Aberrant cell cycle checkpoint function in transformed hepatocytes and ...

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

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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

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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).


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.

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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


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

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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/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


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.

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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


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

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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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