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Expression of differentiated functions in hepatoma cell hybrids
BIOCH1MIE, 1972, 54, 195-201.
Expression of' differentiated functions in hepatoma cell hybrids. VI. Extinction and re-expression of liver alcohol dehydrogenase. R o g e r BERTOLOTTI and Mary C. WEISS. Centre de G~n~tique Mol~culaire du C.N.R.S., 9 1 - Gif-sur-Yvetle. (7/2/1972). Summary. - - Well differentiated rat hepatoma cells, grown both in vitro and in vivo, contain tv¢o forms of alcahol dehydrogenase, one of which corresponds in its electrophoretic mobility to the well knovcn liver enzyme, and the other to the enzyme found in the stomach. Ceils of a line of mouse fibroblasts contain only very small amounts of the s¢omach enzyme, and diploid ra.t epithelial cells show no activiCy. Cells of bo~h of these latter types have been crossed with the rat hepatoma cells, and the result4ng hybrids contain only very small amounts o.f enzyme activity, which has been inc~entified as the stomach type. No activity of liver alcohol dehydrogenase can be detected in the hybrids, and it is concluded that like many other differentiated functions, this enzyme, characteristic of hepatic differen~ia, tion, is extinguished upon hybridization wi.th a cell of different histogenetic origin. However, the failure of the hybrid cells to produce this enzyme is not due to loss or irreversible alteration of genetic material, because one hybrid subclone, which has lost a large number of chromosomes, re-expresses liver alcohol dehydrogenase.
Somatic h y b r i d i z a t i o n , first o b s e r v e d in 1960 [1], has been used e x t e n s i v e l y in r e c e n t y e a r s as a tool for the genetic analysis of s o m a t i c cells. T h e usefulness of s o m a t i c h y d r i d s stems f r o m the o b s e r v a t i o n s that they are viable, possess indefinite g r o w t h potential, can be isolated f r o m crosses of cells d e r i v e d f r o m the same or f r o m different species, that they u n d e r g o c h r o m o s o m e loss or segregation, and that they express the g e n e t i c c h a r acters of both p a r e n t s [for r e v i e w , see ref. 2]. One of the a p p l i c a t i o n s of s o m a t i c h y b r i d i z a t i o n has been the study of the r e g u l a t i o n of expression of d i f f e r e n t i a t e d functions. It has been s h o w n , in n u m e r o u s cases, that h y b r i d cells are c h a r a c t e r ized by c o - e x p r e s s i o n of p a r e n t a l forms of metabolic enzymes, and by c o m p l e m e n t a t i o n of p a r e n t a l e n z y m e deficiencies [2]. H o w e v e r , this is not the case w h e n the fate of a d i f f e r e n t i a t e d f u n c t i o n is e x a m i n e d in h y b r i d s f o r m e d by crossing cells of a p i g m e n t e d S y r i a n h a m s t e r m e l a n o m a a n d those of a p e r m a n e n t line of mouse fibroblasts [3]. T h e resulting h y b r i d s c o n t a i n essentially all of the c h r o m o s o m e s of both p a r e n t a l types, and they produce both p a r e n t a l f o r m s of t w o m e t a b o l i c enzymes, lactate and malate d e h y d r o g e n a s e s . H o w e v e r , these h y b r i d s contain n e i t h e r p i g m e n t n o r activity of the e n z y m e r e s p o n s i b l e for its synthesis (dopa oxidase). F r o m these observations, it Abbreviations : A.D.H. : Alcohol dehydeogenase (Alcohol : NAD oxidoreductase, E.C. 1.1.1.t.). T.A.T. : Tyrosine aminotransferase (L-tyrosine : 2oxoglutarate aminotransferase, E.C. 2.6.1.5.).
was c o n c l u d e d that e x p r e s s i o n of this differentiated f u n c t i o n is subject to extinction, p e r h a p s as a c o n s e q u e n c e of the p r o d u c t i o n by the fibroblast genome of a diffusible r e g u l a t o r y substance, w h o s e final effect is negative [3]. This o b s e r v a t i o n of e x t i n c t i o n of a d i f f e r e n t i a t e d f u n c t i o n w a s soon c o n f i r m e d f o r several d i f f e r e n t tissue specific functions, and w a s found to o c c u r in both i n t r a s p e c i f i c and i n t e r s p e c i f i c h y b r i d s [4]. W i t h r e l a t i v e l y few exceptions, d i f f e r e n t i a t e d f u n c t i o n s d i s a p p e a r upon h y b r i d i z a t i o n w i t h a cell t y p e of different h i s t o g e n e t i c origin [2]. T h e m e c h a n i s m of e x t i n c t i o n of d i f f e r e n t i a t e d f u n c t i o n s r e m a i n s u n k n o w n , but r e c e n t results show that (a) it is not the c o n s e q u e n c e o f an i r r e versible change, such as loss or alteration of genetic i n f o r m a t i o n , since several cases of re-expression of p r e v i o u s l y e x t i n g u i s h e d t u n c t i o n s upon loss of c h r o m o s o m e s have been d e s c r i b e d [5-7], and (b) it is a p p a r e n t l y d e p e n d e n t u p o n gene dosage, since some h y b r i d s r e s u l t i n g f r o m a cross of 2s p i g m e n t e d m e l a n o m a cells w i t h fibroblasts are p i g m e n t e d [8]. T h e s e findings s t r e n g t h e n the e a r l i e r suggestion that e x t i n c t i o n is m e d i a t e d by a diffusible r e g u l a t o r y substance e m e n a t i n g f r o m the fibroblast g e n o m e [3]. In the p r e s e n t series of investigations, we have used cloned p o p u l a t i o n s of w e l l d i f f e r e n t i a t e d rat h e p a t o m a cells, c h a r a c t e r i z e d by the p r o d u c t i o n of a n u m b e r of p r o t e i n s c h a r a c t e r i s t i c of h e p a t i c differentiation. T h e simultaneous e x a m i n a t i o n of s e v e r a l of these p r o d u c t s in a series of s o m a t i c
Roger Bertolotli and M a r y C. W e i s s .
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h y b r i d s pernfits us to deternfine w h e t h e r they are co-ordinately expressed. P r e v i o u s p u b l i c a t i o n s [9, 10, 6] have s h o w n that two enzymes c h a r a c t e r i s t i c of h e p a t i c differentiation, type B fructose-l,6diphosphate aldolase (E.C. 4.1.2.7) a n d t y r o s i n e a m i n o t r a n s f e r a s e (TAT, E.C. 2.6.1.5, a n d its i n d u c i bility w i t h glucocorticosteroid h o r m o n e s ) , fail to be expressed in h y b r i d s formed by crossing rat h e p a t o m a cells w i t h either mouse fibroblasts or diploid rat epithelial cells. However, i n these same h y b r i d s , the p r o d u c t i o n of rat s e r u m a l b u m i n continues, although at a level l o w e r t h a n that characteristic of the p a r e n t a l h e p a t o m a cells [11]. Moreover, it has been observed that aldolase B a n d TAT i n d u c i b i l i t y are i n d e p e n d e n t l y re-expressed in some h y b r i d subclones w h i c h have lost large n u m bers of chromosomes [7, 6], a n d that a cross of 2s rat h e p a t o m a cells w i t h mouse fibroblasts results i n the f o r m a t i o n of some h y b r i d s w h i c h p r o d u c e mouse a l b u m i n [ l l l . The p u r p o s e of the p r e s e n t w o r k has been to characterize the alcohol d e h y d r o g e n a s e (ADH, E.C. 1.1.1.1) isozymes f o u n d i n rat h e p a t o m a cells g r o w n in vitro a n d in vivo, a n d to e x a m i n e the expression of these isozymes in somatic h y b r i d s . ADH may play a role i n the metabolism a n d detoxification of ingested alcohol since its p r i n cipal substrate, ethanol, is not i n v o l v e d i n the well characterized metabolic p a t h w a y s of m a m m a l s [12] ; i n fact the m a j o r ADH activity is f o u n d in the liver and i n the digestive tract [13]. Some activity is p r e s e n t also i n the k i d n e y and the lung [13~. Although purified liver ADH has been well characterized e n z y m a t i c a l l y and p h y s i c a l l y E14~, the origin of the s u r p r i s i n g l y large n u m b e r of m o l e c u l a r forms of this d i m e r i c enzyme r e m a i n s poorly u n d e r s t o o d [15]. Comparative q u a n t i t a t i v e studies of ADH tissue d i s t r i b u t i o n s a n d developmental changes have been c a r r i e d out on a n u m b e r of species El3, 16, 17]. On the other h a n d , electrophoretic characterization of ADH isozymes i n the tissues a n d d u r i n g development has been p r i m a r i l y l i m i t e d to primates El3, 18, 19, 20]. Several authors h o w e v e r have described the p r e s e n c e of only a single b a n d of ADH i n k i d n e y and liver of fetal a n d adull r o d e n t s [13, 21]. MATERIALS AND METHODS.
Cell lines. The cell types used in the p r e s e n t w o r k i n c l u d e a cloned line of rat h e p a t o m a cells (Fu5) a n d four subclones (Fu5-2; Fu5-5, Fu5-7 a n d Fu5-8) derived from it. The cells crossed w i t h the BIOCHIMIE, 1972, 54, n ° 2.
h e p a t o m a i n c l u d e the line of mouse fibroblasts designated 3T3 (clone 4E), a n d a line of diploid epithelial cells derived from Buffalo rat liver (BRL-1). The h y b r i d s used i n c l u d e 3FCa (as well as four other i n d e p e n d e n t l y formed h y b r i d clones from the cross 3T3 × Fu5) a n d BF5 (as well as a n u m b e r of subclones derived from it, isolated from the cross BRL-1 × Fu5,5). The origins of the p a r e n t a l cells a n d the isolation of the h y b r i d s a n d t h e i r k a r y o t y p e s have been p r e v i o u s l y described [9, 6].
Production of cells [or enzyme assays. All cultures have been m a i n t a i n e d as p r e v i o u s l y described [9] in Ham's F12 m e d i u m s u p p l e m e n t e d w i t h 5 p. cent fetal calf serum. Large plastic tissue culture bottles (Falcon) were i n o c u l a t e d w i t h 2.5 X 106 (hepatoma), 1-1.2 X 106 (fibroblasts a n d BRL-1) or 6 X 105 ( h y b r i d ) cells ; 10 cm Petri dishes were i n o c u l a t e d w i t h half these n u m b e r s of cells. After one week of culture, the cells were collected b y t r y p s i n i z a t i o n , w a s h e d three times w i t h phosphate buffered saline a n d counted before the final wash. Tumors. T u m o r s of Fu5-5, o b t a i n e d by subcutaneous i n o c u l a t i o n of 1-4 X 106 cells into 7-14 day old Buffalo rats (Microbiological Associates), were used for enzyme studies after 1-2 m o n t h s (10-14 g). Preparation of extracts. Organs (from Buffalo rats a n d Swiss mice) and tumors were m i n c e d and homogenized m e c h a n i c a l l y i n the presence of 1:1 or 1:4 vol .(w/v) of cold distilled w a t e r or potassium phosphate buffer (0.1 M, pH 7.5), a n d frozen and thawed. Pellets of washed tissue culture cells were s u s p e n d e d in one to three volumes of w a t e r or buffer a n d b r o k e n by freezing a n d t h a w i n g . All p r e p a r a t i o n s were centrifuged for 70 m i n u t e s at 13,000 × g at 0°C ; the clear s u p e r n a t a n t s served as enzyme extracts. Enzyme assays. ADH activity was d e t e r m i n e d by the m e t h o d of Vallee a n d Hoch [22] w h e r e i n the rate of c o n v e r s i o n of NAD to NADH is measured in the presence of ethanol at pH 8.,8. One u n i t of enzyme activity reduces one ~mole of NAD per m i n u t e at 28-29°C u n d e r the c o n d i t i o n s specified. I n every case a b l a n k d e t e r m i n a t i o n was also c a r r i e d out ; the activity f o u n d w i t h o u t a d d i t i o n of substrate was subtracted from the activity f o u n d i n the presence of substrate. P r o t e i n concent r a t i o n s were deternfined by the method of L o w r y et al. [23] using crystalline bovine serum a l b u m i n (Armour) as s t a n d a r d . Electrophoresis. Vertical electrophoresis on starch gel (Connaught) was c a r r i e d out and devel-
E x p r e s s i o n of lioer A.D.H. in h e p a t o m a cell hybrids. o p e d f o r A D H as d e s c r i b e d b y K o e n a n d S h a w [24]. E l e c t r o p h o r e s i s o n c e l l u l o s e a c e t a t e gels (Cellogel, C h e m e t r o n , M i l a n ) w a s p e r f o r m e d i n p o t a s s i u m b a r b i t a l b u f f e r (0.05 M, p H 8.7, c o n t a i n i n g 0.001 M 2 - m e r c a p t o e t h a n o l , a n d i n s o m e e x p e r i m e n t s , 0.005 M E D T A ) at 4°C, u s i n g 250 V d u r i n g 2 1 / 2 h o u r s . T h e gels w e r e i n c u b a t e d at 37°C f o r 30-90 m i n u t e s w i t h t h e s t a i n i n g m i x t u r e u s e d f o r s t a r c h gels, p r e p a r e d i n 0.5 p. c e n t a g a r o s e . T h e staining mixture for blanks contained no substrate.
Reagents. E t h a n o l - f r e e N A D w a s p u r c h a s e d f r o m Nutritional Biochemical Corp., ethanol and acetald e h y d e f r o m M e r c k , a n d all o t h e r r e a g e n t s f r o m Sigma Chemical Corp.
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r e s p o n d to e i t h e r l a c t a t e o r s o r b i t o l d e h y d r o g e n ase. F i n a l l y , it s h o u l d b e p o i n t e d o u t t h a t i n t h e h e p a t o m a cells, t h e r e is a b a n d of a c e t a l d e h y d e d e h y d r o g e n a s e w h i c h c o r r e s p o n d s i n its m i g r a t i o n to s t o m a c h ADH, a n d s i n c e t h e f o r m e r e n z y m e u t i l i s e s as s u b s t r a t e t h e p r o d u c t of t h e l a t t e r e n z y m e ( i n t h e p r e s e n c e of N A D ) , t h i s r e s u l t s i n a n a m p l i f i c a t i o n of t h e s t a i n i n g i n t e n s i t y o f t h e s t o m a c h A D H b a n d (see f o o t n o t e to T a b l e I). T h e p r e s e n c e of s t o m a c h A D H i n r a t h e p a t o m a c e l l s is a s o m e w h a t u n e x p e c t e d f i n d i n g , s i n c e o n l y r a r e l y is a t r a c e of t h i s a c t i v i t y d e t e c t e d i n a d u l t l i v e r . H o w e v e r , t h e o b s e r v a t i o n s of o t h e r a u t h o r s
TABLE I. RESULTS. Preliminary e l e c t r o p h o r e t i c a n a l y s i s of A D H f r o m h e p a t o m a cells r e v e a l e d , i n a d d i t i o n t o l i v e r ADH, t h e p r e s e n c e of a n a n o d a l b a n d of e n z y m e a c t i v i t y (Fig. 1). T h e l a t t e r i s o z y m e c o r r e s p o n d s i n its m i g r a t i o n to s t o m a c h A D H a n d s h o w s a g r e a t e r substrate specificity than the well known liver t y p e : c o n t r a r y t o l i v e r ADH, i t d o e s n o t u t i l i z e (all t r a n s ) r e t i n o l ( S i g m a C h e m i c a l C o r p . ) as a substrate. Moreover, this anodal ADH does not cor-
ADH activity of hepatoma cells and of rat organs. Hepatoma clone FH
5 ...................
Fu Fu Fu Fu
5_2 5_5 5-7 5-8
................. ................. ................. .................
R a t organ Liver . . . . . . . . . . . . . . . . . . Stomach . . . . . . . . . . . . . . . . Kidney . . . . . . . . . . . . . . . . .
Specific activity t 95 (__+ 20) 77 102 ( ! 20)=' 40 (~-~ 4) 98 25 ( ± 5) 9 10
(1) Milliunits of enzyme a e t i v i t y / m g p r o t e i n in the soluble extract. (2) As emphasized by Ohno et al [21], the assay m e t h o d used [22J p r o b a b l y results in a n o v e r e s t i m a t i o n of ADH activity in crude extracts, owing to interference by aldehyde dehydrogenase. Using the series of t h r e e reactions described by Ohno et al. [26], it h a s been d e t e r m i n e d t h a t the activity of acetaldehyde dehydrogenase in these celts is one h a l f of t h a t of ADH.
Fro. 1. - - ADH zymograms. (Cellogel) of r a t organs and h e p a t o m a . The gel on t h e left was stained for 40 m i n u t e s , tha¢ on the r i g h t for 91) m i n u t e s . (a) a n d (d) a d u l t liver, showing a very pale anodaI b a n d and two i n t e n s e l y staini.ng cathodal b a n d s (on starch geIs a t h i r d a n d slower c a t h o d a l b a n d is resolved, as h a s also been described b y Koen a n d Shaw [24]). Cb) and (e) h e p a t o m a cells (clone F u 5 - 5 , grown as a tumor), showing a cathodal b a n d , corresponding in its m i g r a tion to the m o r e r a p i d l y m i g r a t i n g one f r o m liver, a n d a n a n o d a l b a n d . (c) stomach, a n d (f) fetal liver (23 days before b i r t h ) , showing cathodal b a n d s s i m i l a r to those of a d u l t liver, and a clearly resolved a n o d a l b a n d . A l t h o u g h t h e r e appears a slight difference in t h e mig r a t i o n rates of stomach ADH a n d the a n o d a l b a n d f r o m h e p a t o m a , m i x t u r e s of such e x t r a c t s i n v a r i a b l y show a single n a r r o w a n o d a l b a n d of activity. F o r all extracts, identical r e s u l t s are o b t a i n e d w h e t h e r the extracts are p r e p a r e d in buffer or w a t e r and w h a t e v e r the condition of eleetrophoresis (starch gel or Cellogel).
BIOCHIMIE, 1972, 54, n ° 2.
[19, 20] o n t h e d e v e l o p m e n t a l c h a n g e s i n l i v e r A D H s h o w t h a t i n m a n t h e r e a r e (~ f e t a l )> f o r m s of t h e e n z y m e , a n d i t is l i k e l y t h a t t h i s <> A D H is i n d e e d a f e t a l f o r m of t h e e n z y m e i n t h e r a t s i n c e i t s a c t i v i t y i s c l e a r l y v i s i b l e i n 20-23 d a y o l d r a t f e t u s e s (Fig. 1). T h i s i n t e r p r e t a t i o n is c o m p a t i b l e w i t h p r e v i o u s o b s e r v a t i o n s of f e t a l isoz y m e s a n d p r o t e i n s i n h e p a t o m a c e l l s [253. A c l o n e of h e p a t o m a cells, F u 5 , a n d a s e r i e s of s u b c l o n e s d e r i v e d f r o m it, h a v e b e e n e x a m i n e d to d e t e r m i n e t h e v a r i a b i l i t y of e x p r e s s i o n of A D H i n derivatives of the initial population. Table I shows t h e s p e c i f i c a c t i v i t y of A D H f r o m e a c h of f o u r s u b c l o n e s , as w e l l as t h e r a n g e of a c t i v i t y v a l u e s m e a s u r e d i n a n u m b e r of i n d e p e n d e n t a s s a y s of
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Fu5 and Fu5-5. It w i l l be seen that w i t h the exception of Fu5-7, t h e r e is very little v a r i a t i o n in the specific a c t i v i t y of ADH f r o m one s u b c l o n e to another. Fu5-7 h o w e v e r , contains only about half as m u c h activity as Fu5 and the o t h e r subclones. Elect r o p h o r e t i c analyses of these p o p u l a t i o n s , as w e l l as of Fu5-5 cells g r o w n as t u m o r s have s h o w n that all p r e s e n t i n d i s t i n g u i s h a b l e patterns, e x c e p t for Fu5-7 ; cells of the latter t y p e c o n t a i n no t r a c e of the l i v e r f o r m of the enzyme, but the b a n d of stom a c h ADH stains w i t h the same i n t e n s i t y as that f r o m the o t h e r populations. T h e c o m p a r i s o n s bet w e e n FuS-7 a n d the other subclones p e r m i t s the c o n c l u s i o n that in all p o p u l a t i o n s e x c e p t for Fu5-7, r o u g h l y half of the total ADH a c t i v i t y is due to the l i v e r form, and half to the s t o m a c h form. Moreover, these o b s e r v a t i o n s s h o w that ADH a c t i v i t y is a p r o p e r t y w h i c h is r e t a i n e d t h r o u g h clonal passage of the h e p a t o m a cells, and that the s t o m a c h f o r m is e x p r e s s e d in a m o r e r e g u l a r fashion than the h e p a t i c one. T h e cells w h i c h have been crossed w i t h the h e p a t o m a , 3T3 (mouse fibroblasts) a n d BRL-1 ( d i p l o i d epithelial cells d e r i v e d f r o m rat liver), have been e x a m i n e d for t h e i r ADH content. BRL-1 cells c o n t a i n no detectable activity, e i t h e r by q u a n t i t a t i v e assays or by e l e c t r o p h o r e s i s f o l l o w e d
TABLE
In both series of s o m a t i c h y b r i d s (3T3 × Fu5 and BRL-1 × Fu5-5), the b a n d of ADH a c t i v i t y c o r r e s p o n d i n g to the l i v e r f o r m is e n t i r e l y absent, w h i l e a small a m o u n t of the s t o m a c h ADH a c t i v i t y is conserved. T h e specific a c t i v i t y values for the p a r e n t a l a n d h y b r i d cells are given in T a b l e II. It can be seen that the activity of the h y b r i d cells r e p r e s e n t s only a small f r a c t i o n of that p r e s e n t in the h e p a t o m a p a r e n t a l cells. Fig. 2 d e m o n s t r a t e s the isozymes p r e s e n t in these cells. All five clones e x a m i n e d of the rat-mouse h y b r i d cells r e t a i n the s t o m a c h isozymes of b o t h p a r e n t a l types as well as the h y b r i d f o r m e d b e t w e e n the rat and mouse m o n o m e r s . T h e BF5 h y b r i d cells c o n t a i n a single b a n d of (rat) s t o m a c h ADH. H y b r i d clones of the two types h a v e been examined t h r o u g h o u t the g r o w t h c y c l e to d e t e r m i n e w h e t h e r t h e r e exist c y c l i c m o d i f i c a t i o n s in the e x p r e s s i o n of ADH. Specific a c t i v i t y m e a s u r e m e n t s s h o w e d no s i g n i f c a n t fluctuations d u r i n g the g r o w t h cycle, a n d e l e c t r o p h o r e s i s s h o w e d no m o d i f i c a t i o n in the i s o z y m e pattern. Several subclones of h y b r i d BF5 h a v e been e x a m i n e d for ADH c o m p o s i t i o n ; none of these clones, w h e t h e r c h a r a c t e r i z e d by the e x p e c t e d n u m b e r of c h r o m o somes or f e w e r , s h o w either an elevation of ADH a c t i v i t y or e x p r e s s i o n of l i v e r ADH.
II.
Karyotypes and ADH activity of parental and hybrid cells. Cell type 3T3 ............. Fu 5 . . . . . . . . . . . . . 3 F Ga . . . . . . . . . . . BRL_ 1 . . . . . . . . . . Fu 5-5 . . . . . . . . . . . .
BF 5 . . . . . . . . . . . . . B F 5 - 74 . . . . . . . . . .
No of chromosomes t 75.9 52 116.3 42 52 92 55
(70-80) (51-53) (114-126) :~ (51-53) (91-93) (54-55)
Specific activity .2 3 95 (-+- 20) 5 0 lo2 (_-_+_20) 6
22
Activity/t0 6 cells 0.7 10.0 (~-!-2) 1.1 0.0 l o . o ( ± 2) 1.3 5.4
(1) Determined by the analysis of 10-30 metaphases of each clone. The mean and range of chromosome numbers are given for each population. (2) Mfliiunits of enzyme activity/rag protein in the soluble extract or per 106 cells. (3) Cells of this hybrid clone contain 19.9 (16-25) biarmed rat ¢ marker ~> chromosomes. The expected number is 25.7 (24-27).
by specific staining. 3T3 cells do c o n t a i n small a m o u n t s of ADH a c t i v i t y ; e t e c t r o p h o r e s i s reveals that the 3T3 e n z y m e c o r r e s p o n d s in its e l e c t r o p h o r e t i c m o b i l i t y to the e n z y m e f r o m (mouse) stomach, like the a n o d a l band f r o m h e p a t o m a cells ; these cells do not p r e s e n t a b a n d of activity corr e s p o n d i n g to the m o u s e l i v e r enzyme.
BIOCHIMIE, 1972, 54, n ° 2.
T h e s e results suggest that l i v e r ADH, like tyrosine a m i n o t r a n s f e r a s e (and its i n d u c i b i l i t y w i t h s t e r o i d h o r m o n e s ) and aldolase B, is e x t i n g u i s h e d in the h y b r i d cells, and that the s t o m a c h enzyme, although greatly r e d u c e d in a c t i v i t y (.expected activity/106 cells : 5 ; o b s e r v e d : 1.1-1.3) c o n t i n u e s to be expressed. T h e p r e s e n c e of this f o r m of ADH in
Expression o[ liver A.D.H. in hepatoma cell hybrids. m o u s e f i b r o b l a s t s suggests t h a t it m a y h a v e a m u c h m o r e w i d e s p r e a d tissue d i s t r i b u t i o n t h a n the l i v e r f o r m . S i n c e o n e of t h e h e p a t o m a s u b c l o n e s (Fu5-7) fails to p r o d u c e h e p a t i c ADH, it is p o s s i b l e that the h e p a t o m a cells w h i c h f u s e d w i t h p a r e n t a l ceils
199
of t h e o t h e r t y p e to g i v e r i s e to h y b r i d s d i d n o t themselves produce this enzyme. However, this e x p l a n a t i o n c a n be r u l e d out in t h e c a s e of t h e BF5 h y b r i d , b e c a u s e o n e h y b r i d s u b c l o n e (BF5~/-4), w h i c h has lost a l a r g e n u m b e r of c h r o m o -
Fw,. 2. - - A,DH zymograms (Ce]logel) of parental and hybrid cells. Two hepatoma clones (Fu-5-5 and FuS) are shown, as well as parental cell lines BRL-1 and 3T3 (in the latter, a faint band of ADH activity, which remains at the origin and corresponds to mouse stomach ADH, is seen). In the intraspecific hybrid, BF5, a single anodal band of rat stomach ADH is seen, while in the interspecific hybrid, 3FCa, both parental stomach ADH's, as well as a single hybrid band formed between them, are clearly visible. somes, has b e e n f o u n d to r e - e x p r e s s h e p a t i c A D H (Fig. 3, T a b l e II) b u t at a l e v e l l o w e r t h a n t h a t of the Fu5-5 cells. T h i s r e - e x p r e s s i o n of a l i v e r e n z y m e a f t e r loss of l a r g e n u m b e r s of c h r o m o s o m e s , h a s n o w b e e n o b s e r v e d in s e v e r a l cases. I n fact, t h e h y b r i d cells which re-express liver ADH similarly re-express a l d o l a s e B E7], b u t a n o t h e r c l o n e , BF-I-1, w h i c h r e - e x p r e s s e s T A T i n d u c i b i l i t y [6], d o e s n o t ree x p r e s s e i t h e r l i v e r A D t I o r a l d o l a s e B. DISCUSSION.
FIG. 3. - - ADH zymogram (Cellogel) demonstrating re-expression of liver (eathodal) ADH in the hybrid subelone B F S - y 4 (right). On the left is shown the ADH pattern of a subelone of Fu5-5 (Ft~5-5aza967), ineluded as a migration reference, and showing a fainter band of liver ADH than Fu5-5.
BIOCH1MIE, 1972,
54, n ° 2.
E x a m i n a t i o n of t h e e x p r e s s i o n of A D H in h e p a t o m a cell h y b r i d s ( w h i c h c o n t a i n e s s e n t i a l l y all of t h e c h r o m o s o m e s c o n t r i b u t e d by t h e t w o p a r e n t s ) has s h o w n t h a t t h e l i v e r f o r m of t h i s e n z y m e is absent, w h i l e t h e s t o m a c h f r o m c o n t i n ues to be p r o d u c e d , a l t h o u g h at a l e v e l m u c h lower than that which characterizes the parental h e p a t o m a cells. T h i s r e s u l t h a s b e e n o b s e r v e d in both interspecific (rat X mouse) and intraspecific (rat × rat) h y b r i d s . M o r e o v e r , in t h e f o r m e r h y b r i d s , b o t h p a r e n t a l f o r m s of t h e s t o m a c h e n z y m e are p r o d u c e d , as w e l l as a s i n g l e h y b r i d (heterospecif~c) e n z y m e b e t w e e n t h e m , d e m o n s t r a t i n g a h o m o l o g y b e t w e e n t h e t w o s p e c i e s of t h i s e v i d e n t l y d i m e r i c e n z y m e . (It w i l l h a v e b e e n not i c e d t h a t no h y b r i d i z a t i o n o c c u r s b e t w e e n t h e
200
Roger
Bertolotti
liver and the stomach enzymes in the hepatoma cells, e v e n t h o u g h b o t h f o r m s a r e d i m e r i c enzymes). T h e s i m i l a r i t y of t h e A D H s p e c i f i c a c t i v i t y a n d a c t i v i t y / 1 0 6 cells of b o t h k i n d s of h y b r i d s a n d of the 3T3 fibroblast parental cells suggests that prod u c t i o n of a l o w <~b a s e - l i n e >> of t h e s t o m a c h f o r m is n o t a t i s s u e s p e c i f i c f u n c t i o n , w h i l e a h i g h a c t i v i t y of t h i s f o r m s is s u b j e c t t o q u a n t i t a t i v e c o n t r o l . L i k e t h e r e - e x p r e s s i o n of a l d o l a s e B a n d T A T i n d u c i b i l i t y , r e - e x p r e s s i o n of l i v e r A D H i n h e p a t o m a h y b r i d s w h i c h h a v e l o s t l a r g e n u m b e r s of c h r o m o s o m e s c a n b e i n t e r p r e t e d as a n i n d i c a t i o n of t h e s t a b i l i t y of t h e e p i g e n o t y p e of t h e h e p a t o m a p a r e n t a l c e l l s [2]. T h u s , t h e p o t e n t i a l i t y to e x p r e s s these tissue specific functions can be inherited t h r o u g h m a n y h y b r i d cell g e n e r a t i o n s w i t h o u t b e i n g e x p r e s s e d ; w h e n loss of c h r o m o s o m e s ( c a u s i n g e x t i n c t i o n ) o c c u r s , t h e f u n c t i o n is r e - e x p r e s sed. T h i s i n t e r p r e t a t i o n m u s t r e m a i n t e n t a t i v e h o w e v e r u n t i l i d e n t i f i c a t i o n of a s p e c i f i c c h r o m o some, which causes extinction when present and p e r m i t s r e - e x p r e s s i o n w h e n lost; c a n b e m a d e . T h e o b s e r v a t i o n s o f K l e b e , C h e n a n d R u d d l e [5] s u g gest t h a t t h i s w i l l b e p o s s i b l e , s i n c e t h e y w e r e a b l e to f i n d a c o r r e l a t i o n b e t w e e n t h e p r e s e n c e a n d a b s e n c e of t h e h u m a n Clo c h r o m o s o m e a n d t h e r e s p e c t i v e e x t i n c t i o n a n d r e - e x p r e s s i o n of m o u s e k i d n e y ES-2 ( a n e s e r i n e i n s e n s i t i v e e s t e r a s e ) i n hybrids formed by crossing mouse renal adenoc a r c i n o m a c e l l s (ES-2 '+) w i t h h u m a n f i b r o b l a s t s . Acknowledgements. We are g r a t e f u l to Drs. Boris E p h r u s s i and Drew Schwartz for m a n y s t i m u l a t i n g discussions, to Dr. Boris E p h r u s s i for critical r e a d i n g of the m a n u s c r i p t , a n d to M uo Anne Debon for excellent ,technical assistanee. This work was conducted w i t h the aid of a g r a n t to Dr. Boris E p h r u s s i f r o m the D616gation G6n6rale h la Recherche Scientifique et Technique. PRESUME.
Les cellules d ' u n h 6 p a t o m e bien diff6renci4 de rat, eultiv4es in vitro et in vioo, p r 6 s e n t e n t deux formes d'aleool d6shydrog6nase ; la p r e m i 6 r e correspond, p a r sa m i g r a t i o n 61eetrophor6tique, h l ' e n z y m e b i e n eonnu de foie, la seeonde est homologue de l ' i s o e n z y m e mis en 6videnee dans l'estomae. Des eellules de eet h 6 p a t o m e de r a t ont 6td erois6es avec des fibroblastes d ' u n e lign6e p e r m a n e n t e de souris, dont la tr6s faible activit6 alcool d6shydrog6nase est due h l ' i s o e n z y m e stomaca], et avec des cellules 6pith61iales diploides de r a t d o n t l'activit6 alcool d6shydrog6nase est nulle. Dans les h y b r i d e s s o m a t i q n e s a i n s i obtenus, seul l ' i s o e n z y m e h 6 p a t i q u e n'est pas d6celable, ce qui m o n t r e que, comme p o u r b e a u c o u p de fonctions diff6renci6es, son expression es[ sp6cifiquement hloqu6e lorsque les cellules d ' h 6 p a t o m e BIOCHIMIE, 1972, 54, n ° 2.
and Mary
C. W e i s s .
sont crois6es avec des cellules d'une origine histog6n6tique diff6rente. Quc cette e x t i n c t i o n de l'alcool d6shydrog6nase h 6 p a t i q u e n'est pas due h une perte ou u n e alt6ration irr4versible du mat6riel g6n6tique est d6m o n t r 6 p a r le fait que cet isoerrzyme est r6exprim6 dans u n sous-clone d ' h y b r i d e s a y a n t perdu une f r a c t i o n i m p o r t a n t e de leurs chromosomes. ZUSAMMENFASSUNG.
Wohl differenzierte Zellcn eines H'epatoins der Ratte, die in vitro u n d in vivo k u l t i v i e r t ~vurden, e n t h a l t e n zwei F o r m e n der A l k o h o l d e h y d r o g e n a s ' e ; die erste Form entspricht auf Grund ihrer elektrophoretischen W a n d e r u n g dern b e k a n n t e n Leberenzym, die zweite ist dem i m Magen g e f u n d e n e n I s o e n z y m homolog. Zellen dieses R a t t e n h e p a t o m s w u r d e n m i t F i b r o b l a s t e n einer Mfiuselinie, die n u t eine sehr sch~vache Alkoholdehydrogenaseaktivitiit, die dem Magenisoenzym zuzusc h r e i b e n ist, enthiHt, und epithelialen diploiden Zellen der Ratte, deren A l k o h o l d e h y d r o g e n a s e a k t i v i t i i t null ist, gekreuzt. In den so e r h a l t e n e n s o m a t i s c h e n Hyb r i d e n ist allein das Isoenzym d~er Leber n i c h t n a c h weisbar, was zeigt, dass, wie ftir viele differenzierte F u n k t i o n e n , die W i r k u n g dieses E n z y m s u n t e r b u n d e n wird, sobald die Zellen eines H e p a t o m s m i t Zellen u n t e r s e h i e d l i e h e r h i s t o g e n e t i s c h e r H e r k u n f t gekreuzt werden. Dass dieses Verseh~vinden der Leberalkoholdehydrogenase n i e h t e i n e m Verlust oder e i n e r irreversiblen A n d e r u n g des genetisehen Materials zuzuschreib e n ist, zeigt die Tatsache, dass dieses I s o e n z y m in einem Subklon tier Hybride, der eine grosse Z a h l von C h r o m o s o m c n verloren ha.t, w i e d e r z.um Vorschein kommt. REFERENCES. 1. Barski, G., Sorieul, S. ,~ Cornefert, F. (196~) Compt. Rend. Acad. Sci., 251, 182.5. 2. Ephrussi, B., H y b r i d i z a t i o n of Somatic Cells, P r i n c e t o n U n i v e r s i t y Press, P r i n c e t o n , in press. 3. Davidson, R. L., Ephrussi, B. ~ Y a m a m o t o , K. (1966) Proc. Nat. Acaa. Sci. U.S.A., 56, 1437. 4. Davidson, R. L. (1971) I n vitro, 6, 411. 5. KIebe, R., Chen, T. ~ Ruddle, F. (1970) Proc. Nat. Acad. Sci. U.S.A., 66, 1220. 6. Weiss, M. C. a Chaplain, M. (1971) Proc. Nat. Acad. Sci. U.S.A., 68, 3026. 7. Bertolotti, R. ~ Weiss, M. C., Cell D i f f e r e n t i a t i o n , Proceedings of the 1st I n t e r n a t . Conf. on Cell Differentiation (Eds. R. H a r r i s an& D. Viza), in press. 8. Foug6re, C., Ruiz, F. ~ Ephrussi, B. (1972) Proc. Nat. Acad. Sci. U.S.A., 69, 330. 9. Schn,eider, J. ~ "Weiss, M. C. (1971) Proc. Nat. Acad. Sci. U.S.A., 68, 127. 10. Bertolotti, R. ~ Weiss, M. C. J. Cell. Physiol., in press. 11. Peterson, J. A. ~ Weiss, M. C. (1972) Proc. Nat. Acad. Sci. U.S.A., 69, ~571. 12. Krebs, H. A. ~ Perkins, J. R. (197.0~ Biochem. J., 118, 635. 13. Moser, K., Papenberg, J. e Von W a r t b u r g , J. P. (1968) E n z y m . BioL Clin., 9, 447. 14. Theorell, H. (19:67) Harvey Lect., 61, 17. 15. Lutstorf, U. M., Schiirch, P. M. ~ Von W a r t b u r g , J. P. (1970) Eur. J. Biochem., 17, 497. 16. Riiih~i, N. C. R., Kuskinen, M. a P i k k a r a i n e n , P. H. (1967) Biochem. J., 103, 623. 17. Pikkarain'en, P. H. & R/iih~i, N. C. R., (1967~ Pediat. Res., 1, 165. 18. P i k k a r a i n e n , P. H. ~ R/iihii, N. C. R. (196:9) Nature, 222, 563.
Expression of liver A.D.H. in hepatoma cell hybrids. 19. M u r r a y , R. F. ,~ Motulsky, A. G. (1971) Science, 171, 71. 20. Smith, M., H o p k i n s o n , D. A. a Harris, H. (1971) Ann. Hum. Genet. Lond., 34, 251. 21. Ohno, S., Stenius, C. • C h r i s t i a n , L. C. (1970) Clin. Genet., 1, 35. 22. Vallee B. L. a Hoeh, F. L. (1955) Proc. Nat. Acad. Sei., 41, 327.
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23. Lowry, O. H., R o s e b r o u g h , N. O., Farr, A. L. a R a n d a l l , R. J. (1951) J. Biol. Chem., 193, 265. 24. Koen, A. L. & Shaw, C. R. (19~6) Biochim. Biophys. Acta, 128, 48. 25. Schapirn, F., Hatzfeld, A. & Reuber, M. D. (1971) Cancer Res., 31, 1224. 26. Ohno, S., Stenius, C., C h r i s t i a n , L., H a r r i s , C. Ivey C. (1970) Biochemical Genet., 4, 565.
Expression of' differentiated functions in hepatoma cell hybrids. VI. Extinction and re-expression of liver alcohol dehydrogenase. R o g e r BERTOLOTTI and Mary C. WEISS. Centre de G~n~tique Mol~culaire du C.N.R.S., 9 1 - Gif-sur-Yvetle. (7/2/1972). Summary. - - Well differentiated rat hepatoma cells, grown both in vitro and in vivo, contain tv¢o forms of alcahol dehydrogenase, one of which corresponds in its electrophoretic mobility to the well knovcn liver enzyme, and the other to the enzyme found in the stomach. Ceils of a line of mouse fibroblasts contain only very small amounts of the s¢omach enzyme, and diploid ra.t epithelial cells show no activiCy. Cells of bo~h of these latter types have been crossed with the rat hepatoma cells, and the result4ng hybrids contain only very small amounts o.f enzyme activity, which has been inc~entified as the stomach type. No activity of liver alcohol dehydrogenase can be detected in the hybrids, and it is concluded that like many other differentiated functions, this enzyme, characteristic of hepatic differen~ia, tion, is extinguished upon hybridization wi.th a cell of different histogenetic origin. However, the failure of the hybrid cells to produce this enzyme is not due to loss or irreversible alteration of genetic material, because one hybrid subclone, which has lost a large number of chromosomes, re-expresses liver alcohol dehydrogenase.
Somatic h y b r i d i z a t i o n , first o b s e r v e d in 1960 [1], has been used e x t e n s i v e l y in r e c e n t y e a r s as a tool for the genetic analysis of s o m a t i c cells. T h e usefulness of s o m a t i c h y d r i d s stems f r o m the o b s e r v a t i o n s that they are viable, possess indefinite g r o w t h potential, can be isolated f r o m crosses of cells d e r i v e d f r o m the same or f r o m different species, that they u n d e r g o c h r o m o s o m e loss or segregation, and that they express the g e n e t i c c h a r acters of both p a r e n t s [for r e v i e w , see ref. 2]. One of the a p p l i c a t i o n s of s o m a t i c h y b r i d i z a t i o n has been the study of the r e g u l a t i o n of expression of d i f f e r e n t i a t e d functions. It has been s h o w n , in n u m e r o u s cases, that h y b r i d cells are c h a r a c t e r ized by c o - e x p r e s s i o n of p a r e n t a l forms of metabolic enzymes, and by c o m p l e m e n t a t i o n of p a r e n t a l e n z y m e deficiencies [2]. H o w e v e r , this is not the case w h e n the fate of a d i f f e r e n t i a t e d f u n c t i o n is e x a m i n e d in h y b r i d s f o r m e d by crossing cells of a p i g m e n t e d S y r i a n h a m s t e r m e l a n o m a a n d those of a p e r m a n e n t line of mouse fibroblasts [3]. T h e resulting h y b r i d s c o n t a i n essentially all of the c h r o m o s o m e s of both p a r e n t a l types, and they produce both p a r e n t a l f o r m s of t w o m e t a b o l i c enzymes, lactate and malate d e h y d r o g e n a s e s . H o w e v e r , these h y b r i d s contain n e i t h e r p i g m e n t n o r activity of the e n z y m e r e s p o n s i b l e for its synthesis (dopa oxidase). F r o m these observations, it Abbreviations : A.D.H. : Alcohol dehydeogenase (Alcohol : NAD oxidoreductase, E.C. 1.1.1.t.). T.A.T. : Tyrosine aminotransferase (L-tyrosine : 2oxoglutarate aminotransferase, E.C. 2.6.1.5.).
was c o n c l u d e d that e x p r e s s i o n of this differentiated f u n c t i o n is subject to extinction, p e r h a p s as a c o n s e q u e n c e of the p r o d u c t i o n by the fibroblast genome of a diffusible r e g u l a t o r y substance, w h o s e final effect is negative [3]. This o b s e r v a t i o n of e x t i n c t i o n of a d i f f e r e n t i a t e d f u n c t i o n w a s soon c o n f i r m e d f o r several d i f f e r e n t tissue specific functions, and w a s found to o c c u r in both i n t r a s p e c i f i c and i n t e r s p e c i f i c h y b r i d s [4]. W i t h r e l a t i v e l y few exceptions, d i f f e r e n t i a t e d f u n c t i o n s d i s a p p e a r upon h y b r i d i z a t i o n w i t h a cell t y p e of different h i s t o g e n e t i c origin [2]. T h e m e c h a n i s m of e x t i n c t i o n of d i f f e r e n t i a t e d f u n c t i o n s r e m a i n s u n k n o w n , but r e c e n t results show that (a) it is not the c o n s e q u e n c e o f an i r r e versible change, such as loss or alteration of genetic i n f o r m a t i o n , since several cases of re-expression of p r e v i o u s l y e x t i n g u i s h e d t u n c t i o n s upon loss of c h r o m o s o m e s have been d e s c r i b e d [5-7], and (b) it is a p p a r e n t l y d e p e n d e n t u p o n gene dosage, since some h y b r i d s r e s u l t i n g f r o m a cross of 2s p i g m e n t e d m e l a n o m a cells w i t h fibroblasts are p i g m e n t e d [8]. T h e s e findings s t r e n g t h e n the e a r l i e r suggestion that e x t i n c t i o n is m e d i a t e d by a diffusible r e g u l a t o r y substance e m e n a t i n g f r o m the fibroblast g e n o m e [3]. In the p r e s e n t series of investigations, we have used cloned p o p u l a t i o n s of w e l l d i f f e r e n t i a t e d rat h e p a t o m a cells, c h a r a c t e r i z e d by the p r o d u c t i o n of a n u m b e r of p r o t e i n s c h a r a c t e r i s t i c of h e p a t i c differentiation. T h e simultaneous e x a m i n a t i o n of s e v e r a l of these p r o d u c t s in a series of s o m a t i c
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h y b r i d s pernfits us to deternfine w h e t h e r they are co-ordinately expressed. P r e v i o u s p u b l i c a t i o n s [9, 10, 6] have s h o w n that two enzymes c h a r a c t e r i s t i c of h e p a t i c differentiation, type B fructose-l,6diphosphate aldolase (E.C. 4.1.2.7) a n d t y r o s i n e a m i n o t r a n s f e r a s e (TAT, E.C. 2.6.1.5, a n d its i n d u c i bility w i t h glucocorticosteroid h o r m o n e s ) , fail to be expressed in h y b r i d s formed by crossing rat h e p a t o m a cells w i t h either mouse fibroblasts or diploid rat epithelial cells. However, i n these same h y b r i d s , the p r o d u c t i o n of rat s e r u m a l b u m i n continues, although at a level l o w e r t h a n that characteristic of the p a r e n t a l h e p a t o m a cells [11]. Moreover, it has been observed that aldolase B a n d TAT i n d u c i b i l i t y are i n d e p e n d e n t l y re-expressed in some h y b r i d subclones w h i c h have lost large n u m bers of chromosomes [7, 6], a n d that a cross of 2s rat h e p a t o m a cells w i t h mouse fibroblasts results i n the f o r m a t i o n of some h y b r i d s w h i c h p r o d u c e mouse a l b u m i n [ l l l . The p u r p o s e of the p r e s e n t w o r k has been to characterize the alcohol d e h y d r o g e n a s e (ADH, E.C. 1.1.1.1) isozymes f o u n d i n rat h e p a t o m a cells g r o w n in vitro a n d in vivo, a n d to e x a m i n e the expression of these isozymes in somatic h y b r i d s . ADH may play a role i n the metabolism a n d detoxification of ingested alcohol since its p r i n cipal substrate, ethanol, is not i n v o l v e d i n the well characterized metabolic p a t h w a y s of m a m m a l s [12] ; i n fact the m a j o r ADH activity is f o u n d in the liver and i n the digestive tract [13]. Some activity is p r e s e n t also i n the k i d n e y and the lung [13~. Although purified liver ADH has been well characterized e n z y m a t i c a l l y and p h y s i c a l l y E14~, the origin of the s u r p r i s i n g l y large n u m b e r of m o l e c u l a r forms of this d i m e r i c enzyme r e m a i n s poorly u n d e r s t o o d [15]. Comparative q u a n t i t a t i v e studies of ADH tissue d i s t r i b u t i o n s a n d developmental changes have been c a r r i e d out on a n u m b e r of species El3, 16, 17]. On the other h a n d , electrophoretic characterization of ADH isozymes i n the tissues a n d d u r i n g development has been p r i m a r i l y l i m i t e d to primates El3, 18, 19, 20]. Several authors h o w e v e r have described the p r e s e n c e of only a single b a n d of ADH i n k i d n e y and liver of fetal a n d adull r o d e n t s [13, 21]. MATERIALS AND METHODS.
Cell lines. The cell types used in the p r e s e n t w o r k i n c l u d e a cloned line of rat h e p a t o m a cells (Fu5) a n d four subclones (Fu5-2; Fu5-5, Fu5-7 a n d Fu5-8) derived from it. The cells crossed w i t h the BIOCHIMIE, 1972, 54, n ° 2.
h e p a t o m a i n c l u d e the line of mouse fibroblasts designated 3T3 (clone 4E), a n d a line of diploid epithelial cells derived from Buffalo rat liver (BRL-1). The h y b r i d s used i n c l u d e 3FCa (as well as four other i n d e p e n d e n t l y formed h y b r i d clones from the cross 3T3 × Fu5) a n d BF5 (as well as a n u m b e r of subclones derived from it, isolated from the cross BRL-1 × Fu5,5). The origins of the p a r e n t a l cells a n d the isolation of the h y b r i d s a n d t h e i r k a r y o t y p e s have been p r e v i o u s l y described [9, 6].
Production of cells [or enzyme assays. All cultures have been m a i n t a i n e d as p r e v i o u s l y described [9] in Ham's F12 m e d i u m s u p p l e m e n t e d w i t h 5 p. cent fetal calf serum. Large plastic tissue culture bottles (Falcon) were i n o c u l a t e d w i t h 2.5 X 106 (hepatoma), 1-1.2 X 106 (fibroblasts a n d BRL-1) or 6 X 105 ( h y b r i d ) cells ; 10 cm Petri dishes were i n o c u l a t e d w i t h half these n u m b e r s of cells. After one week of culture, the cells were collected b y t r y p s i n i z a t i o n , w a s h e d three times w i t h phosphate buffered saline a n d counted before the final wash. Tumors. T u m o r s of Fu5-5, o b t a i n e d by subcutaneous i n o c u l a t i o n of 1-4 X 106 cells into 7-14 day old Buffalo rats (Microbiological Associates), were used for enzyme studies after 1-2 m o n t h s (10-14 g). Preparation of extracts. Organs (from Buffalo rats a n d Swiss mice) and tumors were m i n c e d and homogenized m e c h a n i c a l l y i n the presence of 1:1 or 1:4 vol .(w/v) of cold distilled w a t e r or potassium phosphate buffer (0.1 M, pH 7.5), a n d frozen and thawed. Pellets of washed tissue culture cells were s u s p e n d e d in one to three volumes of w a t e r or buffer a n d b r o k e n by freezing a n d t h a w i n g . All p r e p a r a t i o n s were centrifuged for 70 m i n u t e s at 13,000 × g at 0°C ; the clear s u p e r n a t a n t s served as enzyme extracts. Enzyme assays. ADH activity was d e t e r m i n e d by the m e t h o d of Vallee a n d Hoch [22] w h e r e i n the rate of c o n v e r s i o n of NAD to NADH is measured in the presence of ethanol at pH 8.,8. One u n i t of enzyme activity reduces one ~mole of NAD per m i n u t e at 28-29°C u n d e r the c o n d i t i o n s specified. I n every case a b l a n k d e t e r m i n a t i o n was also c a r r i e d out ; the activity f o u n d w i t h o u t a d d i t i o n of substrate was subtracted from the activity f o u n d i n the presence of substrate. P r o t e i n concent r a t i o n s were deternfined by the method of L o w r y et al. [23] using crystalline bovine serum a l b u m i n (Armour) as s t a n d a r d . Electrophoresis. Vertical electrophoresis on starch gel (Connaught) was c a r r i e d out and devel-
E x p r e s s i o n of lioer A.D.H. in h e p a t o m a cell hybrids. o p e d f o r A D H as d e s c r i b e d b y K o e n a n d S h a w [24]. E l e c t r o p h o r e s i s o n c e l l u l o s e a c e t a t e gels (Cellogel, C h e m e t r o n , M i l a n ) w a s p e r f o r m e d i n p o t a s s i u m b a r b i t a l b u f f e r (0.05 M, p H 8.7, c o n t a i n i n g 0.001 M 2 - m e r c a p t o e t h a n o l , a n d i n s o m e e x p e r i m e n t s , 0.005 M E D T A ) at 4°C, u s i n g 250 V d u r i n g 2 1 / 2 h o u r s . T h e gels w e r e i n c u b a t e d at 37°C f o r 30-90 m i n u t e s w i t h t h e s t a i n i n g m i x t u r e u s e d f o r s t a r c h gels, p r e p a r e d i n 0.5 p. c e n t a g a r o s e . T h e staining mixture for blanks contained no substrate.
Reagents. E t h a n o l - f r e e N A D w a s p u r c h a s e d f r o m Nutritional Biochemical Corp., ethanol and acetald e h y d e f r o m M e r c k , a n d all o t h e r r e a g e n t s f r o m Sigma Chemical Corp.
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r e s p o n d to e i t h e r l a c t a t e o r s o r b i t o l d e h y d r o g e n ase. F i n a l l y , it s h o u l d b e p o i n t e d o u t t h a t i n t h e h e p a t o m a cells, t h e r e is a b a n d of a c e t a l d e h y d e d e h y d r o g e n a s e w h i c h c o r r e s p o n d s i n its m i g r a t i o n to s t o m a c h ADH, a n d s i n c e t h e f o r m e r e n z y m e u t i l i s e s as s u b s t r a t e t h e p r o d u c t of t h e l a t t e r e n z y m e ( i n t h e p r e s e n c e of N A D ) , t h i s r e s u l t s i n a n a m p l i f i c a t i o n of t h e s t a i n i n g i n t e n s i t y o f t h e s t o m a c h A D H b a n d (see f o o t n o t e to T a b l e I). T h e p r e s e n c e of s t o m a c h A D H i n r a t h e p a t o m a c e l l s is a s o m e w h a t u n e x p e c t e d f i n d i n g , s i n c e o n l y r a r e l y is a t r a c e of t h i s a c t i v i t y d e t e c t e d i n a d u l t l i v e r . H o w e v e r , t h e o b s e r v a t i o n s of o t h e r a u t h o r s
TABLE I. RESULTS. Preliminary e l e c t r o p h o r e t i c a n a l y s i s of A D H f r o m h e p a t o m a cells r e v e a l e d , i n a d d i t i o n t o l i v e r ADH, t h e p r e s e n c e of a n a n o d a l b a n d of e n z y m e a c t i v i t y (Fig. 1). T h e l a t t e r i s o z y m e c o r r e s p o n d s i n its m i g r a t i o n to s t o m a c h A D H a n d s h o w s a g r e a t e r substrate specificity than the well known liver t y p e : c o n t r a r y t o l i v e r ADH, i t d o e s n o t u t i l i z e (all t r a n s ) r e t i n o l ( S i g m a C h e m i c a l C o r p . ) as a substrate. Moreover, this anodal ADH does not cor-
ADH activity of hepatoma cells and of rat organs. Hepatoma clone FH
5 ...................
Fu Fu Fu Fu
5_2 5_5 5-7 5-8
................. ................. ................. .................
R a t organ Liver . . . . . . . . . . . . . . . . . . Stomach . . . . . . . . . . . . . . . . Kidney . . . . . . . . . . . . . . . . .
Specific activity t 95 (__+ 20) 77 102 ( ! 20)=' 40 (~-~ 4) 98 25 ( ± 5) 9 10
(1) Milliunits of enzyme a e t i v i t y / m g p r o t e i n in the soluble extract. (2) As emphasized by Ohno et al [21], the assay m e t h o d used [22J p r o b a b l y results in a n o v e r e s t i m a t i o n of ADH activity in crude extracts, owing to interference by aldehyde dehydrogenase. Using the series of t h r e e reactions described by Ohno et al. [26], it h a s been d e t e r m i n e d t h a t the activity of acetaldehyde dehydrogenase in these celts is one h a l f of t h a t of ADH.
Fro. 1. - - ADH zymograms. (Cellogel) of r a t organs and h e p a t o m a . The gel on t h e left was stained for 40 m i n u t e s , tha¢ on the r i g h t for 91) m i n u t e s . (a) a n d (d) a d u l t liver, showing a very pale anodaI b a n d and two i n t e n s e l y staini.ng cathodal b a n d s (on starch geIs a t h i r d a n d slower c a t h o d a l b a n d is resolved, as h a s also been described b y Koen a n d Shaw [24]). Cb) and (e) h e p a t o m a cells (clone F u 5 - 5 , grown as a tumor), showing a cathodal b a n d , corresponding in its m i g r a tion to the m o r e r a p i d l y m i g r a t i n g one f r o m liver, a n d a n a n o d a l b a n d . (c) stomach, a n d (f) fetal liver (23 days before b i r t h ) , showing cathodal b a n d s s i m i l a r to those of a d u l t liver, and a clearly resolved a n o d a l b a n d . A l t h o u g h t h e r e appears a slight difference in t h e mig r a t i o n rates of stomach ADH a n d the a n o d a l b a n d f r o m h e p a t o m a , m i x t u r e s of such e x t r a c t s i n v a r i a b l y show a single n a r r o w a n o d a l b a n d of activity. F o r all extracts, identical r e s u l t s are o b t a i n e d w h e t h e r the extracts are p r e p a r e d in buffer or w a t e r and w h a t e v e r the condition of eleetrophoresis (starch gel or Cellogel).
BIOCHIMIE, 1972, 54, n ° 2.
[19, 20] o n t h e d e v e l o p m e n t a l c h a n g e s i n l i v e r A D H s h o w t h a t i n m a n t h e r e a r e (~ f e t a l )> f o r m s of t h e e n z y m e , a n d i t is l i k e l y t h a t t h i s <
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Fu5 and Fu5-5. It w i l l be seen that w i t h the exception of Fu5-7, t h e r e is very little v a r i a t i o n in the specific a c t i v i t y of ADH f r o m one s u b c l o n e to another. Fu5-7 h o w e v e r , contains only about half as m u c h activity as Fu5 and the o t h e r subclones. Elect r o p h o r e t i c analyses of these p o p u l a t i o n s , as w e l l as of Fu5-5 cells g r o w n as t u m o r s have s h o w n that all p r e s e n t i n d i s t i n g u i s h a b l e patterns, e x c e p t for Fu5-7 ; cells of the latter t y p e c o n t a i n no t r a c e of the l i v e r f o r m of the enzyme, but the b a n d of stom a c h ADH stains w i t h the same i n t e n s i t y as that f r o m the o t h e r populations. T h e c o m p a r i s o n s bet w e e n FuS-7 a n d the other subclones p e r m i t s the c o n c l u s i o n that in all p o p u l a t i o n s e x c e p t for Fu5-7, r o u g h l y half of the total ADH a c t i v i t y is due to the l i v e r form, and half to the s t o m a c h form. Moreover, these o b s e r v a t i o n s s h o w that ADH a c t i v i t y is a p r o p e r t y w h i c h is r e t a i n e d t h r o u g h clonal passage of the h e p a t o m a cells, and that the s t o m a c h f o r m is e x p r e s s e d in a m o r e r e g u l a r fashion than the h e p a t i c one. T h e cells w h i c h have been crossed w i t h the h e p a t o m a , 3T3 (mouse fibroblasts) a n d BRL-1 ( d i p l o i d epithelial cells d e r i v e d f r o m rat liver), have been e x a m i n e d for t h e i r ADH content. BRL-1 cells c o n t a i n no detectable activity, e i t h e r by q u a n t i t a t i v e assays or by e l e c t r o p h o r e s i s f o l l o w e d
TABLE
In both series of s o m a t i c h y b r i d s (3T3 × Fu5 and BRL-1 × Fu5-5), the b a n d of ADH a c t i v i t y c o r r e s p o n d i n g to the l i v e r f o r m is e n t i r e l y absent, w h i l e a small a m o u n t of the s t o m a c h ADH a c t i v i t y is conserved. T h e specific a c t i v i t y values for the p a r e n t a l a n d h y b r i d cells are given in T a b l e II. It can be seen that the activity of the h y b r i d cells r e p r e s e n t s only a small f r a c t i o n of that p r e s e n t in the h e p a t o m a p a r e n t a l cells. Fig. 2 d e m o n s t r a t e s the isozymes p r e s e n t in these cells. All five clones e x a m i n e d of the rat-mouse h y b r i d cells r e t a i n the s t o m a c h isozymes of b o t h p a r e n t a l types as well as the h y b r i d f o r m e d b e t w e e n the rat and mouse m o n o m e r s . T h e BF5 h y b r i d cells c o n t a i n a single b a n d of (rat) s t o m a c h ADH. H y b r i d clones of the two types h a v e been examined t h r o u g h o u t the g r o w t h c y c l e to d e t e r m i n e w h e t h e r t h e r e exist c y c l i c m o d i f i c a t i o n s in the e x p r e s s i o n of ADH. Specific a c t i v i t y m e a s u r e m e n t s s h o w e d no s i g n i f c a n t fluctuations d u r i n g the g r o w t h cycle, a n d e l e c t r o p h o r e s i s s h o w e d no m o d i f i c a t i o n in the i s o z y m e pattern. Several subclones of h y b r i d BF5 h a v e been e x a m i n e d for ADH c o m p o s i t i o n ; none of these clones, w h e t h e r c h a r a c t e r i z e d by the e x p e c t e d n u m b e r of c h r o m o somes or f e w e r , s h o w either an elevation of ADH a c t i v i t y or e x p r e s s i o n of l i v e r ADH.
II.
Karyotypes and ADH activity of parental and hybrid cells. Cell type 3T3 ............. Fu 5 . . . . . . . . . . . . . 3 F Ga . . . . . . . . . . . BRL_ 1 . . . . . . . . . . Fu 5-5 . . . . . . . . . . . .
BF 5 . . . . . . . . . . . . . B F 5 - 74 . . . . . . . . . .
No of chromosomes t 75.9 52 116.3 42 52 92 55
(70-80) (51-53) (114-126) :~ (51-53) (91-93) (54-55)
Specific activity .2 3 95 (-+- 20) 5 0 lo2 (_-_+_20) 6
22
Activity/t0 6 cells 0.7 10.0 (~-!-2) 1.1 0.0 l o . o ( ± 2) 1.3 5.4
(1) Determined by the analysis of 10-30 metaphases of each clone. The mean and range of chromosome numbers are given for each population. (2) Mfliiunits of enzyme activity/rag protein in the soluble extract or per 106 cells. (3) Cells of this hybrid clone contain 19.9 (16-25) biarmed rat ¢ marker ~> chromosomes. The expected number is 25.7 (24-27).
by specific staining. 3T3 cells do c o n t a i n small a m o u n t s of ADH a c t i v i t y ; e t e c t r o p h o r e s i s reveals that the 3T3 e n z y m e c o r r e s p o n d s in its e l e c t r o p h o r e t i c m o b i l i t y to the e n z y m e f r o m (mouse) stomach, like the a n o d a l band f r o m h e p a t o m a cells ; these cells do not p r e s e n t a b a n d of activity corr e s p o n d i n g to the m o u s e l i v e r enzyme.
BIOCHIMIE, 1972, 54, n ° 2.
T h e s e results suggest that l i v e r ADH, like tyrosine a m i n o t r a n s f e r a s e (and its i n d u c i b i l i t y w i t h s t e r o i d h o r m o n e s ) and aldolase B, is e x t i n g u i s h e d in the h y b r i d cells, and that the s t o m a c h enzyme, although greatly r e d u c e d in a c t i v i t y (.expected activity/106 cells : 5 ; o b s e r v e d : 1.1-1.3) c o n t i n u e s to be expressed. T h e p r e s e n c e of this f o r m of ADH in
Expression o[ liver A.D.H. in hepatoma cell hybrids. m o u s e f i b r o b l a s t s suggests t h a t it m a y h a v e a m u c h m o r e w i d e s p r e a d tissue d i s t r i b u t i o n t h a n the l i v e r f o r m . S i n c e o n e of t h e h e p a t o m a s u b c l o n e s (Fu5-7) fails to p r o d u c e h e p a t i c ADH, it is p o s s i b l e that the h e p a t o m a cells w h i c h f u s e d w i t h p a r e n t a l ceils
199
of t h e o t h e r t y p e to g i v e r i s e to h y b r i d s d i d n o t themselves produce this enzyme. However, this e x p l a n a t i o n c a n be r u l e d out in t h e c a s e of t h e BF5 h y b r i d , b e c a u s e o n e h y b r i d s u b c l o n e (BF5~/-4), w h i c h has lost a l a r g e n u m b e r of c h r o m o -
Fw,. 2. - - A,DH zymograms (Ce]logel) of parental and hybrid cells. Two hepatoma clones (Fu-5-5 and FuS) are shown, as well as parental cell lines BRL-1 and 3T3 (in the latter, a faint band of ADH activity, which remains at the origin and corresponds to mouse stomach ADH, is seen). In the intraspecific hybrid, BF5, a single anodal band of rat stomach ADH is seen, while in the interspecific hybrid, 3FCa, both parental stomach ADH's, as well as a single hybrid band formed between them, are clearly visible. somes, has b e e n f o u n d to r e - e x p r e s s h e p a t i c A D H (Fig. 3, T a b l e II) b u t at a l e v e l l o w e r t h a n t h a t of the Fu5-5 cells. T h i s r e - e x p r e s s i o n of a l i v e r e n z y m e a f t e r loss of l a r g e n u m b e r s of c h r o m o s o m e s , h a s n o w b e e n o b s e r v e d in s e v e r a l cases. I n fact, t h e h y b r i d cells which re-express liver ADH similarly re-express a l d o l a s e B E7], b u t a n o t h e r c l o n e , BF-I-1, w h i c h r e - e x p r e s s e s T A T i n d u c i b i l i t y [6], d o e s n o t ree x p r e s s e i t h e r l i v e r A D t I o r a l d o l a s e B. DISCUSSION.
FIG. 3. - - ADH zymogram (Cellogel) demonstrating re-expression of liver (eathodal) ADH in the hybrid subelone B F S - y 4 (right). On the left is shown the ADH pattern of a subelone of Fu5-5 (Ft~5-5aza967), ineluded as a migration reference, and showing a fainter band of liver ADH than Fu5-5.
BIOCH1MIE, 1972,
54, n ° 2.
E x a m i n a t i o n of t h e e x p r e s s i o n of A D H in h e p a t o m a cell h y b r i d s ( w h i c h c o n t a i n e s s e n t i a l l y all of t h e c h r o m o s o m e s c o n t r i b u t e d by t h e t w o p a r e n t s ) has s h o w n t h a t t h e l i v e r f o r m of t h i s e n z y m e is absent, w h i l e t h e s t o m a c h f r o m c o n t i n ues to be p r o d u c e d , a l t h o u g h at a l e v e l m u c h lower than that which characterizes the parental h e p a t o m a cells. T h i s r e s u l t h a s b e e n o b s e r v e d in both interspecific (rat X mouse) and intraspecific (rat × rat) h y b r i d s . M o r e o v e r , in t h e f o r m e r h y b r i d s , b o t h p a r e n t a l f o r m s of t h e s t o m a c h e n z y m e are p r o d u c e d , as w e l l as a s i n g l e h y b r i d (heterospecif~c) e n z y m e b e t w e e n t h e m , d e m o n s t r a t i n g a h o m o l o g y b e t w e e n t h e t w o s p e c i e s of t h i s e v i d e n t l y d i m e r i c e n z y m e . (It w i l l h a v e b e e n not i c e d t h a t no h y b r i d i z a t i o n o c c u r s b e t w e e n t h e
200
Roger
Bertolotti
liver and the stomach enzymes in the hepatoma cells, e v e n t h o u g h b o t h f o r m s a r e d i m e r i c enzymes). T h e s i m i l a r i t y of t h e A D H s p e c i f i c a c t i v i t y a n d a c t i v i t y / 1 0 6 cells of b o t h k i n d s of h y b r i d s a n d of the 3T3 fibroblast parental cells suggests that prod u c t i o n of a l o w <~b a s e - l i n e >> of t h e s t o m a c h f o r m is n o t a t i s s u e s p e c i f i c f u n c t i o n , w h i l e a h i g h a c t i v i t y of t h i s f o r m s is s u b j e c t t o q u a n t i t a t i v e c o n t r o l . L i k e t h e r e - e x p r e s s i o n of a l d o l a s e B a n d T A T i n d u c i b i l i t y , r e - e x p r e s s i o n of l i v e r A D H i n h e p a t o m a h y b r i d s w h i c h h a v e l o s t l a r g e n u m b e r s of c h r o m o s o m e s c a n b e i n t e r p r e t e d as a n i n d i c a t i o n of t h e s t a b i l i t y of t h e e p i g e n o t y p e of t h e h e p a t o m a p a r e n t a l c e l l s [2]. T h u s , t h e p o t e n t i a l i t y to e x p r e s s these tissue specific functions can be inherited t h r o u g h m a n y h y b r i d cell g e n e r a t i o n s w i t h o u t b e i n g e x p r e s s e d ; w h e n loss of c h r o m o s o m e s ( c a u s i n g e x t i n c t i o n ) o c c u r s , t h e f u n c t i o n is r e - e x p r e s sed. T h i s i n t e r p r e t a t i o n m u s t r e m a i n t e n t a t i v e h o w e v e r u n t i l i d e n t i f i c a t i o n of a s p e c i f i c c h r o m o some, which causes extinction when present and p e r m i t s r e - e x p r e s s i o n w h e n lost; c a n b e m a d e . T h e o b s e r v a t i o n s o f K l e b e , C h e n a n d R u d d l e [5] s u g gest t h a t t h i s w i l l b e p o s s i b l e , s i n c e t h e y w e r e a b l e to f i n d a c o r r e l a t i o n b e t w e e n t h e p r e s e n c e a n d a b s e n c e of t h e h u m a n Clo c h r o m o s o m e a n d t h e r e s p e c t i v e e x t i n c t i o n a n d r e - e x p r e s s i o n of m o u s e k i d n e y ES-2 ( a n e s e r i n e i n s e n s i t i v e e s t e r a s e ) i n hybrids formed by crossing mouse renal adenoc a r c i n o m a c e l l s (ES-2 '+) w i t h h u m a n f i b r o b l a s t s . Acknowledgements. We are g r a t e f u l to Drs. Boris E p h r u s s i and Drew Schwartz for m a n y s t i m u l a t i n g discussions, to Dr. Boris E p h r u s s i for critical r e a d i n g of the m a n u s c r i p t , a n d to M uo Anne Debon for excellent ,technical assistanee. This work was conducted w i t h the aid of a g r a n t to Dr. Boris E p h r u s s i f r o m the D616gation G6n6rale h la Recherche Scientifique et Technique. PRESUME.
Les cellules d ' u n h 6 p a t o m e bien diff6renci4 de rat, eultiv4es in vitro et in vioo, p r 6 s e n t e n t deux formes d'aleool d6shydrog6nase ; la p r e m i 6 r e correspond, p a r sa m i g r a t i o n 61eetrophor6tique, h l ' e n z y m e b i e n eonnu de foie, la seeonde est homologue de l ' i s o e n z y m e mis en 6videnee dans l'estomae. Des eellules de eet h 6 p a t o m e de r a t ont 6td erois6es avec des fibroblastes d ' u n e lign6e p e r m a n e n t e de souris, dont la tr6s faible activit6 alcool d6shydrog6nase est due h l ' i s o e n z y m e stomaca], et avec des cellules 6pith61iales diploides de r a t d o n t l'activit6 alcool d6shydrog6nase est nulle. Dans les h y b r i d e s s o m a t i q n e s a i n s i obtenus, seul l ' i s o e n z y m e h 6 p a t i q u e n'est pas d6celable, ce qui m o n t r e que, comme p o u r b e a u c o u p de fonctions diff6renci6es, son expression es[ sp6cifiquement hloqu6e lorsque les cellules d ' h 6 p a t o m e BIOCHIMIE, 1972, 54, n ° 2.
and Mary
C. W e i s s .
sont crois6es avec des cellules d'une origine histog6n6tique diff6rente. Quc cette e x t i n c t i o n de l'alcool d6shydrog6nase h 6 p a t i q u e n'est pas due h une perte ou u n e alt6ration irr4versible du mat6riel g6n6tique est d6m o n t r 6 p a r le fait que cet isoerrzyme est r6exprim6 dans u n sous-clone d ' h y b r i d e s a y a n t perdu une f r a c t i o n i m p o r t a n t e de leurs chromosomes. ZUSAMMENFASSUNG.
Wohl differenzierte Zellcn eines H'epatoins der Ratte, die in vitro u n d in vivo k u l t i v i e r t ~vurden, e n t h a l t e n zwei F o r m e n der A l k o h o l d e h y d r o g e n a s ' e ; die erste Form entspricht auf Grund ihrer elektrophoretischen W a n d e r u n g dern b e k a n n t e n Leberenzym, die zweite ist dem i m Magen g e f u n d e n e n I s o e n z y m homolog. Zellen dieses R a t t e n h e p a t o m s w u r d e n m i t F i b r o b l a s t e n einer Mfiuselinie, die n u t eine sehr sch~vache Alkoholdehydrogenaseaktivitiit, die dem Magenisoenzym zuzusc h r e i b e n ist, enthiHt, und epithelialen diploiden Zellen der Ratte, deren A l k o h o l d e h y d r o g e n a s e a k t i v i t i i t null ist, gekreuzt. In den so e r h a l t e n e n s o m a t i s c h e n Hyb r i d e n ist allein das Isoenzym d~er Leber n i c h t n a c h weisbar, was zeigt, dass, wie ftir viele differenzierte F u n k t i o n e n , die W i r k u n g dieses E n z y m s u n t e r b u n d e n wird, sobald die Zellen eines H e p a t o m s m i t Zellen u n t e r s e h i e d l i e h e r h i s t o g e n e t i s c h e r H e r k u n f t gekreuzt werden. Dass dieses Verseh~vinden der Leberalkoholdehydrogenase n i e h t e i n e m Verlust oder e i n e r irreversiblen A n d e r u n g des genetisehen Materials zuzuschreib e n ist, zeigt die Tatsache, dass dieses I s o e n z y m in einem Subklon tier Hybride, der eine grosse Z a h l von C h r o m o s o m c n verloren ha.t, w i e d e r z.um Vorschein kommt. REFERENCES. 1. Barski, G., Sorieul, S. ,~ Cornefert, F. (196~) Compt. Rend. Acad. Sci., 251, 182.5. 2. Ephrussi, B., H y b r i d i z a t i o n of Somatic Cells, P r i n c e t o n U n i v e r s i t y Press, P r i n c e t o n , in press. 3. Davidson, R. L., Ephrussi, B. ~ Y a m a m o t o , K. (1966) Proc. Nat. Acaa. Sci. U.S.A., 56, 1437. 4. Davidson, R. L. (1971) I n vitro, 6, 411. 5. KIebe, R., Chen, T. ~ Ruddle, F. (1970) Proc. Nat. Acad. Sci. U.S.A., 66, 1220. 6. Weiss, M. C. a Chaplain, M. (1971) Proc. Nat. Acad. Sci. U.S.A., 68, 3026. 7. Bertolotti, R. ~ Weiss, M. C., Cell D i f f e r e n t i a t i o n , Proceedings of the 1st I n t e r n a t . Conf. on Cell Differentiation (Eds. R. H a r r i s an& D. Viza), in press. 8. Foug6re, C., Ruiz, F. ~ Ephrussi, B. (1972) Proc. Nat. Acad. Sci. U.S.A., 69, 330. 9. Schn,eider, J. ~ "Weiss, M. C. (1971) Proc. Nat. Acad. Sci. U.S.A., 68, 127. 10. Bertolotti, R. ~ Weiss, M. C. J. Cell. Physiol., in press. 11. Peterson, J. A. ~ Weiss, M. C. (1972) Proc. Nat. Acad. Sci. U.S.A., 69, ~571. 12. Krebs, H. A. ~ Perkins, J. R. (197.0~ Biochem. J., 118, 635. 13. Moser, K., Papenberg, J. e Von W a r t b u r g , J. P. (1968) E n z y m . BioL Clin., 9, 447. 14. Theorell, H. (19:67) Harvey Lect., 61, 17. 15. Lutstorf, U. M., Schiirch, P. M. ~ Von W a r t b u r g , J. P. (1970) Eur. J. Biochem., 17, 497. 16. Riiih~i, N. C. R., Kuskinen, M. a P i k k a r a i n e n , P. H. (1967) Biochem. J., 103, 623. 17. Pikkarain'en, P. H. & R/iih~i, N. C. R., (1967~ Pediat. Res., 1, 165. 18. P i k k a r a i n e n , P. H. ~ R/iihii, N. C. R. (196:9) Nature, 222, 563.
Expression of liver A.D.H. in hepatoma cell hybrids. 19. M u r r a y , R. F. ,~ Motulsky, A. G. (1971) Science, 171, 71. 20. Smith, M., H o p k i n s o n , D. A. a Harris, H. (1971) Ann. Hum. Genet. Lond., 34, 251. 21. Ohno, S., Stenius, C. • C h r i s t i a n , L. C. (1970) Clin. Genet., 1, 35. 22. Vallee B. L. a Hoeh, F. L. (1955) Proc. Nat. Acad. Sei., 41, 327.
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