Dedifferentiated variants of a rat hepatoma: Analysis by cell hybridization

Somatic Cell Genetics, Vol. 5, No. 6, 1979, pp. 697-718

Dedifferentiated Variants of a Rat Hepatoma: Analysis by Cell Hybridization

Jean Deschatrette, Emma E. Moore, ~ Monique Dubois, Doris Cassio, and Mary C. Weiss

Centre de Gbnbtique ~tol~culaire du C.N.R.S., 91190 Gif-sur-Yvette, France

Received 9 July 1979

Dedicated to our mentor, Boris Ephrussi

Abstract--Two independent dedifferentiated variants, H5 and FaoflC2, derived from the Reuber H35 hepatoma, produce trans-acting diffusible substance(s) that extinguish the expression of liver-specific proteins when hybridized with a well-differentiated cell line of the same origin (Fao and Fu5-5, respectively). H5 • Fao hybrids show total and stable extinction of four liver functions and clonal variability in the expression of three others. FaoflC2 • Fu5-5 hybrids are initially fiat (like FaoflC2 cells), and die in glucose-free medium where survival requires expression of hepatic gluconeogenic enzymes, but then evolve to hepatoma-like and finally round morphology; these latter cells express all liver functions analyzed including the gluconeogenic enzymes. Two exceptional clones that remained flat long enough for complete analysis showed extinction of all hepatic functions not expressed by FaoflC2 cells. We conclude that this transitory extinction reflects the action and then loss of extinguishing factor(s) contributed by FaoflC2. When crossed with BW1-J mouse hepatoma cells, FaoflC2 causes stable extinction of mouse aldolase B. We propose that production of extinguishing factor(s) is the rule for dedifferentiated variants.

I N T R O D U C T I O N

Different ia ted tumor cells grown in cul ture sometimes give rise to descendant clones tha t no longer produce the protein(s) charac te r i s t ic of their different iat ion (1, 2). The s tudy of such dedif ferent ia ted var iants can be

~Present address: Department of Pathology, University of Colorado Medical Center, Denver, Colorado 80262, USA.

697

0098-0366/79/1100-0697503.00/0 �9 1979 Plenum Publishing Corporation

698 Deschatrette et al.

expected not only to permit identification of the alterations in regulatory mechanisms that result in the absence of expression of differentiated functions, but also to provide clues to the mechanisms involved in the establish- ment and maintenance of the differentiated state. With such material it must be kept in mind that mechanisms unique to aneuploid cells of permanent lines, involving changes in the karyotype and/or structure of the genome (3, 4), could underlie stable changes in gene expression. Whatever mechanisms prove to be involved, clones of dedifferentiated cells will provide precious material for the analysis of gene expression by molecular tech- niques.

While the analysis of "variants" of differentiated cells has been undertaken in several laboratories, most of the cell types studied are permanent lines of determined stem cells, such as Friend erythroleukemia cells, myeloid leukemia cells (5), or myoblasts (6), which give rise to differentiated progeny under certain environmental conditions. With these cell types, it is possible to study the events involved in both the commitment and progression of stem cells to the differentiated state (see for example the review of Eisen et al., ref. 7).

We have chosen to study variants of stably differentiated cells that express a spectrum of specific functions in order to analyze the mechanisms involved in the maintenance of the differentiated state. In an earlier publica- tion (2) we have described the surprising phenotypic stability of Reuber H35 rat hepatoma cells (8) (line H4IIEC3, ref. 9). Among the numerous liver- specific functions examined in many independent clones, the variations in activity are quantitative but not qualitative, and no clone is deficient in expression of only one of the functions. In sharp contrast to the overwhelming majority of clones, we have been able to isolate a few clones, on the basis of altered morphology, which are deficient in the expression of all or most of the liver specific functions tested (2). Such variants, which lack an entire set of tissue-stSecific functions, appear to us to be good candidates for regulatory "mutants" whose defect originates in the inappropriate expression of mechanisms which control the orderly temporal expression of tissue-specific functions during development.

A first step in determining the nature of the changes underlying dedifferentiation of hepatoma cells is to establish the dominance relationship between the differentiated and dedifferentiated states. Such an analysis has already been performed for variant clone p4 (10): hybrids of p4 and well-differentiated Fu5-5 cells present the irregular "dedifferentiated" morphology of p4 cells and extinction of the five specific functions examined (albumin production and activity of four enzymes). These results permitted us to conclude that dedifferentiation of p4 cells is not due to the simple loss of factors necessary for maintenance of the differentiated state.

Dedifferentiated Variants of a Rat Hepatoma 699

Similar crosses have now been performed with two other dedifferentiated clones of independent origin. The results have allowed us to observe a new phenomenon, transitory extinction, and to propose that dedifferentiation is generally correlated with the expression of control mechanisms whose final effects are negative.

MATERIALS AND METHODS

Cell Lines and Cultures. Five parental cell lines have been used in this work; one clone (BW1-J) is derived from the mouse hepatoma line BW1 of Szpirer and Szpirer (11), and the four others are descendants of the rat hepatoma line H4IIEC3 of Pitot et al. (9). Fu5-5 and H5 are two independently derived subclones of H41IEC3 and have no selective markers. Fao (a second-generation clone of Fu5-5) and its subclone FaoflC2 are resistant to 12 ug/ml 8-azaguanine as well as to 3raM ouabain (2), and they have never been observed to revert to HAT-resistance. The karyotypes and liver functions of the various lines are given in tables.

All cell lines were cultivated in modified (12) Ham's F12 medium (13), supplemented with 5% fetal calf serum. Glucose-free (G-) medium, supplemented with 5% dialyzed serum, was prepared as described by Bertolotti (14). Cultures were maintained in Falcon plastic Petri dishes in a gas phase of 7% CO2 and air. Cloning was carried out by inoculating 10 50 cells in 10-cm Petri dishes. The generation times of the different hepatoma clones are 18-24 h, and they all show high cloning efficiency (50 80%).

Cells were detached for transfer with saline solution containing trypsin and EDTA (0.5 and 0.2 g/liter, respectively). For enzyme assays, detached cells were washed with phosphate-buffered saline or, for assay of the gluconeogenic enzymes, with 0.15 M NaCI.

Karyotypes were performed on colchicine-arrested metaphases by the standard air-drying technique (15) followed by staining with acetoorcein.

Hybridization. For all four crosses described below, a complete selective system was used, one parental line being HAT-sensitive and ouabain- resistant, and the other devoid of selective markers. In each case, cell hybridization was carried out by treating variable ratios of 2-10 x 1 0 6

parental cells with 0.5 ml of serum-free medium containing 400-800 hemagglutinating units of UV-inactivated Sendai virus for 10 rain at 4~ and 30 rain at 37~ The treated mixture was then diluted and 10-cm dishes inoculated with 8 • 104 to 2 x 105 cells, in medium containing HAT (10 4M hypoxanthine, 4 x 10-7M aminopterin, 1.7 x 10-SM thymidine) and ouabain (0.6-1.5 raM, depending upon the concentration required to kill the sensitive parent). Hybrid clones were picked from independent dishes, and maintained


in selective medium until freezing; thereafter ouabain but not HAT was omitted.

Utilization of Glucose-free Medium. Glucose-free modified F12 medium ( G ) , is supplemented with 5% extensively dialyzed fetal calf serum and with 2 mM oxaloacetate. Before being challenged with this medium, cells were adapted for several days to growth in G- medium supplemented with glucose (G+). When used to select hybrid cells reexpressing gluconeogenic enzymes, confluent cultures were changed to G- medium; within a week the cell layer had lysed and detached, and surviving colonies were isolated after one month.

As detailed below, newly formed hybrid colonies were tested for their ability to grow in G medium. During the course of these experiments, it was discovered that G + medium is inadequate for the survival of hybrid colonies during the first week or two after fusion. Therefore, crosses were performed in standard F12 selective medium and, at various times thereafter, dishes containing hybrid colonies were adapted to G + medium for two days and then challenged with G- medium (both supplemented with HAT and ouabain).

Assays. Induction of tyrosine aminotransferase (TAT; EC 2.6.1.5) was performed by placing cultures in growth medium supplemented with 10 6 M dexamethasone for 24 h. Control and induced cultures were treated in parallel. Activity was determined by the Diamondstone method (16), on extracts prepared as described (17). One unit of TAT catalyzes the formation of 1 ~tmol ofp-hydroxyphenylpyruvate per minute at 37~

Induction of alanine aminotransferase (AAT; EC 2.6.1.2) was performed by treating cultures for 48 h in growth medium supplemented with 10 7 M dexamethasone; control and induced cultures were treated in parallel. Preparation of the extracts and the enzyme assay were carried out as described (18), using the coupled assay of Segal and Matsuzawa (19). One unit of AAT catalyzes the formation of 0.55 umol of pyruvate per minute at 37~

Alcohol d e h y d r o g e n a s e ( A D H , EC 1.1.1.1) was a s sayed spectrophotometrically, or examined by electrophoresis, using the methods described in references 20 and 21, respectively. One unit of ADH oxidizes 1 umol of NADH per minute at 29 ~ Two different isozymes of ADH are present in differentiated hepatoma clones of line H4IIEC3, one corresponding in its electrophoretic mobility to the liver isozyme (L-ADH) and the other to that of the stomach (21). Zymograms of aldolase isozymes (EC 4.1.2.13) were prepared according to reference 22. The limits of sensitivity o f these electrophoretic tests are given in the tables and have been determined by dilution of positive extracts. For analysis of both ADH and aldolase, extracts were prepared by three, cycles of freeze-thawing (dry ice-alcohol) and centrifugation at 105,000g for 1 h at 4 ~ Extracts for glucose-6-phosphate


dehydrogenase (G6PD; EC 1.4.1.49) analysis were prepared and electrophoresis carried out on gelatinized cellulose acetate strips (Chemetron, Milan) following the method of Meera Khan (23).

Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) and fruc- tose diphosphatase (FDPase, EC 3.1.3.11) were assayed, respectively, by the radioactive method of Ballard and Hanson (24) and the coupled spectrophotometric procedure of Traniello et al. (25), as detailed by Bertolotti (14), with one modification: after arrest of the PEPCK assay, an aliquot of the reaction mixture was dried with a heat lamp, redissolved in distilled water (26), and counted in a Packard scintillation counter.

In all tables, enzyme-specific activities are expressed in milliunits per milligram of protein in the extract. The protein content of the extracts was determined by the method of Lowry et al. (27), using bovine serum albumin (fraction V, Armour) as standard.

Albumin production was determined by the electroimmunodiffusion method of Laurell (28). Nearly confluent cultures were incubated for 48 or 72 h, the medium collected for assay, and the cells counted. When necessary, medium was concentrated on Centriflo membranes (Amicon). Electro- phoresis was carried out at 4 ~ for 4 h at 105 V or overnight at 30 V, using barbital buffer (0.1 M, pH 8.6). Rabbit anti-rat albumin serum was incorporated in the gel at 0.1%, and the standards of rat albumin included on each plate varied from 0.43 to 30 ug/ml. Under these conditions, 0.1 ~g/ml of rat albumin can be detected. Preparation and use of immunoadsorbed rabbit anti-mouse and anti-rat albumin are detailed in ref. 29.

RESULTS

Differentiated Parental Clones. The differentiated clones derived from line H4IIEC3 present the following liver specific properties: (1) morphology resembling that of hepatocytes in culture, i.e., very regular epithelial cells, firmly attached to the culture dish, presenting a clear round nucleus with one or two large nucleoli, and phase-dense cytoplasm decorated with granules and lipid droplets (see Fig. 1 ); (2) activity of TAT, AAT, and of the liver isozymes of aldolase (type B) and ADH; (3) inducibility of TAT and AAT by glucocorticosteroid hormones; (4) secretion of serum albumin; and (5) the ability to proliferate in glucose-free ( G ) medium, reflecting activity of the gluconeogenic enzymes (PEPCK and FDPase). These cells are hyperdiploid (usual mode: 52), and the differentiated clones show a great karyological homogeneity. The clones used in this work are Fu5-5 and Fao; their properties are given in Tables 1 and 3.

BW1-J mouse hepatoma cells present hepatocyte-like morphology only in dense culture. They are subtetraploid (mode: 64), and of the liver functions


Fig. 1. Phase-contrast photomicrographs of living cultures of Fao (1), HF1 (2), H5 (3), and of segregant clone HF1-5(4). The bar in panel 1 corresponds to 83 Izm.

given in the preceding paragraph, they express only aldolase B and albumin, as well as alpha-fetoprotein, as detailed in Table 5.

Dedifferentiated Parental Clones: H5 and FaoflC2. H5 celfs, isolated directly from H4IIEC3, show irregular epithelial morphology and form piled-up disorganized colonies (Fig. 1). They express none of the above- mentioned hepatic functions. The H5 karyotype is very similar to that of the majority of hepatoma clones (Table 1).

The other dedifferentiated cell line, FaoftC2, was derived from the clone Fao. The cells retain a regular epithelial morphology, but are flat and lack the large nucleolus, cytoplasmic granules, and lipid droplets characteristic of the differentiated cells (Fig. 3). Although the majority of liver functions are undetectable in these cells, they have retained AAT inducibility and essentially normal activity and inducibility of TAT (Table 3). These cells present a karyotype much like that of the other clones; a fraction of the metaphases contain one or two new marker metacentric chromosomes, accompanied by a reduced number of telocentrics, such that the number of chromosome arms does not differ for the metaphases with and without the new markers.

H5 x Fao. HF Hybrids. Since a complete selective system was available for this cross, it was possible to examine several hundred hybrid colonies for morphological traits in the original hybridization dishes. The hybrid colonies were homogeneous in morphology and resembled the H5 parent much more closely than the Fao parent (Fig. 1). Owing to this morphological homogeneity, only six independent hybrid clones have been characterized (Table 1 ).

The mean chromosome number for cells of each clone is equal to or close

7

Tab

le 1

. K

aryo

logi

cal

and

ph

eno

typ

ic p

rop

erti

es o

f F

ao,

H5,

an

d H

F h

ybri

d ce

lls ~

AD

H c

TA

T

AA

T

Tot

al

Ser

um

N

um

ber

of

spec

ific

al

bu

min

C

ell

line

ch

rom

oso

mes

b B

1

B

1 L

-AD

H

acti

vity

A

ldol

ase

B d

(#

g/1

06

cel

l/2

4 h

)

Gro

wth

in

gluc

ose-

free

m

ediu

m

Fao

52

(5

0 55

) 54

.0

380.

0 93

28

0 +

32

,2

+

2,5

H5

52

(50

-55

) 1.

0 1.

0 5

5 -

<5

.0

<0.

001

HF

I 10

6(10

1 11

2)

2.9

6.3

35

98

- 5.

2 -

<0.

003

HF

3 10

7 (1

01

-11

2)

6.3

9.3

42

71

W

14.2

-

0.53

H

F4

10

3 (9

6-1

05

) 1.

4 1.

3 37

44

W

12

.7

- 0,

9 H

F5

102

(99

-10

4)

2.8

7.7

16

35

W

11.7

2.

0 H

F6

10

3 (9

7 ll

O)

5.7

11.7

<

5

<5

+

24

.9

- 1.

1 H

F8

103

(96

-10

7)

3,1

9.7

39

140

W

9.2

- <

0,0

02

"Sym

bols

: +

in

dica

tes

pres

ence

of

a st

ron

g b

and

(s)

of a

ctiv

ity

on z

ym

og

ram

s;

indi

cate

s ab

sen

ce o

f ac

tivi

ty;

W i

ndic

ates

pre

senc

e of

def

init

e b

ut

wea

k ac

tivi

ty.

B a

nd 1

ref

er t

o ba

sal

and

indu

ced

spec

ific

act

ivit

ies,

res

pect

ivel

y.

bMea

n, w

ith

rang

e gi

ven

in p

aren

thes

es.

Th

e an

tici

pat

ed

nu

mb

er o

f ch

rom

oso

mes

for

HF

hy

brid

s is

10

4 (1

00

110)

. T

he

nu

mb

er o

f m

etap

has

es

anal

yzed

for

eac

h cl

one

is f

rom

13

to 2

3.

CT

he l

imit

of

sens

itiv

ity

of t

he e

lect

roph

oret

ic t

est

|br

L-A

DH

act

ivit

y co

rres

po

nd

s to

10%

of

the

acti

vity

pre

sen

t in

Fao

ext

ract

s. T

he

spec

ific

act

ivit

y co

rres

pond

s to

the

su

m o

f bo

th L

-AD

H a

nd

"st

om

ach

" A

DH

iso

zym

es (

see

in F

ig.

2 th

e st

ain

ing

int

ensi

ty f

or b

oth

isoz

ymes

).

dThe

lim

it o

f se

nsit

ivit

y of

the

elec

trop

hore

tic

met

ho

d c

orr

esp

on

ds

to 7

% o

f th

e ac

tivi

ty p

rese

nt

in F

ao e

xtra

cts.


Fig. 2. ADH (top) and aldolase (bottom) zymograms of extracts of parental and HF hybrid cells. ADH: all extracts show activity of the "stomach" isozyme that migrates to the anode, while activity of L-ADH (cathode) is seen only for Fao, HF6, and HFI-I . For the latter extract, the staining intensity of L-ADH is greater than that of Fao. Aldolase: all exlracts show a band corresponding to aldolase A 4 which remains at the origin. Extracts of HF6 and H 5 present a band of A3C heterotetramer (anodal side of the origin). The presence of cathodal AB heterotetramers is limited to extracts of Fao and HFI - i .

to the sum of the parental karyotypes, Some of the liver-specific functions of Fao cells are entirely extinguished while the expression of others varies from one hybrid clone to another. Among the functions subject to quasi-total extinction are aldolase B and TAT. In all clones, aldolase B activity is undetectable (<7% of that of Fao; Fig. 2), and TAT activity ranges from 5 to 11% of that of Fao and is very slightly inducible. The hybrid clones are unable to survive in G medium, indicating that they do not express significant amounts of FDPase and/or PEPCK. Enzymatic assays have confirmed that the expression of both of these gluconeogenic enzymes is extinguished in the two hybrid clones tested (Table 4).

In contrast, the expression of AAT, ADH, and albumin continues in many hybrid clones, although there is marked variation from clone to clone. Furthermore, the expression of these functions is not coordinate; i.e, the done


(HF6) expressing the highest ADH activity has no significant AAT activity and the two clones (HF1 and HFS) that do not secrete albumin have higher AAT activity than the clone (HF5) with maximal albumin production. There is no apparent correlation between the phenotype and karyotype of the various hybrid clones.

A search for hybrid clones reexpressing functions of Fao parental cells was carried out in order to determine whether the various functions reappear independently of one another and if any karyotypic changes accompany reexpression.

The reports that the X chromosome is responsible for certain cases of extinction in hybrid cells (30, 31) led us to undertake selection against the X chromosome contributed by the H5 parent. The H5 chromosome coding for HGPRT can be eliminated from the hybrids by selecting survivors in 8-azaguanine-containing medium. In all mammals thus far investigated, HGPRT and G6PD are both encoded on the X chromosome. Since only H5 cells produce G6PD(2) and this enzyme is produced by all of the hybrid clones, loss of the H5 X chromosome should be accompanied by loss of G6PD activity from the hybrid cells. Cells of clones HF6 and HF8 were subjected to two cycles of selection in medium containing 15 ~g/ml of 8-azaguanine; the surviving cells of both clones are killed in HAT medium and lack activity of G6PD. No increase in activity or inducibility of TAT, nor of production of L-ADH or aldolase B was observed in these cells. Thus, unlike the case reported by Croce et al. (30) for rat hepatoma-human fibroblast hybrids, loss of the X chromosome of the noninducible parent does not result in reexpression of TAT inducibility.

Hybrid cells reexpressing FDPase and PEPCK can be isolated selectively in glucose-free medium. Therefore, large numbers of cells of two hybrid clones, representing the extremes of expression of hepatic functions (HF1 and HF6), were challenged in glucose-free medium. No survivors were observed among 144 x 1 0 6 HF6 cells. However, five colonies were isolated from 48 • 1 0 6 HF1 cells. Although each colony was isolated from a different culture bottle, it cannot be affirmed that they represent independent segregants since all derive ultimately from the same hybrid clone.

All five of the HF1 subclones present similar properties, detailed in Table 2. Unlike the HF1 cells of origin, they show the specific morphological traits of liver cells in culture (Fig. 1). Their ability to proliferate in G medium indicates that these cells reexpress FDPase and PEPCK activities, as demonstrated for subclone HF1-3 (Table 4). Moreover, all the subclones reexpress significant activities of TAT (and a 4- to 17-fold inducibility), L-ADH, and aldolase B, as well as production of albumin (Table 2 and Fig. 2). HF1 cells express significant basal and induced levels of AAT, and these activities are essentially unchanged in the subclones (Table 2). The mean

O',

Tab

le 2

. K

aryo

logi

cal

and

phen

otyp

ic p

rop

erti

es o

f H

E1

and

its

five

sub

clon

es s

elec

ted

in g

luco

se-f

ree

med

ium

"

Cel

l li

ne

TA

T

AA

T

Ser

um

G

row

th i

n N

um

ber

of

alb

um

in

gluc

ose-

free

ch

rom

oso

mes

b B

1

B

I L

-AD

H c

A

ldol

ase

B a

(~g

/10

6 c

e11/

24 h

) m

ediu

m

HF

I 10

6 (9

7-1

12

) 2.

9 6.

3 35

98

-

- <

0.00

3 H

FI-

1

73 (

72

75)

12.8

15

8.3

55

94

+

+

0.2

+

HF

1-2

74

(6

6-8

0)

72.6

31

9.4

45

105

+

+

0.5

+

HF

1-3

78 (

75

81)

3

2.3

55

0.4

14

61

§ +

2.

2 +

H

FI-

4

71 (

67

75)

17.2

19

7.6

70

107

+

+

0.3

+

HF

1-5

69

(68

71

) 14

.2

208.

0 13

23

+

+

0.

3 +

"Sym

bols

: +

in

dica

tes

pres

ence

of

a st

ron

g b

and

(s)

of

acti

vity

on

zym

og

ram

s; -

in

dica

tes

abse

nce

of

acti

vity

. B

and

I r

efer

to

basa

l an

d in

duce

d sp

ecif

ic a

ctiv

itie

s re

spec

tive

ly.

bMea

n, w

ith

ran

ge

give

n in

par

enth

eses

. F

or e

ach

subc

lone

, 15

met

aph

ases

wer

e an

alyz

ed.

CT

hc li

mit

of

sens

itiv

ity

of

the

elec

trop

hore

tic

met

hod

for

L-A

DH

act

ivit

y co

rres

po

nd

s to

10%

of

the

acti

vity

pre

sent

in

Fao

ext

ract

. d

Het

ero

tetr

amer

s A

3B~,

A2

B 2

, an

d A

~B 3

are

obs

erve

d fo

r al

l H

FI

subc

lone

s.


Fig. 3. Phase contrast photomicrographs of living cultures of Fu5-5 (panel 1), FaoflC2 (panel 2), and of the three types of Flof hybrids (panels 3 to 6). The "flat hybrids" of panel 5 present, like FaoflC2 cells, pale cytoplasm and rather small nucleoli. The "hepatoma-like" and "round" hybrids (panels 4 and 3, respectively) show dark granular cytoplasm and large, often centrally located nucleoli (compare to panel l). Panel 6 shows the edge of an original hybrid colony where a focus of round cells has appeared within the hepatoma-like borders. The bar in panel 6 corresponds to 83 ~tm.

Tab

le 3

. K

aryo

logi

cal

and

ph

eno

typ

ic p

rop

erti

es o

f F

u5-5

, F

aofl

C2,

and

Flo

f h

yb

rid

cel

ls ~

TA

T

AA

T

Ser

um

G

row

th i

n N

um

ber

of

alb

um

in

gluc

ose-

free

C

ell

line

M

orp

ho

log

y

chro

mo

som

es b

B

I B

l

L-A

DH

c

Ald

olas

e B

(t

tg/1

06 c

e11/

24 h

) m

ediu

m

Fu5

-5

Hep

ato

ma

52 (

5t-

53

) 35

22

3 15

0 45

0 +

+

0.

5 +

F

aofl

C2

Fla

t 52

(49

54

) 20

24

0 <

10

73

-

<0

.00

2

-

Flo

f e a

F

lat

100

(99

-10

i)

31

136

<8

<

18

W

-

<0

.00

4

Flo

fd a

Fla

t 10

1 (9

8-1

04

) 14

14

4 <

9

<1

3

W

0.02

-

Flo

fg a

Hep

ato

ma

90

(89

10

3)

36

216

20

80

+

+

0.03

F

lof

5 R

ou

nd

85

(8

4-8

7)

18

980

80

279

+

+

0.9

+

Flo

f7

Ro

un

d

92

(87

-95

) 34

16

8 15

9 40

0 +

+

0.

2 +

F

lof9

R

ou

nd

10

4 (9

8-1

07

) 33

17

4 10

4 28

6 +

+

0.

3 +

F

lof

13

Ro

un

d

100

(96

102)

26

21

9 52

14

9 +

+

2.

5 +

F

lof

14

Ro

un

d

99 (

90

-10

1)

45

188

68

242

+

+

3.8

+

Flo

f 15

R

ou

nd

10

2 (1

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number of chromosomes in all 5 subclones is reduced by 27-35%. This chromosome loss corresponds to about half of the chromosomes of H5 cells, or a quarter from each parent. Because of the extensive chromosome loss, it is not possible to conclude that the simultaneous reexpression of the hepatic functions in these subclones reflects a coordinate regulation.

FaoflC2 • Fu5-5: Flof Hybrids. Hybrids from this cross fall into three distinct morphological and phenotypic classes. We will begin by presenting the properties of these different kinds of hybrids and then turn to a consideration of the time of appearance of each type, as well as the evolution of one from another. The logic of this presentation will become clear as we progress.

The three types of hybrids observed are shown in Fig. 3: panel 5, very flat cells, similar in morphology to FaoflC2 parental cells; panel 4, refractile phase dense cells that closely resemble Fu5-5 parental cells (hereafter referred to as "hepatoma-like"; and panel 3, extremely refractile cells that attach poorly to the substrate and tend to detach in grape-like clusters. The latter "round" cells resemble neither parent.

Hybrid clones of each type were isolated and grown up for further characterization. However, clones initially displaying flat or hepatoma-like morphology were unstable and eventually partially or completely converted to round morphology. Therefore, only two homogeneous flat clones have been completely characterized (Tables 3 and 4; Fig. 4). The expression of liver- specific functions by these clones is either very low or undetectable, with the exception of TAT which is expressed by both parental cells. Characterization of one hepatoma-like clone indicates that its properties are intermediate;

Table 4. Specific activities of gluconeogenic enzymes of parental and hybrid cells a

Cell line FDPase PEPCK

Fao 13.0 80 H5 <0.5 19 HF6 <0.2 3 HF1 <0.2 2 HFI-3 6.9 88

Fu5-5 2.5 39 FaoflC2 <0.1 19 Flof d <0.4 1.2 Flof 5 0.9 69 Flof 14 2.1 49 Flof 18 16.6 58

aOne milliunit of FDPase causes the reduction of 1 rr~mol of NADP per min at 28~ one milliunit of PEPCK activity catalyzes the fixation of 1 nmol of [~4C]bicarbonate per min at 37 ~ A low PEPCK activity is found in nearly all cultured cells and may reflect activity of the ubiquitous mitochondrial enzyme. In accordance with the results described by Bertolotti (14), we find that the ability of cells to grow in glucose-free medium is correlated with PEPCK activities greater than 20.


Fig. 4. Zymograms of ADH (top) and aldolase (bottom) of parental and Flof hybrid cell extracts. The left part of the figure permits comparison of the activities of parental cells, and one hybrid clone of round cells. L-ADH and aldolase AB heterotetramers are seen only in FuS-5 and Flof 14 extracts. The right part of the figure shows these activities of fiat (Flofd), hepatoma-iike (Flof g), and round (Flof 18) hybrid cells. As stated in the text and Table 3, lhe flat hybrid cells show faint L-ADH activity and no aldolase; the hepatoma-like cells normal L-ADH and only one band of AB heterotetramers, while the round cells present activities clearly superior to those of FuS-5 cells. Interpretation of the bands is explained in Fig. 2.

some, but not all, of the hepat ic functions are expressed. In contrast , all nine c~ones of round morplaology express the ent ire spec t rum of hepat ic functions (Table 3, Fig. 4). Karyotyp ic analysis confirms the hybr id na ture of these clones, and no obvious chromosomal differences among the various types of hybrids can be discerned (Table 3).

The morphological evolution of these hybrids was sys temat ica l ly followed from very ear ly stages af ter fusion, beginning on day six. At this t ime, no colonies of round ceils were observed. Eighteen colonies of hepa toma- like morphology were marked and observed every few days. The major i ty of these colonies degenerated, some of them leaving behind fiat colonies; it therefore appears probable that they were surviving FuS-5 cells, or mixed colonies. Thi r ty-one colonies of flat cells were also marked on day six, in order to follow their morphological evolution. F igure 5 shows that between 14 and 20 days af ter fusion, the major i ty (70%) of these switched from fiat to


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Fig. 5. Kinetics of the morphological evolution of Flof hybrids. As detailed in the text, six days after fusion of FaoflC2 x Fu5-5 cells, 31 colonies of "flat" cells were marked and examined every 1 3 days. o Cumulative curve of the fraction of flat colonies that converted to hepatoma-like morphology; ~ cumulative curve of the appearance of loci of round cells, observed only within hepatoma-like colonies and never in flat colonies.

hepatoma-like morphology. The subsequent death of most of the remaining flat colonies suggests that they represent slowly degenerating FaoflC2 cells. At 22 days after fusion, loci of round cells (Fig. 5) began to appear in the recently converted hepatoma-like colonies (see panel 6 of Fig. 3), and spread until eventually such colonies consisted exclusively of round cells. These observations show that the initial hybrids of FaoflC2 and Fu5-5 cells display the flat morphology characteristic of the FaoflC2 parent, but that this form is unstable and evolves first to hepatoma-like and later to round morphology.

In view of the morphological evolution of these hybrids and the apparent correlation between hybrid morphology and the expression of liver-specific functions (Table 3), experiments were undertaken to determine whether the differentiated state of the hybrids varies with their age. For this experiment it was necessary to analyze the hybrids for the expression of liver-specific


functions very soon after fusion and preferably on a colony-by-colony basis. Therefore, the criterion used to determine the differentiated state of the hybrids was their ability to grow in glucose-free (G-) medium. Only those hybrids expressing both of the liver-specific gluconeogenic enzymes (FDPase and PEPCK) will survive under these conditions. As a control, hybrids prepared between two well-differentiated cell lines (Fao and Fu5-5) were subjected to the same analyses.

The fusion mixtures were prepared as usual and inoculated into 50 plates (105 cells/10-cm plate) containing the standard selective medium: F12 supplemented with HAT and ouabain (see Materials and Methods). HAT and ouabain were maintained throughout the experiment to ensure selective growth of the hybrids. (To verify that this selective medium was effective, equivalent numbers of parental cells were plated together in selective medium without virus. One survivor appeared in the FaoflC2-Fu5-5 mix and two suvivors in the Fu5-5-Fao mix.). On day five, and every two days thereafter, groups of four plates from each cross were changed from standard selective medium to selective medium containing dialyzed rather than whole serum (G+). Two days later, the medium on two of these four plates was replaced with G- medium, also containing serum previously dialyzed to remove free glucose. The plates were fixed and stained 50 days after fusion and the number of surviving hybrid colonies determined. The frequency of hybrids resulting from fusion between the two well-differentiated hepatoma cell lines, Fu5-5 and Fao, is always small. Furthermore, these hybrids grow slowly, and until approximately 12-16 days after fusion are extremely sensitive to the deleterious effects of dialyzed serum (Fig. 6). However, the survival of these hybrids in G- medium closely parallels their survival in dialyzed serum. Strikingly different results were obtained for the hybrids formed between FaoflC2 and Fu5-5; even for early changes to dialyzed serum about half of the potential hybrid colonies survive and proliferate, whereas it is only after 12-16 days that any hybrids survive in G- medium (Fig. 6). Furthermore, the maximum number of hybrids capable of growth without glucose is attained at approximately 20 days after fusion, and represents only 35% of the total number of hybrids surviving in dialyzed serum. This apparent plateau at 35% holds only for young hybrids; after being picked and grown for another 20 generations, practically all hybrids grow in G- medium (Table 3).

We conclude that extinction occurs in the initial hybrids formed between FaoflC2 and Fu5-5, as evidenced by their flat, "FaoflC2-1ike" morphology, and their inability to grow in G- medium. The morphological instability of the flat hybrids, and the abrupt appearance of hybrid colonies capable of growth in G- medium, shows that for a significant proportion of hybrids, extinction is only transitory. Possible explanations for this "transitory extinction" are considered below.

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Fig. 6. Curves of the survival of hybrid colonies, as a function of time after fusion, in G § and G- medium. For both crosses, groups of plates were changed from normal selective medium to G + selective medium at the times indicated by the points. Two days later, half of the G + plates were switched to G selective medium. Plates were maintained in the respective media, with renewals every 3-4 days, until 50 days after fusion, when all were fixed, stained, and counted. Top curves: control cross between two well-differentiated hepatoma lines, Fao and Fu5-5. In all plates, hybrid colonies were mostly small, and failed to show vigorous growth (survival of these hybrids requires reduction by a factor of 10 in the amounts of aminopterin used for HAT). The hybrids survive well in medium containing dialyzed serum only 10 or more days after fusion; thereafter the numbers of colonies in G + (o) and G (O) are nearly identical. Bottom curves: FaoflC2 x Fu5-5; same experimental conditions as above. Hybrid colonies surviving in G (O) appear only 12-16 days after fusion. The star near the ordinate of each graph indicates the number of colonies surviving in normal FI2 selective medium.


Table 5. Karyological and phenotypic properties of FaoflC2, BW1-J, and Flob hybrid cells

Albumin 0zg/106 cells/24 h) Aldolase B b TAT c

Cell Number of line chromosomes ~ Rat Mouse Rat Mouse B I

FaoflC2 52 (49-54) <0,002 23.0 104.0 BWI -J 64 (60-72) 13.0 + 3.0 6.0

Flob 1 108 (94-122) <0,7 't 18.8 'l - - 1.0 6.8 Flob 3 110 (106-119) <0.4 10.7 - 0.4 1.4 Flob 4 110 (106-119) <0.3 7.9 - - 1.0 6.0 Flob 5 106 (91-117) <0.2 4.6 - - 1.0 3.8 Flob6 100(89 I15) <0.2 4.8 - W 0.7 1.9 Flob 8 114 (109-116) <0.2 17.0 - - 3.5 26.9

a Mean, with range given in parentheses, based upon the analysis of at least 15 metaphases. bThe limit of sensitivity of the electrophoretic method for aldolase B corresponds to 10% of the

activity of BWI-J. W signifies very weak activity. CB and I: basal and induced, respectively. Values given are specific activities (milliunits per mg

protein in the extracts). 80-90% of the TAT activity of BW1-J cells is due to the liver-specific enzyme, as demonstrated by neutralization with a specific antiserum (32). Enzyme induction was achieved by exposing cultures to 10 7 M dexamethasone for 20 h, observed as optimal for BW l-J cells.

aAssays for the hybrids were performed with immunoadsorbed non-cross-reacting antisera on unconcentrated spent media. For this reason the sensitivity of the test for rat albumin is less than that given in other tables.

FaoflC2 • BWI-J." Flob Hybrids. The indication from the preceding experiments is that FaoflC2 cells impose extinction on the Fu5-5 genome, but that for a significant fraction of the hybrids this effect is only transitory. A second cross was carried out to determine whether or not FaoflC2 cells extinguish liver-specific functions of hepatoma cells from a different line. For this experiment, cells of the mouse hepatoma line BW1-J were chosen. BW1-J cells display a pattern of phenotypic expression like that of fetal liver (32): they produce albumin and aldolase B, while the other liver enzymes are not produced at all or only in trace amounts (e.g., TAT).

Hybrid colonies from this cross show striking morphological homogeneity and no tendency towards evolution to a new form was observed during the 30-40 generations that six independent clones were maintained in culture. The characterization of these six clones gave the results summarized in Table 5. Only mouse albumin is produced by cells of each of the clones, and in quantities varying from 40% to 150% of those of BW1-J cells; there is thus neither extinction of mouse albumin nor activation of rat albumin synthesis in these hybrids, a surprising observation since albumin production is partially or totally extinguished in the flat Flof hybrid clones (Table 3). Aldolase B, produced only by the mouse parental cells, is extinguished totally in five out of six of the hybrid clones (10% of the aldolase B of BWI-J cells can be detected and the marginal aldolase B activity of Flob 6 cells probably corresponds to


this value). Finally, the high TAT activity and inducibility of FaoflC2 cells is reduced in nearly all cases to the level of activity of BWl-J cells, and L-ADH, produced by neither parent, is absent from the hybrid cells (data not shown). In conclusion, this sample of six hybrid clones demonstrates a fairly homogeneous pattern of expression, including absence of extinction for albumin synthesis, and total or partial extinction of aldolase B synthesis. It is important to note that both aldolase B and albumin are coexpressed in hybrids of Fao • BW1-J (32).

DISCUSSION

This paper describes the genetic characterization of two phenotypically distinct dedifferentiated variants, H5 and FaoflC2, derived from the Reuber H35 hepatoma. Whereas H5 cells express none of the liver functions examined and show an entirely chaotic growth habit, FaoflC2 cells continue not only to express TAT activity and inducibility, but also retain normal epithelial morphology. The behavior of hybrids between each variant and well-differentiated hepatoma cells indicates that regulatory element(s) acting in a negative and transdominant fashion have been activated in cells of both variant lines, but that the initial lesion responsible for dedifferentiation is probably not the same for the two variants.

Hybrids prepared between H5 and Fao fail to express most of the liver-specific functions, and the extinguishing effect of the H5 parent is stable through many cell generations. These results resemble those previously reported for a third dedifferentiated variant, p4 (10). However, for all of the H5 x Fao hybrids, significant amounts of at least one liver protein are produced, most often albumin or AAT, suggesting that extinction of the set of hepatic functions is not coordinated. In contrast, only a few of the p4 • Fu5-5 hybrids failed to show total extinction, and these cells produced small amounts of several liver specific proteins.

The stability of the extinction imposed by H5 cells is demonstrated by the rarity of hybrids reexpressing the gluconeogenic enzymes: segregants able to grow in G medium were detected at a frequency of 1 x 10 -7 for HF1, and none was obtained from HF6 (frequency <7 • 10 9). Moreover, not even a small fraction of hybrids escapes the extinguishing effect of the H5 parent. Newly arisen hybrid colonies were tested for their ability to proliferate in G medium: in equal numbers of dishes, over 500 colonies grew in G + while not a single colony survived in G .

Hybridization of FaoflC2 with Fu5-5 cells has given rather complicated results that have been analyzed in some detail, for this appears to be the first case documented of transitory extinction. Since the period of extinction is too brief to be analyzed by classical assay methods, we have drawn this conclu-


sion from the following correlations: (1) the morphological evolution of hybrid cells and the similarity of the kinetics of evolution to that of the appearance of hybrid cells capable of growth in G- medium, and (2) the morphological similarity between newly formed hybrid cells and the two flat hybrid clones (Flof d and Flof e) that show extinction of the various liver functions.

Three morphological types of hybrid cells can be identified, and the same order of appearance of the different types has been observed in several independently performed crosses. The initial hybrids are mostly if not uniquely "flat" like the FaoflC2 parent; this form is unstable, and entire colonies convert to Fu5-5-1ike or "hepatoma-like" morphology. This form is also unstable and a second change occurs, that is the appearance within hepatoma-like colonies of foci of "round" cells that spread slowly to fuse with other loci until eventually the entire colony consists of round cells. This final round form, that resembles neither parent, is characteristic of all hybrids from this cross, even though in exceptional cases it is observed only after several transfers. We conclude that the flat hybrids show extinction of the majority of liver functions because (1) they all fail to survive in G medium, and (2) the two exceptional clones that remained uniformly flat long enough to be characterized showed extinction of nearly all of the functions not expressed by parental FaoflC2 cells.

Two possible interpretations of this morphological and phenotypic evolution can be offered. While the most obvious explanation might seem to be chromosome loss, the rate and high frequency of the changes do not seem compatible with this explanation. Moreover, in the cases where it has been possible to compare the karyotypes of flat and round hybrid cells, no differences were apparent. Therefore, we are led to propose that FaoflC2 cells produce extinguishing factor(s) and that inactivation or greatly reduced production of such factor(s) occurs in a significant proportion of hybrids, probably owing to some element contributed by Fu5-5. The kinetics of the morphological and phenotypic evolution of the hybrids could be explained in many ways. Assuming that the extinguishing factor(s) are extremely stable but no longer produced in the hybrids, the "transitory extinction" would represent the time required for dilution of these molecules by cell division and/or slow, but eventual, degradation. Alternatively, the extinguishing factor(s) may be quickly inactivated in the hybrids, either by direct interac- tion with some Fu5-5 element or by decreased production followed by rapid degradation. The period of "transitory extinction" would then represent the time necessary for the hybrids to resume expression of the liver-specific functions which may be a function of absolute time, the number of cell divisions, or a critical cell density. The variations in the rates at which the apparently inevitable morphological and phenotypic evolution occurs could be due to heterogeneity within one or both of the parental populations,


When the properties of h,ybrids from the two crosses involving FaoflC2 cells are compared, there is an apparent contradiction in the results. Thus, although aldolase B production by the mouse hepatoma BW l-J genome is stably and permanently extinguished, the extinction of the group of functions of the Fu5-5 genome is only transitory. This situation closely parallels that described by Forquignon and Ephrussi (1) for an amelanotic variant of hamster melanoma cells, which causes extinction of melanogenesis when crossed with pigmented mouse melanoma cells but fails to impose even a transitory extinction when hybridized with pigmented cells of the original hamster melanoma line.

These apparently contradictory situations may in fact enlighten us concerning the nature of the changes responsible for the loss of expression of differentiation by cells of permanent lines. While the most simple explanation for such a change would appear to be the loss of some factor necessary to maintain the differentiated state, our analyses of lines P4, H5, and FaoftC2 have not lent support to this hypothesis, nor have the experiments of Forquignon and Ephrussi (1). Thus, for each variant analyzed, at least one cross with differentiated cells of the same histotype has produced hybrids showing extinction, leading us to propose that whether extinction is observed in such hybrids depends upon whether or not extinguishing factor(s) continue to be produced by the variant genome after fusion. We conclude that the absence of expression of differentiated functions in such variant cells results, directly or indirectly, from production of the factor(s) responsible for extinction.

ACKNOWLEDGMENTS

Emma E. Moore was a recipient of fellowships from the Fondation pour la Recherche M6dicale and the Philippe Foundation. We thank Nathalie Goussef for performing the albumin assays and Linda Sperling for critical reading of the manuscript.

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Dedifferentiated variants of a rat hepatoma: Analysis by cell hybridization

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Transcript of Dedifferentiated variants of a rat hepatoma: Analysis by cell hybridization