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From the Division of Cellular and Molecular Biology,*
Ontario Cancer Institute, and Department of Laboratory Medicine and
Pathobiology and Department of Medical
Biophysics,§
Toronto Hospital/Princess Margaret
Hospital and University of Toronto, Toronto, Ontario, Canada; and
Department of Molecular Pathology
and First
Department of Surgery,
Tohoku University
School of Medicine, Sendai, Miyagi, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
Met/hepatocyte growth factor receptor (HGFR), vascular
endothelial growth factor (VEGF), and keratinocyte growth
factor (KGF). They also expressed high levels of amphiregulin but did
not express EGF and cripto. The expression levels were similar in
primary normal HPDE cells and those expressing transfected E6E7 genes
of human papilloma virus-16, but their immortalization appeared
to enhance the expression of EGFR and Met/HGFR. In comparison,
pancreatic carcinoma cell lines commonly demonstrated overexpression of
EGFR, erbB2, TGF-, Met/HGFR,
VEGF, and KGF, but they consistently showed marked
down-regulation of amphiregulin mRNA expression. In contrast to all
carcinoma cell lines that showed deletions of the p16
gene, HPDE cells consistently demonstrated normal
p16 genotype and its mRNA expression. This is the first
report that compares the phenotypic expression of cultured pancreatic
ductal carcinoma cells with epithelial cell lines derived from normal
human pancreatic ducts. The findings confirm that malignant
transformation of human pancreatic duct cells commonly results in a
deregulation of expression of various growth factors and
receptors.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, the molecular basis of aggressive and resistant phenotypes of pancreatic cancer remains unclear. Many investigators have demonstrated oncogene activation, tumor suppressor gene inactivation, and growth factor and receptor overexpression in these tumors.5 Some of these molecular changes do not correlate with prognosis,6-8 whereas others have been suggested to correlate with poor patient survival.9-15 Thus, further understanding on the functional roles of these genotypic and phenotypic changes during human pancreatic carcinogenesis and in the biology of pancreatic cancer cells may provide new clues for developing strategies to prevent and treat this disease.
Among the various phenotypic changes occurring in human pancreatic ductal adenocarcinoma, overexpression of tyrosine kinase receptors (RTKs), such as family members of epidermal growth factor receptor (EGFR) and their ligand molecules,16 and the Met/hepatocyte growth factor receptor (HGFR) have been reported.13, 16-28 These studies compared their expression in normal pancreas and primary pancreatic cancer tissue using total mRNA or protein extracts or by immunohistochemistry. Studies on the expression of various RTKs and/or their ligands in pancreatic carcinoma cell lines have also been published.29, 30 These studies, however, did not compare the expression in cancer cells with that in corresponding control cell lines derived from normal human pancreatic duct epithelium, thus imposing some limitation on the ability to interpret the data.
Our laboratory31 has recently reported a successful establishment of primarily cultured and immortal epithelial cell lines from normal human pancreatic duct epithelium (HPDE). We have further characterized the phenotype of these HPDE cell lines by comparing the expression of various growth factors, tyrosine kinase receptors, and p16INK4A cyclin inhibitor in these cells with their expression in several previously uncharacterized human pancreatic ductal adenocarcinoma cell lines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The explant method for establishing primary cultures of normal HPDE cells has previously been published.31 The life span of primarily cultured HPDE cells could be prolonged by infection with LXSN16-E6E7 retroviral expression vector for the E6 and E7 genes of human papilloma virus (HPV)-16 virus. These cells, however, eventually also developed crisis at passages 9 to 12 (approximately 45 to 60 population doublings). Maintenance of these senescent cells by frequent fresh medium replacement eventually led to the emergence of immortal cell lines. These immortal lines have continuously proliferated for more than 20 passages (approximately 100 population doublings). HPDE cells were cultured routinely in keratinocyte serum-free (KSF) medium supplemented by bovine pituitary extract and epidermal growth factor (Gibco-BRL, Grand Island, NY). These HPDE cell lines do not proliferate in serum-containing medium.
Human Pancreatic Carcinoma Cell Lines
The establishment of cell lines PK-1, -8, and -9 were reported previously.32 PK-9 was established from a primary tumor, whereas PK-1 and PK-8 were established from liver metastases of pancreatic ductal adenocarcinomas. PK-45 and PK-59 were subsequently established using the same method. They were established and routinely maintained in RPMI-1640 containing 10% (v/v) fetal bovine serum (Wisant, Quebec, Canada). These carcinoma cell lines do not proliferate in supplemented or basal KSF medium.
RNA Isolation and Northern Blot Analyses
Total cellular RNA was isolated from cultured cells using the
method previously described.33
RNA was isolated from cells
that have reached confluent growth for 2 to 3 days and also from
proliferating cells in cultures that were approximately 60 to 70%
confluent. The medium of the cultures was always renewed 24 hours
before isolation. Twenty-microgram samples of RNA were separated
electrophoretically, transferred onto Hybond-N membrane in 10X SSC (20X
SSC contains 2 mol/L NaCl and 0.3 mol/L sodium citrate, pH 7.0), and
then cross-linked by exposure to the ultraviolet light of a
transilluminator. The blots were probed with cDNA probes for EGFR,
transforming growth factor (TGF)-, erbB2/neu/HER-2, EGF, and
amphiregulin, as reported previously.33
The Met/HGFR mRNA
expression was probed with an 841-bp Bg1I-XhoI
insert of the plasmid POK6 containing full-length c-met
cDNA.34
Vascular endothelial growth factor (VEGF) mRNA was
probed with a 3.36-kb VEGF189 cDNA.35
Cripto
mRNA was probed with an 890-bp EcoRI fragment
cDNA.36
The cDNA probe for keratinocyte growth factor (KGF)
spanning nucleotides 997 to 1672 was cloned using the reverse
transcriptase polymerase chain reaction (RT-PCR) technique and
subcloned into pGEM-4Z plasmid. EGF mRNA was probed with a 960-bp
PstI fragment of plasmid pmEGF-26F12 (American Type Culture
Collection, Rockville, MD). The p16INK4A
gene/mRNA was probed by a 0.8-kb XbaI-ApaI
fragment of RC/CMV/p16 plasmid. Probes for the 18 S and 28 S ribosomal
RNA were also obtained from American Type Culture Collection. The cDNA
probes were labeled by [32P]dCTP using the oligolabeling
kit from Pharmacia (Piscataway, NJ). The hybridized membranes were
stringently washed at 60°C in 0.2X SSC solution containing 0.1% SDS.
Membranes were exposed to Kodak XAR-5 film at -80°C for 3 to 5 days.
Southern Blot Analyses
Genomic DNA was isolated according to standard procedure. Ten micrograms of each DNA sample was digested with EcoRI. After separation in 1% agarose gel and Southern blot transfer, hybridization was carried out as previously reported.31 The cDNA probe for c-myc was a 1.4-kb EcoRI-HindIII fragment of PKSC-myc plasmid.
Protein Immunoblot Analyses
Protein extracts were prepared from preconfluent or confluent cultures of HPDE6-E6E7 (greater than 16) and PK-9 cell lines. Before protein isolation, the HPDE cells were starved for 24 hours in basal KSF (supplement-free) medium. This was done because the KSF supplement contained EGF and bovine pituitary extract that would down-regulate the levels of various growth factor receptors. Similarly, the PK cells were starved for 24 hours in serum-free RPMI-1640 medium. After washing in ice-cold phosphate-buffered saline (PBS), the cultured cells were lysed in 1 ml of aqueous solution containing 50 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 8.0), 1% Triton X-100, 10% glycerol, 150 mmol/L NaCl, 10 mmol/L EDTA, 100 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 100 µg phenylmethylsulfonyl fluoride (PMSF). After a centrifugation at 13,000 x g for 15 minutes, the supernatants were collected. Protein concentrations were determined using the BioRad protein assay kit (BioRad Canada, Oakville, Ontario, Canada).
The protein levels of EGFR and Met/HGFR were estimated using the Western blot technique. Fifty-microgram protein samples were electrophoretically separated in an 8% SDS-polyacrylamide gel and then transferred onto a polyvinylidene difluoride membrane (Boehringer Mannheim Canada, Dorval, Quebec, Canada). After blocking with a 5% skim milk solution in a buffer containing 10 mmol/L Tris/HCl (pH 7.0), 0.1% Tween 20, 2.5 mmol/L EDTA, and 50 mmol/L NaCl, the membrane was incubated with polyclonal rabbit antibodies against EGFR (sc-003) or h-Met (sc-161). These were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunoreactivity was subsequently revealed using the BM chemiluminescence western blotting kit (Boehringer Mannheim Canada).
To assay the protein levels of erbB2/neu, 0.6 mg of protein lysates was immunoprecipitated with the 9G6 mouse monoclonal antibody against Neu (sc-008, Santa Cruz Biotechnology). After incubation for 1 hour at 4°C, 50 µl of protein-A-Sepharose (Pharmacia) was added, and the suspensions were rocked for 1 hour at 4°C. The antigen-antibody complex was pelleted by microcentrifugation and subsequently washed three times in the same lysis buffer. The immunoprecipitated pellet was then dissolved in 25 µl of Laemmli sample buffer and electrophoretically separated as described above. After transfer onto a polyvinylidene difluoride membrane, it was immunoblotted using the same Neu antibody, with the reaction detected by chemiluminescence.
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Among the various HPDE cell lines studied, HPDE6 was the only primarily cultured cells that have not been infected with LXSN16-E6E7 retrovirus, and hence represented a truly normal HPDE cell line. The HPDE(1,4,5,6)-E6E7 cell lines were cells that were infected with LXSN16-E6E7 and expressed the E6E7 genes of HPV-16 genes.31 The lines that were at or less than passage (P)9 represented pre-crisis cells, whereas those at P18 were post-crisis immortal cell lines. Both immortalized HPDE4-E6E7 (P18) and HPDE6-E6E7 (P18) were anchorage dependent in their growth and were nontumorigenic for 4 months in the subcutaneous tissue of SCID mice.31
mRNA Expression in Confluent Cultures
Figures 1 and 2
show the levels of mRNA expression for
EGFR, erbB2, TGF-, AR, Met/HGFR, VEGF, and KGF among the various
HPDE cell lines established from different individuals.
Inter-individual differences were slight, but it appears that for some
genes (EGFR and Met/HGFR), the immortalized HPDE (4,6)-E6E7 (P18) cell
lines showed slightly increased levels of expression as compared with
their pre-immortalized (P9) cells. In turn, the expression levels in
the latter cells were similar to those of normal HPDE6 cell line. These
HPDE cell lines uniformly demonstrated a relatively high level of AR
mRNA expression.
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, Met/HGFR, VEGF, and KGF
(Figures 1 and 2)
. Enhanced expression for both EGFR and TGF- was
seen in three of five carcinoma lines, and for erbB2 in four of five
lines (Figure 1)
. All five PK lines demonstrated overexpression of
Met/HGFR (Figure 2)
. Four carcinoma cell lines also demonstrated
overexpression of VEGF and KGF mRNA. The overexpression of various
receptors was not associated with gene amplification (Figure 3)
. All five PK lines demonstrated marked
and consistent down-regulation of AR as compared with the HPDE cell
lines (Figure 1)
. None of the HPDE and PK cell lines showed significant
mRNA expression for EGF and cripto (data not shown).
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To confirm the differential expression of EGFR, erB2, and Met/HGFR
at protein levels, we performed immunoblot assays on HPDE6-E6E7 (P23)
and PK9 cells that were in proliferative (preconfluent) and in
relatively quiescent (confluent) culture conditions (Figure 4)
. The results indicated that levels of
all three RTK proteins were higher in the cancer cells as compared with
HPDE cells. The results were consistent with the findings at the mRNA
level. Although the levels of EGFR and Met/HGFR proteins in both
HPDE6-E6E7 and PK-9 cell lines were not significantly influenced by the
proliferative state of the cells, those of erbB2 were lower in
preconfluent as compared with postconfluent cells.
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A great majority of pancreatic ductal carcinomas are known to have
deletion of the p16INK4A gene.37, 38
Four of the PK carcinoma cell lines also demonstrated a homozygous
deletion of the p16 gene and, the loss of its mRNA
expression (Figure 3, A and B)
. In contrast, all HPDE cells showed a
normal p16 genotype and mRNA expression, which appeared not
to be influenced by the immortalization of these cells. In the PK-59
tumor cell line, the p16 gene copy number was approximately
one-half of that in HPDE cells, suggesting a heterozygous deletion.
This p16 mRNA, however, remained expressed in this cell
line.
In contrast to p16, the c-myc genotype was not significantly different between the HPDE and adenocarcinoma cell lines. The variation between cell lines was most likely due to slightly unequal loading of the DNA samples, as the same pattern of variation was also detected with the c-met and EGFR genes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of protein and/or mRNA for EGFR, TGF-, erbB2, and
Met/HGFR in HPDE cells is consistent with reports that indicated low
levels of immunoreactivity for these proteins in the normal duct
epithelium of human pancreas.17-23, 27
In situ
hybridization studies have also demonstrated the expression of mRNAs
for these genes in the normal duct epithelium of human
pancreas.17, 20
Some of these studies reported enhanced
immunoreactivity for EGFR, TGF-, and Met/HGFR in ducts within areas
of chronic pancreatitis.18, 24-27
As chronic pancreatitis
is most likely associated with duct cell regeneration and
proliferation, the latter findings suggest a partial regulation of
expression of these genes by the proliferative state in these duct
cells. Except for erbB2, the expression of which was up-regulated, we
were unable to detect significant differences between the mRNA and/or
protein levels of EGFR, TGF-, and Met/HGFR in relatively quiescent
cells in confluent cultures, as compared with proliferative cells in
preconfluent cultures. The findings were also noted in the cancer cell
line studied. Unfortunately, due to the limited number of cells that we
could obtain from primary cultures of HPDE6 cells before their entering
senescence, we were unable to perform similar studies on these truly
normal cells. It has been reported, however, that the E6E7 genes of HPV
may increase the levels of EGFR protein, and this appears to be
regulated at the posttranscription level.41
Although there is a heterogeneity of overexpression for EGFR, erbB2,
and TGF- among cell lines, all five carcinoma lines we studied
demonstrated overexpression as compared with HPDE cells on these genes.
Furthermore, all five carcinoma cell lines also showed marked
down-regulation of amphiregulin expression. The EGF family agonist
genes consist of several members, including EGF, TGF-, betacellulin,
cripto, amphiregulin, and heparin-binding EGF.36, 42
In
contrast to other family members of EGF, amphiregulin is a bifunctional
growth factor with predominantly a stimulatory effect on the growth of
normal cells, but an inhibitory effect on tumor cells.43, 44
Furthermore, the EGF receptor (EGFR) also has several
homologous family members including erbB2, erbB3, and
erbB4.45
These erbB family receptors can inter-dimerize,
but they demonstrate different binding affinities to the different
receptor forms.42
Therefore, our findings suggest that a
net enhancement of the expression, hence, putatively, the activity of
EGFR family autocrine loops, is a commonly occurring biochemical event
during human pancreatic ductal carcinogenesis. Additional studies are
required to confirm their roles in the biology of pancreatic ductal
carcinomas.
These in vitro findings are consistent with the in
vivo data on expression levels of EGF and EGFR family gene
products in pancreatic ductal carcinoma. A strong immunostaining of
cancer cells and overexpression of EGFR, EGF, heparin-binding EGF, and
TGF- mRNA and/or protein in primary cancerous as compared with
normal pancreatic tissues have been reported.17, 18, 23, 25, 27
Expression of amphiregulin mRNA in both normal and cancerous
pancreatic tissue has also been reported.46
This study also
reported a positive nuclear staining in normal cells, whereas cancer
cells stained cytoplasmically. The significance of this is currently
unknown. We were unable to detect EGF and cripto mRNA expression
in vitro in both the HPDE and PK carcinoma cell lines.
Similar findings were reported by Oikawa et al29
who failed
to detect any mRNA expression of EGF, heparin-binding EGF, and cripto
in all 12 carcinoma cell lines they studied. In contrast, Tsutsumi et
al22
and Friess et al23
have reported an
immunoreactivity to cripto antibody in duct epithelium of normal
pancreas and chronic pancreatitis and also commonly in pancreatic
ductal adenocarcinoma.
Our result indicating frequent overexpression of erbB2 among pancreatic carcinoma cell lines was similar to that reported by Oikawa et al.29 Although normal pancreatic duct epithelium consistently demonstrates absent or very low levels of reactivity with various erbB2 antibodies, strong immunoreactivity in cancer cells was detected in 45 to 70% of ductal adenocarcinoma cases.13, 19-21 Overexpression of erbB2 appears to be more frequent in well or moderately differentiated than poorly differentiated tumors, and it is overexpressed in an even greater (>80%) percentage of hyperplastic and preneoplastic pancreatic ductal lesions.21 The role of erbB2 overexpression in human pancreatic ductal carcinogenesis requires additional investigations.
The overexpression of Met/HGFR appears to be common in human pancreatic cancer cell lines, which is consistent with previous reports that pancreatic ductal carcinoma cells in vivo overexpressed Met/HGFR as compared with normal duct epithelium.26, 28 DiRenzo et al31 have also reported expression of Met/HGFR mRNA and/or protein in all 31 pancreatic carcinoma cell lines studied, but their findings could be evaluated only in a relative term, as normal HPDE cells were not available for comparison.
The current study also demonstrated that when compared with the HPDE cells, pancreatic cancer cell lines commonly overexpress KGF and VEGF. Siddiqi et al47 detected KGF mRNA expression by the RT-PCR method in four of seven pancreatic carcinoma cell lines, and they reported increased KGF mRNA levels in 44% of cancerous as compared with normal pancreatic tissue. In our hands, four of five cancer cell lines showed KGF mRNA overexpression. Itakura et al48 recently reported the expression of VEGF mRNA and protein in all six human pancreatic carcinoma cell lines they studied. In situ hybridization and immunohistochemistry also demonstrated VEGF mRNA and protein overexpression in primary pancreatic ductal carcinoma cells as compared with normal duct epithelium.
The cyclin inhibitor p16INK4A/MTS-1/CDKN2 is a critical cell cycle regulator protein that is inactivated in almost all pancreatic ductal adenocarcinomas.38 Inactivation occurs by homozygous deletion in approximately one-half of these tumors and by heterozygous deletion accompanied by intragenic mutation or methylation silencing of the remaining allele in the remaining cases. The inactivation of the p16 gene putatively leads to deregulation of the G1-phase cell cycle control. Consistent with these results, the p16 gene is also homozygously deleted in four of five PK adenocarcinoma cell lines studied, with the fifth cell line showing a heterozygous deletion.
In summary, we have demonstrated that, in contrast to pancreatic ductal carcinoma cells, the phenotypic and genotypic characteristics of HPDE cells resemble more those of normal pancreatic ductal epithelium. We believe the cultured HPDE cells and immortalized cell lines derived from them are valuable for in vitro research in human pancreatic carcinogenesis.
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Acknowledgements |
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Footnotes |
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Supported by the Medical Research Council of Canada grant MT-14359.
Accepted for publication April 14, 1998.
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