma, especially in the light of
a poor prognosis after late diagnosis. Generally, the presence of
autoantibodies directed against oncogenic proteins in cancer patient
sera predict a poor prognosis.40 However, several studies
showed that antibodies are already detectable in sera of patients
without obvious tumor formation. The presence of antibodies against p53
have been detected in the serum of patients several months before the
diagnosis of a squamous cell lung carcinoma, proposing that the
detection of specific autoantibodies may be an ideal tool for early
diagnosis, especially for early detection of squamous cell lung
carcinoma.5
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FOOTNOTES |
Submitted November 6, 1998; accepted January 10, 1999.
Supported by grants from the Deutsche Krebshilfe (Grant No.
10-11-1-Me2) and from the Deutsche Forschungsgemeinschaft (SFB 399, A1). A.R. was a recipient of a grant from the Landesgraduierten Förderungsprogramm des Saarlandes.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Eckart Meese, PhD, Department of Human
Genetics, Medical School, University of Saarland, Building 60, 66421 Homburg/Saar, Germany; e-mail: hgemee{at}med-rz.uni-sb.de.
| |
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