Received 8/1 1/97; revised 12/10/97; accepted 12/15/97.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 Supported by the Institut National de Ia Sante et de Ia RechercheMedicale, by EEC Grant BlOT-CT 960052, and by the Ugue Nationalecontre le Cancer, Comite d’llle et Vilaine. S. C. is the recipient of afellowship from the Association pour Ia recherche contre Ic Cancer.2 To whom requests for reprints should be addressed. Phone:0299543737; Fax: 0299540137.

Vol. 9, 165-1 76, February 1998 Cell Growth & Differentiation 165

Cell Cycle Gene Regulation in Reversibly Differentiated NewHuman Hepatoma Cell Lines1

Denise Glaise, Gennady P. llyin,2 Pascal Loyer,Sandrine Canou, Marc Bilodeau, Josiane Lucas,Alain Puisieux, Mehmet Ozturk, andChnstiane Guguen-Guillouzo

INSERM U49 [D. G., G. P. I., P. L, S. C., M. B., C. G-G.] andLaboratoire de G#{233}n#{233}tiqueet Biologie Cellulaire [J. L], H#{244}pitalPontchaillou, 35033 Rennes, France, and INSERM U453, Centre LeonB#{233}rard,69373 Lyon, France [A. P., M. 0.]

AbstractSeveral novel differentiated cell lines have beenderived from a human hepatocarcinoma named HBG.Analysis of their functional properties evidenced agradual differentiation process as they becameconfluent and a remarkable stability of the wholequiescent population for at least 6 weeks. However,when replated at low density after several weeks ofquiescence, the differentiated cells were able to rapidlyreverse to active proliferation, accompanied bytransient dedifferentiation. Demonstration that thedifferentiated hepatic cells were growth-arrested in G1phase was provided by the increased number of cellswith 2C DNA content and decreased expression of S-phase markers. Characteristic features of oncogenesand cell cycle genes were defined during thedifferentiation process: (a) a biphasic expression of c-myc, with the latter wave covering the quiescenceperiod; (b) opposite kinetics of c-Ki-ras and of N-msexpression with a pattern of changes paralleling that ofc-myc; and (c) a decrease of cyclin Dl proteinexpression and of the cyclin Dl -associated kinaseactivity. The mechanisms by which quiescentdifferentiated cells might reinitiate active proliferationwere analyzed by studying several genes involved incell growth and death regulation. We found: (a) a pointmutation and loss of the specific activity of the tumorsuppressor gene p53 without alteration of theapoptotic response to transforming growth factor flu;(b) a gradual decrease of retinoblastoma protein, whichwas constantly present, mainly in ahyperphosphorylated form; and (c) an increase ofcyclin-dependent kinase inhibitor p27 expression inconfluent differentiating cells, as expected, whereas,

surprisingly, a disappearance of the p21 protein wasobserved in parallel. These data may reflect specificmechanisms of cell cycle regulation in liverparenchymal cells through which these cells canproceed to control their reversible differentiationprogram.

IntroductionA number of studies have reported changes in tissue-specificgene expression occurring in vitro in various cell types such

as myocytes, adipocytes, and neuronal and hemopoieticcells, which undergo terminal differentiation after severalrounds of division, as normally observed in vivo. Most ofthese cells, when becoming differentiated, ultimately losetheir ability to divide and finally die. This leads to the generalassumption that a balance between proliferation, differenti-ation, and cell death does exist, and that a cross-talk be-tween the regulatory molecules that control each of themmight be precisely orchestrated to make it possible (1). A

number of permanent rodent and human cell lines can also

undergo their irreversible terminal differentiation program like

their normal cell counterparts (2-4).

Unlike the above-mentioned types of terminally differenti-ated cells, mature hepatocytes maintain their high prolifera-tive potential after a long-term quiescent state, manifested

by the ability of the liver in vivo to regenerate at the adultstage after necrosis or partial hepatectomy (5). To date,because of the lack of suitable in vitro model systems, little

is known about the regulation of this hepatic feature. Isolatedhepatocytes, although highly differentiated at plating, have a

limited lifetime and proliferating capacity and fail to rediffer-

entiate after division under conventional culture conditions(6). A few studies have reported enhanced expression ofliver-specific functions in the course of the hepatic differen-

tiation process in permanent hepatoma cell lines, parallelinga decrease in their proliferation activity (7, 8). However, noclear definition of the cell cycle phase at which hepatoma

cells undergo differentiation has been reported, and no evi-dence has clearly been provided that they are able to reversetoward an active proliferative status after differentiation. In

this paper, we describe a series of novel differentiated cell

lines derived from a human hepatoma named HBG that

undergo highly stable differentiation at confluence and can

reverse to active proliferation when replated, making themideal candidates for studying the mechanisms of growth

arrest in relation with differentiation and the balance with cell

death in liver parenchymal cells.

It is well documented that various cell types, when made

quiescent by serum deprivation or at confluence or by addi-

tion of differentiating agents, stop to proliferate and remain

arrested in G1 phase. This is evidenced by arrest of cellgrowth, inhibition of DNA synthesis, or accumulation of cells

with 2C DNA content and expression of cell cycle markers

165 Cell Cycle Regulation in Reversibly Differentiated Hepatoma Cells

characteristic of the G1 phase (9). Indeed, G1 phase arrest

generally corresponds to one of the checkpoints that prevent

the completion of G1 and thus the entry into S phase and

further progression through G2-M phases. These differentcheckpoints correlate with activation or inhibition of proteincomplexes such as cyclins and cdks3 (1 0-1 2). Among them,the D-type cyclins associated with their two major partners,cdk4 and cdk6, are mainly involved in the progressionthrough G1 phase (1 2, 13). In addition, several studies dem-

onstrated cell type-specific changes of cell cycle-relatedgene expression during in vitro differentiation (3, 14-17). Inparallel, changes in the expression of some proto-onco-genes have been reported to accompany entry into the ter-minal differentiation program of different cell types. Thus,

c-Myc associated with early/mid-G1 phase was accumulatedin rat myoblasts and mouse erythroleukemia cells undergo-

ing terminal differentiation (1 8-20). In the present paper, we

analyzed the expression of different proto-oncogenes andcell cycle-related proteins to define the phase of the cell

cycle at which HBG cells were growth-arrested during dif-ferentiation and to understand the phenomenon of reversible

differentiation of hepatic cells.

Depending on cell types, the differentiation program maybe terminated by irreversible growth arrest and sequentialcell death or may lead to reversion, associated with reinitia-

tion of cell proliferation and partial retrodifferentiation, as in

liver parenchymal cells. The regulatory processes that con-

trol these different cell behavior patterns remain poorly un-

derstood. Over the past years, it has gradually emerged thatproducts of two tumor suppressor genes, namely Rb andp53, as well as members of the family of cdk inhibitors serveas safeguards of the control of cell differentiation, prolifera-tion, and death (21-25). However, several potent inducers ofcell growth arrest and death, such as TGF-31 , may trigger

different types of responses, depending on the conditions

and the cell type concerned, acting through p53- and Rb-dependent or -independent pathways (26-28). This was as-sessed with differentiated hepatoma cell lines that can un-dergo growth arrest and apoptosis when exposed to TGF-j3although not expressing the wild-type p53 gene (29-31),indicating the possibility of other regulatory pathways inwhich the Rb gene product might play a critical role (31). Italso appeared that recently discovered cdk inhibitors, inparticular, members of the cip/kip family such as p21 (Cipl/Wafl/Sdil) and p27 (Kipi), contribute to the regulation of cellcycle progression, apoptosis, and differentiation throughmultiple distinct molecular mechanisms and stimuli (24-25,32, 33).

In this study, we have defined the growth characteristics ofthe clones selected from the HBG hepatoma in relation to

their differentiated status. Our results show that these cells,

when undergoing spontaneous differentiation at confluence,were arrested in G� phase and retained the capacity of activeproliferation after several weeks of quiescence. HBG cells

3 The abbreviations used are: cdk, cyclin-dependent kinase; PK, pyruvatekinase; Rb, retinoblastoma; TGF-j31 , transforming growth factor f31 ; mAb,monoclonal antibody; lP, immunoprecipitation.

exhibited a characteristic pattern of proto-oncogene- andcell cycle-associated gene expression during the differenti-ation process, suggesting that specific regulatory mecha-nisms are involved in the control of reversible differentiationof liver cells.

ResultsMorphology, Karyological Analysis, and Tumongenicityof HBG Cell Lines. Cells isolated from the tumor by colla-genase dissociation were distributed in 20 Petri dishes. Pri-mary cultures consisting mostly of hepatocyte-like cells re-mained stable and quiescent for 5 weeks. At that time,

growth of polygonal cells started at the periphery of fewcolonies (Fig. 1A), whereas the initially plated cells graduallyretracted forming brown clumps. The new colonies of slowlyproliferating hepatocyte-like cells grew out and became pre-

dominant after 3-4 months. The largest colonies were de-tached by trypsination, transferred to new flasks, and serially

propagated for several months. Thus, stable cell lines Bi-B20 were obtained (Fig. 1D). Two main types of cells could

be observed: (a) cells morphologically resembling granularliver parenchymal cells; and (b) those resembling bile ductu-lar epithelial cells. Depending on the proportion of the two

cell types, the lines were divided into two main groups: (a)one group, represented by Bi and Bi 6, mainly containedcells closely resembling primary hepatocytes (Fig. 1 B); and(b) the other group, represented by B9, exhibited a majorityof large flat cells. In addition, three clonal colonies were

selected from Bi cells, giving rise to the subclones BC1,BC2, and BC3, which are very similar in their morphologicalfeatures and are composed exclusively of hepatocyte-likecells (Fig. 1C). Bi 6 and these three subclones were selectedfor further characterization (Fig. 1D).

BC2 cells were more extensively used for karyologicalstudies. These cells seemed to be pseudotriploid (modalnumber, 58) and exhibited several chromosomal abnormal-

ties with a significant frequency (more than 75% mitoses;Table 1). Among the characteristic gains and losses, wefound polysomy of chromosome 7, loss of chromosome Y,tnsomy 1 p31 qter, monosomy of chromosome 9, deletiondel (4) (p1 5 pter) and del (1 7) (p1 2 pter).

The tumorigenicity of HBG hepatoma cells was evaluatedby their capacity to grow in soft agar and to induce tumors innude mice. The different clones were found to grow weakly

in soft agar and seemed poorly tumorigenic in vivo. Only 1 of1 5 injected mice developed a slow-growing tumor.

Growth Arrest and Reinitiation of Cell Growth after aLong Period of Quiescence. After seeding cells attachedto plastic with a plating efficiency of 60-80% within 5 h (Fig.

2�4), they grew for about 1 week, and during this time, theyunderwent nearly four population doublings (Fig. 2B). Thenthey went through a critical period characterized by a re-

duced proliferation activity, which occurred after 1 week ofculture, and reached a plateau at which they remained alive

for a long period of at least 6 weeks. Fig. 2C shows that

confluent cells exhibited a very weak proliferative activity.Flow cytometric analysis of DNA content in both BC1 andBC2 cells after a 4-6-week quiescent period confirmed thelow proportion of cells in S phase, which never exceeded

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Cell Growth & Differentiation 167

Fig. 1 . Phase-contrast micro-graphs of HBG cells during estab-lishment of the cell lines and aftercloning. A, 6-week-old primaryculture of Bi cells with a new col-ony of hepatocyte-like cells grow-ing from a clump of brown primaryhepatocarcinoma cells on the leftside; B, subculture of the 61 cellline, passage 3, showing hepato-cyte-like cells surrounded by clearepithelioid cell; C, culture of theBC1 cell line after cloning from Bi,passage 5, showing pure popula-tion of regular polygonal cells. D,derivation of the hepatoma celllines described in this study. Bar,100 �tm.

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Table 1 Cytogenetic characteristics of HBG, clone BC2�

Chromosomes with abnormalities

Frequency ..Chromosome (% of mitoses) Abnormalities

1 100 Partial trisomy del(1)(p31 pter)surnumerary

7 100 Complete polysomy7 100 Partial polysomy del(7)(q23 qter)

surnumerary4 100 Partial monosomy del(4)(pl 5 pter)

1 7 82 Partial monosomy del(1 7)(p12 pter)

20 68 Complete trisomy2 59 Complete trisomy

10 59 Complete trisomy18 50 Complete trisomy

6 45 Complete trisomy16 1 5 Complete trisomy1 9 32 Complete trisomy

9 77 Complete monosomy8 50 Complete monosomy

y 100 Nullosomyx 68 Complete disomy

a Chromosomes 5, 1 1 , 12, 1 3, 1 4, 1 5, 21 , and 22 did not have abnormal-

ities.

7%. Moreover, the percentage of cells with 2C DNA content

(G1 phase) increased from 55-60% in proliferating cells to75-90% in 4-6-week quiescent cells (Fig. 3A).

The ability of the cells to reinitiate an active proliferation

after a long quiescent period was analyzed in parallel. Cells

were growth-arrested at confluence for 1 , 3, and 5 weeks,

and then they were detached by trypsin and seeded at the

same density used for routinely subcultured cells. A gradualincrease in the delay of cell attachment and spreading was

observed with time of confluence, but the plating efficiencyof 5-week growth-arrested cells reached 60-80% at 8-10 h,

a level similar to that observed in cells obtained from 1-week-old cultures (Fig. 2D). Then, after a relatively long G1 pro-

gression, the population of cells in S phase was strongly

increased, as shown at 42 h after seeding (Fig. 3B), and no

significant difference could be seen in the growth curves

between cells replated after 1 or 5 weeks of culture (Fig. 2E).

Spontaneous Differentiation of Hepatoma Cells atConfluence. After 2 weeks of culture, the parental B1 and

B16 cells and the three subclones BC1 , BC2, and BC3, butnot the B9 line, spontaneously exhibited gradual changes in

their morphological features, characterized by a very regular

epithelial shape, clear round nuclei with one or two dense

nucleoli, and phase-dense cytoplasm. The extent of differ-

entiation was monitored by comparing in B9, B1 6, and one

subclone, BC1 , the kinetics of appearance of the PKL

isozyme known to be the specific form of the hepatic glyco-

lytic pathway in adult hepatocytes (Fig. 4A). Indeed, the

electrophoretic pattern of the enzyme zymogram showed

that only homotetramers of PKL were expressed in freshly

isolated adult hepatocytes used as control, whereas only

homotetramers of PKM2 characteristic of fetal hepatocytes

were synthesized in the undifferentiated B9 cells. In contrast,

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168 Cell Cycle Regulation in Reversibly Differentiated Hepatoma Cells

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in BC1 and Bi 6 cells, homotetramers of PKM2 were first

formed in dividing cells, whereas heterotetramers composedof both PKL and PKM2 and characterized by intermediatemigrating pattern appeared in arrested cells after 2 weeks ofculture. These results correlated well with the increased 1ev-els of PKL transcripts observed after 10 days in the threesubclones (Fig. 4B). In parallel, a gradual accumulation ofalbumin transcripts was also observed, whereas a-fetopro-

tein was always barely detectable.Proto-oncogene, Cyclin, and cdk Gene Expression as a

Function of HBG Status of Differentiation. A biphasic pat-tern of c-myc mRNA expression was observed in subclonesBC1 , BC2, and BC3 maintained in culture for 6 weeks; a firstwave occurred during the first week, and then, after animportant drop down to undetectable levels from day 10 today 18, an accumulation of c-myc transcripts was foundagain in confluent differentiated cells (Fig. 5A). Similar kinet-ics with a biphasic accumulation of the Myc protein wasfound by Western blotting (Fig. 5B). The expression of Maxprotein, a partner of c-Myc, was also evidenced. However,only a slight change in its level could be observed during theculture time (Fig. 5B). Increased levels of Ki-ras transcriptswere observed during the differentiation process, whereas,

Fig. 2. Plating efficiency andgrowth characteristics of HBGhepatoma cells. A, plating effi-ciency. Cells (10#{176})were plated in35-mm Petri dishes. Every 30 mm,attached cells were trypsmnizedand counted. Plating efficiencywas expressed as the percentageof attached cells relative to thenumber of initially plated cells. B,growth curves. Cells (10#{176})wereplated in 35-mm Petri dishes. Attimes indicated, cells weretrypsmnized, and viable cells werecounted by the trypan blue exclu-sion method. C, �H]methylthyrni-dine incorporation into cellularDNA. Cuftures of hepatoma cellswere pulsed with 4 �CVml rH]m-ethyl thymidmne for 24 h. At thetimes indicated, radiolabeled cellswere collected, and incorporatedradioactivity was determined asdescribed in “Materials and Meth-ods.” BC1 (Es, BC2 (0), and BC3(t cell lines. D, plating efficiencyof growth-arrested hepatomacells. BC1 cells were maintainedin culture for 1 week (E’, 3 weeks(0), and 5 weeks (� and then de-tached with trypsin and reseededin 35-mm Petri dishes (i0� cells/dish). Plating efficiency was de-termined as desCribed in A. Sim-ilar resufts were obtained fromthree independent experiments.E, growth efficiency of hepatomacells after a long quiescence pe-nod. BC1 cells were maintained inculture for 1 week � and 5 weeks(� and then detached with tryp-sin and reseeded in 35-mm Petridishes. Growth curves were de-termined as described in B.

interestingly, the N-ms mRNA levels decreased, indicating acomplete opposite kinetics. No c-fos transcript could bedetected at any time during culture. The same results wereobtained with the three subclones (Fig. 5A).

In parallel, we also defined the kinetic expression of say-eral cell cycle genes for a 3-4-week culture period of BC1cells. Expression of cdc2 and cyc!in A mRNAs, both asso-ciated with S phase and DNA synthesis, was clearly evi-denced early after seeding, but it gradually decreased there-after in confluent differentiated cultures (Fig. 6A). Cyclin B, aprotein mainly associated with the G2-M phase, showed asimilar decrease of expression during the culture time (Fig.6D). Conversely, when 3-week growth-arrested cultureswere replated, both cdc2 and cydlln A transcripts werestrongly induced at 48 h after seeding (Fig. 6B). The reentryof differentiated hepatoma cells in the cell cycle was accom-panied by transient decrease of albumin expression duringthe period of active proliferation (Fig. 6B).

In normal hepatocytes, cyclin Dl might play a critical rolein G1 progression and G1-S-phase transition (34). No signif-icant changes in the levels of cyclin Dl transcripts and of thatof its partner, cdk4, were observed in differentiating BC1cells, even after a long quiescence period (Fig. 6C). However,

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Cell Growth & Differentiation 169

Fig. 3. Flow cytometric analysis of the cell cycle of HBG cells. A, comparison between exponentially growing (1) and 5-week quiescent (2) BC2 cells. B,reentry in cell cycle of quiescent differentiated BC2 cells. Cells were growth-arrested for 5 weeks, detached with trypsin, plated in 60-mm Petri dishes, andanalyzed at 20 (1), 42 (2), and 70 h (3) after seeding.

the level of cyclin Dl protein was high in proliferating cells

during the first week and was slightly diminished thereafter,

whereas that of cyclin D3 was elevated in growth-arrested

cells (Fig. 6D). Using a kinase assay with Rb as a substrate,

the functional activity of immunoprecipitated cyclin Dl -as-

sociated complexes was evidenced during the first week of

culture and strongly decreased thereafter to a weak level in

confluent differentiating cells (Fig. 6E).

Altered p53 Activity Associated with p53 Mutation. Asan attempt to define the mechanism by which the cells might

reinitiate cell proliferation after a period of quiescence, we

first studied the activity of the p53 gene. RNA blot analysis

showed constantly high levels of p53 transcripts during the

culture time (Fig. 7A). Furthermore, immunoblot analysis ev-

idenced high levels and abnormal migration of p53 protein in

BC1 and B1 6 cells, suggesting that the gene was mutated

(Fig. 7B). Indeed, functional analysis of this form demon-

strated that cells expressed a nonfunctional p53 protein,

because no transcriptional activity of the reporter plasmidcarrying p53-binding sites was detected when transfected

into both cell lines (Fig. 7C). In confirmation of these obser-

vations, direct sequencing of the entire p53 cDNA from BC1

and B1 6 cells revealed that both cell lines display a missense

point mutation at codon 21 4 (CAT-�CGT), leading to the

substitution of histidine for arginine. The mutation occurred

at the DNA-binding region of p53 protein.

Maintenance of Cell Sensitivity to TGF-�1 . To deter-

mine whether the p53 mutation was correlated with an im-

pairment of the apoptotic process, we analyzed the sensi-

tivity to TGF-�1 of the BC2 cells cultured with or without

serum. The addition of 5 ng/ml TGF-j31 rapidly induced

fibroblast-like morphological changes and growth arrest. In

addition, daily cell counts clearly indicated a decrease of

viable cells that seemed more pronounced in the absence of

serum (Fig. 8A). The TGF-�-induced growth inhibition was

accompanied by an increased number of apoptotic figures

(Fig. 8B), demonstrating that the sensitivity to TGF-pl , and

therefore at least one pathway of apoptotic regulation, was

conserved in HBG cells. Similar results were obtained with

BC1 cells (data not shown).

Decreased Expression of Rb and p21 Proteins afterProlonged Quiescence. We then studied the expression

of other factors known to play a critical role in the balance

between proliferation and differentiation, such as Rb and

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Fig. 4. Expression of liver-specific genes during differentiation of HBGcells. A, pattems of PK isozymes in BC1 , B9, and Bi 6 hepatoma cells. Thepositions of L- and M2-type homotetramers after migration are indicatedby arrowheads. B, RNA blot analysis of albumin and PKL mRNA levels inBC1 (Lanes 1), BC2 (Lanes 2), and BC3 (Lanes 3) cells at different timesafter seeding. Total RNA (20 �g) was analyzed by blot hybridization withthe cDNA probes mentioned above. Controls: T0, freshly isolated humanhepatocytes; and G2, HepG2 hepatoma cells.

the cdk-inhibitors p21 , p27, p1 6, and p1 5. The levels of Rb

transcripts were stable during the culture period (data not

shown), whereas the Rb protein was abundant during the

first week, and its level gradually diminished thereafter

with time of culture (Fig. 98). Analysis of the electro-

phoretic mobility of the protein evidenced that Rb was in

the two hypo- and hyperphosphorylated states. However,

throughout the period of culture, it remained primarily in

the hyperphosphorylated form.

Surprisingly, the transcript levels of the different cdk in-

hibitors studied showed no accumulation during differentia-

tion of BC1 cells, and a decrease in the levels of most of them

was observed in cells growth-arrested after 2 weeks of cul-

ture (Fig. 9A). Western blotting revealed that the p21 protein

was present for the first week of culture, but, unexpectedly,

its expression dropped drastically to a very low level in

growth-arrested cells, whereas, on the other hand, p27 was

weakly expressed during proliferation and was abundant in

quiescent differentiated cells (Fig. 9B).

DiscussionSeveral novel differentiated cell lines have been derived from

a human hepatoma named HBG. Three of them, BC1 , BC2,

and BC3, are pure populations obtained by two complemen-

tary strategies of cell cloning. They are defined as

pseudotriploid cells with some characteristic structural and

numerical chromosomal abnormalities.

B

3 7 11 15 20 25 29 (days)

c.myc � . �

max � � � � � -�

Fig. 5. Expression of oncogenes during differentiation of HBG cells.A, RNA blot analysis. Total RNA was extracted from BC1 (Lanes 1),BC2 (Lanes 2), and BC3 (Lanes 3) cells at different times after seedingand analyzed by blot hybridization with c-myc, N-ras, Ki-ras, and c-foscDNA probes. Controls: T0, freshly isolated human hepatocytes; andG2, HepG2 hepatoma cells. B, Western blot analysis. BC1 total cellextracts (100 .tg) were assayed for the presence of c-Myc and Maxproteins.

An interesting feature of HBG cells is their ability to un-

dergo stable spontaneous differentiation at confluence. This

was assessed by studying different specific markers such as

albumin and PKL-specific isoform expression. Indeed, two

isozymic forms of glycolytic enzyme PK are present in the

liver, the expression and ratio of which vary according to

physiological conditions such as regeneration and carcino-

genesis, and PKL is the only PK isozyme expressed in normal

mature hepatocytes (35). Our results confirmed and ex-

tended our previous observations, showing a time-depen-

dent increased expression of different liver-specific proteins,

including a large set of plasma proteins, enzymes involved in

xenobiotic metabolism, and liver-specific transcription fac-

tors (36), in these clones and other HBG cell lines. Evidence

for a positive role of insulin in differentiation of BC1 cells has

also been recently reported (37). However, the most original

traits of HBG cells compared to other human hepatoma celllines are their slow progression toward differentiation, their

high stability when differentiated at confluence (at least 6

weeks), and their capacity to reverse to active proliferation.

These studies show that long-term confluent HBG cells are

blocked in the cell cycle in G1 phase, as argued by the

following data: (a) flow cytometric analysis of DNA content in

HBG cells maintained for 4-6 weeks at confluence revealedthat 75-90% of cells exhibited 2C DNA content; (b) expres-sion of S-phase markers and DNA synthesis level were dra-

matically decreased in confluent cultures; (c) accumulation of

170 Cell Cycle Regulation in Reversibly Differentiated Hepatoma Cells

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c-Myc, previously shown to be restricted to G1 -phase-ar-

rested differentiated erythroleukemia cells and myoblasts

(1 8-20), was observed in differentiating quiescent HBG cells;and (a) a strong decrease of the cyclin Dl -associated kinaseactivity was found in parallel.

All of these observations agree with a number of studies

demonstrating that the differentiation program of various cell

types is intimately coupled to the cell cycle and is associated

with dramatic changes of proto-oncogenes and cyclins to-

gether with their cdk partners (1 4, 1 5, 38-40). The biological

significance of the accumulation of c-Myc in G1-phase-

arrested differentiating cells is not yet elucidated. In differ-

entiating L6 myoblasts, constitutive overexpression of c-Myc

was shown to block cell differentiation (19, 41). However, it

Cell Growth & Differentiation 171

seems that a transient decrease of c-Myc is required only for

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pRb �

Fig. 6. Cell cycle-related gene expression during differentiation andgrowth reinitiation of HBG cells. A-C, RNA blot analysis. Total RNA wasextracted from BC1 cells at different times after seeding of exponentiallygrowing cells (A and C) or of growth-arrested cells for 32 days (B). Blotswere hybridized with cdc2, cdk4, cyclin A, cyclmn Dl , and albumin (ALB)probes as indicated on the left. A: T0, freshly isolated human hepatocytes;and G2, HepG2 hepatoma cells. B: T32, BC1 cells maintained in culturefor 32 days before trypsinization; and T0, the same cells after trypsiniza-tion and washing. D, Westem blot analysis. BC1 total cell extracts (1 00 �g)were assayed for the presence of cyclmns B, Dl , and D3 proteins. E,kinetics of cyclin Dl -associated kinase activity. Lysates prepared at theindicated times from differentiated BC1 cells were immunoprecipitatedwith mAb to cyclin Dl and assayed for pRb kinase activity. Control beadswere prepared with an irrelevant antibody (C).

Fig. 7. Analysis of p53 gene expression in hepatoma cells. A, Northernblot analysis. Total RNA was extracted from BC1 cells at different timesafter seeding, and hybridization was performed with p53 cDNA probe.Controls: T0, freshly isolated human hepatocytes; and G2, HepG2 hepa-toma cells. B, Western blot analysis. Total cell lysates were prepared fromHepG2 cells (G2) untreated (-) or treated with 0.2 �g/ml Adriamycin (+),BC1, and B16 cells, and probed with mAbs against human p53. C,transcriptional activity of endogenous p53. Cotransfections of hepatomacells were performed using a reporter plasmid containing a p53 bindingsite (pRGC�Fos lacZ) and a pCMv-CAT plasmid as a control of transfec-tion. The galactosidase activity was determined by a colorimetric test andis expressed as units of absorbance at 420 nm. Controls: G2, HepG2; andHuH7, hepatoma cells.

A

.� 1,o�

;;- 0,80

I- 0,6�x

E�C’

a)C) 0,0 I I I0 2 4 6

BDays in culture

8

A

3 10 15 20 25 (days)

p21

p27 0 �

�‘ �4

15 -

p16 #{149}#{149}s#{149}OlBs

B

3 7 ! I 15 20 25 29 (days)

Rb � � � ppRb

p2! �

p27 �. � � -�

Fig. 9. Expression of cdk inhibitors in differentiating BC1 cells. A, RNAblot analysis of p21 , p27, p1 5, and p1 6. Total RNA was extracted fromBC1 cells at different times after seeding, and 20 �g were applied to eachline. Blots were probed for the transcripts on the left. B, Western blotanalysis. BC1 total cell extracts (1 00 �g) were assayed for the presenceof pRb, p21 , and p27 proteins.

mia cells (47). This observation could be interpreted in light of

a recent report showing the possibility that a functional anal-ogy between ras and c-myc oncogenes might exist, because

they can induce both proliferation and growth arrest or ap-optosis, with the modulation of their effects depending on

complex intracellular signalings (48).

In contrast to different cell types undergoing terminal dif-

ferentiation and culminating in cell death, differentiated HBG

cells were capable of growth reinitiation even after 6 weeks

of confluence with induction of cyclin A and cdc2 and tran-

sient dedifferentiation during the period of active prolifera-

tion. This property could reflect, to some extent, the liver

regenerative process in vivo, in which all growth-arrested

differentiated hepatocytes reverse to proliferation activity (5).

To our knowledge, the ability of cells to reverse to active

proliferation after several weeks of growth arrest with se-

quential loss of differentiated phenotype has never been

described in other established hepatoma cell lines.

To further understand their mechanism of reversible dif-

ferentiation, analysis of several genes involved in regulation

of cell growth and death, such as p53, Rb, and cdk inhibitors,

was performed. Substantial recent data suggest that two keytumor suppressor genes, Rb and p53, exert direct effects onboth cell cycle progression and cell death (21 , 22), and thesegenes were found to be frequently mutated in various human

Fig. 8. TGF-#{216}induced growth inhibition and apoptosis in BC2 cells. A,growth curves. Cells (0.5 x 1 06) were seeded in the presence of 1 0% FCSin 12-well plates. The next day, the cells were exposed to TGF-j31 (5ng/ml) under serum-free conditions (C)) or in the presence of 10% FCS 4.The control cultures were also maintained without 4 or in the presenceof 1 0% FCS (EJ). At 1 , 2, 4, and 7 days after seeding, cells were trypsinized,and viable cells were counted by the trypan blue exclusion method. B,nuclear morphology of BC2 cells treated with TGF-j31 . Hepatorna cellswere cultured in serum-free medium in the presence of TGF-(31 (5 ng/ml)for 7 days and then fixed and stained with Hoechst 33258. Fluorescencemicrographs of control (left panel) and TGF-f3-treated BC2 cells (rightpanel). Bar, 15 �m.

commitment of cells to terminal differentiation, and that once

this stage is overcrossed, its reexpression cannot suppress

established differentiated phenotype (42, 43). A similar con-

clusion could be drawn for HBG cells. Complexity is greatly

increased by the fact that stability and biological activity of

c-Myc could be strongly modulated by association with sev-eral proteins. Among them, the Max protein was shown to

form heterodimers with c-Myc and to increase its site-

dependent transcriptional activation (44). Expression of Max,

however, was found to be relatively stable during HBG cell

differentiation, raising the possibility that other proteins, such

as Mad proteins, previously shown to be up-regulated duringthe differentiation process might antagonize Myc-Max func-

tions in liver cells (45, 46).

Another intriguing feature of the differentiated HBG cells is

the pattern of ras proto-oncogene expression showing op-

posite kinetics of Ki-ras and N-ras transcripts, but whose

changes were concomitant with those of c-Myc during dif-ferentiation. Modulation of ras protein expression has been

also reported during differentiation of human erythroleuke-

172 Cell Cycle Regulation in Reversibly Differentiated Hepatoma Cells

Cell Growth & Differentiation 173

malignancies (49, 50), including human hepatocellular carci-

noma (51-53). A point mutation in the p53 gene was found inBC1 and Bi 6 cells, resulting in the loss of transcription

activity. This mutation (CAT-+CGT) localized at codon 214, aregion beyond the described hot spot ofp53 gene alterationsin hepatocellular carcinoma (52), occurs in the specific DNA-

binding region of p53. However, cells from the three clonesseemed to be poorly tumongenic and sensitive to TGF-�1 , asdemonstrated by its ability to induce growth arrest and ap-optosis in HBG clones. It has previously been shown thatTGF-f31 could mediate apoptotic death in a p53-deficienthuman hepatoma cell line, Hep3B (29), and this responsewas not affected by expression of the mutant p53-249serprotein (30). Thus, it is unlikely that p53 is directly involved inTGF-� apoptotic signaling, at least in hepatoma cells. Otherregulators, such as Rb protein, cdk, and cdk-inhibitors, seemto be implicated in TGF-(3-mediated responses (31 , 54).

We show that the Rb protein levels were high in prolifer-ating cells but gradually diminished with long-term quies-cence. The presence of both hyper- and hypophosphory-lated forms provides strong arguments for its functional

activity. Moreover, the hyperphosphorylated form was themajor form observed in both actively proliferating andgrowth-arrested cells. This contrasts with increased expres-sion of the hypophosphorylated form that was found to ac-company the terminal differentiation of myoblasts to myo-tubes (55). However, it emphasizes the observations onregenerating liver and proliferating HuH7 hepatoma cells alsoreporting a predominance of the hyperphosphorylated form,

(56) and leads us to postulate that the hyperphosphorylatedRb might exhibit particular functional properties constitutinga characteristic feature of liver cells.

Important recent progress in the understanding of cdkinhibitor functions has greatly helped to explain how antipro-liferative signals arrest cells in G1 to enable, for example,

either terminal differentiation, cell senescence, or reversiblygrowth-arrested cells (24, 25). p21 and p27 are consideredtwo major regulators of the levels of cdk-inhibitory activityinvolved in these processes. Whereas p27 tends to accumu-late in quiescent cells and declines in response to mitogenstimulation, p21 levels are generally low in quiescent cellsbut rise in response to mitogen treatment (57, 58). However,

p21 levels remain elevated in irreversibly nondividing senes-cent cells or terminally differentiated cells, with these statesof growth arrest being considered as different from that of

quiescent cells that retain a proliferative activity (15, 32, 33,59-61). Interestingly, our observation showed in HBG cellsthat these two inhibitors followed kinetics of expressioncloser to confluent cells capable of active proliferation thanto terminally differentiated cells. Indeed, p27 protein wassignificantly increased in growth-arrested cells undergoingdifferentiation at confluence, whereas p21 , which would havebeen expected to increase during differentiation, was ex-pressed in dividing cells but drastically fell to very low levelsin differentiating cells. Thus, it may be speculated that ab-sence of accumulation of p21 during the course of sponta-neous hepatic differentiation in HBG cells may reflect, tosome extent, the in vivo situation and may primarily account

for the mechanisms that provide the capacity to reverse toactive proliferation to differentiated liver cells.

Taken together, our results show that the three clones ofHBG studied here are able to undergo differentiation in long-term confluence being arrested in G1 phase and to reverse toactive proliferation, thus mimicking, in some, aspects, thenormal regenerating process of adult liver in vivo. Analysis ofthe expression of various cell cycle-related genes leaves thepossibility open that liver-specific regulation of some ofthem, mainly Rb and cdk inhibitors, during the differentiation

process could contribute to the genetically determined re-versible differentiation program in liver. New experiments arein progress to better define the regulatory mechanisms thatcontrol the balance between proliferation and differentiationin liver cells. The new human HBG cell lines, which are

capable of spontaneous and reversible differentiation andremained functionally stable over a 4-year study, shouldprovide useful tools for these studies.

Materials and MethodsCell culture and Cloning. The tumor tissue was obtained from a liver

biopsy of a 61-year-old human male suffering from alcoholic cirrhosis andhepatomegaly. Clinical examination revealed the presence of a hepatictumor in the left lobe with a histopathological diagnosis of hepatocarci-

noma. The tumor was found to be a-fetoprotein-positive and hepatitis Bvirus-negative. The tumor tissue was resected, and the samples were

minced into small pieces, washed with HEPES buffer [(pH 7.7); 140 m�NaCI, 2.68 mM KCI, 0.2 mp�i Na�HPO4, and 10 m�i HEPES], and digestedwith 0.025% collagenase A (Boehringer Mannheim) diluted in the same

buffer supplemented with 0.075% CaCI2 under gentle stirring at 37#{176}C.Thecell suspension was washed twice in HEPES buffer and resuspended in a

mixture of 75% MEM and 25% Medium 199 supplemented with 10%FCS, 100 units/mI penicillin, 50 �g/ml streptomycin, 5 �g/ml insulin, 1mg/mI BSA, and 7 x i0� M hydrocortisone hemisuccinate. Cell suspen-

sion was distributed in 20 Petri dishes without any coating feeder layer.

Cloning of BC1 , BC2, and BC3 was performed by selective detachmentand selective adhesion, using the differences in sensitivity to trypsmn andin attachment efficiency of the different cell types to plastic dishes: hep-

atocyte-like cells are less sensitive to trypsinization and attached moreslowly to the support than the nonparenchymal cells. The cell lines ob-tamed were then further cloned by limited dilution.

Plating Efficiency and Population Doublings. For plating efficiency,

the cells were plated after trypsinization in 35-mm Petri dishes (106

cells/dish). Then, every 30 mm, some dishes were trypsmnized, and theviable cells were counted with a hemocytometer. For determination ofpopulation doublings, the cells were counted every 2 days for up to 2weeks. The stability of these growth features has been evaluated over 4years for the three clones.

[‘H]Thymldine Incorporation. DNA replication was estimated bymeasuring of �H]methyl thymidmne incorporation. Briefly, cells were incu-bated with 4 pcVml [�H]methyl thymidine (5 CVmmol; Amersham) for 24 hat the indicated times. The samples were washed with PBS, scraped, and

frozen in 1 ml of PBS. Thereafter, cells were sonicated, and aliquots were

taken for protein determination. DNA was precipitated with 1 5% trichlo-roacetic acid, washed with 10 and 5% trichloroacetic acid, successively,and dissolved in formic acid. Incorporated radioactivity was detected byliquid scintillation counting and expressed as cpm/�g protein.

Flow Cytometry. Cell monolayers were washed once with PBS, de-tached with trypsin, and fixed with 70% ethanol. The fixed cells in sus-

pension were exposed to RNase A (0.1 mg/mI) for 30 mm at 375C andstained with propidium iodide (0.05 mg/mI). For determination of the DNAcontent of the cells, an EPICS Elite flow cytometer (Coultronics, Hialeah,FL) equipped with an argon laser (15 mW at 488 nm) was used. Theproportion of cells in the different phases of the cycle was calculated fromthe experimental fluorescence histograms by a polynomial fit using Mul-ticycle software (Phoenix Flow Systems, San Diego, CA).

174 Cell Cycle Regulation in Reversibly Differentiated Hepatoma Cells

Enzyme Assays. At indicated times, cells were washed with HEPES

bufter(pH 7.5), scraped off the dishes, and collected in a buffer composedof 10 m� Tris-HCI (pH 8.0), 5 m�i Mg504, 1 m� EDTA, and 0.1 mp.i�3-mercaptoethanoI. The suspension was frozen and thawed twice and

clarified by centrifugation. Total PK activity was measured by the methodof B#{252}cherand Pfleiderer (62). Samples of cell lysates containing equalactivities of total PK were separated in nondenaturatlng polyacrylamide

gel, and the position of isoenzymes was visualized with specific stainingaccording to Susor and Rutter (63).

RNA Isolation and Blot Analysls Hepatoma cell monolayers werewashed in PBS, and total RNA was extracted by the thiocyanate guani-

dine procedure. Twenty �g of RNA were separated by electrophoresisthrough a 1.2% agarose gel in 10 m� phosphate buffer(pH 7.4) containing1 .1 M formaldehyde and transferred onto nitrocellulose (Hybond C; Am-ersham) or nylon fliters (Hybond N�; Amersham) by capillary blotting.Hybridization was performed in the presence of 32P-labeled probefor 16 hat 65C. Filters were washed once with 3x SS� and 0.1 % SDS for 1 h andthen washed three times with 1 x SSC and 0.1 % SDS. Autoradlographywas performed using a hyperfilm MP or X-Omat film with DuPont Ught-

ening Plus intensifying screens at -80#{176}C. As controls, we used total RNAprepared from HepG2 cells and freshly isolated human hepatocytes.HepG2 cells were obtained from B. Knowles, and human hepatocytes

were prepared by enzymatic dissociation of liver biopsies from surgicalresections.

The cDNA probes used were as follows: human PKL and albumin

cDNAs were kindly provided by A. Kahn; the human odc2 probe was

obtained from P. Nurse; and the murine cyclmn Dl and cdk4 probes wereobtained from C. Sherr. The human cyclin A and p21 probes were pro-vided by C. Brechot and W. Harper, respectively. The human p53 cDNA

was provided by E. May. The c-myc probe was the 1 .6-kb fragmentcorresponding to the third exon of the human gene (64), the N-ras probewas the 4-kb human genomic fragment (65), and the Ki-ras probe was the1.2-kb fragment of v-Ki-ras (66). The human p15 and p16 cDNA probeswere kindly provided by 0. Beach, and human p27 probe was obtained by

reverse transcription-PCR based on the sequence data (67). The 18 SrRNA (human genomic 5.7-kb fragment) was used as a control.

Western Blot Analysis. Antihuman cyclin Dl (clone DCS-6) was ob-tamed from NeoMarkers (Fremont CA). The antibodies against humancyclin B, cyclin D3, Cipl(p21), Kipl(p27), and max were purchased fromTransduction Laboratories (Lexington, KY). Antihuman c-myc (clone 9E1 0)

and anti-Rb (clone 63-245) were from Santa Cruz Biotechnology (SantaCruz, CA) and PharMingen (San Diego, CA), respectively. Immunodetec-tion of p53 was performed using HR231 mAb (68). The second antibody

was peroxidase-conjugated Affipure F(ab’)2 fragment goat antimouse lgG(Jackson lmmunoReseach Laboratories, West Grove, PA). Peroxidaseactivity was detected by enhanced chemiluminescence according to themanufacturer’s instructions (Amersham). For immunodetection of p53,whole-cell lysates (100 gig) were boiled in sample-loading buffer, sepa-

rated by SDS-PAGE electrophoresis, and transferred to polyvinylidenedifluoride lmrnobilon membrane (Millipore, Saint-Quentin, France). Other-

wise, cell monolayers were lysed directly in buffer [50 m�.i HEPES (pH 7.5),150 m� NaCl, 15 m� MgCI2, 2 m� EDTA, 2 m�.i EGTA, and 0.1% Tween20]contamning 1 mM DII, 0.1 m� sodium orthovanadate, 1 m� NaF, 10 mp.i/3-glycerophosphate, 0.1 m� phenylmethylsulfonyl fluoride, and 100

�g/ml benzamidine and protease inhibitor mixture (5 �ig/ml aprotmnin,leupeptin, pepstatin, and soybean trypsin inhibitor) and sonicated In ice.Lysates were clarified by centrifugation at 10,000 x g for 15 mm andstored at -80#{176}C.Total cellular extracts (100 �g) were separated bySDS-PAGE electrophoresis and transferred to nitrocellulose membrane

(Amersham).Immune Complex Kinase Assay. IP of cyclin Dl and kinase activity

of the complex were performed as described by Matsushime et al. (69)with minor modifications. Briefly, cells were lysed in P buffer [50 m�iHEPES (pH 7.5), 150 mM NaCI, 1 m� EDTA, 2.5 m� EGTA, 1 m�i Dli, and0.1 % Tween 20] containing 10% glycerol, 10 m�,i f3-glycerophosphate, 1

mM NaF, 0.1 mM sodium orthovanadate, 0.1 m� phenylmethylsulfonyl

fluoride, and 10 �&g/ml leupeptin and aprotinin and sonicated in ice.Lysates were clarified by centrifugation at 10,000 x g for 5 mm, andsupematants (500 M9 protein/assay) were further precleared with 5 �J ofnormal mouse serum, followed by incubation with protein A-Sepharose

(Pharmacia, Uppsala, Sweden), which was pretreated with rabbit anti-mouse immunoglobulmn G (Jackson lmmunoresearch Laboratories). Pre-

cleared lysates were immunoprecipitated with mouse mAb to human

cyclmn Dl (clone DCS-1 1 ; NeoMarkers) for 12 h and then incubated withpretreated protein A-Sepharose. As a negative control, Irrelevant mAb to

Crry/p65 (PharMingen) was used. Immunoprecipitated proteins werewashed four times with 1 ml of P buffer and three times with 50 mt�i

HEPES (pH 7.5) containing 1 m�i DTT. The beads were suspended In 30�iJ of kinase buffer [50 mM HEPES (pH 7.5), 10 m� MgCl2, and 1 m� Dir]

containing 2.5 mM EGTA, 10 m�i �3-glycerophosphate, 0.1 mr�i sodium

orthovanadate, 1 mM NaF, 20 ATP, 10 �Ci of [�,-�PJATP (Amersham),and 1 �g of soluble glutathione S-transferase-pRb fusion protein. Afterincubation of reaction mixture for30 mm at 305C, the samples were boiledin sample buffer and separated in 10% PAGE gel. Phosphorylated pro-

teins were visualized by autoradiography of the dried slab gel.

For preparation of pRb substrate, the pGEX-2T plasmid containing aportion of the Rb cDNA encoding amino acid residues 379-928 waselectroporated into the Escherichia coil. The glutathione S-transferase-Rbfusion protein was produced according to the protocol published byMatsushime et aL (69).

p53 Functional Analysis. The reporterplasmid pRGCAF0s lacZ (kind-ly provided by Dr. T. Fr#{233}bourg)contains two copies of the RGC-p53

binding sites in head-to-head orientation upstream of a minimal fos pro-moter that controls the expression of the bacterial lacZ gene to produce

13-galactosidase protein (70). Cells were plated at subconfluent density in100-mm2 dishes and transfected with 10 �g of plasmid DNA, according

to Puisieux eta). (71). The �-gaIactosidase activityoftransfected cells was

analyzed 72 h after DNA transfection, as described previously (70).Sequence-based Analysis of p53. Total RNA was prepared from the

frozen cell samples under stringent conditions to avoid degradation and

contammnationfollowed by enzymatic conversion ofthe RNAto cDNA. p53was amplified from cDNA by PCR using four overlapping primer pairscovering the complete coding region of the p53 gene. Biotin-labeled PCRproducts were generated with one of the primers (in each pair) modified

with a biotin molecule, which facilitates solid-phase sequencing. Solid-phase sequencing was carried out using AutoLoad Solid Phase Sequenc-ing Combs and 17 DNA polymerase (Pharmacia). The sequencing prod-

ucts generated were analyzed using an automated laser fluorescence ALF

DNA sequencer (Pharmacia).

Cytogenetlc Analysis. BC2 cells were karyotyped at passages 6 and12. The cells were grown for 24-48 h in RPMI 1640 supplemented with10% FCS and blocked in metaphase by Colcemid exposure(1O �g/m� for

45 mm. The cells were then treated with hypotonic solution (0.1 M M9CI2),fixed with the Camoy acetic solution, and stained for RHG bandings.

Treatment with TGF-fil. The hepatoma cells were plated onto 12-well plates at a density of 0.5 x 106 cells/well in medium supplementedwith 10% FCS. After 24 h, the medium was removed and replaced withfresh medium with or without 10% FCS. Human natural TGF-�31 (BritishBiotechnology) was added at a concentration of 5 ng/ml. After treatment,

the cells were detached with trypsin, and the viability was determined by

the trypan blue exclusion method. The cells were then fixed in a mixtureof ethanol and acetic acid (3:1, v/v) and stained with Hoechst 33258 (0.5�g/ml).

Tumorlg.nlclty Tests. Athymic Nu/Nu mice (Swiss-nu; ta Credo,Arbresle, France) were given 1 x 1o� cells s.c. Fifteen mice were injectedwith each clone. The animals were maintained in a sterile environment andexamined every week for the formation oftumors during a period of 1 year.

AcknowledgmentsWe thank J-G. Delcros for cytofluorimetric analysis. We are also gratefulto Dr. A. Guillouzo for helpful suggestions and critical reading of themanuscript.

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