Cancer Research Annual Meeting 2010 Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
[Cancer Research 59, 5565-5571, November 1, 1999]
© 1999 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohnami, S.
Right arrow Articles by Yoshida, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohnami, S.
Right arrow Articles by Yoshida, T.
[Cancer Research 59, 5565-5571, November 1, 1999]
© 1999 American Association for Cancer Research

Molecular Biology and Genetics

Identification of Genes Showing Differential Expression in Antisense K-ras-transduced Pancreatic Cancer Cells with Suppressed Tumorigenicity1

Shumpei Ohnami, Nobuyuki Matsumoto, Masaru Nakano, Kazunori Aoki, Koichi Nagasaki, Takashi Sugimura, Masaaki Terada and Teruhiko Yoshida2

Genetics Division [S. O., N. M., M. N., K. A., T. S., M. T., T. Y.] and Growth Factor Division [K. N.], National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
K-ras point mutation occurs in >80% of pancreatic cancer. We reported previously that the transduction of an antisense K-ras RNA expression vector suppressed the growth of pancreatic cancer cells with K-ras point mutations in vitro and in vivo. The RNA differential display method (DD) was used to compare the mRNA expression profile of the pancreatic cancer cell line AsPC-1 and that of the antisense K-ras-transduced, growth-retarded AsPC-1 cells. cDNA fragments were isolated from 20 bands on the DD gel, and their differential expression between the two cell lines was confirmed. A sequence analysis revealed that all of the 11 clones up-regulated in the antisense-transduced cells were mitochondrial genes. The other nine cDNA clones that were down-regulated in the antisense-transduced AsPC-1 cells included an oncogene PTI-1 (prostate tumor inducing gene-1), matrix metalloproteinase (MMP)-7, the {beta}3 chain of laminin-5, lysosome-associated membrane protein-2, the H chain of apoferritin, ribosomal protein S6, proteasome subunit XAPC7, and two cDNA fragments with no homology to the GenBank database. In addition to the AsPC-1 cells, reverse transcription-PCR analysis on surgical specimens of pancreatic cancer revealed that the PTI-1 and MMP-7 genes were overexpressed in three and four cases, respectively, of five cases examined. This method offers a unique opportunity to identify a set of genes that may be modulated by K-ras activation, at least in a subset of the pancreatic cancer. The information on such genes may facilitate our understanding of the spectrum of the functional genetic changes in pancreatic cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Various genetic abnormalities accumulate during multistage carcinogenesis. The changes include structural aberrations such as point mutation, amplification, and deletion, as well as functional alterations such as overexpression and down-regulation (1 , 2) . Although the type and incidence of genetic alteration differs according to the type of human cancer, the most striking example may be the mutations of the K-ras gene, the activation of which by point mutation is found at characteristically high frequencies of 70–90% in pancreatic cancer (3, 4, 5, 6) . That mutation incidence is then followed by 40–50% in colon and thyroid tumors and 20–30% in lung cancers. In other types of cancers such as gastric (4 , 7) , esophageal, and liver cancers (4) , however, the mutation is observed at low frequencies or not at all.

Like most other cancers, pancreatic cancer does not seem to arise from a single genetic change; other than K-ras mutation, structural abnormalities have been reported for the p53 (8, 9, 10) , p16 (10) , DPC4 (11) , c-myc (12) , APC (13) , RB1 (14) , BRCA2 (15) , and akt2 (16) genes. Abnormal expression has been noted for basic fibroblast growth factor (17) , c-met (18) , and DCC (9) . However, among the constellation of genetic abnormalities that a single pancreatic cancer cell may harbor, the K-ras mutation appears to be essential for expression of the malignant phenotype, because we reported previously that the expression of antisense K-ras RNA alone effectively suppressed the in vitro and in vivo growth of pancreatic cancer cell lines with K-ras mutation and other genetic abnormalities (19 , 20) . Moreover, the K-ras mutation is considered as an early event in pancreatic carcinogenesis, because it has been found in intraductal pancreatic cancer, ductal hyperplasia, and even chronic pancreatitis (21) .

Considering the almost ubiquitous expression of the K-ras gene, the unusual dependency of pancreatic ductal carcinogenesis on K-ras activation is intriguing but may be explained by one of the following: (a) an unidentified carcinogen/mutagen is uniquely present in the pancreatic tissue and specifically causes K-ras point mutation in the duct; (b) the point mutational activation of this housekeeping gene is too toxic to most cells except a few "K-ras-mediated transformation sensitive" tissues, such as pancreatic duct or colorectal epithelium; or (c) the K-ras mutation leads to the activation of a special set of cancer-related genes only in those "K-ras sensitive" tissues. A signaling cascade downstream of the K-ras protein leading to the mitogen-activated protein kinase activation has been well elucidated. However, if this is the only outcome of the K-ras activation, the K-ras point mutation should have been found in many types of cancers, because the K-ras gene is expressed ubiquitously as a housekeeping gene.

The antisense K-ras-transduced AsPC-1 pancreatic cancer cells with a "revertant" phenotype (19 , 20) offer a unique opportunity to analyze the gene expression that is dependent on the K-ras activation. We identified 20 cDNA fragments that showed a differential expression between the parental and antisense K-ras-transduced AsPC-1 cells. All of the 11 clones up-regulated in the antisense-transduced cells were mitochondrial genes. By contrast, the remaining nine clones that were down-regulated by antisense K-ras effect included an oncogene that has not been described before in the context of pancreatic carcinogenesis, a matrix metalloproteinase gene, and two unknown genes.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Tissue Specimens.
Human pancreatic cancer cell lines AsPC-1, MIAPaCa-2, BxPC-3, PSN-1, Panc-1, Hs 700T, and Hs 766T were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS, 1 mM NaHCO3, 2 mM L-glutamine, and penicillin-streptomycin in a humidified atmosphere of 95% air-5% CO2 at 37°C. The AsPC-1, MIAPaCa-2, PSN-1, and Panc-1 cell lines have mutations in codon 12 of the K-ras gene, whereas the BxPC-3, Hs 700T, and Hs 766T cell lines contain wild-type K-ras genes. The K-ras mutation in the AsPC-1 cells is GGT to GAT transition at codon 12, the mutation observed in >50% of the pancreatic cancer patients in Japan (5 , 6) . AsPC-1-AS (4.3), AsPC-1-AS (4.4), and AsPC-1-AS (4.9) are three independent clones of the AsPC-1 cells transduced with the AS-K-ras-LNSX expression vector. The level of the K-ras p21 protein expression and the growth rate is more suppressed in AsPC-1-AS (4.4) and AsPC-1-AS (4.9) than in AsPC-1-AS (4.3) cells (19) . Samples of pancreatic cancer tissues and corresponding noncancerous tissues were obtained from five patients at surgery at National Cancer Center Hospital. The cancerous and noncancerous portions were macroscopically identified and excised by an experienced pathologist. All patients had histologically confirmed adenocarcinoma of the pancreas. The nonpancreatic cancer cells and tissues used in this study were gastric cancer cells, TMK-1 (22) , KATO III (23) , and MKN28 (24) ; hepatomas, Alexander and HepG2 (American Type Culture Collection); an erythroleukemia, HEL (American Type Culture Collection); HUVEC3 (Clonetics), and the human muscle (Clontech). Exons 1 and 2 of the K-ras gene were sequenced for all those nonpancreatic samples and found to be wild-type (data not shown).

Differential Display Analysis.
Total cellular RNA was extracted from parental AsPC-1 cells and from pooled single-cell clones of antisense K-ras-transduced AsPC-1 cells as described (19) . Twenty-five µg of total RNAs were treated at 37°C for 30 min with 20 units of DNase I (TaKaRa, Kyoto, Japan) in 50 µl of 1x PCR buffer containing 2 units of RNase inhibitor (Promega). The reaction was stopped by extraction with phenol:chloroform (1:1, v/v), and the RNA was ethanol precipitated and resuspended in diethylpyrocarbonate-treated H2O. Single-stranded cDNA was synthesized from 0.5 µg of total RNA by incubation with 200 units of Superscript reverse transcriptase (Life Technologies, Inc.) at 37°C for 60 min in a 50-µl reaction volume containing 100 mM DTT, 250 µM dNTP and 10 µM one-base anchored oligo-dT primers (T11M, where M denotes G, C, or A; Ref. 25 , 26 ). cDNA (0.2 µg) was amplified in a 40-µl reaction volume containing 4 mM anchored primer (T11 G, 5'-TGCCGAAGCTTTTTTTTTTTG-3'; T11 C, 5'-TGCCGAAGC-TTTTTTTTTTTC-3'; or T11 A, 5'-TGCCGAAGCTTTTTTTTTTTA-3'), 4 mM arbitrary primer (LH-A1, 5'-TGCCGAAGCTTACCAGTC-3'; LH-A2, 5'-TGCCGAAGCTTGATCGCT-3'; LH-A3, 5'-TGCCGAAGCTTGAGCCTG-3'; LH-A4, 5'-TGCCGAAGCTTCGAGATC-3'; LH-A5, 5'-TGCCGAAGCTTCGATGCA-3'; LH-A7, 5'-TGCCGAAGCTTGCGATCA-3'; or LH-A10, 5'-TGCCGAAGCTTATGCGAC-3'), 74kBq [32P]dCTP (220 TBq/mmol; Amersham), 40 pmol dNTP, and 2.5 units of AmpliTaq DNA polymerase. The cycling parameters were: 94°C for 1 min, 40°C for 3 min, 72°C for 1 min for three cycles, then 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min for 35 cycles, followed by 72°C for 5 min. Samples of 3.5 µl of amplified cDNA and 2 µl of loading dye were denatured at 80°C for 10 min and electrophoresed on 6% polyacrylamide sequencing gels. After electrophoresis, the gels were transferred to Whatmann 3M paper, dried at 80°C for 2 h, and exposed to X-ray film for 3–5 h at room temperature. The experiment was repeated at least twice, and only the bands that showed a reproducible difference between the parental and antisense K-ras-transduced AsPC-1 cells were further analyzed by excision from the dried gel, reamplified by PCR with the same primer set, and subcloned using the TA cloning kit (Invitrogen).

cDNA Southern Blot Analysis.
The differential expression of the DD clones was confirmed by hybridization to the cDNA pools of the respective cell lines, because it was difficult to obtain a sufficient amount of RNA from the growth-retarded antisense K-ras-transduced AsPC-1 cells without risking the loss of the growth-suppressive transgene expression over an extended passage in vitro. cDNA was synthesized from 5 µg of total RNA using the cDNA Synthesis System Plus (Amersham) with some modification (27) . After the second-strand cDNA synthesis, cDNA was blunt ended at 37°C for 15 min with 8 units of T4 DNA polymerase and ligated to 10 pmol of EcoRI-NotI-BamHI adapter (TaKaRa) at 16°C for 12 h with 350 units of T4 DNA ligase (TaKaRa) in a 20-µl reaction volume containing 10 mM ATP. cDNA was purified by the SpinBind kit (TaKaRa) and amplified by 30 cycles of 1 min at 95°C and 3 min at 72°C. For cDNA Southern blot analysis, 5 or 10 µg of the amplified cDNA were electrophoresed on 1% agarose gels and transferred to nylon membrane filters. Membranes were prehybridized at 42°C for 2 h in a hybridization buffer containing 50% formamide and then hybridized with cloned cDNA probe, labeled with 1.85 MBq of [32P]dCTP using a random hexanucleotide primer at 42°C for 12 h. Membranes were washed in 0.1x SSC/0.1% SDS for 30 min at room temperature, followed by three washes in 0.1x SSC/0.1% SDS for 15 min at 65°C and exposed to X-ray film at room temperature.

DNA Sequencing.
DNA sequencing of cloned cDNA was carried out using T7 and M13 primers, Thermo Sequenase fluorescent labeled primer cycle sequencing kit, and an A.L.F. Express DNA sequencer (Pharmacia LKB, Uppsala, Sweden). The sequences were analyzed by the FASTA program (W. R. Pearson, University of Virginia).

RT-PCR Analysis on Cell Lines.
Amplified cDNA from various sources (AsPC-1, MIAPaCa-2, PSN-1, BxPC-3, Panc-1, Hs 700T, Hs 766T, KATO III, MKN28, TMK-1, HepG2, Alexander, HUVEC, HEL, and human muscle) were synthesized as described above and subjected to PCR. The primer sequences (and expected sizes of the PCR products) were: PTI-1 (28) , 5'-ATGGGGGTAGAGCACTG-3' and 5'-AACACCAGCAG CAACAATCAG-3' (252 bp); MMP-7 (29) , 5'-TCTTTGGCCTACCTATAACTGG-3' and 5'-CTAGACTGCTACCATCCGTC-3' (420 bp); and {beta}-actin (30) , 5'-GACTACCTCATGAAGATCCT-3' and 5'-GCGGATGTCCACGTCACACT-3' (313 bp). PCR amplification for PTI-1 was performed as 30 cycles of 94°C for 45 s, 60°C for 1 min, and 72°C for 1 min. The thermal cycle profile for MMP-7 was 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min. Samples were subjected to electrophoresis on 2% agarose gels and stained with ethidium bromide.

Semiquantitative RT-PCR Analysis on Surgical Specimens.
One µg of total RNA extracted from the tissues was incubated at 25°C for 10 min, 42°C for 60 min, and 70°C for 15 min with 200 units of Superscript reverse transcriptase (Life Technologies, Inc.) in a 40-µl reaction volume containing 10 mM DTT, 1 mM dNTP, 2 units of rRNasin (Promega), and 10 µM random hexadeoxynucleotide primer (TaKaRa, Kyoto, Japan). PTI-1 and MMP-7 expression was examined by semiquantitative RT-PCR based on comparison with the {beta}-actin expression as an internal reference. The 5' ends of the sense primers were labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase, and cDNA from AsPC-1 cells was amplified by PCR for 15–30 cycles with each primer set as described above. The PCR products were separated on 6% polyacrylamide gels, and radioactivity levels of the bands were quantified by Bio-Imaging Analyzer (BAS 2000; Fujix, Tokyo, Japan). The range of linear amplification for both the target (PTI-1 or MMP-7) and the reference {beta}-actin genes was determined, and the optimal number of amplification cycles for semiquantitative PCR was found to be 20. Under this condition, a semiquantitative RT-PCR was performed on surgical specimens using the unlabeled primers. For PTI-1 expression analysis, the PCR products were hybridized with the PTI-1-specific Bridge Region probe (Fig. 2A)Citation to further assure the specificity of the analysis. The relative increase in the band intensity was confirmed using the BAS 2000 imaging analyzer.



View larger version (59K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. A, structure of PTI-1 cDNA and its relationship with the EF-1{alpha} gene and M. hyopneumoniae genome. BR, Bridge Region, is the PTI-1-specific fragment aimed as a target of PCR. B, RT-PCR analysis of Bridge Region to examine PTI-1 expression in parental AsPC-1 cells and the antisense K-ras-transduced AsPC-1 clones. M, 100-bp ladder size marker; Lane 1, parental AsPC-1 cells; Lane 2, AsPC-1-AS (4.4); Lane 3, AsPC-1-AS (4.3). C, cDNA Southern blot analysis of parental AsPC-1 cells and the antisense K-ras-transduced AsPC-1 clones. Probes are: PTI-1 fragment (upper panel) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; lower panel). Lanes 1–3, same as B; Lane 4, parental AsPC-1 cells; Lane 5, AsPC-1-AS (4.9). D, cDNA Southern blot analysis of parental AsPC-1 cells and the antisense K-ras-transduced AsPC-1 clones. Probes are: MMP-7 fragment (upper panel) or GAPDH (lower panel). Lanes 1–5, same as C.

 
Detection of the K-ras Mutations in Surgical Specimens.
The K-ras cDNA fragment was amplified by RT-PCR from total RNAs obtained from surgical specimens of pancreatic cancer as described (19) . Mutations at codons 12, 13, and 61 were subsequently determined by direct sequencing using the Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer Corp., Foster City, CA) and the ABI PRISM 310 DNA sequencer (Perkin-Elmer Corp., Foster City, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DD and cDNA Southern Blot Analysis of Antisense K-ras-transduced AsPC-1 Cells.
The bands that showed significant differences on the DD analysis between the parental and antisense K-ras-transduced AsPC-1 cells were cloned and hybridized to cDNA from the respective cells to confirm the difference in the expression level. The bands marked by an arrow in Fig. 1ACitation were cloned and hybridized to cDNA (Fig. 1, B and C)Citation .



View larger version (115K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. DD (A) and cDNA Southern blot (B and C) analyses of parental AsPC-1 cells (Lanes 1) and the antisense K-ras-transduced clone, AsPC-1-AS (4.4) (Lanes 2). A, arrow, band that showed a significant difference between the parental AsPC-1 cells and antisense K-ras-transduced AsPC-1 cells. Each sample was loaded in duplicated lanes to exclude the electrophoresis artifact. B and C, PCR-amplified cDNA Southern blot analysis gives a smear-like signal. Upper panel, hybridization with the 32P-labeled clones that are up-regulated (B) or down-regulated (C) in the antisense K-ras-transduced probe; lower panel, hybridization with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe.

 
In total, 20 DD clones were found to be differentially expressed before and after antisense K-ras transduction, and their identity is listed in Table 1Citation . Remarkably, all of the 11 clones that were up-regulated in the antisense K-ras-transduced, transformation-suppressed cells were the mitochondrial genes (Fig. 1B)Citation . On the other hand, the genes down-regulated by antisense K-ras included seven known genes (Fig. 1C)Citation and two clones, DD53 and DD134, which showed no homology to the current database. Cloning of full-length cDNAs for the two unknown clones are under way, and we are analyzing the PTI-1 and MMP-7 genes, which have been implicated in transformation and tissue invasion, respectively.


View this table:
[in this window]
[in a new window]

  		Table 1 List of clones that showed significant differences in the level of expression in the antisense K-ras-transduced AsPC-1 cells

 
PTI-1 Expression and cDNA Southern Blot Analysis of PTI-1 and MMP-7.
The reported full-length PTI-1 cDNA has a unique structure consisting of 630-bp 5' UTR with significant homology with Mycoplasma hyopneumoniae 23S rRNA gene fused to a truncated and mutated EF-1{alpha} (31) . DD17 corresponds to the 5' UTR of PTI-1, nucleotide positions 431–605, and it appears that the anchored primer T11C annealed to the sequence 5'-AAAAAG-3', at positions 431–436. To confirm that DD17 represents the PTI-1 oncogene and not the contaminating Mycoplasma, PTI-1-specific RT-PCR primers were used to amplify the "Bridge Region" (28) of PTI-1, which spans the Mycoplasma-homology UTR and truncated EF-1{alpha} coding region (nucleotide positions 537–788; Fig. 2A)Citation . The product of the expected size (252 bp) was amplified from the RNA of the AsPC-1 cells (Fig. 2B)Citation , and its sequence was confirmed to be identical to the sequence of the Bridge Region. The Bridge Region sequence was not detected by RT-PCR in RNAs of the antisense K-ras-transduced clones AsPC-1-AS (4.4) and AsPC-1-AS (4.3) (Fig. 2B)Citation . By cDNA Southern blot analysis, however, the Bridge Region probe weakly hybridized to the amplified cDNA of the AsPC-1-AS (4.3) cells, but not to that of the AsPC-1-AS (4.4) and AsPC-1-AS (4.9) cells (Fig. 2C)Citation . A similar finding also obtained for the MMP-7 (clone 135 in Table 1Citation ) probe (Fig. 2D)Citation . We have reported previously that the degree of p21 K-ras protein suppression and hence the growth inhibition was less in the AsPC-1-AS (4.3) cells than the AsPC-1-AS (4.4) and AsPC-1-AS (4.9) cells (19) . The Bridge Region sequence of the PTI-1 gene was detected in the genomic DNAs from AsPC-1, MIAPaCa-2, BxPC-3, and PSN-1 cell lines as well as the human peripheral blood lymphocytes by PCR, and the amplified bands hybridized correctly to the Bridge Region probe (data not shown).

Expression of PTI-1 and MMP-7 in Pancreatic Cancer Cell Lines and Various Cell Lines.
In contrast to the AsPC-1 cells, the 252-bp Bridge Region fragment of the PTI-1 gene was not detected by RT-PCR in other six pancreatic cancer cell lines examined (MIAPaCa-2, Panc-1, PSN-1, BxPC-3, Hs 700T, and Hs 766T; Fig. 3ACitation ), suggesting that PTI-1 is expressed only in a subset of the pancreatic cancers. A very small amount of multiple PCR products >252 bp were observed in MIAPaCa-2, PSN-1, and Hs 700T cells (Fig. 3A)Citation . Although those bands hybridized to the Bridge Region probe, their significance remains to be shown. In cells other than the pancreatic cancer cells, the PTI-1 expression was detected by RT-PCR in TMK-1 and Alexander cells derived from a gastric cancer and hepatoma, respectively, but it was not detectable in the KATO III and MKN28 (gastric cancers), HepG2 (hepatoma), HEL (erythroleukemia) cell lines, HUVEC, or in human muscle (Fig. 3A)Citation . In addition to AsPC-1, MMP-7 was expressed in two other pancreatic cancer cell lines, Panc-1 and BxPC-3, as well as in a gastric cancer cell line KATO III and HUVEC (Fig. 3B)Citation . We confirmed that BxPC-3, Hs 700T, Hs 766T, and all of the nonpancreatic cells and tissues have a wild-type K-ras gene (data not shown).



View larger version (43K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Expression of PTI-1 (A) and MMP-7 (B) in pancreatic cancer and other cell lines. In A, the PTI-1-specific Bridge Region fragment was detected. {beta}-Actin was analyzed as an internal control for both A and B. M, 100-bp ladder size marker; Lane 1, AsPC-1; Lane 2, MIAPaCa-2; Lane 3, PSN-1; Lane 4, BxPC-3; Lane 5, Panc-1; Lane 6, Hs 700T; Lane 7, Hs 766T; Lane 8, KATO III; Lane 9, MKN28; Lane 10, TMK-1; Lane 11, HepG2; Lane 12, Alexander; Lane 13, HUVEC; Lane 14, HEL; Lane 15, human muscle.

 
Expression of PTI-1 and MMP-7 in Surgical Specimens.
To explore the involvement of the PTI-1 and MMP-7 gene activation in human pancreatic cancer in vivo, five surgical specimens were analyzed by semiquantitative RT-PCR. The PTI-1 expression was elevated in cancerous pancreatic tissues as compared with the noncancerous tissues in patients #2 (GGT->GAT mutation in codon 12), #3 (GGT->GAT), and #4 (no mutation in codon 12, 13, or 61 of the K-ras gene). PTI-1 expression was not increased in cancerous tissues of patients #1 (GGT->CGT) and #5 (GGT->CGT) (Fig. 4A)Citation . MMP-7 expression in the cancerous tissues were higher than the normal counterparts in all of the cases except patient #4 (Fig. 4B)Citation .



View larger version (56K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. K-ras point mutation status (A) and expression of PTI-1 (B) and MMP-7 (C) in human pancreatic cancer tissues. In B, the Bridge Region fragments amplified in the exponential phase of PCR were transferred onto a nylon membrane and hybridized with a 32P-labeled Bridge Region probe. wt, wild-type K-ras sequence. For cases 1 to 5, N and T, noncancerous and cancerous tissues, respectively. M,R 100-bp ladder size marker.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is noteworthy that all of the 11 DD clones that were up-regulated in the antisense K-ras-transduced AsPC-1 cells turned out to be mitochondrial genes, such as the 16S rRNA, cytochrome oxidase, or NADH dehydrogenase genes. One of the hallmarks of cancer cells is their propensity to depend on glycolytic metabolism and diminution in mitochondrially mediated oxidative phosphorylation, and it was demonstrated previously that highly glycolytic tumor cells had a markedly deficient mitochondrial content (32) . In colon cancers, even the cancer risk and progression in vivo were found to be associated with an abnormally low expression of the mitochondrial cytochrome c oxidase subunit 3 (33) . Although no previous study has reported the down-regulation of mitochondrial genes during the course of pancreatic carcinogenesis, two ultrastructural studies, one on human pancreatic carcinomas (34) and the other on hamster pancreatic carcinomas (35) , described a paucity of mitochondria as a feature of the pancreatic duct adenocarcinoma. However, the role of mitochondria appears more complex, and the cancer phenotype, at least a part of it, depends on the multifunctional organelles (36 , 37) .

More recently, mitochondrion was found to be the reservoir for key components involved in apoptosis (38) , probably playing a pivotal role in coordinating cell proliferation and death. Our data suggest that alteration of the mitochondrion is one of the major elements in K-ras-induced transformation in AsPC-1 cells. It remains to be shown whether other pancreatic cancer cells with or without K-ras mutation show a similar change in mitochondrial gene expression during their carcinogenic transformation.

Among the genes that were down-regulated in the antisense K-ras-transduced AsPC-1 cells, we focused first on the PTI-1 gene, because it is an oncogene that has not been implicated in pancreatic cancer before. The antisense K-ras-induced suppression of PTI-1 gene expression was observed in three independent antisense-transduced clones, and the suppression of PTI-1 gene expression was greater in the clones AsPC-1-AS (4.4) and (4.9), which showed a higher degree of K-ras p21 protein suppression. Moreover, infection of the AsPC-1 cells with an antisense K-ras RNA expression adenovirus vector induced growth suppression and decreased expression of the PTI-1 gene as compared with the parental AsPC-1 cells.4 Thus, we conclude that the PTI-1 gene expression is indeed functionally modulated by the K-ras activation status in the AsPC-1 cells, and the clonal variation is not the reason for the decreased PTI-1 expression in the three transfected AsPC-1 clones.

PTI-1 was originally isolated as a transforming gene from human prostate carcinoma DNA (39) . PTI-1 has a unique fusion structure consisting of the 5' UTR with a high homology to the 23S rRNA gene from Mycoplasma hyopneumoniae, juxtaposed to a sequence that encodes a truncated and mutated human EF-1{alpha} (31) . A genomic PCR using primers to amplify the 5'-untranslated, Mycoplasma-homology region of PTI-1 demonstrated that PTI-1 is present in human brain and kidney tissues (28) . Moreover, our PCR-Southern blot analysis showed the presence of the Bridge Region, which spans the 5' Mycoplasma region and the 3' EF-1{alpha} region of PTI-1 in genomic DNAs obtained from human pancreatic cancer cell lines and peripheral blood mononuclear cells. These genomic analyses indicate that the transforming gene PTI-1 is an authentic human gene.

In addition to prostate cancer, PTI-1 expression has been reported in breast, colon, and lung carcinoma cell lines (28 , 31) . Our study has added the PTI-1 gene to the list of the possible genes involved in pancreatic carcinogenesis. It seems that the PTI-1 gene is activated only in a subset of pancreatic cancer; three of five surgical specimens of pancreatic cancer showed increased PTI-1 gene expression, and one of the three cases had no K-ras mutation at codons 12, 13, or 61. The elevated expression of the gene should not be the result of the relative enrichment of ductal cells in the cancerous tissues, because six cell lines and two similar surgical specimens of pancreatic ductal carcinoma showed low or undetectable PTI-1 gene expression. The role of PTI-1 in the pancreatic carcinogenesis may be a modulation of the cancer phenotype, the nature of which awaits further studies.

Another apparently cancer-related gene in Table 1Citation was MMP-7, one of the matrix metalloproteinases implicated in cancer invasion. Recently, a study showed that mRNA expression of MMP-7 was higher in pancreatic carcinoma than in the normal pancreas (40) , an observation congruous to our data. As in the case with PTI-1, MMP-7 expression was also observed in the cells without K-ras mutation, and some pancreatic cancer cells with K-ras mutation did not show an increased MMP-7 transcription, suggesting the presence of a molecular pathway other than the K-ras mutation to modulate the expression of these genes.

Laminin-5 is also considered to play a role in tissue invasion of cancer cells. The anchoring filament protein has been shown to be synthesized and deposited by pancreatic cancer cells in the basement membrane in an abnormal manner (41) . In contrast to MMP-7 and laminin-5 {beta}3, the other four known genes found to be down-regulated in the antisense-transduced cells have not been described in the context of pancreatic carcinogenesis. However, available information suggests that involvement in a malignant phenotype is also conceivable for the lysosomal membrane-associated glycoproteins 1 and 2 (42, 43, 44) , apoferritin (45) , ribosomal protein S6 (46) , and proteasome subunit XAPC7 (47 , 48) .

In sum, 20 clones have been isolated from the DD gel to compare the parental and growth-suppressed, antisense K-ras-transduced pancreatic cancer cells. Eighteen clones turned out to be known genes, and two of them, those encoding MMP-7 and the laminin-5 {beta}3 chain, have been reported by others to be overexpressed in pancreatic cancers. The other 16 clones corresponded to nine distinct genes, including four mitochondrial genes. A literature survey suggests that all of them could play a role in cancers and/or cellular transformation, although no report except the present study has suggested their involvement in pancreatic cancers.

On the other hand, this analysis also revealed a heterogeneity of the pancreatic cancer cells, because K-ras-induced overexpression of the PTI-1 and MMP-7 genes was not a universal phenomenon among the pancreatic cancers; some pancreatic cancer cells and tissues with K-ras mutation did not overexpress those genes, and some with the wild type K-ras gene appeared to have increased expression. Although it is conceivable that PTI-1 and MMP-7 contribute to the expression of a certain phenotype in a subset of pancreatic cancers, they may not belong to a set of genes that are crucial and indispensable for the process of pancreatic carcinogenesis. The biological significance of those genes in the pancreatic cancer should be determined by further investigation.

The experimental strategy of antisense K-ras transduction to pancreatic cancer cells and mRNA DD has been generating more clones that await characterization, and it may enhance our understanding of the oncogenic mechanisms of K-ras activation and/or pancreatic carcinogenesis.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by a Health Sciences Research Grant on Human Genome and Gene Therapy from the Ministry of Health and Welfare of Japan (MHW), by a grant-in-aid for the 2nd Term Comprehensive 10-Year Strategy for Cancer Control from MHW, by grants-in-aid for Cancer Research from MHW, and from the Ministry of Education, Science, Sports and Culture of Japan. Back

2 To whom requests for reprints should be addressed, at Genetics Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. Back

3 The abbreviations used are: HUVEC, human umbilical vein endothelial cell; dNTP, deoxynucleotide triphosphate; RT-PCR, reverse transcription-PCR; PTI-1, prostate tumor-inducing gene 1; MMP, matrix metalloproteinase; DD, differential display; UTR, untranslated region; EF, elongation factor. Back

4 Unpublished observation. Back

Received 3/23/99. Accepted 9/ 8/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sugimura T. Multistep carcinogenesis: a 1992 perspective. Science (Washington DC), 258: 603-607, 1992.[Abstract/Free Full Text]
  2. Howe J. R., Conlon K. C. The molecular genetics of pancreatic cancer. Surg. Oncol., 6: 1-18, 1997.[Medline]
  3. Almoguera C., Shibata D., Forrester K., Martin J., Arnheim N., Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell, 53: 549-554, 1988.[Medline]
  4. Bos J. L. ras oncogenes in human cancer: a review. Cancer Res., 49: 4682-4689, 1989.[Abstract/Free Full Text]
  5. Mariyama M., Kishi K., Nakamura K., Obata H., Nishimura S. Frequency and types of point mutation at the 12th codon of the c-Ki-ras gene found in pancreatic cancers from Japanese patients. Jpn. J. Cancer Res., 80: 622-626, 1989.[Medline]
  6. Motojima K., Urano T., Nagata Y., Shiku H., Tsunoda T., Kanematsu T. Mutations in the Kirsten-ras oncogene are common but lack correlation with prognosis and tumor stage in human pancreatic carcinoma. Am. J. Gastroenterol., 86: 1784-1788, 1991.[Medline]
  7. Lee K. H., Lee J. S., Suh C., Kim S. W., Kim S. B., Lee J. H., Lee M. S., Park M. Y., Sun H. S., Kim S. H. Clinicopathologic significance of the K-ras gene codon 12 point mutation in stomach cancer. An analysis of 140 cases. Cancer (Phila.), 75: 2794-2801, 1995.[Medline]
  8. Ruggeri B. A., Huang L., Berger D., Chang H., Klein-Szanto A. J., Goodrow T., Wood M., Obara T., Heath C. W., Lynch H. Molecular pathology of primary and metastatic ductal pancreatic lesions: analyses of mutations and expression of the p53, mdm-2, and p21/WAF-1 genes in sporadic and familial lesions. Cancer (Phila.), 79: 700-716, 1997.[Medline]
  9. Simon B., Weinel R., Hohne M., Watz J., Schmidt J., Kortner G., Arnold R. Frequent alterations of the tumor suppressor genes p53 and DCC in human pancreatic carcinoma. Gastroenterology, 106: 1645-1651, 1994.[Medline]
  10. Kaino M. Alterations in the tumor suppressor genes p53, RB, p16/MTS1, and p15/MTS2 in human pancreatic cancer and hepatoma cell lines. J. Gastroenterol., 32: 40-46, 1997.[Medline]
  11. Hahn S. A., Schutte M., Hoque A. T., Moskaluk C. A., da Costa L. T., Rozenblum E., Weinstein C. L., Fischer A., Yeo C. J., Hruban R. H., Kern S. E. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science (Washington DC), 271: 350-353, 1996.[Abstract]
  12. Yamada H., Sakamoto H., Taira M., Nishimura S., Shimosato Y., Terada M., Sugimura T. Amplifications of both c-Ki-ras with a point mutation and c-myc in a primary pancreatic cancer and its metastatic tumors in lymph nodes. Jpn. J. Cancer Res., 77: 370-375, 1986.[Medline]
  13. Horii A., Nakatsuru S., Miyoshi Y., Ichii S., Nagase H., Ando H., Yanagisawa A., Tsuchiya E., Kato Y., Nakamura Y. Frequent somatic mutations of the APC gene in human pancreatic cancer. Cancer Res., 52: 6696-6698, 1992.[Abstract/Free Full Text]
  14. Huang L., Lang D., Geradts J., Obara T., Klein-Szanto A. J., Lynch H. T., Ruggeri B. A. Molecular and immunochemical analyses of RB1 and cyclin D1 in human ductal pancreatic carcinomas and cell lines. Mol. Carcinog., 15: 85-95, 1996.[Medline]
  15. Goggins M., Schutte M., Lu J., Moskaluk C. A., Weinstein C. L., Petersen G. M., Yeo C. J., Jackson C. E., Lynch H. T., Hruban R. H., Kern S. E. Germline BRCA2 gene mutations in patients with apparently sporadic pancreatic carcinomas. Cancer Res., 56: 5360-5364, 1996.[Abstract/Free Full Text]
  16. Ruggeri B. A., Huang L., Wood M., Cheng J. Q., Testa J. R. Amplification and overexpression of the AKT2 oncogene in a subset of human pancreatic ductal adenocarcinomas. Mol. Carcinog., 21: 81-86, 1998.[Medline]
  17. Korc M. Role of growth factors in pancreatic cancer. Surg. Oncol. Clin. N. Am., 7: 25-41, 1998.[Medline]
  18. Ebert M., Yokoyama M., Friess H., Buchler M. W., Korc M. Coexpression of the c-met proto-oncogene and hepatocyte growth factor in human pancreatic cancer. Cancer Res., 54: 5775-5778, 1994.[Abstract/Free Full Text]
  19. Aoki K., Yoshida T., Sugimura T., Terada M. Liposome-mediated in vivo gene transfer of antisense K-ras construct inhibits pancreatic tumor dissemination in the murine peritoneal cavity. Cancer Res., 55: 3810-3816, 1995.[Abstract/Free Full Text]
  20. Aoki K., Yoshida T., Matsumoto N., Ide H., Sugimura T., Terada M. Suppression of Ki-ras p21 levels leading to growth inhibition of pancreatic cancer cell lines with Ki-ras mutation but not those without Ki-ras mutation. Mol. Carcinog., 20: 251-258, 1997.[Medline]
  21. Yanagisawa A., Ohtake K., Ohashi K., Hori M., Kitagawa T., Sugano H., Kato Y. Frequent c-Ki-ras oncogene activation in mucous cell hyperplasias of pancreas suffering from chronic inflammation. Cancer Res., 53: 953-956, 1993.[Abstract/Free Full Text]
  22. Ochiai A., Yasui W., Tahara E. Growth-promoting effect of gastrin on human gastric carcinoma cell line TMK-1. Jpn. J. Cancer Res., 76: 1064-1071, 1985.[Medline]
  23. Sekiguchi M., Sakakibara K., Fujii G. Establishment of cultured cell lines derived from a human gastric carcinoma. Jpn. J. Exp. Med., 48: 61-68, 1978.[Medline]
  24. Sakakibara K., Suzuki T., Motoyama T., Watanabe H., Nagai Y. Biosynthesis of an interstitial type of collagen by cloned human gastric carcinoma cells. Cancer Res., 42: 2019-2027, 1982.[Abstract/Free Full Text]
  25. Liang P., Pardee A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science (Washington DC), 257: 967-971, 1992.[Abstract/Free Full Text]
  26. Liang P., Zhu W., Zhang X., Guo Z., O’Connell R. P., Averboukh L., Wang F., Pardee A. B. Differential display using one-base anchored oligo-dT primers. Nucleic Acids Res., 22: 5763-5764, 1994.[Free Full Text]
  27. Akiyama N., Sasaki H., Ishizuka T., Kishi T., Sakamoto H., Onda M., Hirai H., Yazaki Y., Sugimura T., Terada M. Isolation of a candidate gene, CAB1, for cholesterol transport to mitochondria from the c-ERBB-2 amplicon by a modified cDNA selection method. Cancer Res., 57: 3548-3553, 1997.[Abstract/Free Full Text]
  28. Sun Y., Lin J., Katz A. E., Fisher P. B. Human prostatic carcinoma oncogene PTI-1 is expressed in human tumor cell lines and prostate carcinoma patient blood samples. Cancer Res., 57: 18-23, 1997.[Abstract/Free Full Text]
  29. Ishikawa T., Ichikawa Y., Mitsuhashi M., Momiyama N., Chishima T., Tanaka K., Yamaoka H., Miyazakic K., Nagashima Y., Akitaya T., Shimada H. Matrilysin is associated with progression of colorectal tumor. Cancer Lett., 107: 5-10, 1996.[Medline]
  30. Ide H., Katoh M., Sasaki H., Yoshida T., Aoki K., Nawa Y., Osada Y., Sugimura T., Terada M. Cloning of human bone morphogenetic protein type IB receptor (BMPR-IB) and its expression in prostate cancer in comparison with other BMPRs. Oncogene, 14: 1377-1382, 1997.[Medline]
  31. Shen R., Su Z. Z., Olsson C. A., Fisher P. B. Identification of the human prostatic carcinoma oncogene PTI-1 by rapid expression cloning and differential RNA display. Proc. Natl. Acad. Sci. USA, 92: 6778-6782, 1995.[Abstract/Free Full Text]
  32. Pedersen P. L. Tumor mitochondria and the bioenergetics of cancer cells. Prog. Exp. Tumor Res., 22: 190-274, 1978.[Medline]
  33. Heerdt B. G., Halsey H. K., Lipkin M., Augenlicht L. H. Expression of mitochondrial cytochrome c oxidase in human colonic cell differentiation, transformation, and risk for colonic cancer. Cancer Res., 50: 1596-1600, 1990.[Abstract/Free Full Text]
  34. Satake K., Shim K., Sowa M., Umeyama K. Electron microscopic studies of human pancreatic adenocarcinoma. Eur. J. Surg. Oncol., 11: 125-135, 1985.[Medline]
  35. Takahashi M., Arai H., Kokubo T., Furukawa F., Kurata Y., Ito N. An ultrastructural study of precancerous and cancerous lesions of the pancreas in Syrian golden hamsters induced by N-nitrosobis(2-oxopropyl)amine. Gann, 71: 825-831, 1980.[Medline]
  36. Golshani-Hebroni S. G., Bessman S. P. Hexokinase binding to mitochondria: a basis for proliferative energy metabolism. J. Bioenerg. Biomembr., 29: 331-338, 1997.[Medline]
  37. Cavalli L. R., Varella-Garcia M., Liang B. C. Diminished tumorigenic phenotype after depletion of mitochondrial DNA. Cell Growth Differ., 8: 1189-1198, 1997.[Abstract]
  38. Susin S. A., Lorenzo H. K., Zamzami N., Marzo I., Snow B. E., Brothers G. M., Mangion J., Jacotot E., Costantini P., Loeffler M., Larochette N., Goodlett D. R., Aebersold R., Siderovski D. P., Penninger J. M., Kroemer G. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature (Lond.), 397: 441-446, 1999.[Medline]
  39. Su Z., Goldstein N. I., Fisher P. B. Antisense inhibition of the PTI-1 oncogene reverses cancer phenotypes. Proc. Natl. Acad. Sci. USA, 95: 1764-1769, 1998.[Abstract/Free Full Text]
  40. Bramhall S. R., Neoptolemos J. P., Stamp G. W., Lemoine N. R. Imbalance of expression of matrix metalloproteinases (MMPs) and tissue inhibitors of the matrix metalloproteinases (TIMPs) in human pancreatic carcinoma. J. Pathol., 182: 347-355, 1997.[Medline]
  41. Tani T., Lumme A., Linnala A., Kivilaakso E., Kiviluoto T., Burgeson R. E., Kangas L., Leivo I., Virtanen I. Pancreatic carcinomas deposit laminin-5, preferably adhere to laminin-5, and migrate on the newly deposited basement membrane. Am. J. Pathol., 151: 1289-1302, 1997.[Abstract]
  42. Sawada R., Lowe J. B., Fukuda M. E-selectin-dependent adhesion efficiency of colonic carcinoma cells is increased by genetic manipulation of their cell surface lysosomal membrane glycoprotein-1 expression levels. J. Biol. Chem., 268: 12675-12681, 1993.[Abstract/Free Full Text]
  43. Skrincosky D. M., Allen H. J., Bernacki R. J. Galaptin-mediated adhesion of human ovarian carcinoma A121 cells and detection of cellular galaptin-binding glycoproteins. Cancer Res., 53: 2667-2675, 1993.[Abstract/Free Full Text]
  44. Saitoh O., Wang W. C., Lotan R., Fukuda M. Differential glycosylation and cell surface expression of lysosomal membrane glycoproteins in sublines of a human colon cancer exhibiting distinct metastatic potentials. J. Biol. Chem., 267: 5700-5711, 1992.[Abstract/Free Full Text]
  45. Cermak J., Balla J., Jacob H. S., Balla G., Enright H., Nath K., Vercellotti G. M. Tumor cell heme uptake induces ferritin synthesis resulting in altered oxidant sensitivity: possible role in chemotherapy efficacy. Cancer Res., 53: 5308-5313, 1993.[Abstract/Free Full Text]
  46. Lott J. B., Mackie G. A. Isolation and characterization of cloned cDNAs that code for human ribosomal protein S6. Gene (Amst.), 65: 31-39, 1988.[Medline]
  47. Huang J., Kwong J., Sun E. C., Liang T. J. Proteasome complex as a potential cellular target of hepatitis B virus x protein. J. Virol., 70: 5582-5591, 1996.[Abstract/Free Full Text]
  48. Kumatori A., Tanaka K., Inamura N., Sone S., Ogura T., Matsumoto T., Tachikawa T., Shin S., Ichihara A. Abnormally high expression of proteasomes in human leukemic cells. Proc. Natl. Acad. Sci. USA, 87: 7071-7075, 1990.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 CarcinogenesisHome page
M. M. Jellicoe, S. J. Nichols, B. A. Callus, M. V. Baker, P. J. Barnard, S. J. Berners-Price, J. Whelan, G. C. Yeoh, and A. Filipovska
Bioenergetic differences selectively sensitize tumorigenic liver progenitor cells to a new gold(I) compound
Carcinogenesis, June 1, 2008; 29(6): 1124 - 1133.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
S. Ueda, K. Fukamachi, Y. Matsuoka, N. Takasuka, F. Takeshita, A. Naito, M. Iigo, D. B. Alexander, M. A. Moore, I. Saito, et al.
Ductal origin of pancreatic adenocarcinomas induced by conditional activation of a human Ha-ras oncogene in rat pancreas
Carcinogenesis, December 1, 2006; 27(12): 2497 - 2510.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Qian, J. Niu, M. Li, P. J. Chiao, and M.-S. Tsao
In vitro Modeling of Human Pancreatic Duct Epithelial Cell Transformation Defines Gene Expression Changes Induced by K-ras Oncogenic Activation in Pancreatic Carcinogenesis
Cancer Res., June 15, 2005; 65(12): 5045 - 5053.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Dong, W. Chen, A. Welford, and A. Wandinger-Ness
The Proteasome {alpha}-Subunit XAPC7 Interacts Specifically with Rab7 and Late Endosomes
J. Biol. Chem., May 14, 2004; 279(20): 21334 - 21342.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
P. J. Ross, M. George, D. Cunningham, F. DiStefano, H. J. N. Andreyev, P. Workman, and P. A. Clarke
Inhibition of Kirsten-ras Expression in Human Colorectal Cancer Using Rationally Selected Kirsten-ras Antisense Oligonucleotides
Mol. Cancer Ther., November 1, 2001; 1(1): 29 - 41.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
H. Fukushima, H. Yamamoto, F. Itoh, H. Nakamura, Y. Min, S. Horiuchi, S. Iku, S. Sasaki, and K. Imai
Association of matrilysin mRNA expression with K-ras mutations and progression in pancreatic ductal adenocarcinomas
Carcinogenesis, July 1, 2001; 22(7): 1049 - 1052.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
K. M. Anderson and J. E. Harris
Selected Features of Nonendocrine Pancreatic Cancer
Experimental Biology and Medicine, June 1, 2001; 226(6): 521 - 537.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Agnihotri, H. C. Crawford, H. Haro, L. M. Matrisian, M. C. Havrda, and L. Liaw
Osteopontin, a Novel Substrate for Matrix Metalloproteinase-3 (Stromelysin-1) and Matrix Metalloproteinase-7 (Matrilysin)
J. Biol. Chem., July 20, 2001; 276(30): 28261 - 28267.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohnami, S.
Right arrow Articles by Yoshida, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohnami, S.
Right arrow Articles by Yoshida, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online