Carcinogenesis vol.22 no.8 pp.1257–1269, 2001
Aberrant cell cycle checkpoint function in transformed
hepatocytes and WB-F344 hepatic epithelial stem-like cells
William K.Kaufmann1,2,4, Cynthia I.Behe1,
Vita M.Golubovskaya1, Laura L.Byrd1,
Craig D.Albright3, Kristen M.Borchet1,
Sharon C.Presnell1, William B.Coleman1,2,
Joe W.Grisham1,2 and Gary J.Smith1,2
1Departments
of Pathology and Laboratory Medicine, 2Lineberger
Comprehensive Cancer Center and 3Department of Nutrition, University of
North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA
4To
whom correspondence should be addressed at: Lineberger
Comprehensive Cancer Center, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599-7295, USA
Email: [email protected]
Cell cycle checkpoints are barriers to carcinogenesis as
they function to maintain genomic integrity. Attenuation
or ablation of checkpoint function may enhance tumor
formation by permitting outgrowth of unstable cells with
damaged DNA. To examine the function of cell cycle
checkpoints in rat hepatocarcinogenesis, we analyzed the
responses of the G1, G2 and mitotic spindle assembly
checkpoints in normal rat hepatocytes, hepatic epithelial
stem-like cells (WB-F344) and transformed derivatives of
both. Normal rat hepatocytes (NRH) displayed a 73%
reduction in the fraction of nuclei in early S-phase 6–8 h
following 8 Gy of ionizing radiation (IR) as a quantitative
measure of G1 checkpoint function. Chemically and virally
transformed hepatocyte lines displayed significant attenuation of G1 checkpoint function, ranging from partial to
complete ablation. WB-F344 rat hepatic epithelial cell lines
at low, mid and high passage levels expressed G1 checkpoint
function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant
attenuation of G1 checkpoint function. Attenuation of G1
checkpoint function in transformed hepatocytes and WBF344 cells was associated with alterations in p53, ablated/
attenuated induction of p21Waf1 by IR, as well as aberrant
function of the spindle assembly checkpoint. NRH displayed
93% inhibition of mitosis 2 h after 1 Gy IR as a quantitative
measure of G2 checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G2 checkpoint. Moreover, the parental WBF344 line displayed significant age-related attenuation of
G2 checkpoint function. Abnormalities in the function
of cell cycle checkpoints were detected in transformed
hepatocytes and WB-F344 cells at stages of hepatocarcinogenesis preceding tumorigenicity, sustaining a hypothesis
that aberrant checkpoint function contributes to carcinogenesis.
Abbreviations: BrdU, bromodeoxyuridine; FACS, fluorescence-activated cell
sorter; FITC, fluorescein isothiocyanate; HBSS, Hank’s balanced saline
solution; IR, ionizing radiation; MEM, minimal essential medium; MPF,
M-phase promoting factor; NHF, normal human fibroblasts; NRH, normal rat
hepatocytes; PB, phenobarbital; PCR, polymerase chain reaction; SSCP, singlestrand conformational polymorphism.
© Oxford University Press
Introduction
Cell cycle checkpoints serve as guardians of the genome and
suppress carcinogenesis (1,2). A checkpoint is a point of
control where cell division can pause before proceeding to the
next cycle phase. DNA damage checkpoints provide more
time for DNA repair before DNA synthesis and mitosis
thereby protecting against mutagenesis and clastogenesis (1,2).
Dependence checkpoints ensure the proper timing of essential
events in the cell cycle (3). Checkpoint responses suppress
tumor formation by preventing the induction and outgrowth
of unstable cells with altered content and/or structure of DNA.
The G1 checkpoint may slow or even arrest entry into Sphase. Key gene products for G1 checkpoint response to
ionizing radiation (IR)-induced DNA damage include ATM,
p53, p21Waf1 and Rb. The ATM gene is mutated in the familial
cancer syndrome ataxia telangiectasia (AT). ATM has protein
kinase activity and can be induced to phosphorylate p53 by
DNA damage (4,5). The activation of p53 leads to increased
levels of p21Waf1, GADD45, MDM2 and BAX. BAX induction
and the subsequent inhibition of BCL-2 may trigger apoptosis
(6). The higher levels of p21Waf1 inhibit G1 cyclin-dependent
kinases (CDKs) and arrest or delay the entry of cells into
S-phase (7,8). Inhibition of G1 CDKs by p21Waf1 preserves Rb
binding to E2F and enforces G1 arrest. Cells that lack p53
function by genomic mutation or viral gene expression are
unable to induce p21Waf1 in response to DNA damage and,
consequently, are unable to halt progression from G1 to S-phase
(7). Cells with Rb function inactivated by human papilloma
virus E7 gene product also display defective G1 checkpoint
function (9).
Progression from G2 to mitosis is regulated by the G2
checkpoint. This checkpoint monitors the genome for altered
DNA structure and delays the onset of mitosis in response to
DNA double-strand breaks, incompletely replicated replicons,
or insufficiently decatenated replicons (10). The phosphorylation status and compartmentalization of the M-phase promoting
factor (MPF) mediates this checkpoint (11). MPF contains a
catalytic subunit, CDK1 (p34CDC2), and a regulatory subunit,
cyclin B1 (12). Phosphorylation of MPF substrates causes
nuclear lamin disassembly, nuclear envelope vesicularization,
condensation of chromosomes and spindle formation (13).
The G2 checkpoint delays mitosis by preventing activation
of nuclear MPF kinase activity. Key gene products include
ATM, BRCA1, CHK1, CDC25C and 14-3-3. ATM is an
integral part of the G2 checkpoint as AT cells display reduced
sensitivity to radiation-induced G2 delay (14,15). Expression
of a natural splice variant of BRCA1, which deletes exon
11, fully ablated G2 checkpoint function in mouse embryo
fibroblasts without affecting G1 checkpoint function (16).
Inhibition of CHK1, a kinase that can phosphorylate CDC25C,
also inactivated G2 checkpoint response (17). CDC25C is a
phosphatase that removes inhibitory phosphates in the ATPbinding domain of MPF. Phosphorylation of CDC25C generates a 14-3-3 binding site, resulting in sequestration of CDC25C
1257
W.K.Kaufmann et al.
in the cytoplasm (18) and inhibition of phosphatase activity
(19). Mutation of the 14-3-3 binding site in CDC25C attenuated
G2 checkpoint response to DNA damage (18). Cells with
damaged DNA accumulate in G2 with inactive MPF.
The spindle assembly checkpoint is activated not by damage
to DNA but rather damage to the spindle apparatus in mitotic
cells. The spindle assembly checkpoint is a dependence checkpoint that delays anaphase and chromosome segregation until
metaphase has been completed. Completion of metaphase is
sensed when all chromosomal kinetochores are attached to the
bipolar spindle (20). The identification of a spindle assembly
checkpoint came when Saccharomyces cerevisiae mutants
failed to undergo mitotic arrest in response to spindle damage
(21,22). Defective spindle assembly signals include lack of
chromosome attachment to the spindle and absence of tension
on the spindle (20). Seven genes (BUB1-3, MAD1-3 and Mps1)
have been identified in yeast strains that are required for arrest
after damage to the mitotic spindle. Studies of human colorectal
cancer lines showed that BUB1 mutations can inactivate the
spindle assembly checkpoint (23). Cancer lines with mutations
in BUB1 fail to accumulate in metaphase when incubated with
spindle poisons and simply pass through mitosis without
segregating chromosomes.
Recent studies also have implicated p53 as a possible
component of the spindle assembly checkpoint. Both mouse
and human fibroblasts that are p53-deficient do not display
sustained growth arrest after treatment with microtubule destabilizing agents such as colcemid and nocodazole. They instead
undergo a new round of DNA synthesis in the absence of cell
division and become polyploid (24–26). In contrast to cells
with MAD or BUB mutations, p53-defective cells that are
treated with colcemid or nocodazole first arrest at prometaphase
due to the spindle assembly checkpoint. This arrest is not
stable however, and after a variable interval arrested cells
collapse out of mitosis into a G1-like state with reformation
of nuclear envelope around decondensed chromosomes. Cells
appear to re-enter G1 but with twice normal DNA content. In
cells expressing wild-type p53, the G1 checkpoint is then
activated in these 4N interphase nuclei and a G1 arrest occurs
(27). Cells with mutations in p53 and defective G1 checkpoint
function initiate DNA synthesis from this 4N G1 compartment,
initiating two rounds of DNA synthesis without completing
the intervening mitosis.
Inactivation or attenuation of cell cycle checkpoint function
is associated with enhanced growth and genetic instability.
Immortal Li–Fraumeni cells expressing only mutant p53 and
human papilloma virus type 16 E6-transformed human
fibroblasts lacking p53 function fail to undergo G1 arrest when
DNA is damaged and display severe genetic instability (28,29).
Cells lacking p53 and Rb function also display an extension
of proliferative lifespan and bypass the replicative senescence
checkpoint (30). Cells from AT patients display chromosomal
fragility and enhanced recombination (31,32). Mouse cells
expressing the natural splice variant of BRCA1 which ablates
G2 checkpoint function displayed chromosome number instability (16). These observations suggest that defects in cell cycle
checkpoints may enhance carcinogenesis by allowing cell
division under inappropriate conditions and by inducing genetic
instability. To test this hypothesis, we examined the functions
of the G1, G2 and mitotic spindle assembly checkpoints in
rat hepatocytes, rat hepatic epithelial stem-like cells and
transformed derivatives. These studies indicated that trans1258
formation of rat hepatic epithelial cells was associated with
significant defects in cell cycle checkpoint function.
Materials and methods
Cell culture
The properties of the hepatic epithelial cell types and lines used in this study
are listed in Table I. Hepatocytes were isolated from male F344 rats (Charles
River Breeding Laboratories, Raleigh, NC). Livers were perfused through the
vena cava with a 0.1% collagenase solution to dissociate hepatocytes (33).
Purification of isolated primary hepatocytes was done by sedimentation
through Percoll (34). Freshly isolated hepatocytes were cultured in growth
medium (Eagle’s minimal essential medium supplemented with 10 mM
HEPES, 2 µM FeCl3, 0.7 µM insulin, 100 µM L-proline, 1 mM L-glutamine,
1 mM non-essential amino acids, 0.5 µM zinc sulfate, 1 µM vitamin B12, 26
mM sodium bicarbonate) with 5% fetal bovine serum for 6 h to allow cellular
adherence to plastic dishes. After 6 h, the media was replaced with growth
medium including 3 ng/ml TGF-α and 10 ng/ml norepinephrine.
All cells were incubated in a humidified atmosphere of 5% CO2 at 37°C.
Established cell lines were grown in growth medium with 10% fetal bovine
serum and 50 mg/ml gentamycin. The phenobarbital-dependent hepatocyte
line 6/27C1 and the tumorigenic hepatocyte line 6/15 (35–37) had 2 mM
phenobarbital added to their growth media and were passaged at a split ratio
of 1:6. All other rat hepatic cell lines were passaged each week at a 1:12 split
ratio. Rat hepatic cell lines included the following: diploid WB-F344 epithelial
stem-like cells at passage levels 5–228 (38); two selectively cycled but nontumorigenic WB-F344 lines, L10C10 and L18C10 (39); four selectively cycled
tumorigenic WB-F344 lines, L2C10, L6C8, L14C8 and L20C10 (39); clonal
lines derived from tumors that grew after transplantation of the preceding
lines, L2.3.2, L2.3.5, L6.3.1, L6.3.2, L14.1.1 and L20.6.5 (39–41); a rat
hepatocellular carcinoma line, RLE-57 (33,35) and an SV40-transformed
hepatocyte line, CWSV1 (42,43).
G1 checkpoint function
G1 checkpoint function was quantified using flow cytometry (44). Primary
hepatocyte cultures at the peak of DNA synthesis after addition of TGF-α
and established hepatic cell lines in logarithmic growth were treated with 2
or 8 Gy 137Cs γ-rays (Gammacell 40) and returned to the incubator. Shamtreated controls were taken in and out of the incubator with the irradiated
samples but not exposed to γ-rays. Six hours after treatment, BrdU (10 µM
final concentration) was added to the media for 2 h. Cells were harvested with
trypsin, washed in Hank’s balanced salt solution (HBSS) and fixed with 70%
ethanol overnight. The fixed cells were incubated with 0.08% pepsin in 0.1
N HCl for 20 min at 37°C followed by a 20 min incubation with 2 N HCl.
To neutralize the suspension, 0.1 M sodium borate was added. Nuclei were
washed with 10 mM HEPES pH 7.4, 150 mM NaCl, 4% fetal calf serum,
0.1% sodium azide and 0.5% Tween 20 (flow buffer) then incubated with
FITC-labeled anti-BrdU antibody (Becton Dickinson, Bedford, MA) in flow
buffer for 30 min in the dark. The nuclei were washed again and then
resuspended in flow buffer. Propidium iodide (50 µg/ml) and 5 µg/ml RNase
A were added to stain the DNA and degrade RNA. Samples were then
analyzed by two-parameter flow cytometry using a Becton-Dickinson FACScan
analyzer. Quantification of G1 checkpoint function was done by determining
the radiation-induced reduction in the percentage of cells in the first half of
the S-phase 6–8 h after IR (44).
SSCP analysis
Total cellular RNA was extracted from primary hepatocytes or established cell
lines with guanidine isothiocyanate by standard methods (45). Complimentary
DNA (cDNA) was generated by reverse transcription (RT) from mRNA. Each
reaction for cDNA synthesis contained 1 µg total RNA, 5 mM MgCl2, 1 mM
each dNTP, 0.5 U RNAase inhibitor, 2.5 µM random hexamers and 1.25 U
reverse transcriptase (Promega, Madison, WI). The cDNA synthesis reaction
was performed as follows: 42°C for 15 min, 99°C for 5 min and 5°C for 5
min. Double-stranded DNA was amplified from cDNA by PCR. A 10 µl
aliquot from the cDNA synthesis reaction was combined with 40 µl 2 mM
MgCl2 containing 0.5 µCi [α-32P]dCTP (Amersham), 1.25 U Taq polymerase
(Promega) and 0.5 µM p53 primers described below. Twenty eight cycles of
denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at
72°C for 1 min were performed in a thermal cycler.
PCR products were heated to 95°C for 6 min in 1⫻ sample buffer (5⫻
sample buffer contained 750 µl formamide, 250 µl glycerol, 40 µl 0.5 M
EDTA pH 8.0, 2.5 µg bromophenol blue and 2.5 µg xylene cyanol) and
chilled on ice for 8 min. The reaction mixtures were immediately loaded on
a 6% acrylamide/0.12% bisacrylamide gel containing 10% glycerol. Gels were
run at 30 W for 8–9 h at room temperature. Autoradiography was performed
with an intensifying screen for 16 h. A rat nasal squamous carcinoma cell
Aberrant cell cycle checkpoint function
Table I. F344 rat hepatic epithelial cells and cell lines used in this study
Cell line
Cell type
Properties
References
–
6/27C1
CWSV1
6/15
RLE-57
WB-F344
WBL10C10, –18C10
WBL2C10, –6C8, –14C8, –20C10
WBL2.3.2, –2.3.5, –6.3.1, –6.3.2, –14.1.1, –20.6.5
Hepatocytes
Hepatocytes
Hepatocytes
Hepatocytes
Hepatocytes
Hepatic stem-like
Hepatic stem-like
Hepatic stem-like
Hepatic stem-like
Primary culture
Chemically initiated, promoter-dependent
SV40-transformed
Chemically initiated, tumorigenic
Chemically initiated, tumorigenic
Secondary cultures
Selectively cycled, non-tumorigenic
Selectively cycled, tumorigenic
Tumor clones
33
35,37
42,43
35,37
35
38,56
39
39
41
line, FAT 7 (American Type Culture Collection), with a known mutation in
exon 8 of p53 (271 codon, transversion from CGT→CAT) (46) was used as
a positive control.
The following primers for amplification of p53 exons 5–9 were used: exon
5, 5⬘-CAGCCAAGTCTGTTATGTGC-3⬘ and 3⬘-CGGATTTCCTTCCCACCGGA-5⬘; exons 6 and 7, 5⬘-CCTGGCTCCTCCCCAACATC-3⬘ and 3⬘TCCCGTCCCAGAAGATTCCC-5⬘; exons 8 and 9, 5⬘-CTTACCATCATCACGCTG-3⬘ and 3⬘-GCTCACGCCCACGGATCTTAA-5⬘.
Detection of p53 mutations
Messenger RNA was isolated by cesium chloride gradient followed by
separation utilizing a biotinylated oligo (dT) primer and streptavidin-coupled
magnetic beads (PolyATract® mRNA Isolation System; Promega). Poly A
mRNA (2 µg) was reverse transcribed utilizing an oligo (dT) primer and
MMLV reverse transcriptase according to the manufacturer’s instructions
(Advantage™ RT for PCR kit; Clontech, Palo Alto, CA). PCR reactions were
carried out in Easy Start-50 PCR tubes (Molecular Bioproducts, San Diego,
CA) using 1 µl template, 2.5 U AmpliTaq polymerase (Perkin Elmer Applied
Biosystems, Foster City, CA) and primers at 0.15 µM. Primers for the p53
coding region and a fragment corresponding to bases 581–822 of the coding
region (F4) of rat p53 (GenBank accession number X13058) were previously
described (47). Full-length p53 coding region was amplified over 30 cycles
(95°C for 60 s, 54°C for 90 s, 72°C for 60 s) using 1 µl of a 1:10 dilution of
each RT reaction. The PCR product was visualized on an agarose gel and the
1235 bp band was excised and purified (Qiaex II Gel Extraction kit; Qiagen,
Valencia, CA). The F4 region was amplified from the purified full-length
coding region over 30 cycles (95°C for 60 s, 62°C for 90 s, 72°C for 60 s),
using the nested primers, and the 242 bp product was visualized, excised and
purified. This fragment was then ligated into the pGEM®-T Easy Vector System
and transformed into JM109 High Efficiency Competent Cells (Promega).
Following overnight growth, plasmids were purified (Wizard® Plus Miniprep
DNA Purification System; Promega) and diagnostic digests were performed
to confirm the plasmids contained the 242 bp fragment of interest. DNA was
sequenced at the UNC-CH Automated Sequencing Facility on a Model 377
DNA Sequencer (Perkin Elmer Applied Biosystems, Foster City, CA) using
the ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction kit
with AmpliTaq DNA polymerase, FS (Perkin Elmer Applied Biosystems).
Sequences were compared using Sequencher™ Version 4.0.5 (Gene Codes,
Ann Arbor, MI).
Western immunoblot analysis
Control and IR-treated cell cultures were washed with HBSS and harvested
with 0.1% trypsin. Whole cell pellets were then suspended in 2⫻ lysis buffer
(2% SDS, 20% glycerol, 2% β-mercaptoethanol and 0.02% bromophenol blue
in 62.5 mM Tris–HCl pH 6.8) at a concentration of 5⫻106 cells/ml. Lysates
were boiled for 5 min and protein was then separated on a 12% SDS–PAGE
gel. Proteins were transferred to 0.45 µm PROTRAN nitrocellulose (Schleicher
& Schuell, Keene, NH) for western immunoblot analysis using anti-p21 (C-19;
Santa Cruz Biotechnology, Santa Cruz, CA) (48) antibody and anti-β-actin
antibody (clone AC-15; Sigma, St Louis, MO). Specific proteins were detected
using a chemiluminescent substrate (ECL Western Blotting Detection Reagents;
Amersham, Buckinghamshire, UK).
Spindle assembly checkpoint function
Logarithmic cell cultures were incubated for 24 h with 100 ng/ml colcemid
to depolymerize spindle microtubules. BrdU was added for the final 2 h of
incubation and the cells then harvested for flow cytometry as described above.
The colcemid-induced increase in the percentage of BrdU-labeled nuclei with
4–8 N DNA was quantified as a measure of spindle assembly checkpoint
function.
G2 checkpoint function
G2 checkpoint function was quantified using fluorescence microscopy (44).
Cells in log-phase growth were treated with 1 Gy IR or sham-treated (controls)
and then fixed with 3:1 (v/v) methanol:acetic acid various times later.
Propidium iodide was used to stain the nuclei of the cells so mitotic figures
could be counted using fluorescence microscopy. At least 2000 cells were
counted for each sample and the percentage of mitotic cells was determined
as the mitotic index. G2 checkpoint function was quantified as the percentage
of G2 cells that evaded radiation-induced mitotic delay (mitotic indextreated/
mitotic indexcontrol) 2 h after 1 Gy.
Results
G1 checkpoint function
DNA strand breaks induced by ionizing radiation activate the
p53-dependent G1 checkpoint (7). The G1 checkpoint response
results in a quantifiable emptying of the early S-phase compartment seen in diploid human fibroblasts as a 92% reduction in
the fraction of early S-phase cells 6–8 h following 8 Gy of IR
(49). A flow cytometric method was used to investigate G1
checkpoint function in rat hepatocytes at various stages of
transformation. Primary rat hepatocytes were isolated and
incubated with TGF-α to induce proliferation. DNA synthesis
assay using [3H]thymidine incorporation showed a peak in
DNA synthesis 36 h after the addition of the growth factor
(data not shown). Therefore, primary hepatocyte cultures were
treated with 8 Gy γ-rays 36 h post addition of TGF-α during
active cell division and then incubated for 6 h. Subsequently,
BrdU was added for 2 h and cells were harvested for flow
cytometric assessment of S-phase nuclei. Primary cultures of
normal rat hepatocytes expressed a 73% reduction in the early
S-phase compartment in response to 8 Gy γ-rays (Figure 1A
and B) (Table II). This emptying of the early-S compartment
was seen in both diploid and tetraploid nuclei in primary
hepatocyte cultures.
Having shown that primary cultures of rat hepatocytes
displayed a quantifiable G1 checkpoint response to DNA
damage, we then tested other hepatocyte lines. The CWSV1
line is an SV40-transformed hepatocyte line (42,43). SV40large T antigen is known to bind and inactivate p53 (50).
Accordingly, SV40-transformed human fibroblasts lack p53dependent G1 checkpoint function (51). The CWSV1 cell line
showed no emptying of the early-S compartment (Figure 1I
and J), suggesting that p53 is required for the G1 checkpoint
response in rat hepatocytes.
Phenobarbital (PB) is a promoter of liver carcinogenesis
(52). Effects of PB include inhibition of normal hepatocyte
growth (53) and inhibition of apoptotic cell death in normal
and initiated hepatocytes (54). Phenobarbital has recently been
shown to attenuate G1 checkpoint response in primary cultures
1259
W.K.Kaufmann et al.
Fig. 1. G1 checkpoint function in rat hepatocyte lines. Cells were sham-treated or irradiated with 8 Gy γ-rays. BrdU was added 6 h later for the final 2 h of
incubation to label DNA in S-phase cells. FITC-labeled anti-BrdU antibody was used to identify S-phase nuclei. Nuclei were counterstained with propidium
iodide. The frequency plots of the number of nuclei in each stage of the cell cycle are shown. G0–G1 (bottom left), S-phase (top), G2–M (bottom right). The
box encloses cells in early S-phase. The altered tone (gray scale) of the images in (C) and (D) is due to an upgrade in computer software.
Table II. Defective G1 checkpoint function in transformed rat hepatocytes
Cell line
Normal rat hepatocytes
WB-F344 cells (passages 5–20)
6/27C1 (immortalized hepatocytes)
6/15 (tumorigenic hepatocytes)
CWSV1 (SV40-transformed hepatocytes)
RLE-57 (tumorigenic hepatocytes)
Percent reduction in early S-phasea
2 Gy
8 Gy
–
69
6
34
–
14
73
75
31
60
0
14
⫾ 19 (9)
⫾ 21* (3)
⫾ 14* (5)
⫾ 16* (2)
⫾
⫾
⫾
⫾
⫾
⫾
8 (7)
18 (10)
14** (3)
8 (6)
11** (2)
14** (2)
⫾ standard deviation (n ⫽ number of experiments).
*P ⬍ 0.01 by Student’s t-test (versus WB cells passages 5–20 after 2 Gy).
**P ⬍ 0.05 by Student’s t-test (versus NRH and WB cells passages 5–20
after 8 Gy).
Cells were treated with 2 or 8 Gy of γ-rays then incubated at 37°C for 8 h.
BrdU was added for the final 2 h of incubation. Nuclei were prepared and
analyzed by flow cytometry. Radiation-induced reduction of the early
S-phase fraction was determined as a measure of G1 checkpoint function.
aMean
of mouse hepatocytes (55). An immortalized, PB-dependent
hepatocyte line, 6/27C1, expressed only a 31% reduction in
the early-S compartment after 8 Gy of IR (Figure 1C and D)
in comparison to the 73% reduction seen in normal hepatocytes
(P ⬍ 0.05). The 6/15 tumorigenic hepatocyte line which was
also grown in the presence of PB showed a 60% reduction in
early S-phase nuclei after 8 Gy, which was not significantly
different from normal hepatocytes (Figure 1E and F). Rat
hepatocellular carcinoma line RLE-57 grown in the absence
1260
of PB showed a significant attenuation of G1 checkpoint
response with only a 13% reduction in early S-phase after 8
Gy of IR (Figure 1G and H). By flow cytometric assay, primary
rat hepatocytes and the tumorigenic hepatocyte line, 6/15,
appeared to have a functional G1 checkpoint (Table II).
However, the SV40-transformed hepatocyte line CWSV1, an
immortal, PB-dependent line, 6/27, and a PB-independent rat
hepatocellular carcinoma line, RLE-57, all displayed reduced
G1 checkpoint function.
WB-F344 rat hepatic epithelial stem-like cells have often
been used in the study of hepatocarcinogenesis (38–40,56,57).
Low-passage WB cells displayed a time-dependent emptying
of the early-S compartment after IR (Figure 2). Two hours
following 8 Gy, there was a distinct reduction in early S-phase
cells. The emptying of the S-phase compartment increased at
4 and 6 h with progressive losses of nuclei with increasing
DNA content. These data showed that during the G1 checkpoint
response in rat hepatic epithelial stem-like cells, S-phase
emptied from beginning to end, as cells that were in S-phase
at the time of irradiation continued with and completed DNA
synthesis. As the WB cells were aged in culture, there was no
significant alteration in G1 checkpoint function (Figure 3A–F)
(Table III). At passage 58, the population had shifted to
predominantly tetraploid DNA content. Both the diploid and
tetraploid populations emptied early S-phase post-irradiation.
WB cells at passage 228 also displayed an intact G1 checkpoint.
These were interesting findings as WB-F344 cells and similar
rat epithelial cells were found to become tumorigenic spontaneously by passage 25 (58,59).
Aberrant cell cycle checkpoint function
Fig. 2. Time-dependent emptying of the early S-phase compartment in WB cells. BrdU was added at 2, 4 or 6 h post IR or sham-treatment for the final 2 h of
incubation to label DNA in S-phase cells.
Fig. 3. G1 checkpoint function in WB cells and transformed derivatives. Conditions of cell treatment and analysis were as described in the legend to Figure 1.
1261
W.K.Kaufmann et al.
Table III. Normal G1 checkpoint function in WB cells and most
transformed derivatives
Cell line
Normal rat hepatocytes
WB-F344 (passage 5–20)
WB-F344 (passage 22–59)
WB-F344 (passage 104–228)
WBL10C10 and L18C10
(non-tumorigenic lines)
WBL2C10 (parental, tumorigenic line)
WBL2.3.2 and L2.3.5 (tumor-derived
clones)
WBL6C8 (parental, tumorigenic line)
WBL6.3.1 and L6.3.2
(tumor-derived clones)
WBL14C8 (parental, tumorigenic line)
WBL14.1.1 (tumor-derived clone)
WBL20C10 (parental, tumorigenic line)
WBL20.6.5 (tumor-derived clone)
Percent reduction in early S-phasea
2 Gy
8 Gy
–
69
64
54
79
73
75
72
73
82
⫾19 (9)
⫾ 19 (11)
⫾ 23 (8)
⫾ 4 (2)
⫾
⫾
⫾
⫾
⫾
8 (7)
18 (10)
22 (11)
15 (8)
9 (2)
67 ⫾ 11 (5)
74 ⫾ 7 (7)
81 ⫾ 9 (5)
70 ⫾ 7 (7)
72 ⫾ 9 (5)
60 ⫾ 8 (4)
77 ⫾ 10 (5)
81 ⫾ 3 (4)
61
61
79
18
⫾
⫾
⫾
⫾
2 (2)
4 (2)
8 (4)
9* (4)
80
70
88
12
⫾
⫾
⫾
⫾
2 (2)
1 (2)
6 (4)
17* (4)
⫾ standard deviation (n ⫽ number of experiments).
*P ⬍ 0.001 by Student’s t-test (versus WB passage 5–20 after 2 Gy and
8 Gy).
G1 checkpoint function was quantified as in Table II.
aMean
In addition to the normal WB-F344 cells, several transformed
lines were examined. These cell lines were derived from WBF344 cells following a protocol of spontaneous transformation
in vitro (39,58). Cells were grown to confluence during week 1.
The next 3 weeks the cells were held at confluence with fresh
media being replaced each week. After the 4 week period, the
cells were trypsin-harvested, replated and the selection cycle
repeated. WB-F344 cell lineages were subjected to 8–10 cycles
of selection. Cells from each lineage were then injected into
F344 rats at various cycles of selection to evaluate their
tumorigenic potential (39), and cell lines were derived from
tumors as clonal isolates (39,41,60). Tested here were two
non-tumorigenic cell lines that were cycled 10 times (L10C10
and L18C10) and four tumorigenic lines that were cycled 8
or 10 times (Table III). G1 checkpoint function was determined
for the tumorigenic lines prior to transplantation and for clonal
lines isolated from tumors. The non-tumorigenic cell lines
displayed G1 checkpoint function equivalent to the low passage
WB-F344. Additionally, most of the tumorigenic lines and
tumor-derived lines had normal G1 checkpoint function. Data
for two sets of the tumor-derived clonal lines (L2.3.2 and
L2.3.5, L6.3.1 and L6.3.2) were combined as they did not
differ appreciably. The only WB cell line with defective
G1 checkpoint response to IR was the tumor-derived line
WBL20.6.5. Following 8 Gy, WBL20.6.5 cells displayed
significantly less reduction in early-S nuclei both in comparison
to WB-F344 cells at low passage (Figure 3J versus B) and the
parental line, WBL20C10 (Table III). The WB-F344 hepatic
cell model suggests that the loss of G1 checkpoint function
was unnecessary for progression to tumorigenicity.
A reduced dose of 2 Gy was administered to all established
lines to determine whether the 8 Gy dose saturated the response.
WB cells responded to the 2 Gy dose with a reduction of early
S-phase cells nearly equal to that seen after 8 Gy (Table III).
This suggests that the 8 Gy dose was saturating for the G1
checkpoint response. The tumorigenic and tumor-derived WB
lines also had a G1 arrest at 2 Gy nearly equivalent to the
arrest seen at 8 Gy (Table III). However in the 6/27C1 and 6/15
1262
Fig. 4. SSCP analysis of p53 in rat hepatocyte and WB cell lines. Total
RNA was extracted from hepatocytes and WB cell lines, and cDNA was
synthesized by PCR using [α-32P]dCTP and p53 primers encompassing
exons 5, 6–7 and 8–9. Denatured products of the PCR amplification were
analyzed by polyacrylamide gel electrophoresis. Autoradiography was
performed with an intensifying screen for 16 h. FAT 7, a rat nasal squamous
carcinoma cell line, was used as a methodologic control.
hepatocyte lines, the G1 arrest following 2 Gy was significantly
reduced from the level seen in low passage WB cells and
these lines given 8 Gy (Table II).
SSCP and sequence analysis of p53
To investigate the p53-dependent G1 checkpoint further, we
looked for alterations in p53 structure by single-strand conformational polymorphism (SSCP) analysis and direct sequencing. SSCP analysis examined exons 5–9 which display
mutations or deletions in many different cancers (61). Normal
rat hepatocytes were considered to display wild-type p53
(Figure 4). FAT 7, a rat nasal squamous carcinoma cell line
with a known mutation in exon 8 of p53, was used as a
methodologic control (46). FAT 7 cells displayed altered
mobility of the exons 8⫹9 amplimer and attenuated G1
checkpoint response by the flow cytometry assay (data not
shown). The PB-dependent 6/27C1 cell line had a significantly
attenuated G1 checkpoint response to DNA damage. However,
SSCP did not detect a p53 alteration in the exons examined.
The 6/15 line with measurable G1 checkpoint response after
IR also showed no p53 alteration by SSCP. The rat hepatocellular line RLE-57 with defective G1 checkpoint function did not
yield an amplified product from exons 5–7 suggestive of an
intragenic deletion (Figure 4). WB-F344 cells displayed a
functional G1 checkpoint function at all passage levels and
no alteration was observed in p53 by SSCP analysis. The
tumorigenic parental line WBL20C10 that displayed an intact
G1 checkpoint by flow cytometric analysis also had no p53
alteration by SSCP. However, the tumor-derived cell line
WBL20.6.5 with severely attenuated G1 checkpoint function
displayed an alteration in exons 6⫹7 producing two bands
with altered mobility. The lack of bands with wild-type
mobility suggests that wild-type mRNA was not present in the
WBL20.6.5 line. Upon sequence analysis, 20.6.5 was found
to have a mutation in the coding region of p53 at base 762
(C→T). This codon 247 mutation caused an amino acid change
Aberrant cell cycle checkpoint function
from arginine to tryptophan. The presence of this mutation
was confirmed in 10/10 additional 20.6.5 clones sequenced.
In most of the cell lines the SSCP and mutation results were
correlated with the functional analysis of the G1 checkpoint.
The FAT 7, RLE-57 and WBL20.6.5 lines with attenuated G1
checkpoint function displayed an alteration in p53 by SSCP
and the WB20.6.5 line was found to have a mutation in codon
247. Only the 6/27C1 line with attenuated G1 checkpoint
function displayed apparently normal p53 structure.
Analysis of p21Waf1 induction
An important component of the p53-dependent G1 checkpoint
response is p21Waf1. Activation of p53 by ATM and other
effectors leads to increased levels of p21Waf1 and subsequent
inhibition of G1 CDKs. Cells that lose p53 function are unable
to induce p21Waf1 and do not arrest progression from G1 to S
after IR. WB cells at passage 16 and 28 induced p21Waf1 6 h
after 8 Gy IR (Figure 5A). The parental WBL20C10 line
responded as the low passage WB cell lines with induction of
p21Waf1 6 h after IR (Figure 5B). No p21Waf1 protein was
detected 6 h following 8 Gy in the WBL20.6.5 cells. These
results show that WB cell lines with normal G1 checkpoint
function also display induction of the p21Waf1 protein following
IR, and the one WB line with mutant p53 did not express
p21Waf1 nor induce it after IR.
The results with transformed hepatocytes were similar to
those with WB cells. The immortal 6/27C1 hepatocyte line
displayed a significantly attenuated G1 checkpoint response to
DNA damage. Although p21Waf1 was detected in unirradiated
controls, there was little induction of p21Waf1 protein 6 h after
8 Gy IR (Figure 5C). Although the tumorigenic 6/15 hepatocyte
line had apparently normal G1 checkpoint function following
8 Gy of IR, only modest p21Waf1 induction was seen in these
cells. RLE-57 displayed both attenuation of G1 checkpoint
function and altered p53 by SSCP analysis. No p21Waf1 protein
was detected in the sham or irradiated RLE-57 cell lysates.
The p21Waf1 results in the WB and hepatocyte models associated
loss of G1 checkpoint function with loss of p53-mediated
induction of p21Waf1. The only exception was the 6/15 hepatocyte line which showed modest induction of p21Waf1 after 8
Gy of IR.
Spindle damage checkpoint function
The p53 tumor suppressor gene product appeared to be a
crucial component of DNA damage checkpoint function in rat
hepatic epithelial cells. Several studies have shown that p53
inhibits cell division after disruption of the mitotic spindle
(24–26). Colcemid causes depolymerization of microtubules
thereby disrupting assembly of the mitotic spindle. Cells with
wild-type p53 first arrest in metaphase of mitosis when
incubated with colcemid then collapse into a G1-like state with
restitution of interphase nuclear structure where they remain
arrested. Cells with dominant-negative mutations in p53 and
cells lacking expression of wild-type p53 also arrest in metaphase when incubated with colcemid but then, after collapse
to the restitution G1, these cells with polyploid DNA content
re-initiate DNA synthesis. The spindle damage checkpoint,
therefore, provides another measure of p53-dependent signaling.
The p53-dependent spindle damage checkpoint was monitored in cell lines with normal or altered p53 as shown by
SSCP. Cells were incubated with colcemid for 24 h and then
incubated with BrdU to identify cells synthesizing DNA. Flow
cytometry was used to quantify the fraction of cycling cells
Fig. 5. Induction of p21Waf1 protein in irradiated WB cells and hepatocyte
lines. Cells were either sham-treated or γ-irradiated with 8 Gy ionizing
radiation and harvested 2 or 6 h later. Cell lysates were separated by
polyacrylamide gel electrophoresis and p21Waf1 protein levels were
demonstrated by Western immunoblot analysis. β-Actin was immunolabeled to show protein loading.
that underwent a shift to higher ploidy during the incubation
with colcemid. Many of the unlabeled cell nuclei seen in the
colcemid-treated cultures had less than 4N DNA content and
therefore represented restitution nuclei (62) (Figure 6). The
WBL20C10 line with intact G1 checkpoint function showed
no alterations in p53 by SSCP, and when incubated with
colcemid, did not undergo endoreduplication (Figure 6E and
F). Similarly, WB-F344 cells with apparently wild-type p53
also had few endoreduplicating cells when incubated with
colcemid (Table III). The RLE-57 hepatocellular carcinoma
cell line had an apparent deletion in p53 exons 5–7 associated
with defective G1 checkpoint function. When colcemid was
added to RLE-57 cells, a significantly increased fraction of
polyploid cells were found to be synthesizing DNA (Figure
6C and D). The WBL20.6.5 line with a mutation in p53 codon
1263
W.K.Kaufmann et al.
Fig. 6. Spindle assembly checkpoint function in hepatic cell lines. Cells in log-phase growth were incubated for 24 h with colcemid. BrdU was added for the
final 2 h of incubation to label DNA in S-phase cells. The box encloses the tetraploid S/G2/M nuclei.
Table IV. Spindle assembly checkpoint function in rat hepatic cell lines
Cell line
Percent of cells endoreduplicatinga
Sham
WB-F344
WBL20C10
WBL20.6.5
RLE-57
6/15
4
2
6
4
4
⫾
⫾
⫾
⫾
2
1
1
2
Colcemid
8
4
23
18
4
⫾ 5 (13)
⫾ 2 (6)
⫾ 4* (6)
⫾ 7* (5)
(1)
⫾ standard deviation (n ⫽ number of experiments).
*P ⬍ 0.0005 by Student’s t-test (versus sham control).
Cells were incubated for 24 h with colcemid. Sham controls were incubated
in parallel without colcemid. BrdU was added for the final 2 h of incubation
and the cells then harvested for flow cytometry as described in the Materials
and methods. The fraction of cells in the tetraploid S, G2 and M
compartments was determined as a measure of endoreduplication. Defective
spindle assembly checkpoint function was recognized by the significantly
increased percentage of colcemid-treated cells that underwent
endoreduplication.
aMean
247 was comparable to RLE-57 in its response to colcemid,
with over 20% of cells proceeding through two rounds of
DNA synthesis without an intervening mitosis (Figure 6G and
H). The 6/15 hepatocyte line showed no p53 alteration by
SSCP and arrested growth in G1 when damaged by 8 Gy IR.
Accordingly, colcemid-treated 6/15 cells arrested in the diploid
G2/M (tetraploid G1) compartment (Figure 6A and B; Table
IV). The percentage of 6/15 cells found in the tetraploid
S/G2/M was not increased after 24 h incubation with colcemid.
These results indicated that hepatic epithelial cells with intact
1264
G1 checkpoint function and wild-type p53 first gather in mitosis
when exposed to colcemid, then fall out of mitosis and
become restitution nuclei which do not initiate DNA synthesis.
Inactivation of p53 function by mutation or exon deletion
appeared to enable tumorigenic hepatic cells to go through
two rounds of DNA synthesis without completing mitosis
(Table IV).
G2 checkpoint function
Ionizing radiation was also used to assess the G2 checkpoint
response to DNA damage. Cells were treated with 1 Gy and
then incubated for 2 h. This incubation allowed time for cells
in mitosis and past the G2 checkpoint to finish mitosis and
move into G1. Thus, when the cells were fixed 2 h post IR
any mitotic figures that were seen represented cells that
were exposed in G2 but evaded the checkpoint. Normal rat
hepatocytes were again treated 36 h after the addition of
growth factor as in the G1 checkpoint analysis. Mitosis in
normal rat hepatocytes was inhibited by 90% 2 h after 1 Gy
γ-rays (Figure 7). Six hours later the mitotic index had
recovered to the level of the sham-treated control. The stringent
mitotic delay response seen in NRH was comparable with that
seen in NHF (e.g. see Figure 7). The combined results from
six independent analyses indicated that, on average, only 7%
of G2 phase NRH evaded the G2 checkpoint and entered
mitosis 2 h after 1 Gy (Table V).
Both the PB-dependent 6/27C1 line and the tumorigenic
6/15 line displayed an attenuation of G2 checkpoint response
in comparison with NRH (Table V). For the 6/27C1 cells 79%
evaded mitotic delay, while 24% of 6/15 hepatocytes evaded
Aberrant cell cycle checkpoint function
Table V. Defective G2 checkpoint response in transformed hepatocytes
Cell line
Number of cells
counted (n)
Normal rat hepatocytes 12 000
6/27C1
6000
6/15
6000
CWSV1
2000
RLE-57
4000
γ/shama
Fraction evading
mitotic delay
10/141
129/164*
22/90*
13/20*
31/58*
0.07
0.79
0.24
0.65
0.53
of mitotic cells in γ-irradiated cultures divided by the number of
mitotic cells in sham-treated control cultures.
*P ⬍ 0.025 by χ2 test (versus NRH).
Cells in log phase were treated with 1 Gy γ-rays (or sham-treated as
controls) and then incubated for 2 h before fixation with 3:1 (v/v) methanol/
acetic acid. Nuclei were stained with propidium iodide and mitotic figures
were counted by fluorescence microscopy. At least 2000 cells were counted
for each sample and the percentage of mitotic cells was determined as the
mitotic index. G2 checkpoint function (γ/sham) was quantified as the
percentage of G2 cells that evaded radiation-induced mitotic delay (mitotic
indextreated/mitotic indexcontrol) 2 h after 1 Gy.
aNumber
Fig. 7. G2 checkpoint response in rat hepatocytes and WB cells. Cells were
treated with 1 Gy γ-rays and 2 h later fixed with 3:1 (v/v) methanol:acetic
acid. Propidium iodide was used to stain the nuclei for detection of mitotic
figures by fluorescence microscopy. G2 checkpoint function was quantified
as the percentage of G2 cells that evaded radiation-induced mitotic delay
(mitotic indextreated/mitotic indexcontrol). Normal rat hepatocytes (NRH) were
tested in primary culture (open box). Results for WB-F344 cells represent
mean ⫹ SD (n ⫽ 3) for passages 艋9 (checked box). Diploid human
fibroblasts were tested in secondary culture as described in Kaufmann et al.
(51) (gray box).
Table VI. Defective G2 checkpoint response in WB-F344 rat hepatic
epithelial stem-like cells and transformed derivatives
Cell line
Number of cells γ/shama
counted (n)
Fraction evading
mitotic delay
Normal rat hepatocytes
WB (passage 艋9)
WB (passage 10–19)
WB (passage 艌20)
WBL10C10 and L18C10
WBL2C10
WBL2.3.2 and L2.3.5
WBL6C8
WBL6.3.1 and L6.3.2
WBL14C8
WBL14.1
WBL20C10
WBL20.6.5
12 000
26 000
36 000
42 000
12 000
2000
12 000
2000
12 000
4000
8000
2000
8000
0.07
0.29
0.66
0.81
0.68
0.86
0.30
1.00
0.61
0.32
0.34
0.51
0.97
10/141
97/335*
406/615*
889/1092*
204/299*
57/66*
49/161*
41/39*
91/148*
20/62*
81/241*
21/41*
278/287*
Fig. 8. Age-dependent attenuation of G2 checkpoint function in WB cells.
Logarithmic cells were dosed with 1 Gy γ-rays and fixed 2 h later. G2
checkpoint function was quantified as the percentage of G2 cells that evaded
radiation-induced mitotic delay (mitotic indextreated/mitotic indexcontrol).
Linear regression showed a significant correlation between G2 checkpoint
function and WB passage level (R2 ⫽ 0.43, P ⬍ 0.01).
aNumber of mitotic cells in γ-irradiated cultures divided by the number of
mitotic cells in sham-treated control cultures.
*P ⬍ 0.025 by χ2 test (versus NRH).
G2 checkpoint function was quantified as in Table V.
the checkpoint. Both fractions were significantly increased
over NRH (P ⬍ 0.025). The SV40-transformed CWSV1 cells
and the hepatocellular carcinoma line, RLE-57, also displayed
significant attenuation of G2 checkpoint response in comparison
to NRH. These results suggest that G2 checkpoint function
may be frequently altered in hepatocarcinogenesis.
G2 checkpoint function was also examined in the WB-F344
cell line and transformed derivatives. Low-passage WB cells
(passage 艋9) displayed mitotic inhibition and subsequent
recovery after treatment with 1 Gy IR as was seen in NRH
and NHF (Figure 7). However, as the WB cells were aged in
culture, G2 checkpoint function was lost progressively (Figure
8). The degree of attenuation fluctuated substantially but
increased as the WB cells aged. There was a highly significant
correlation between inactivation of G2 checkpoint function and
passage level (R2 ⫽ 0.43, P ⬍ 0.01). Degradation of G2
checkpoint function occurred early in the lifespan of WBF344 rat hepatic epithelial stem-like cells (Table VI). In
Table VII. Defective cell cycle checkpoint function at stages of rat
hepatocarcinogenesis
Stage of transformation
G1 checkpoint
G2 checkpoint
Primary hepatocytes
Promoter-dependent, chemically
initiated hepatocytes
SV40-transformed hepatocytes
Tumorigenic hepatocytes
Secondary cultures of WB-F344 cells
Selectively cycled, non-tumorigenic
Selectively cycled, tumorigenic
Tumor-derived WB clones
Intact
Attenuated
Intact
Attenuated
Ablated
Intact/ablated
Intact
Intact
Intact
Intact/ablated
Attenuated
Attenuated
Intact/attenuated
Attenuated
Attenuated/ablated
Attenuated/ablated
Primary cultures of rat hepatocytes and WB-F344 cells at passages ⬍6
responded to DNA damage with significant cell cycle delays. Checkpoints
were judged to be intact. Cell cultures in which ⬍15% of cells responded to
DNA damage were considered to have ablated checkpoint function. Cell
lines with checkpoint responses that were significantly different than NRH
and low passage WB-F344 cells but not fully ablated were considered to
have attenuated checkpoint function.
1265
W.K.Kaufmann et al.
comparison to NRH, the non-tumorigenic, parental and tumorigenic WB lines all displayed significant attenuation of G2
checkpoint function. Attenuation of G2 checkpoint function
appeared to precede tumorigenicity in WB-F344 cells and
transformed derivatives.
Discussion
The goal of this study was to determine whether transformation
of rat hepatocytes and hepatic epithelial stem-like cells was
associated with alterations in cell cycle checkpoint function.
Cell cycle checkpoint systems represent complex signaling
networks that integrate the machinery of the cell cycle with
DNA repair pathways and lifespan controls (1–4). Because
functional defects in checkpoint response both enhance growth
and destabilize the genome, such defects are expected to
accelerate multistep carcinogenesis (63). It is prohibitively
costly and time-consuming to determine mutation in every
gene known to affect cell cycle checkpoint function in a survey
such as this. Quantitative functional assays therefore were
used to assess the integrity of G1 and G2 checkpoint signaling
pathways in the two models of rat hepatocarcinogenesis
(Table VII). Checkpoint dysfunction was a common event in
hepatocarcinogenesis, with significant deficits in checkpoint
function being observed in both tumorigenic and non-tumorigenic cell lines in both models. Future studies will be devoted
to determining the genetic and/or epigenetic alterations that
account for these functional defects.
The cell lines used in this study were all derived from livers
of F344 male rats and were chosen to express a range of
transformation-related traits including promoter dependence,
immortality and tumorigenicity. Significant deficits in checkpoint function were observed even in the earliest stages
represented, the promoter-dependent but non-tumorigenic 6/
27C1 hepatocyte line and the WB-F344 line at passage levels
⬍25. A second chemically initiated hepatocyte line (RLE-57)
also displayed a defect in G1 checkpoint function, suggesting
that inactivation or attenuation of p53-dependent signaling
in response to DNA damage occurred during hepatocyte
transformation. Three of four tumor-derived WB-F344 lineages
displayed normal G1 checkpoint response to IR, implying that
inactivation of the p53-dependent signaling pathway was not
required for tumorigenic progression in the stem-like cell
model. All transformed hepatocytes and WB-F344 cells displayed some degree of attenuation of G2 checkpoint function,
however. According to current models, reduced checkpoint
function renders cells resistant to growth arrest and apoptosis,
and enhances genetic instability. Enhanced growth and genetic
instability should fuel malignant progression by increasing the
numbers of cells acquiring mutations and the rate that mutations
are acquired (63).
Both primary cultures of rat hepatocytes and secondary
cultures of WB-F344 hepatic epithelial stem-like cells displayed a G1 arrest response to IR. Transformation of hepatocytes with SV40 viral DNA inactivated this response
presumably through large T antigen binding to p53 and Rb.
Treatment with IR induced p21Waf1 in WB-F344 cells, and
alterations in p53 seen by SSCP and sequencing were associated
with an absence of basal p21Waf1 expression and no induction
of protein after IR. The two types of hepatic epithelial cells
in culture appeared to express a stereotypic G1 checkpoint
response. Under conditions of IR-induced G1 checkpoint
response in primary cultures of rat hepatocytes, apoptosis also
1266
was observed and radiation-induced apoptosis was significantly
reduced in the SV40-transformed hepatocytes (64). We previously demonstrated that a mid-thoracic dose of 6 Gy IR given
to young adult F344 rats at 4 h after partial hepatectomy
inhibited hepatocyte entry to S-phase by about 20 h (65).
During this G1 delay large numbers of apoptotic hepatocytes
were seen (W.K.Kaufmann, unpublished data). Thus, in this
biological model system, p53-dependent G1 checkpoint
response is also associated with radiation-induced apoptosis.
Attenuation and inactivation of G1 checkpoint function might
reduce hepatic cell sensitivity to certain apoptotic signals,
contributing to clonal expansion.
The 6/27C1 immortal line and 6/15 tumorigenic line were
both derived from chemically initiated hepatocytes cultivated
in medium containing the tumor promoter PB (35). A single
treatment with the chemical carcinogen, DMN-OAc, in vivo
resulted in a population of initiated hepatocytes that, when
cultured in vitro with PB, were promoted to immortality, and
in the case of the 6/15 hepatocytes, progressed to tumorigenicity
(35). Immortal 6/27C1 hepatocytes displayed a significantly
attenuated G1 checkpoint response to IR indicating that this
function may be lost at a stage of hepatocarcinogenesis
preceding tumorigenicity. This cell line has remained PBdependent in colony formation assays (data not shown). The
6/15 hepatocytes displayed PB-dependent colony formation at
passage 55, but at passage 90 showed less requirement for PB
and produced hepatocellular carcinomas in animals not fed PB
(35). At passages 119–159, when used in this study, 6/15
hepatocytes were PB-independent by colony formation assay
but still tumorigenic (data not shown). G1 checkpoint function
in 6/15 hepatocytes appeared to be normal after 8 Gy of IR.
Moreover these cells did not undergo endoreduplication when
incubated with colcemid. These results that suggest there was
normal p53-dependent G1 checkpoint function in the 6/15 line
are tempered by the reduced G1 arrest in 6/15 cells after 2 Gy
and the lack of induction of p21Waf1 after 8 Gy of IR.
Phenobarbital had been shown previously to delay induction
of p53 in primary cultures of normal mouse hepatocytes
after exposure to the radiomimetic chemotherapeutic drug,
bleomycin (55). When PB was withdrawn from culture medium
6/15 cells continued to express G1 arrest after the high dose
of 8 Gy IR (data not shown), implying that PB did not inhibit
G1 checkpoint response after a saturating dose of IR. It is
nevertheless conceivable that the apparent reduced G1 checkpoint response to the lower 2 Gy dose and the modest induction
of p21Waf1 in the 6/15 line were related to the presence of
PB in culture medium. Phenobarbital may attenuate the G1
checkpoint response after a low dose of IR, but not after a
high IR dose that saturates the signaling pathway. The 6/27C1
hepatocytes die when deprived of PB for ⬎4 days (37). G1
checkpoint function remained attenuated when the 6/27C1
hepatocytes were deprived of PB for 24 or 48 h (data not
shown) suggesting that their reduced G1 checkpoint function
was not rapidly reversed after removal of PB. A recent study
indicated that epidermal growth factor produced a repression
of ATM mRNA and protein expression in human fibroblasts
and lymphoblasts through reduction in the SP1 transcription
factor (66). As phenobarbital appears to sustain clonal expansion by chemically initiated hepatocytes through a TGF-αassociated signaling pathway that may include the epidermal
growth factor receptor (36), it is conceivable that G1 checkpoint
function was attenuated in the 6/27C1 line through reduced
expression of ATM. RLE-57 cells were initiated under the
Aberrant cell cycle checkpoint function
same conditions as 6/27C1 and 6/15, but were subjected to
57 weeks promotion with PB in vivo before isolation and
establishment into cell culture (33). The observation that
in vitro culture of 6/15 hepatocytes and WB-F344 cells did
not select for loss of p53 function suggests that p53 was
altered during, not after, malignant transformation of the RLE57 line.
The stem cell model of hepatocarcinogenesis involved
WB-F344 hepatic epithelial stem-like cells and transformed
derivatives. When WB-F344 cells were transplanted into the
intrascapular fat pads of syngeneic rats, aggregates of cells
that resembled hepatocytes and bile ducts were formed (67).
Continuous passaging of rat liver epithelial cells such as WBF344 results in spontaneous transformation at passage levels
above 25 (58,68). WB-F344 cells are also susceptible to
chemical carcinogenesis. When exposed to 11 treatments
with N-methyl-N⬘-nitro-N-nitrosoguanidine, WB-F344 cells
underwent malignant transformation producing a range of
tumor types, including hepatocellular carcinomas, when transplanted into syngeneic rats (56,57). In this study, we focused
on the use of repetitive cycles of growth followed by prolonged
confluence arrest to generate tumorigenic segregants (39).
WB-F344 cells displayed normal G1 checkpoint function at
low and high passage number. Transformed lines derived by
selection with confluence arrest also had normal G1 checkpoint
function. The tumor-derived line WBL20.6.5 with a mutation
in p53 was the only WB cell line in this series that displayed
an attenuated G1 checkpoint response. The parental line
WBL20C10 with apparently wild-type p53 had normal G1
checkpoint response, suggesting that tumorigenic outgrowth
in vivo may have been associated with mutation in p53.
Mutations at codon 247 have been reported in rats with liver
tumors induced by aflatoxin B1 (47) and in rats fed ethionine
with a methyl deficient diet (69). Codon 247 in the rat
corresponds to codon 249 in the human (70). Other studies on
the set of transformed lines selected by confluence arrest
indicated aneuploidy was a frequent event during malignant
progression (39). Moreover, when seven tumor-derived lines
were tested for their ability to arrest nuclear replication in the
presence of cytochalasin B, which disrupts microfilaments,
three lines including WBL20.6.5 failed to arrest growth in
cytochalasin B (G.J.Smith, unpublished data). When tested for
induction of p21Waf1 by IR, these three lines displayed no
induction. Although inactivation of p53-dependent G1 checkpoint function does not appear to be required for tumorigenicity
of WB-F344 cells, it was clearly demonstrable in a subset of
tumor-derived lines.
None of the transformed WB cell lines had a normal G2
checkpoint response. WB cells that were passaged 1:12 each
week beginning at passage 4 displayed significant attenuation
of G2 checkpoint function by passage 10. WB-F344 cells
rapidly lost G2 checkpoint function during in vitro passaging
while retaining G1 checkpoint function. A mechanistic explanation for this phenomenon is not apparent at this time, although
during the same interval WB cells also lost expression of
telomerase (41). Evidently gene products whose expression is
required to sustain G2 checkpoint function and expression of
telomerase are unstable in secondary cultures of WB cells. G2
checkpoint function and expression of telomerase (41) also
were lost in the WB cells that underwent the selective growth
protocol. It is conceivable that during normal WB-F344 cell
culture, as well as in the protocol of selective growth from
confluence, there was selection for cells that repressed expres-
sion of telomerase and G2 checkpoint function. As the G2
checkpoint also appears to be an important barrier protecting
against chromosomal destabilization (44), the aging-related loss
of G2 checkpoint function may contribute to the chromosomal
instability noted during malignant transformation of WB-F344
cells (39,40).
The combined results with hepatocyte and WB-F344 models
suggest that loss of G2 checkpoint function may be an early
event in hepatocarcinogenesis. Inactivation of G2 checkpoint
function in transformed human fibroblasts was associated with
increased levels of cyclin B1 (15,51). A study of patients
with hepatocellular carcinoma found that 15% had serum
autoantibodies reactive with cyclin B1 (71), suggestive of
overexpression and release from the carcinoma. Further studies
need to be done to examine the mechanisms of defective G2
checkpoint response in transformed hepatocytes and hepatic
epithelial stem-like cells.
The G2 checkpoint also ensures that mitosis is not initiated
until intertwined sister chromosomes are sufficiently decatenated by topoisomerase II (72). Failure of the chromatid
catenation-sensitive G2 checkpoint may permit entry of cells
into mitosis with incompletely decatenated chromosomes. The
tangled chromosomes cannot be segregated properly resulting
in aneuploidy through non-disjunction errors or polyploidy
after mitotic collapse.
Polyploidization is a normal process in both human and rat
livers. At birth, a majority of hepatocytes are diploid. As the
liver ages, there is a shift of ploidy towards tetraploidy. The
majority of the hepatocytes in an adult rat liver are tetraploid
as was seen in the cytometric profile of normal rat hepatocytes.
All of the chemically transformed hepatocyte lines had a large
fraction of tetraploid cells. The WB cells also underwent
polyploidization during aging in vitro. It remains to be determined how the acquisition of polyploidy in parenchymal
hepatocytes in vivo is integrated with cell cycle checkpoints
that act to suppress polyploidization
The spindle assembly checkpoint also ensures the fidelity
of chromosome segregation by monitoring attachment of
chromosomes to the spindle and their subsequent movement
to the poles of the dividing cell. Chemical and physical damage
to the spindle could lead to cells with aneuploid or polyploid
chromosome number. Cells that are defective in the spindle
assembly checkpoint do not collect in mitosis when spindles
are damaged but instead undergo additional rounds of DNA
replication without completing mitosis (endoreduplication)
(23). Hepatic cells with normal G1 checkpoint function were
found to have normal spindle assembly checkpoint function.
Tumorigenic hepatic cells with defective G1 checkpoint function (RLE-57 and WBL20.6.5) had a defective response to
spindle damage and endoreduplicated in colcemid. Failure of
the spindle assembly and G1 checkpoints also may contribute
to polyploidization during hepatocarcinogenesis.
In summary, aberrant cell cycle checkpoint function appears
to occur early in the multi-step process of hepatocarcinogenesis.
Loss of G2 checkpoint function preceded tumorigenicity in
both the hepatocyte and stem cell models. Loss of G1 checkpoint function associated with alterations in p53 occurred
in both models but did not always precede or accompany
tumorigenicity. The results suggest that alterations in cell cycle
checkpoint function occur frequently and early in the process
of hepatocarcinogenesis.
1267
W.K.Kaufmann et al.
Acknowledgements
We thank Dr Harriet Isom for providing the CWSV1 hepatocyte line. We are
grateful to UNC-CH graduate students who contributed to these studies,
Chi-Liang Yen and Cheryl Cistulli. This study was supported by PHS grants
CA59496 (WKK), CA29323 (JWG) and CA59486 (GJS).
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Received July 19, 2000; revised March 29, 2001; accepted April 19, 2001
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