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Regular Articles |
From the Laboratory of Cancer Genetics,*
Institute of
Medical Technology, University of Tampere and Tampere University
Hospital, Tampere, Finland, and the Department of
Oncology,
University Hospital, Lund, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Within the 8q arm, two independently amplified subregions, 8q21 and 8q23-q24, have been identified,4,8 suggesting the presence of several target genes. The c-myc oncogene, cloned over 10 years ago, is considered the putative target gene at 8q24.1. However, according to several studies, c-myc amplification is not always associated with its overexpression.11,12 Large amplifications at 8q occur also without c-myc amplification, suggesting that multiple target genes might be involved, as with other amplicons in breast cancer.11-13
The two minimal commonly amplified regions (8q21 and 8q23-q24) comprise approximately 60 Mb of DNA containing possibly up to 1000 genes. We used 8q suppression subtractive hybridization (SSH)14 between breast cancer cell lines with and without the 8q-amplification2 to identify overexpressed target genes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breast cancer cell lines SK-Br-3, ZR-75-1, MDA436, and MCF-7 and prostate cancer cell lines PC-3, DU145, and LNCaP were obtained from the American Type Culture Collection (Manassas, VA) and cultured in recommended conditions. Formalin-fixed, paraffin-embedded hormone-refractory prostate carcinomas (n = 44) were obtained from transurethral resections, which were done to relieve urethral obstruction. The average time from diagnosis (the beginning of hormonal therapy) to progression was 44 months (range, 8–113 months). The second set of tumors comprised 39 freshly frozen primary invasive breast carcinomas taken from patients before any treatment. In addition, 19 breast carcinoma imprint touch preparations were obtained from the Department of Oncology, University of Lund (Lund, Sweden). These tumors were selected because they were known to contain c-myc amplification according to Southern analysis.15
Suppression Subtractive Hybridization (SSH)
SSH was done with PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, CA) with minor modifications, as earlier described.14 Total RNAs were isolated from SK-Br-3 and ZR-75-1 breast cancer cell lines by TRIzol Reagent (Gibco BRL, Grand Island, NY), from which mRNAs were isolated using Dynabeads (Dynal A.S., Oslo, Norway) and used for cDNA synthesis. cDNA from SK-Br-3 was used as tester and cDNA from ZR75-1 as driver in the subtraction hybridization. The resulting subtracted cDNAs were subcloned into pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA). The inserts were amplified by polymerase chain reaction (PCR) using adapter-specific primers (Clontech) from randomly picked clones and sequenced using ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer, Foster City, CA) and ABI310 sequencer (Perkin-Elmer).
Fluorescence in Situ Hybridization (FISH)
Metaphase and interphase cell preparations from the cancer cell
lines, normal blood lymphocytes, and nuclei isolated from prostate and
breast carcinomas were used for the FISH analysis. Metaphase and
interphase FISH were performed as described in detail
elsewhere.16
After identification of the p40 subunit of
eukaryotic translation initiation factor 3 (eIF3-p40), a
locus-specific genomic probe was obtained by screening human PAC
library with PCR using primers specific to eIF-p40
(5'-GCCCAGGCTCTTCAAGAATAC-3' and 5'ATAGCCAAAATCGGCAATGA-3'). A genomic
P1-probe for c-myc was obtained from RMC (RMC08P001,
Berkeley, CA). The probes were labeled with biotin-16-dUTP or
digoxigenin-11-dUTP (Boehringer Mannheim, Mannheim, Germany) using
nick-translation. Texas Red-labeled chromosome 8 
-satellite
(centromere) probe was used as a reference probe (CEP8, Vysis, Inc.,
Downers Grove, IL). Before hybridization, prostate cancer samples were
pretreated by heating in 59% glycerol/0.1x standard saline citrate
(SSC, pH 7.5) solution at 90°C for 3 minutes to improve hybridization
efficiency of the probes. Slides were denatured in a 70% formamide-2x
SSC solution at 73°C for 3 minutes. After hybridization the probes
were detected immunochemically with avidin-fluorescein isothiocyanate
and anti-digoxigenin rhodamine. Slides were counterstained with 0.1
µmol/L 4,6-diaminido-2-phehylindole in an antifade solution. Signal
copy numbers were counted from 100 randomly chosen nonoverlapping
nuclei. Control hybridizations included normal lymphocytes and
formalin-fixed, paraffin-embedded benign prostate hyperplasia (BPH)
samples (n = 10). These experiments showed that
the probes recognized a single copy target and that the hybridization
efficiencies were similar. The mean averages ± SD of p40 and
c-myc signals in the BPH samples were 2.2 ± 0.3 and
2.1 ± 0.2, respectively. Digital images were captured with a
Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss Jena GmbH,
Jena, Germany) equipped with a Hamamatsu C9585 camera (Hamamatsu
Photonics, Hamamatsu, Japan) and ISIS software program (Metasystems
GmbH, Altslusheim, Germany). Tumors that showed >20% of nuclei with
increased copy number of either eIF3-p40 or c-myc were
considered to have amplification. In these cases, the level of
amplification was determined counting only nuclei with an increased
number of signals. The tumors were classified into three groups:
nonamplified (no increase in p40 or c-myc copy number),
low-level amplification (3–5 copies per cell), and high-level
amplification (
5 copies of the genes per cell or gene/centromere
ratio >2).
Southern Blot Analysis
Genomic DNAs were extracted according to standard protocols.
Following restriction enzyme digestion with BclI (MBI
Fermentas, Vilnius, Lithuania), DNAs were separated on a 0.7% agarose
gel and blotted onto a nylon membrane (Hybond-N, Amersham, Arlington
Heights, IL). 32P-labeled probes (Random
Primed DNA labeling kit, Boehringer Mannheim) for eIF3-p40 (670-bp
fragment corresponding to eIF3-p40 sequences from exon 3 to exon 5) and
hypoxantine-guanine phosphoribosyltransferase (insert of EST 270419;
GenBank accession no. N33128) were hybridized sequentially
using standard protocols. The hybridization signals were detected with
Phosphoimager (Molecular Dynamics, Inc., Sunnyvale, CA).
Northern Blot Analysis
Total RNAs from cancer cell lines were isolated by TRIzol Reagent
(Gibco BRL). Twenty micrograms of total RNA were electrophoresed and
transferred to a nylon membrane. 32P-labeled
probes for eIF3-p40 (1.2-kb insert of EST 346021; GenBank accession no.
W72146), c-myc (2.2-kb insert of EST 51699; GenBank
accession no. H24033), and ß-actin (Clontech) were hybridized
sequentially using standard protocols. The hybridization signals were
detected and quantitated with Phosphoimager and ImageQuaNT software
program (Molecular Dynamics).
Expression of eIF3-p40 in Clinical Tumors Studied with mRNA in Situ Hybridization
EcoRI-HincII-fragment (780 bp) from EST-clone 595376 (GenBank accession no. AA173710) was subcloned into pBluescript SK vector (Stratagene, La Jolla, CA) and used for in vitro transcription of eIF3-p40 to obtain antisense and sense cRNA probes. A cytokeratin antisense probe derived from a EcoRI-SmaI fragment (690 bp) of EST-clone 487868 (GenBank accession no. AA044589) was used to control the quality of RNA and hybridization of the samples. We hybridized 27 formalin-fixed, paraffin-embedded hormone-refractory prostate carcinomas, 34 primary breast carcinomas, 1 normal breast tissue, and 3 BPHs with 33P-dUTP-labeled cRNA probes. Deparaffinized sections were rinsed in phosphate-buffered saline and digested with proteinase K (10 µg/ml) for 20 minutes at 37°C in phosphate-buffered saline. The sections were covered with hybridization solution (50% formamide, 2xSSC, 20 mmol/L Tris, 1 mmol/L EDTA, 1x Denhart's, 10% dextran sulfate, and 500 µg/ml yeast tRNA) containing the probe with the final activity of 107 cpm/ml and hybridized overnight at 55°C. After hybridization, the sections were washed and digested with RNase A (20 µg/ml) for 30 minutes at 37°C in 0.5 mol/L NaCl, 10 mmol/L Tris (pH 8.0), and 1 mmol/L EDTA.
For quantitation, the hybridized sections were exposed to Amersham ß-max Hyperfilm for 3 days. After the film was developed, it was scanned using Personal Densitometer SI (Molecular Dynamics). The expression levels were quantitated with ImageQuaNT software using the volume quantitation option. First, representative objects of equal size were selected from each slide. The quantitation results were given as integrated intensity of all pixels in the objects excluding the background. For microscopic examination, the hybridized sections were immersed in autoradiographic emulsion NTB2 (Kodak) and exposed for 4 weeks at 4°C. After developing the autoradiographic signals (grains), the sections were counterstained with hematoxylin and examined in a Nikon Microphot-SA (Nikon Corp., Tokyo) microscope equipped with an epipolarization filter allowing simultaneous visualization of grains (using polarized epi-illumination light) and morphology by hematoxylin staining (using transmitted light).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SSH was used to identify overexpressed transcripts in breast cancer cell line SK-Br-3. cDNAs from SK-Br-3 were subtracted against those from ZR-75-1. Database searches with BLASTN revealed that the first redundant clone, named A8, recognized an EST clone 595376 (accession no. AA173710), which, according to the Unigene database, was located in the region of interest between marker D8S276 (8q22.3) and D8S1799 (8q24). Next, we verified by Northern analysis that A8 was differentially expressed in SK-Br-3 and ZR-75-1.
Database search showed that the sequence of A8 was identical to the
recently cloned gene, eukaryotic translation initiation factor 3
subunit p40 (eIF3-p40).17
To map the gene precisely, we
obtained genomic clone for eIF3-p40 by screening human PAC library.
Using the PAC-probe and FISH, we localized the eIF3-p40 to 8q23, about
12 Mb centromeric from c-myc (Figure 1A)
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To study the p40 gene copy number status in breast and prostate
cancer, we first analyzed three prostate (PC-3, DU-145, LNCaP) and four
breast (SK-Br-3, MDA-436, MCF-7, ZR-75-1) cancer cell lines by FISH.
High-level amplification (5 or more copies of the gene or p40 and
centromere ratio >2) of p40 was found in PC-3 (Figure 1B)
, SK-Br-3
(Figure 1C)
, MDA-436, and in MCF-7, in concordance with the gain of 8q
found by CGH in these cell lines.5,18
The amplification of
eIF3-p40 gene in SK-Br-3 was also confirmed by Southern analysis
(Figure 1C
, inset). Figure 1, D and E
, illustrates examples of
high-level amplifications of eIF3-p40 in prostate and breast carcinomas
determined by FISH. Table 1
summarizes
the results in clinical tumor material obtained with FISH. Thirteen of
44 (30%) of the hormone-refractory prostate carcinomas showed
high-level amplification of p40, and the remaining cases showed a
low-level copy number gain (3 to 4 copies) of p40. In prostate tumors
with amplification, the mean (± SD) copy number of p40 was 6.7 (±
1.5). Seven of 39 (18%) of the breast cancers showed high-level
amplification of p40, 17 of 39 (43%) showed a low-level gain, and the
remaining 15 tumors (39%) showed two copies of the p40. The mean copy
number of p40 was 8.5 (± 2.9) in the breast tumors with amplification.
Next, we analyzed 19 selected breast carcinomas with high-level
c-myc amplification demonstrated by Southern
blot.15
Sixteen of these tumors showed high-level
amplification of p40 with the mean copy number 21.8 (± 21.12).
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To analyze coamplification of eIF3-p40 and c-myc
oncogene, copy number of c-myc was also studied in the same
samples by FISH. In breast and prostate cancer cell lines the copy
numbers of c-myc and p40 were identical, except in PC-3,
where p40 was present in 15 copies and c-myc in 9 copies per
cell. All hormone-refractory prostate carcinomas showed similar copy
number of p40 and c-myc. One of the unselected breast
carcinomas showed high-level amplification of c-myc, but
only a low-level amplification of p40. Three selected breast carcinomas
showed ~5 times higher copy number of p40 than c-myc,
whereas one case showed ~2 times more c-myc than p40
signals (Figure 2)
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The amplification of the target proto-oncogenes is thought to lead
to their overexpression. We compared the expression levels of p40 and
c-myc in cancer cell lines using Northern blot analysis.
Whereas there was no clear association between the expression and
amplification status of c-myc, the expression of p40 was
related to its gene copy number (Figure 3A)
. The expression of p40 was examined
in prostate and breast tumors with semiquantitative mRNA in
situ hybridization (Figure 3, B–E)
. The hormone-refractory
prostate carcinomas expressed over four times more p40 than benign
prostate hyperplasia tissues (Figure 4)
(Mann-Whitney U test; P = 0.0021). The level
of p40 expression was higher in breast carcinomas with high-level
amplification than low-level or no amplification (Kruskal-Wallis test;
P = 0.028).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The well-characterized proto-oncogene c-myc and eIF3-p40 were coamplified in a vast majority of tumors and cancer cell lines studied. The copy number for both genes was also similar in most tumors with coamplification. Thus, the amplicon either extends from eIF3-p40 to c-myc (approximately 12 Mb) or consists of several separate amplification peaks with nonamplified DNA in between, as has been described in 20q12-q13 amplification in breast cancer.19 Whatever the structure of the 8q23-q24 amplicon, it is clear based on the present study that the amplification contains several target genes. Thus, the amplification resembles those in 11q13, 17q23-q25, and 20q12-q13, where several target genes are often coamplified.13,19-21
Many genes may also be amplified simply because they are located near the target gene. The commonly used criterion for a putative target oncogene is that the amplification of the gene leads to its overexpression.22-25 Therefore, we used SSH to identify transcripts that were overexpressed in the SK-Br-3 cancer cell line containing high-level amplification of 8q21 and 8q23-q24 regions. After identification of the eIF3-p40 by SSH, we confirmed differential expression pattern by Northern blot in the cancer cell lines. Subsequently, we screened more breast and prostate cancer cell lines for eIF3-p40 copy number and expression. The cancer cell lines containing high-level amplification expressed more of the gene than those with no amplification. Because the clinical material consisted of paraffin-embedded tumor blocks, we chose to use mRNA in situ hybridization to detect the gene expression in breast and prostate tumors. We have previously shown that mRNA in situ hybridization is a reliable technique for the quantitation of gene expression in formalin-fixed, paraffin-embedded tissue.26 Breast carcinomas with the high-level amplification expressed significantly more p40 than tumors with low-level or no amplification, although there were individual cases with high-level expression without the gene amplification. The hormone-refractory prostate carcinomas expressed more p40 than BPHs. However, significant differences between the p40 expression levels in prostate tumors with high- and low-level amplification were not observed. Altogether, the results suggest that gene amplification is not the only mechanism that leads to overexpression of the eIF3-p40 gene. Alterations occurring in the regulation of eIF3-p40 expression could also be involved in the overexpression of the gene. Similarly, it has previously been shown that overexpression of ERBB2 oncogene, which is usually due to the high-level amplification of the gene, can also take place without the gene copy number changes.27 Nevertheless, the expression studies demonstrated that all tumors containing eIF3-p40 amplification expressed the gene at high levels. There was also a trend between increased copy number of eIF3-p40 and enhanced expression of the gene, as would be expected for the true target gene of the amplification.
In the prostate cancer and most of the breast cancers the gene copy number of p40 often remained relatively low (<10 copies) when compared with copy numbers reported, eg, in 20q13 amplification in breast cancer.19 This may be due to the fact that, according to CGH studies, the 8q amplification usually comprises the whole chromosome arm. It may well be that even a few additional copies of p40 (and c-myc) are enough to cause overexpression and thereby contribute to the tumorigenesis. However, in the selected breast cancer material found to contain c-myc amplification by Southern analysis,15 very high copy numbers of p40 (50–100 copies) were also found. Three tumors showed clearly higher copy number of eIF3-p40 than c-myc, suggesting that the genes may belong to separate subamplicons. However, the fact that the most frequent finding was coamplification with equal copy numbers suggests that both genes may contribute to and perhaps cooperate in the progression of breast and prostate cancer.
The finding that eIF3-p40 is amplified in breast and prostate cancer
suggests a new functional category of amplified oncogenes in breast and
prostate cancer. Most of the known oncogenes are involved in signal
transduction (eg, EGFR, erbB-2, ras), regulation of
transcription (eg, fos, jun, myc), or cell cycle regulation (cyclinD1).
Several lines of recent evidence imply that aberrant regulation of
translation could also be involved in the tumorigenesis. Overexpression
of initiation factors eIF-4E and eIF-4G have been shown to transform
NIH 3T3 cells as well as rat embryo fibroblasts in collaboration with
v-myc or E1A.28-30
It has also been suggested
that the mitogenic and oncogenic activities of eIF-4E are mediated by
ras oncogene.31
On the other hand, the
expression of eIF-4E and eIF-2 are regulated by
c-myc.32
Thus, amplified c-myc may
increase cell growth by regulating the expression of these translation
initiation factors. Translational apparatus also contains elongation
factors. The constitutive expression of elongation factor 1
(EF-1) causes cells to become susceptible to transformation by UV
light or 3-methylcholantrene.33
In addition, the PTI-1
gene, which, based on sequence analysis, is a mutated and truncated
form of EF-1, was recently cloned from LNCaP prostate cancer cell
line using differential RNA display.34
There is limited
in vivo evidence on the role of translation initiation
factors in the development and progression of cancer. Allelic imbalance
of eIF3-p48 subunit, which is encoded by INT6, has been detected in
breast carcinomas.35,36
Amplification of eIF-4G has been
found in a few squamous cell lung carcinomas.37
In
addition, overexpression of eIF-4E has been detected in breast cancer
and it may be associated with the recurrence of the
disease.38
The eIF3-p40 gene, which we found amplified and overexpressed in breast and prostate cancer, has not been implicated in the development or progression of cancer before. It is a subunit of the largest (~600 kd) eukaryotic translation initiation factor protein complex, which has a central role in the initiation of translation. eIF3 complex binds to 40S ribosomal subunits in the absence of other initiation factors and preserves the dissociated state of 40S and 60S ribosomal subunits. It also stabilizes eIF2-GTP-Met-tRNA binding to 40S subunits and mRNA binding to ribosomes.39 Very little is known about the p40 subunit itself. Based on the sequence homology, it seems to be related to mouse protein Mov-34.17 The gene product of human homologue of Mov-34 is a component of the 26S proteasome. However, due to the fragmentary nature of the current knowledge, it is not possible to draw conclusions about how eIF3-p40 could be involved in the tumorigenesis. Therefore, functional studies, in addition to studies aiming at defining the clinical significance, are required to elucidate the role of eIF3-p40 in the progression of breast and prostate cancer.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Pirkanmaa Cancer Society, the Irja Karvonen Cancer Foundation, the Cancer Society of Finland, the Reino Lahtikari Foundation, Academy of Finland, the Medical Research Fund of Tampere University Hospital and CaPCURE.
Accepted for publication March 12, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to growth induction by c-myc. Proc Natl Acad Sci USA 1993, 90:6175-6178 determines susceptibility to transformation. Nature 1992, 359:333-336[Medline]
is encoded by an amplified gene and induces an immune response in squamous cell lung carcinoma. Hum Mol Genet 1997, 6:33-39This article has been cited by other articles:
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