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
1
Departments of Pathology and Laboratory Medicine,
2
Lineberger Comprehensive Cancer Center and
3
Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA
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Abstract
|
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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 G
1
, G
2
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 68 h following 8 Gy of ionizing radiation (IR) as a quantitative measure of G
1
checkpoint function. Chemically and virally transformed hepatocyte lines displayed significant attenuation of G
1
checkpoint function, ranging from partial to complete ablation. WB-F344 rat hepatic epithelial cell lines at low, mid and high passage levels expressed G
1
checkpoint function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant attenuation of G
1
checkpoint function. Attenuation of G
1
checkpoint function in transformed hepatocytes and WB-F344 cells was associated with alterations in
p53
, ablated/attenuated induction of p21
Waf1
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 G
2
checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G
2
checkpoint. Moreover, the parental WB-F344 line displayed significant age-related attenuation of G
2
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
,
single-strand conformational polymorphism.
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Introduction
|
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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 G
1
checkpoint may slow or even arrest entry into S-phase. Key gene products for G
1
checkpoint response to ionizing radiation (IR)-induced DNA damage include ATM, p53, p21
Waf1
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 p21
Waf1
, GADD45, MDM2 and BAX. BAX induction and the subsequent inhibition of BCL-2 may trigger apoptosis (
6
). The higher levels of p21
Waf1
inhibit G
1
cyclin-dependent kinases (CDKs) and arrest or delay the entry of cells into S-phase (
7
,
8
). Inhibition of G
1
CDKs by p21
Waf1
preserves Rb binding to E2F and enforces G
1
arrest. Cells that lack p53 function by genomic mutation or viral gene expression are unable to induce p21
Waf1
in response to DNA damage and, consequently, are unable to halt progression from G
1
to S-phase (
7
). Cells with Rb function inactivated by human papilloma virus E7 gene product also display defective G
1
checkpoint function (
9
).
Progression from G
2
to mitosis is regulated by the G
2
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 (p34
CDC2
), 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 G
2
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 G
2
checkpoint as AT cells display reduced sensitivity to radiation-induced G
2
delay (
14
,
15
). Expression of a natural splice variant of BRCA1, which deletes exon 11, fully ablated G
2
checkpoint function in mouse embryo fibroblasts without affecting G
1
checkpoint function (
16
). Inhibition of CHK1, a kinase that can phosphorylate CDC25C, also inactivated G
2
checkpoint response (
17
). CDC25C is a phosphatase that removes inhibitory phosphates in the ATP-binding domain of MPF. Phosphorylation of CDC25C generates a 14-3-3 binding site, resulting in sequestration of CDC25C in the cytoplasm (
18
) and inhibition of phosphatase activity (
19
). Mutation of the 14-3-3 binding site in CDC25C attenuated G
2
checkpoint response to DNA damage (
18
). Cells with damaged DNA accumulate in G
2
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 G
1
-like state with reformation of nuclear envelope around decondensed chromosomes. Cells appear to re-enter G
1
but with twice normal DNA content. In cells expressing wild-type p53, the G
1
checkpoint is then activated in these 4N interphase nuclei and a G
1
arrest occurs (
27
). Cells with mutations in
p53
and defective G
1
checkpoint function initiate DNA synthesis from this 4N G
1
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 LiFraumeni cells expressing only mutant p53 and human papilloma virus type 16 E6-transformed human fibroblasts lacking p53 function fail to undergo G
1
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 G
2
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 G
1
, G
2
and mitotic spindle assembly checkpoints in rat hepatocytes, rat hepatic epithelial stem-like cells and transformed derivatives. These studies indicated that transformation of rat hepatic epithelial cells was associated with significant defects in cell cycle checkpoint function.
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Materials and methods
|
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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 FeCl
3
, 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 B
12
, 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-
and 10 ng/ml norepinephrine.
All cells were incubated in a humidified atmosphere of 5% CO
2
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 5228 (
38
); two selectively cycled but non-tumorigenic 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
).
G
1
checkpoint function
G
1
checkpoint function was quantified using flow cytometry (
44
). Primary hepatocyte cultures at the peak of DNA synthesis after addition of TGF-
and established hepatic cell lines in logarithmic growth were treated with 2 or 8 Gy
137
Cs
-rays (Gammacell 40) and returned to the incubator. Sham-treated controls were taken in and out of the incubator with the irradiated samples but not exposed to
-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 G
1
checkpoint function was done by determining the radiation-induced reduction in the percentage of cells in the first half of the S-phase 68 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 MgCl
2
, 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 MgCl
2
containing 0.5 µCi [
-
32
P]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 1x sample buffer (5x 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 89 h at room temperature. Autoradiography was performed with an intensifying screen for 16 h. A rat nasal squamous carcinoma cell line, FAT 7 (American Type Culture Collection), with a known mutation in exon 8 of
p53
(271 codon, transversion from C
G
T
C
A
T) (
46
) was used as a positive control.
The following primers for amplification of
p53
exons 59 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 (AdvantageTM 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 581822 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 PRISMTM Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase, FS (Perkin Elmer Applied Biosystems). Sequences were compared using SequencherTM 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 2x lysis buffer (2% SDS, 20% glycerol, 2% ß-mercaptoethanol and 0.02% bromophenol blue in 62.5 mM TrisHCl pH 6.8) at a concentration of 5x10
6
cells/ml. Lysates were boiled for 5 min and protein was then separated on a 12% SDSPAGE 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-ß-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 48 N DNA was quantified as a measure of spindle assembly checkpoint function.
G
2
checkpoint function
G
2
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. G
2
checkpoint function was quantified as the percentage of G
2
cells that evaded radiation-induced mitotic delay (mitotic index
treated
/mitotic index
control
) 2 h after 1 Gy.
 |
Results
|
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G
1
checkpoint function
DNA strand breaks induced by ionizing radiation activate the p53-dependent G
1
checkpoint (
7
). The G
1
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 68 h following 8 Gy of IR (
49
). A flow cytometric method was used to investigate G
1
checkpoint function in rat hepatocytes at various stages of transformation. Primary rat hepatocytes were isolated and incubated with TGF-
to induce proliferation. DNA synthesis assay using [
3
H]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
-rays 36 h post addition of TGF-
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
-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.

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Fig. 1.
G
1
checkpoint function in rat hepatocyte lines. Cells were sham-treated or irradiated with 8 Gy -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. G
0
G
1
(bottom left), S-phase (top), G
2
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.
|
|
Having shown that primary cultures of rat hepatocytes displayed a quantifiable G
1
checkpoint response to DNA damage, we then tested other hepatocyte lines. The CWSV1 line is an SV40-transformed hepatocyte line (
42
,
43
). SV40-large T antigen is known to bind and inactivate p53 (
50
). Accordingly, SV40-transformed human fibroblasts lack p53-dependent G
1
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 G
1
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 G
1
checkpoint response in primary cultures 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 of PB showed a significant attenuation of G
1
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 G
1
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 G
1
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 G
1
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 G
1
checkpoint function (
Figure
3AF
) (
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 G
1
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
).

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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.
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Fig. 3.
G
1
checkpoint function in WB cells and transformed derivatives. Conditions of cell treatment and analysis were as described in the legend to
Figure
1
.
|
|
In addition to the normal WB-F344 cells, several transformed lines were examined. These cell lines were derived from WB-F344 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 810 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
). G
1
checkpoint function was determined for the tumorigenic lines prior to transplantation and for clonal lines isolated from tumors. The non-tumorigenic cell lines displayed G
1
checkpoint function equivalent to the low passage WB-F344. Additionally, most of the tumorigenic lines and tumor-derived lines had normal G
1
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 G
1
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 G
1
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 G
1
checkpoint response. The tumorigenic and tumor-derived WB lines also had a G
1
arrest at 2 Gy nearly equivalent to the arrest seen at 8 Gy (
Table
III
). However in the 6/27C1 and 6/15 hepatocyte lines, the G
1
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 G
1
checkpoint further, we looked for alterations in
p53
structure by single-strand conformational polymorphism (SSCP) analysis and direct sequencing. SSCP analysis examined exons 59 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 G
1
checkpoint response by the flow cytometry assay (data not shown). The PB-dependent 6/27C1 cell line had a significantly attenuated G
1
checkpoint response to DNA damage. However, SSCP did not detect a
p53
alteration in the exons examined. The 6/15 line with measurable G
1
checkpoint response after IR also showed no
p53
alteration by SSCP. The rat hepatocellular line RLE-57 with defective G
1
checkpoint function did not yield an amplified product from exons 57 suggestive of an intragenic deletion (
Figure
4
). WB-F344 cells displayed a functional G
1
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 G
1
checkpoint by flow cytometric analysis also had no
p53
alteration by SSCP. However, the tumor-derived cell line WBL20.6.5 with severely attenuated G
1
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 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 G
1
checkpoint. The FAT 7, RLE-57 and WBL20.6.5 lines with attenuated G
1
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 G
1
checkpoint function displayed apparently normal
p53
structure.
Analysis of p21
Waf1
induction
An important component of the p53-dependent G
1
checkpoint response is p21
Waf1
. Activation of p53 by ATM and other effectors leads to increased levels of p21
Waf1
and subsequent inhibition of G
1
CDKs. Cells that lose p53 function are unable to induce p21
Waf1
and do not arrest progression from G
1
to S after IR. WB cells at passage 16 and 28 induced p21
Waf1
6 h after 8 Gy IR (
Figure
5A
). The parental WBL20C10 line responded as the low passage WB cell lines with induction of p21
Waf1
6 h after IR (
Figure
5B
). No p21
Waf1
protein was detected 6 h following 8 Gy in the WBL20.6.5 cells. These results show that WB cell lines with normal G
1
checkpoint function also display induction of the p21
Waf1
protein following IR, and the one WB line with mutant
p53
did not express p21
Waf1
nor induce it after IR.

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Fig. 5.
Induction of p21
Waf1
protein in irradiated WB cells and hepatocyte lines. Cells were either sham-treated or -irradiated with 8 Gy ionizing radiation and harvested 2 or 6 h later. Cell lysates were separated by polyacrylamide gel electrophoresis and p21
Waf1
protein levels were demonstrated by Western immunoblot analysis. ß-Actin was immuno- labeled to show protein loading.
|
|
The results with transformed hepatocytes were similar to those with WB cells. The immortal 6/27C1 hepatocyte line displayed a significantly attenuated G
1
checkpoint response to DNA damage. Although p21
Waf1
was detected in unirradiated controls, there was little induction of p21
Waf1
protein 6 h after 8 Gy IR (
Figure
5C
). Although the tumorigenic 6/15 hepatocyte line had apparently normal G
1
checkpoint function following 8 Gy of IR, only modest p21
Waf1
induction was seen in these cells. RLE-57 displayed both attenuation of G
1
checkpoint function and altered
p53
by SSCP analysis. No p21
Waf1
protein was detected in the sham or irradiated RLE-57 cell lysates. The p21
Waf1
results in the WB and hepatocyte models associated loss of G
1
checkpoint function with loss of p53-mediated induction of p21
Waf1
. The only exception was the 6/15 hepatocyte line which showed modest induction of p21
Waf1
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 G
1
-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 G
1
, 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 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 G
1
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 57 associated with defective G
1
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 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 G
1
when damaged by 8 Gy IR. Accordingly, colcemid-treated 6/15 cells arrested in the diploid G
2
/M (tetraploid G
1
) compartment (
Figure
6A
and
B
;
Table
IV
). The percentage of 6/15 cells found in the tetraploid S/G
2
/M was not increased after 24 h incubation with colcemid. These results indicated that hepatic epithelial cells with intact G
1
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
).

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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/G
2
/M nuclei.
|
|
G
2
checkpoint function
Ionizing radiation was also used to assess the G
2
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 G
2
checkpoint to finish mitosis and move into G
1
. Thus, when the cells were fixed 2 h post IR any mitotic figures that were seen represented cells that were exposed in G
2
but evaded the checkpoint. Normal rat hepatocytes were again treated 36 h after the addition of growth factor as in the G
1
checkpoint analysis. Mitosis in normal rat hepatocytes was inhibited by 90% 2 h after 1 Gy
-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 G
2
phase NRH evaded the G
2
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 G
2
checkpoint response in comparison with NRH (
Table
V
). For the 6/27C1 cells 79% evaded mitotic delay, while 24% of 6/15 hepatocytes evaded 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 G
2
checkpoint response in comparison to NRH. These results suggest that G
2
checkpoint function may be frequently altered in hepatocarcinogenesis.
G
2
checkpoint function was also examined in the WB-F344 cell line and transformed derivatives. Low-passage WB cells (passage
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, G
2
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 G
2
checkpoint function and passage level (
R
2
= 0.43,
P
< 0.01). Degradation of G
2
checkpoint function occurred early in the lifespan of WB-F344 rat hepatic epithelial stem-like cells (
Table
VI
). In comparison to NRH, the non-tumorigenic, parental and tumorigenic WB lines all displayed significant attenuation of G
2
checkpoint function. Attenuation of G
2
checkpoint function appeared to precede tumorigenicity in WB-F344 cells and transformed derivatives.

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Fig. 8.
Age-dependent attenuation of G
2
checkpoint function in WB cells. Logarithmic cells were dosed with 1 Gy -rays and fixed 2 h later. G
2
checkpoint function was quantified as the percentage of G
2
cells that evaded radiation-induced mitotic delay (mitotic index
treated
/mitotic index
control
). Linear regression showed a significant correlation between G
2
checkpoint function and WB passage level (
R
2
= 0.43,
P
< 0.01).
|
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Table VI. Defective G
2
checkpoint response in WB-F344 rat hepatic epithelial stem-like 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 G
1
and G
2
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 G
1
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 G
1
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 G
2
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 G
1
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 p21
Waf1
in WB-F344 cells, and alterations in
p53
seen by SSCP and sequencing were associated with an absence of basal p21
Waf1
expression and no induction of protein after IR. The two types of hepatic epithelial cells in culture appeared to express a stereotypic G
1
checkpoint response. Under conditions of IR-induced G
1
checkpoint response in primary cultures of rat hepatocytes, apoptosis also 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 G
1
delay large numbers of apoptotic hepatocytes were seen (W.K.Kaufmann, unpublished data). Thus, in this biological model system, p53-dependent G
1
checkpoint response is also associated with radiation-induced apoptosis. Attenuation and inactivation of G
1
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 G
1
checkpoint response to IR indicating that this function may be lost at a stage of hepatocarcinogenesis preceding tumorigenicity. This cell line has remained PB-dependent 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 119159, when used in this study, 6/15 hepatocytes were PB-independent by colony formation assay but still tumorigenic (data not shown). G
1
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 G
1
checkpoint function in the 6/15 line are tempered by the reduced G
1
arrest in 6/15 cells after 2 Gy and the lack of induction of p21
Waf1
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 G
1
arrest after the high dose of 8 Gy IR (data not shown), implying that PB did not inhibit G
1
checkpoint response after a saturating dose of IR. It is nevertheless conceivable that the apparent reduced G
1
checkpoint response to the lower 2 Gy dose and the modest induction of p21
Waf1
in the 6/15 line were related to the presence of PB in culture medium. Phenobarbital may attenuate the G
1
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
). G
1
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 G
1
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-
-associated signaling pathway that may include the epidermal growth factor receptor (
36
), it is conceivable that G
1
checkpoint function was attenuated in the 6/27C1 line through reduced expression of ATM. RLE-57 cells were initiated under the 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 RLE-57 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 WB-F344 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 G
1
checkpoint function at low and high passage number. Transformed lines derived by selection with confluence arrest also had normal G
1
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 G
1
checkpoint response. The parental line WBL20C10 with apparently wild-type p53 had normal G
1
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 p21
Waf1
by IR, these three lines displayed no induction. Although inactivation of p53-dependent G
1
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 G
2
checkpoint response. WB cells that were passaged 1:12 each week beginning at passage 4 displayed significant attenuation of G
2
checkpoint function by passage 10. WB-F344 cells rapidly lost G
2
checkpoint function during
in vitro
passaging while retaining G
1
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 G
2
checkpoint function and expression of telomerase are unstable in secondary cultures of WB cells. G
2
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 expression of telomerase and G
2
checkpoint function. As the G
2
checkpoint also appears to be an important barrier protecting against chromosomal destabilization (
44
), the aging-related loss of G
2
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 G
2
checkpoint function may be an early event in hepatocarcinogenesis. Inactivation of G
2
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 G
2
checkpoint response in transformed hepatocytes and hepatic epithelial stem-like cells.
The G
2
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 G
2
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 G
1
checkpoint function were found to have normal spindle assembly checkpoint function. Tumorigenic hepatic cells with defective G
1
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 G
1
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 G
2
checkpoint function preceded tumorigenicity in both the hepatocyte and stem cell models. Loss of G
1
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.
 |
Notes
|
---|
4 To whom correspondence should be addressed at: Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA
Email: bill_kaufmann{at}med.unc.edu 
 |
Acknowledgments
|
---|
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.