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From the Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan
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Introduction |
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In the present study, to identify and isolate differentially expressed genes during oxystress-induced renal carcinogenesis, we have developed and used modified fluorescent differential display (FDD) technique. We identified 15 genes that revealed either significant increase, decrease, or lack of expression, and also found five novel transcripts. Most of the identified genes were associated either with stress-response or cellular proliferation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RCCs were induced in F1 hybrid rats between Wistar strain (Shizuoka Laboratory Animal Center, Shizuoka, Japan) and Long-Evans strain (originally outbred from Ben May Laboratory for Cancer Research, University of Chicago, Chicago, IL) by administrating Fe-NTA as previously described.13-15 RCCs or nontumorous renal tissues were carefully dissected with razor blades and kept frozen at -80°C until use. FRCC001 cell line was established from a Fe-NTA-induced renal cell carcinoma (F1 hybrid rat, No. 10-21-4, grade-2, granular cell subtype, solid structure, INF-ß with lung metastasis)15 using a procedure previously described,16 and were grown in Dulbecco’s modified Eagle’s medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and antibiotics/antimycotics (GIBCO; penicillin G 100 U/ml; streptomycin sulfate 100 µg/ml; amphotericin B 0.25 µg/ml). This cell line formed typical epithelial monolayers on plastic, revealed anchorage-independent growth after confluence, and proliferated well in the subcutis of nude mice with no evidence of metastasis.
RNA Isolation and Northern Blotting
Total RNA was isolated from each frozen tissue or cells by means
of a modified acid guanidinium/phenol/chloroform method (Isogen, Nippon
Gene, Tokyo). Poly(A)-rich RNA was isolated by oligo(dT)-latex beads
(Nippon Roche, Tokyo). For Northern blot analysis, poly(A)-rich RNA
sample (2 µg) was separated on 1% agarose gel containing
formaldehyde and transferred onto nylon membrane as previously
reported.17
We used as probes the isolated cDNA clones by
FDD, which were labeled with [
-32P]dCTP by
Megaprime DNA labeling system (Amersham Pharmacia Biotech, Tokyo).
Autoradiography was performed using an imaging plate (Fuji Film, Tokyo)
and the visualization and quantitation were done with BAS2000 (Fuji
Film). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used
as a standard for quantitation.
Differential Display
Total RNA was treated with RNase-free DNase using a Message clean kit (GenHunter Corp., Brookline, MA). Random oligonucleotide primers (20–22 mer) synthesized were used as arbitrary primers. Anchor primers were synthesized as follows: 5'-CCCGGATCC (dT)15N-3' (where N is C, G, or A; this primer was designed to include BamHI site) and were labeled with rhodamine at the 5'-end of the oligonucleotides. A 500-ng aliquot of total RNA was reverse transcribed in 1x reverse transcriptase buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl) with 5 mmol/L MgCl2, 1 mmol/L deoxynucleotide phosphates (dNTP), 20 U RNase inhibitor, and 50 pmol of each anchor primer. Each sample was incubated for 10 minutes at 70°C and then cooled on ice. This was followed by addition of 1.5 mU avian myeloblastosis virus reverse transcriptase (Takara Bio, Shiga, Japan) in a total volume of 20 µl, which was incubated at 30°C for 10 minutes and at 55°C for 30 minutes. Reverse transcriptase was inactivated by incubation at 95°C for 5 minutes. For each PCR reaction, 2 µl of cDNA was dissolved in a volume of 20 µl in PCR buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2) in the presence of 0.1 mmol/L dNTP, 10 pmol of arbitrary primer, 5 pmol of anchor primer, and 0.5 U Taq polymerase (Takara Bio). PCR conditions were 94°C for 5 minutes, 37°C for 5 minutes, 72°C for 5 minutes, which was followed by 34 cycles of 94°C for 30 seconds, 37°C for 2 minutes, and 72°C for 1 minute. A final extension reaction was carried out at 72°C for 5 minutes. Five microliters of each PCR product was analyzed by electrophoresis in 7 mol/L urea/4% polyacrylamide gel at 40 W constant power for 2 to 3 hours. Gels were visualized with a fluorescence image analyzer (FMBIO-100, Takara Bio). The fragments showing differential expression were excised from the gel with razor blade, extracted by heating in water, and then reamplified by PCR using the corresponding primer pair.
Cloning and cDNA Sequencing of Fragments Obtained by Differential Display
An aliquot of reamplified cDNA was checked for a single band by urea/polyacrylamide gel electrophoresis. In case of multiple bands, the procedures were repeated until single band was obtained. Then the rest of the product was digested with BamHI (Takara Bio) to remove rhodamine labeling as well as to obtain cohesive end, and was loaded to 1% agarose gel. The band was carefully excised and extracted from the gel by the use of QIAEX II gel extraction kit (QIAGEN, Tokyo). The product was finally cloned into the pBluescript II SK(+) vector (Stratagene, La Jolla, CA) after treating the vector first with HincII digestion (Takara Bio), then with terminal deoxynucleotidyl transferase (Takara Bio) for adding dT at the blunt end and finally with BamHI. The plasmid DNAs were sequenced with the AmpliTaq Cycle Sequence kit (Perkin Elmer, Foster City, CA) according to the manufacturer’s protocol using an ABI PRISM 377 DNA sequencer. The homology search was performed with Genomenet World Wide Web server (http://www.genome.ad.jp).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To isolate genes differentially expressed in oxystress-induced rat RCCs, we have used five samples as a set: cortical portion of normal kidney (1-year-old F1), cortical non-neoplastic portion of Fe-NTA-treated kidney (1-year-old), two independent RCCs (a grade 2 RCC with lung metastasis and a grade 1 RCC with no metastasis), and a RCC cell line (FRCC001) established from Fe-NTA-induced RCC of grade 2. Total RNA was isolated from each sample and was reverse transcribed to cDNA. Using three kinds of anchor primers in combination with 280 arbitrary primers, an average of approximately 100 cDNA transcripts was amplified and displayed in each lane of the gel. Thus, in total, approximately 84,000 clones were compared on display for the set of samples.
Our strategy was to select only clones that showed different expression
pattern between the former two samples (normal control and
non-neoplastic after Fe-NTA administration) and the latter three
samples (two RCC tissues and one RCC cell line) to avoid false positive
clones. We found a total of 290 clones to be differentially expressed.
Examples of representative FDD runs are shown in Figure 1
. Each band was carefully excised from
the gel by a razor blade, purified, and reamplified. Among these, 142
clones were successfully subcloned and sequenced. Using each cDNA clone
as a probe, 72 selected clones (more than fivefold increase or decrease
by FDD) were subjected to reverse Northern blotting for which a
different set of samples (normal renal cortex of a 1-year-old male F1
rat, renal cortex of a 12-week-old male Wistar rat after 6 weeks of
repeated Fe-NTA administration, two pairs of RCCs and their nontumorous
counterparts: a grade 1 RCC with no metastasis and a grade 3 RCC with
lung metastasis and peritoneal invasion) were prepared (Figure 2)
. Finally, 20 cDNA clones were
confirmed to be differentially expressed (Figure 1, A–F
, and Figure 2, A–F
); the remaining cDNA clones either showed no signal or difference
in expression by Northern blots or exhibited no difference between RCCs
and its non-neoplastic counterparts. These were interpreted as false
positive and excluded from the present study.
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Sequence analyses revealed clones 28, 29, 256, 418, and 435 as
novel gene fragments (Figure 3)
. Clone 28
was overexpressed, whereas the others were deleted in expression in the
Fe-NTA-induced RCCs. Homology search through GenBank and EMBL databases
using BLAST programs revealed no significant homology with any
published genes in the five clones. Each clone showed single band by
Northern blots; the sizes were 2.6 kb in clone 28, 2.8 kb in clone 29,
1.6 kb in clone 256, 2.0 kb in clone 418, and 2.4 kb in clone 435. All
of the fragments were flanked by DNA sequence of the corresponding
oligo(dT) primer and arbitrary primer used for FDD and further revealed
polyadenylation signal sequence (AAUAAA) upstream of the putative polyA
tail.
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In the present FDD study, we have identified 15 genes as
differentially expressed. Increased in the Fe-NTA-induced RCCs were
homologues of Y-box binding protein, heat shock protein (HSP) 86, DNA
polymerase catalytic subunit and glycoproteins of 96 kd (gp96);
ribosomal proteins (RPs) S13, S15 and L19; non-muscle caldesmon and
annexin II (calpactin I heavy chain) as shown in Table 1
. We estimated the increase of
expression in the RCCs as 7.8- to 14.3-fold (1.3- to 2.3-fold) for the
homologue of Y-box binding protein, 1.7- to 1.8-fold (0.93- to
1.4-fold) for the homologue of HSP 86, 2.8- to 4.0-fold (0.8- to
1.5-fold) for RP S15, 3.1- to 3.3-fold (1.4- to 2.7-fold) for RP S13,
1.7- to 2.4-fold (1.1- to 1.3-fold) for RP L19, and 2.3- to 2.6-fold
(1.6- to 1.8-fold) for the homologue of gp96 as compared with a control
sample of normal age-adjusted kidney. (Data for kidney after 6 weeks of
repeated Fe-NTA administration are shown in parentheses.) Expression of
the homologue of DNA polymerase catalytic subunit , nonmuscle
caldesmon (Figure 2C)
and annexin II in the samples of non-neoplastic
kidneys was under the detection limit of Northern blot.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and found five novel transcripts (Figure 3)
. Y-box binding protein (YB-1) is a member of DNA-binding protein family with a unique cold shock domain, which is highly conserved from prokaryotes to eukaryotes.18 YB-1 regulates gene expression through binding to Y-box sequence, 5'-CTGATTGG-3', which is contained in the cis-regulatory elements of several genes, including thymidine kinase, cyclin-dependent kinase 1, proliferating cell nuclear antigen (PCNA), DNA polymerase, epidermal growth factor receptor, multidrug resistance 1 (MDR1),19,20 and OxyR, a thiol-containing transcriptional activator21 whose redox status controls the expression of downstream genes.22 YB-1 is an integral component of redox signaling pathway. Recently it was reported that cisplatin resistance was proportionally associated with the expression of YB-1,23 that YB-1 binds preferentially to cisplatin-modified DNA24 and further that increased amounts of nuclear YB-1 was associated with a poor prognosis of ovarian cancer.25 Therefore, it is possible that cells with high expression of YB-1 were selected during carcinogenesis in consideration of the fact that cancer cells, especially adenocarcinoma, are exposed to persistent oxidative stress.26
Ribosomes catalyze protein synthesis with a complex structure including both RNA and protein components. There is increasing evidence that the ribosomal RNA molecules play a central role in its catalytic activities; many of the ribosomal proteins have been relatively poorly conserved, so it has been suggested that the ribosomal proteins mainly enhance the function of the ribosomal RNAs. Overexpression of RP S13 is observed in actively growing cells such as human colorectal carcinoma,27 and overexpression of RP L19 was reported in human breast tumors that overexpress erbB-2.28 There are many reports on the overexpression of other RPs in human cancer: L31 in colorectal tumors,29 P0 in hepatocellular carcinoma and colon carcinoma,30 and L7a and L37 in prostate cancer.31 As far as we know, there are no data available that deal with the association of RP S15 and tumors. Moreover, it was reported that overproduction of mycobacterial RP S13 induces catalase/peroxidase activity, presumably as a stress response which, in this situation, may cause hypersensitivity to isoniazid in Mycobacterium smegmatis.32 Our results support the idea that increased expression of certain ribosomal proteins is associated with oxidative stress and is a common feature of cancer.
Glycoprotein of 96 kd (gp96) and heat shock protein (HSP) 86 belong to the HSP family. Exposure to stress such as heat shock, oxygen deprivation, and calcium or glucose limitation results in the cellular induction of HSPs. These two proteins are involved in stress response, working as a molecular chaperon in the cytoplasm. For example, irradiation and interleukin-6 administration induced gp96 in human cervical and breast cancers, respectively.33,34 These two proteins are coexistent with a broad range of antigen peptides derived from that particular cell and are named as tumor rejection antigen for gp 9635 or tumor-specific transplantation antigen for HSP86,36 and autologous tumor-derived gp96 was used successfully for immunotherapy of tumors.35 HSP86 has been constitutively induced in murine embryonal carcinoma, but is decreased on differentiation,37 suggesting induction of these proteins as a common molecular event in cancers.
Annexin II (also known as calpactin I, p36, or lipocortin II), a
Ca2+-binding protein, is a substrate for an
oncogene growth factor-associated protein tyrosine kinase. Increased
expression of annexin II is observed in pancreatic adenocarcinoma of
Syrian hamsters,38
human hepatocellular
carcinoma,39
human brain tumor,40
and cell
lines of hereditary RCC from Eker rats.41
Recent evidence
indicates a role for annexin II in DNA synthesis by interacting
with DNA polymerase ,42
cell
proliferation,43
and differentiation of F9 teratocarcinoma
cells.44
Moreover, it has been reported that expression of
annexin II was induced in several sets of transformed cells
irrespective of the nature of oncogene products, including v-H-ras-,
v-mos-, and SV40-transformed cells.45
Nonmuscle caldesmon, an actin-binding and phosphorylation-activated
protein, is closely associated with mitosis; caldesmon stays
dissociated from microfilaments from prometaphase until at least the
early stages of cytokinesis, suggesting that continued dissociation of
caldesmon may be required for mitosis-specific organization of
microfilaments, including disassembly of stress fibers and formation
and activation of contractile rings for cell division.46
In this context, induction of DNA polymerase catalytic subunit and
down-regulation of protein-tyrosine phosphatases (PTPases) were
consistent in the metabolism of RCCs in the present study.
PTPases regulate tyrosine phosphorylation, which plays a key role in cellular proliferation, differentiation, and oncogenesis.47,48 PTPases can be structurally classified into transmembrane and non-transmembrane isozymes.48 One of the functions of PTPases is to reverse the effect of protein tyrosine kinases, many of which are oncogenes, suggesting that they may act as tumor suppressors.49 Indeed, recently it was reported that expression of PTPase were not observed in 55% of grade 4 glioblastomas, whereas all grade 1 gliomas showed expression50 and were dramatically decreased in lung adenocarcinomas.51 Furthermore, PTPases were found to play a role in cell-to-extracellular matrix communication as well as cell-to-cell communication.52 Our results suggest that decrease in PTPase expression might work for promotion or progression of the initiated cells.
Selenoprotein P is a glycoprotein containing selenocysteine that has been purified from rat and human plasma.53 Selenium is an essential trace element in mammals, and its remarkable biological effect in eukaryotes may be related to a unique function of various selenoproteins such as glutathione peroxidase and thioredoxin reductase.54 Selenoprotein P has been postulated to serve as an extracellular oxidant defense.53 So far there is no report on the association of selenoprotein P and cancer. Deleted expression of selenoproteins in RCCs might lead to more oxidative stress than that of adjacent non-neoplastic tissue, which in turn may contribute to higher oxidative stress and, therefore, genomic instability in cancer.
Trefoil peptides are stable, secreted molecules containing a conserved 6-cysteine motif and are known to be expressed in the gastrointestinal tract and kidney.55 Induction of human spasmolytic polypeptide and porcine spasmolytic polypeptide in the trefoil protein family has been reported in association with peptic ulcers and mucosal injury in inflammatory bowel disease.55 In gastric cell lines, the trefoils were shown to act in a manner suggestive of immediate-early genes capable of auto- and cross-induction through cis-acting regulatory regions: trefoil-mediated transcriptional regulation required activation of the Ras/MEK/MAP kinase signal transduction pathway. Intestinal trefoil factor (ITF) stimulation of gastric cell lines led to phosphorylation of epidermal growth factor receptor.56 Further, it was shown recently that transfection of ITF-3 induced apoptosis in human colon adenocarcinoma cells by perturbing the complexes between E-cadherin, ß-catenin, and associated proteins.57 Although function of this protein in the kidney is still unknown, the same mechanism may work for RCCs. However, we cannot rule out the possibility of "cell population effect," because ITFs were reported to be localized to the collecting ducts of the kidney,58 and the majority of Fe-NTA-induced rat RCCs are thought to arise from proximal tubular epithelial cells.
The mac25 gene is a newly discovered member of the insulin-like growth factor-binding protein (IGFBP) family and renamed as IGFBP-7.59 The IGFBP family regulates the interaction between insulin-like growth factor (IGF)-1 and -2, and their receptors. IGF-1 and -2 play important roles in normal growth and development and have been implicated as growth regulators and potent mitogens in some cancer. It has been reported that decreased expression of mac25/IGFBP-7 during progression of breast carcinomas was associated with allelic loss at human chromosome 4q12–13, where mac25/IGFBP-7 is located.60 This report and our data suggest that this gene may have tumor suppressor or associated function in renal carcinogenesis, and loss of expression may lead to tumor cell growth.
A chemokine, stromal cell-derived factor 1 (SDF-1), is an important regulator of leukocyte and hematopoietic precursor migration and pre-B cell proliferation.61 There are few reports about function and expression of SFD-1 in solid organ and carcinoma. In a recent report, the chemokine receptor CXCR4, which is G-protein-coupled receptor for the CXC chemokine SDF-1, is essential for vascularization of the gastrointestinal tract,62 suggesting SDF-1 and CXCR4 are involved in a new signaling system of organ vascularization. Our results suggest that no expression of SDF-1 mRNA may lead to abnormal angiogenesis in RCCs.
NADH dehydrogenase plays a fundamental role in cellular respiration in both eukaryotes and prokaryotes. This protein is an integral membrane protein whose activity is crucial to the buildup and maintenance of a transmembrane proton gradient, sustained by the reduction of dioxygen to water and by coupling with translocations across the mitochondrial membrane. The genome is present in the mitochondrial DNA. There are reports that expression of NADH dehydrogenase or cytochrome c oxidase is up-regulated in several kinds of cancers.63,64 Our result was the opposite. Of note is the fact that human RCCs show a high incidence of mitochondrial DNA deletion.65 The frequency with which this phenomenon occurs in RCCs, but not in other types of cancers, suggests that this may be an important phenotype associated with renal cell neoplastic transformation. Further study is necessary to elucidate the significance of our results.
The purpose of the present study was to find genes differentially
expressed in oxystress-induced cancer based on a hypothesis that either
alteration in transcriptional control or genomic deletion may
contribute directly to carcinogenesis. We performed a careful selection
and identified 15 different clones by screening approximately 84,000
transcripts. It was interesting to find that most of these clones were
associated with either stress response or cellular proliferation, but
that multiple genetic pathways are apparently involved, as shown in
Table 1
. One transcription factor, YB-1, was included. Most of the
clones could be classified into "information" and
"communication" categories by a proposed genome
classification.66
We believe that these transcriptional
controls of the identified genes or genomic deletion are working in the
promotion and progression processes of carcinogenesis. Further study is
now in progress to differentiate between transcriptional shutoff and
genomic deletion in the genes with no expression in the RCCs, to find
links among the identified genes, and to find earlier changes in
combination with morphological approach. Our data also warrant
investigation of human cancers for the expression of genes reported in
the present study, to test a hypothesis that oxystress-induced cancers
share a common panel of gene expression.
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Acknowledgements |
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Footnotes |
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Supported in part by a Grant-in-Aid from the Japanese Ministry of Education, Science, Sports and Culture, a grant from the Program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN), and a grant from the Japanese Owner’s Association.
Accepted for publication February 9, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J Biol Chem 1991, 266:5169-5176
is a candidate tumor suppressor gene at human chromosome region 3p21. Proc Natl Acad Sci USA 1991, 88:5036-5040This article has been cited by other articles:
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