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Regular Articles |
From the Ontario Cancer Institute*
and the
Department of Laboratory Medicine and
Pathobiology,
University Health
Network-Princess Margaret Hospital, Toronto; and the Department of
Medical Biophysics,
University of Toronto,
Toronto, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiation to up-regulate p53 and
p21waf1/cip1 protein. The p110/Rb protein level was also
not detectable consistent with the expression of E7 protein and haploid
loss of Rb1 gene. Despite this, the
proliferation of both c7 and c11 cells were markedly inhibited by
transforming growth factor-ß1. This was associated with up-regulation
of p21cip1/waf1 but not p27kip1. Further
studies showed that p130/Rb2 and cyclin D3 were expressed,
suggesting that p130/Rb2 may have partially assumed the maintenance of
G1 cell cycle checkpoint regulation. These results indicate
that except for the loss of p53 functional pathway, the two
clones of HPDE6-E6E7 cells demonstrated a near normal genotype and
phenotype of pancreatic duct epithelial cells. These cell lines will be
useful for future studies on the molecular basis of pancreatic duct
cell carcinogenesis and islet cell differentiation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our laboratory has previously reported the establishment of primary and immortal epithelial cell lines from normal human pancreatic ducts.6 We also reported that in comparison with the pancreatic cancer cell lines, the human pancreatic duct epithelial (HPDE) cells demonstrated a gene expression pattern that more consistently resembled the phenotype of normal cells rather than cancerous duct cells in vivo.7 These similarities included relatively low expression levels of various tyrosine kinase receptors, a wild-type Ki-ras genotype, and the retention and expression of p16INK4A gene. We have subsequently isolated several clones of these cell lines. We report here the phenotypic and genotypic characteristics of two of these cell lines that demonstrate anchorage-dependent growth requirement and that are nontumorigenic in immune-deficient mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The establishment of the HPDE6E76E7 (HPDE6) cell line was previously reported,6 and it was routinely cultured in keratinocyte serum-free (KSF) medium supplemented by epidermal growth factor and bovine pituitary extract (Life Technologies, Inc., Grand Island, NY). Clones of HPDE6 cells were isolated using cloning rings. Primary cultures of normal human bronchial epithelial cells were also established in the supplemented KSF medium, as previously reported.8
Cell Growth Assays
To determine the effect of transforming growth factor-ß1
(TGF-ß1; R & D Systems, Minneapolis, MN),
104
trypsin-dissociated cells were plated in
replicate wells of Nunc’s 6-well tissue culture plates (Life
Technologies, Inc.). Two days after plating, the culture medium was
replaced with fresh supplemented KSF medium. Replicate plates were also
replaced with the same medium containing 10 ng/ml of TGF-ß1. Cells in
triplicate wells were counted during the subsequent 6 to 8 days, using
the Coulter’s ZM Cell/Particle Counter (Hialeah, FL). The effect of 10
ng/ml of TGF-ß1 was also assessed at the colony-forming level by
seeding 60-mm tissue culture plates with 500 cells. The plates were
stained with 4% Giemsa solution (Sigma Chemical Co., St. Louis, MO),
and colonies with >10 cells were counted using a dissecting
microscope.
The effect of TGF-ß1 concentration on the proliferation of HPDE cells was assessed by the [3H]-thymidine uptake assay. Briefly, 4,000 cells were plated into each well of Nunc’s 12-well tissue culture plates. Two days later, the medium of groups of four replicate wells was changed to fresh medium containing various concentrations of TGF-ß1. The cells were then cultured for additional 4 days. One µCi/ml of [3H]-thymidine (NEN Life Science Products, Boston, MA) was then added to the medium of each well, and the cells were cultured for an additional 20 to 24 hours. To estimate the incorporation of [3H]-thymidine into the DNA, the cells were washed three times in cold phosphate-buffered saline, then were fixed in three changes of cold 5% aqueous solution of trichloroacetic acid (Sigma Chemical Co.) The cells were then serially dehydrated in ice-cold 70%, 95%, and 100% ethanol, and air-dried for at least 1 day. The incorporated radioactivity in each well was solubilized by an overnight incubation in 1 ml of 0.3 N NaOH at 37°C. After neutralization with 0.1 ml 3 N HCl, the solute was transferred into scintillation vials. After the addition of 5 ml Ecolite scintillation liquid (ICN, Costa Mesa, CA), they were counted using the Beckmann’s scintillation counter model LS6000SC.
The ability of cells to grow anchorage-independently was assayed by suspending them in KSF medium containing 0.3% Bacto-agar (Difco, Detroit, MI), as described previously.9 Cells were seeded at 104 or 5 x 104 cells per 60-mm plates, and allowed to form colonies in 4 weeks.
G-Banding, Spectral Karyotyping (SKY), and Fluorescent in Situ Hybridization (FISH)
Cytogenetic preparations were made according to standard protocols using colcemid and KCl hypotonic treatment.10 The slides were G-banded with trypsin (Difco) and Leishman’s stain (Sigma) and scanned for metaphase spreads.10 For each cell line, 20 G-banded metaphases were karyotyped and their descriptions were provided according to ISCN 95 nomenclature.11
The SKY kit probe cocktail from Applied Spectral Imaging (ASI, Carlsbad, CA) was hybridized to the cytogenetic preparation from the C7 clone according to standard protocols12,13 and the manufacturer’s instructions (ASI). Briefly, the slide was treated with pepsin, fixed with 1% formaldehyde, dehydrated using an ascending ethyl alcohol series, and denatured for 2 minutes in 70% formamide/2x standard saline citrate at 70°C. The SKY probe was denatured for 7 minutes at 75°C, re-annealed at 37°C for 1 hour, and hybridized to denatured slide for 36 hours at 37°C. The posthybridization washes and detection steps were performed per manufacturer’s instructions (ASI). The slide was counterstained by 4,6-diamidino-2-phenylindole (Sigma).
The metaphase images were captured using a SD 200 spectral bio-imaging system (ASI Ltd., MigdalHaemek, Israel) attached to a Zeiss microscope (Axioplan 2) and stored on a SKY image-capture workstation. The images were analyzed using the SKYView software version 1.5 (ASI), which resolves individual fluorochrome spectra by Fourier spectroscopy and distinguishes the spectral signatures for each chromosome to provide a unique pseudocolor for each chromosome (classified image). The determination of the position of rearrangement breakpoints was performed according the G-banding as well as spectral patterns.
Single color metaphase and interphase FISH analysis using different centromeric probes was performed on routine cytogenetic preparations from three different passages (P4, P14, P35) of clone C7 to determine the proportion of aneusomies as an overall measure of segregation anomalies associated with chromosomal stability. Probes for centromeres of chromosome 1 and 17 from Roche-Boehringer Mannheim (Dorval, Québec, Canada) were used for interphase and metaphase FISH. For each probe and each passage, 200 metaphase/interphase nuclei were scanned and the number of signals per cell counted independently by two individuals. To determine copy number of the Rb1 gene, the retinoblastoma DNA probe from Ventana was hybridized to a cytogenetic preparation derived from P35 of clone C7.
Comparative Genomic Hybridization (CGH) and Imaging
Cell line DNA was extracted using the standard phenol-chloroform extraction method. Phytohemagglutinin-stimulated normal lymphocytes were prepared as targets for CGH experiments using standard protocol.14 Slides were aged for 2 weeks before denaturation and proteinase K treatment. CGH was preformed according to the standard protocols.14,15 Briefly, reference and test DNA were labeled with digoxigenin and biotin, respectively. Biotinylated DNA was detected using avidin-fluorescein isothiocyanate (Oncor Inc., Gaithersburg, MD) whereas digoxigenin-labeled DNA was detected with anti-dig-rhodamine (Boehringer Mannheim, Mannheim, Germany). At least five images were captured per case using a Nikon Microphot microscope connected to a Photometrics (Tucson, AZ) SenSys KAF 1400 charge-coupled device (CCD) camera for the analysis. The QuipXL Genetics Workstation (Vysis Inc., Downers Grove, IL) was used for the image analysis. The image analyzing software calculates an average ratio of fluorescein isothiocyanate:rhodamine and expressed it as a green:red ratio for each metaphase with a 95% confidence limit. Gains or losses of chromosomal regions were detected when the fluorescent intensity ratio deviated from 1. The lower and upper limit of gain and loss was established by performing the control CGH experiments with DNA derived from normal male and female tissues. Based on these findings, the cut-off values were set at 1.25 and 0.80, with a 95% confidence limit. Analysis was excluded from the following regions: centromere, acrocentric p-arms, teleomere, and heterochromatic-rich areas.16-18
RNA Expression Assays
Total cellular RNA was isolated using the acid guanidinium thiocyanate-phenol-chloroform technique, as previously reported. The RNA was digested with RNase-free DNase I and reverse-transcribed using the murine Moloney leukemia virus reverse transcriptase, and aliquots of the product was used as template for polymerase chain reaction. The quality of reverse transcriptase reaction was checked first by amplification using the ß-actin primers.19 The primer set for carbonic anhydrase-II consists of forward primer (nucleotides 56 to 77): 5'-GCTCTAGACCATGTCCCATCAC-3' and reverse primer (nucleotides 541 to 520): 5'-GGAATTCTGAAGGCCCGGTTTAG-3'. The primers for MUC-1 were forward primer (nucleotides 2958 to 2979) 5'-GAAGATCTGCATCAGGCTCAGC-3' and reverse primer (nucleotides 3645 to 3624) 5'-GGAATTCTTTCGGCGGCACTGAG-3'.
Global gene expression was also studied using the Atlas Human Cancer cDNA Expression Array filter (Clonetech, Palo Alto, CA) as per instruction provided in user’s manual. 32P-labeled cDNA was generated by reverse-transcription using 5 µg of total cellular RNA as template. The labeled probe was purified with a ChromaSpin-200 (Clontech) column, and hybridized to the Atlas membrane at 68°C overnight. After repeated washing as recommended, the filter was exposed to X-ray film overnight.
Protein Immunoblot and Immunoprecipitation Assays
Antibodies to HPV16-E7 (ED17), p21waf1 (C-19), and Smad4 (B-8) were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechology, Santa Cruz, CA). Antibodies to Rb, Rb2/p130, and p27Kip1/p2, cyclin D3, cyclin B, and cdk-4 were obtained from Transduction Laboratory (Mississauga, Ontario, Canada). Western blot was performed as previously described.7
Antibodies to p53 protein were purified from supernatants of hybridoma clones. These included pAb 240 that was specific for mutant p53 protein, and pAb 421 and pAb 1620 that were reactive only to wild-type p53 protein. To perform immunoprecipitation, cellular proteins were extracted with cell lysis buffer, and the cleared supernatant was incubated with the appropriate p53 antibody at 4°C for 2 hours, then 30 µl of protein A Sepharose was added. After a further 1 hour of incubation at 4°C, the protein-A beads were centrifuged and washed, and then boiled in 30 µl of loading buffer containing 100 mmol/L dithiothreitol. After electrophoretic separation in polyacrylamide gel, the proteins were transferred onto polyvinylidene difluoride filter membrane (Boehringer Mannheim), then immunoblotted with p53 antibody, and revealed by the Chemiluminescence Western Biotin kit of Boehringer Mannheim.
-Irradiation
Five hundred thousand cells were grown to confluence in replicate
60-mm tissue culture dishes, then cultured for a further 2 days but
with a daily change of fresh medium. The culture plates were then
exposed to 5-Gy -irradiation from a 137Cs
source Gammacell-40 Exactor irradiator (Nordion International Inc.,
Kanata, Ontario, Canada). Sham control plates were brought to
room temperature for the same length of time. The plates were then
returned to the tissue culture incubator, and paired irradiated and
control plates were removed at varying times for protein extraction.
Biochemical Analysis of TGF-ß1-Treated Cells
Replicate plates of cultured cells growing to 80% confluence
were used in this series of experiments. Treatment was initiated by
replacing the medium with fresh medium containing 20ng/ml TGF-ß1 (R &
D Systems). The control plates were concurrently replaced with fresh
medium only. Total cellular protein from both TGF-ß1-treated and
control plates was serially collected at 3, 6, 9, and 24 hours later.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As previously reported, the HPDE6-E6E7 cells underwent growth crisis at approximately passage 12, but immortal clones emerged and have subsequently been propagated for >40 passages.6 At passage 18, the cells were plated at a colony forming density of 100 cells per 100-mm plate. The colonies that formed were isolated using steel cloning rings, and four clones were chosen randomly for further studies. Two clones (c7 and c11) that showed paradiploid DNA index by flow cytometry were also characterized for their karyotypic changes and growth properties. These two clones have been cultured for up to 35 passages and have shown no appreciable changes in both the morphology and growth characteristics. These cell lines also demonstrate telomerase activity as detected by the telomere repeat amplification (TRAP) assay (data not shown). These clones are herewith referred to as HPDE6c7 and HPDE6c11 cell lines.
Gene Expression of HPDE6-E6E7 Clones
Reverse transcriptase-polymerase chain reaction demonstrated that
the pre-immortalized HPDE6-E6E7 cells at passage 6 (P6), its immortal
line (P17), and clones isolated from the latter expressed mRNAs of
carbonic anhydrase-II and MUC-1 (Figure 1A)
. Northern blot analyses confirmed the
mRNA expression of p16INK4A and c-myc (Figure 1C)
. The levels of p16INK4A mRNA expression
in cells transduced with the E6E7 genes were much higher than that in
the primary cultured HPDE6 cells, but the transcript was normal in
size. Southern blot analyses demonstrated that all clones retained a
normal copy number of p16, c-myc, and
p53 genes (Figure 1D)
. Using the Atlas cDNA array analyses,
we also demonstrated high-level expression of cytokeratin-7, -14, and
-18 (Figure 1B)
. Lower level expression of cytokeratin-2, -8, and -19
was also noted.
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The chromosomal compositions of HPDE6c7 and HPDE6c11
cells were studied in more detail using both G-banding and SKY.
Both clones demonstrated an almost identical hypodiploid karyotype
(45/44, XX) with numerical and some structural changes (Figure 2, A and C)
. The common aberrations were
der (3;14) (q10;q10), del(10p)(p11), der
(17)t(13;17)(q21;p13), and +20. The c7 clone additionally
demonstrated a der (8)t(8;9)(q24;?), whereas c11 clone showed a
-22. The CGH studies were compatible with the karyotypic findings and
demonstrated chromosomal losses on 3p, 10p12, and 13q14 in both cell
lines, but a loss of chromosome 22 in c11 only (Figure 2, B and D)
. A
gain on 20 was also evident in both clones.
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FISH analysis using centromeric DNA probes of two chromosomes (1
and 17) on P4, P14, and P35 cells of HPDE6c7 line showed relatively
stable karyotype with passaging (Figure 3, A–C)
. P35 cells of HPDE6c7 line
showed relatively stable karyotype with passaging (Figure 3, A–C)
.
Some cells with one to four chromosome 1 signals were noted but the
frequency did not change with increasing passage number. Such variation
was not seen with chromosome 17. FISH analysis also confirmed the
loss of one Rb1 allele as a consequence of an
unbalanced translocation of 13q to chromosome 17 (Figure 3D)
.
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In KSF medium, the HPDE6c7 and c11 clones proliferated with
population doubling times of 24 to 32 hours. Similar to the parental
HPDE6-E6E7 cells, the growth of these immortal HPDE clones were
inhibited by TGF-ß1 (Figure 4, A and B)
. Inhibition was evident starting at 0.1 ng/ml, and became maximal at
1 ng/ml concentration (Figure 4C)
. At 4 x
104
cells/60-mm plate seeding density, both
clones failed to form colonies in soft agar. The implantation of two
million cells into the neck fat pad of SCID mice did not result in
tumor formation for up to 6 months.
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HPDE6c7 cells demonstrated a very low level of p53 protein that
was detectable by immunoprecipitation (Figure 5A)
, and both p53 and
21wafl/cipl proteins (Figure 5B)
and mRNA (data not shown)
were not inducible by -ray irradiation. As a control, primary
cultured normal human bronchial epithelial cells showed normal
up-regulation of p53 and p21 after radiation (Figure 5B)
. The low-level
p53 protein that was detectable by immunoprecipitation was reactive
with antibodies pAb1620 and pAb 421 that detect wild-type p53, but was
not recognized by antibody pAb 240 that reacts exclusively with p53
protein with mutations in amino acids 213 to 217 (Figure 5A)
. These
results indicated that HPDE6c7 cells and most likely its sister clones
expressed wild-type protein but at a markedly low level resulting from
the expression of E6 gene that was transduced during the establishment
of the HPDE6-E6E7 cell line.
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Western blot analysis demonstrated a marked reduction of Rb
protein level in HPDE6c7 cells (Figure 6A)
, but high levels of Rb2/p130 protein.
The low level of Rb protein is consistent with the presence of HPV16E7
protein (Figure 6B)
. Interestingly, the p16INK4A mRNA levels
in all HPDE6-E6E7 clones were markedly higher than that in the parental
primary cultured HPDE6 cells (Figure 1C)
.
|
. Growth inhibition by TGF-ß1 (10 ng/ml) was associated
with an up-regulation of the p21wafl/cipl protein 3 hours
after exposure to TGF-ß1, and of the p27kipl 24 hours
after treatment (Figure 6C)
. No change in the levels of cyclin D3,
cyclin B, and Cdk4 was noted. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported that as compared to a series of pancreatic duct carcinoma cell lines, HPDE cell lines expressed significantly lower levels of several growth factors and tyrosine kinase receptors commonly overexpressed in pancreatic ductal carcinoma.7 The findings indicated that the phenotypes of HPDE cells more closely resembled the normal duct epithelium than carcinoma cells in vivo. We demonstrate here that the HPDE cells also expressed several characteristic structural and functional genes expressed in normal pancreatic duct epithelium. These included the CK-7, -8, -18, and -19; carbonic anhydrase-II; and MUC-1 apomucin genes.23-25 The expression of these CK genes are characteristic of simple epithelial cells, and was also demonstrated in primary cultured normal human pancreatic duct cells.26-28 Our HPDE6c7 cells additionally also expressed high levels of CK14, which apparently was expressed only in <5% cells of normal duct epithelium. Several possibilities can be postulated to explain the expression of CK14 gene in HPDE-E6E7 cells. It is possible that this was an adaptive change in gene expression as a consequence of long-term culture, or as the result of HPV16-E6E7 gene expression. It is also possible that clone 7 cells originated from a subpopulation of human pancreatic ductal cells that constitutively expressed CK14. Note that epithelial cell lines established from adult rat pancreas expressed CK14 additional to CK7 and CK8, but adult rat pancreas is not known to express CK14. Further studies are necessary to clarify the importance of CK14 expression in immortal HPDE cell lines.
We have used both conventional and modern cytogenetic techniques to characterize the karyotypic and genomic changes in HPDE6-E6E7 c7 and c11 cells. Except for one copy gain of chromosome 20 and an additional copy loss of chromosome 22 in HPDE6c11 cells, other karyotypic changes involved small chromosomal losses that are associated with structural changes on 3p, 10p12, and 13q14. The latter included a haploid loss of the Rb gene. It is of interest to note that gain of whole chromosome 20 and deletions on 3p and 13q have been reported to occur commonly in primary pancreatic ductal carcinoma.29-33 Homozygous intragenic deletions of the FHIT gene located in 3p14.2 have been reported in approximately one third of primary tumor and cell lines of pancreatic cancer.34-36 Our results suggest that at least these chromosomal losses and gain in combination with HPV16-E6E7 genes are not sufficient for malignant transformation of human pancreatic duct cells.
The expression of E6E7 genes putatively should inactivate the p53 and Rb pathways, and these abnormalities clearly were not sufficient to induce malignant transformation of HPDE cells. Both clones of HPDE6E6E7 cells were neither tumorigenic in immune-deficient mice, nor were anchorage-independent in their growth requirement. This is consistent with previous results on the effect of E6E7 genes in various other human epithelial cells, and the need of other genetic abnormalities such as ras oncogene as a co-factor for the malignant transformation of human epithelial cells.22,37 Previous reports, however, indicated that an intact Rb pathway is essential for mediating the mito-inhibitory effect of TGF-ß1.38 The proliferation of both HPDE6 clone 7 and 11 cells were partially but significantly inhibited by TGF-ß1, and this was associated with the up-regulation of p21cip1 and p27kip1. The expression of Smads-2/3/4 was consistent with this preservation of the TGF-ß1-signaling pathway. In most normal epithelial cells including Mv1 Lu mink lung epithelial cells and primary cultured human keratinocytes, growth inhibition by TGF-ß1 resulted from increased expression of p15INK4B, p27kip1, and p21cip1 that caused the inhibition of the cyclinD:Cdk4/6 and cyclinE:Cdk2 activities.38-40 The former requires the presence of Rb, hence HPDE6-E6E7 cells are expected to be resistant to TGF-ß1. An Rb family member p130/Rb2 gene may also exert similar G1/S cell cycle regulatory function, and it is also phosphorylated by G1 cyclin-associated Cdks.41 TGF-ß1-induced p21cip1/waf1 up-regulation can also increase the levels and stability of hyperphosphorylated p130/Rb, hence induce G1 growth arrest.42 In contrast to Rb, however, binding of E7 to p130/Rb does not lead to increase proteolytic degradation.43 The results suggest that the G1 cell-cycle checkpoint regulation may still be intact in HPDE6-E6E7 cells, and the p130/Rb2 may have assumed this important function in place of the p110/Rb. This hypothesis remains to be tested.
We have also shown that despite the loss of wild-type p53 protein function, the HPDE6c7 cells demonstrated chromosomal stability. Chromosomal instability is defined as alterations of chromosome number involving losses or gains of whole chromosomes.44-46 In contrast to microsatellite instability which is a recessive trait and is caused by defects in mismatch repair genes resulting in genomic losses at the nucleotide sequence level, chromosomal instability seems to be a dominant phenotype that is consistently associated with the loss of spindle checkpoint control.44,45 The p53 gene has been considered as a "guardian" of the genome and its loss may result in genomic and/or chromosomal instability.47 Gualberto et al48 have reported that certain missense p53 mutants may cause the failure of cells to arrest at 4n after treatment with colcemid. The generation of polyploid cells may result in chromosomal instability through random losses of chromosomes and generation of aneuploidy. On the other hand, the loss of p53 protein caused by expression of the HPV16-E6 gene seemed to result in the maintenance of normal spindle checkpoint control. Our results also suggests that loss of the wild-type p53 protein and its function is not sufficient to induce chromosomal instability.
In conclusion, aside from the apparent loss of wild-type p53 protein function and the presence of a limited number of genetic changes, the chromosomally stable clones of HPDE6-E6E7 cells show phenotypic resemblance to normal pancreatic duct epithelial cells. These immortal cell lines will be useful as in vitro models for future studies in pancreatic duct cell carcinogenesis and differentiation.
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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 (to M. S. T.) and the Academic Enrinchment Fund of UHN Pathology Department.
Accepted for publication July 17, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced cell cycle arrest through the p21waf1/cip1-G1 cyclin/cdks-p130 pathway in gastric carcinoma cells. Int J Cancer 1999, 83:512-517[Medline]
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H. Matsubayashi, N. Sato, N. Fukushima, C. J. Yeo, K. M. Walter, K. Brune, F. Sahin, R. H. Hruban, and M. Goggins Methylation of Cyclin D2 Is Observed Frequently in Pancreatic Cancer but Is Also an Age-related Phenomenon in Gastrointestinal Tissues Clin. Cancer Res., April 1, 2003; 9(4): 1446 - 1452. [Abstract] [Full Text] [PDF]
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 B. Terris, E. Blaveri, T. Crnogorac-Jurcevic, M. Jones, E. Missiaglia, P. Ruszniewski, A. Sauvanet, and N. R. Lemoine Characterization of Gene Expression Profiles in Intraductal Papillary-Mucinous Tumors of the Pancreas Am. J. Pathol., May 1, 2002; 160(5): 1745 - 1754. [Abstract] [Full Text] [PDF]
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