Characterization of aldehyde dehydrogenase from HTC rat hepatoma cells

180

Biochimica et Biophvs'ica A ~ta ~43 (1985) 1gO I g5 t-lsevier

BBA 22172

Characterization of aldehyde d e h y d r o g e n a s e from H T C rat h e p a t o m a cells R o n a l d L i n d a h l a,,, D a v i d W. B a g g e t t b a n d A l v i n L. W i n t e r s b Biochemistry Program and Departments of ~ Biology and h Microbiology, The Universi(v of Alabama, Universi(v, .4 L 35486 (U.S.A.)

(Received May 16th, 1985)

Key words: Aldehyde dehydrogenase; Multiple forms; (Rat hepatoma cell)

We have proposed developing rat hepatoma cell lines as an in vitro model for studying the regulation of changes in aldehyde dehydrogenase activity occurring duringhepatocarcinogenesis. Aldehyde dehydrogenase purified in a single step from HTC rat hepatoma cells is identical to the aldehyde dehydrogenase isolated from rat hepatocellular carcinomas. HTC aldehyde dehydrogenase is a 110 kDa dimer composed of 54-kDa subunits, prefers NADP + as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes but not phenylacetaldehyde. The substrate and coenzyme specificity, effects of disulfiram, pH profile and isoelectric point of HTC aldehyde dehydrogenase are also identical to these same properties of the tumor aldehyde dehydrogenase. In immunodiffusions, both isozymes are recognized with complete identity by anti-HTC aldehyde dehydrogenase antibodies. Having established that HTC aldehyde dehydrogenase is very similar, if not identical, to the aldehyde dehydrogenase found in hepatoceilular carcinomas, simplifies the development of molecular probes for examination of the regulation of tumor aldehyde dehydrogenase activity in vivo and in vitro.

Introduction Work in our laboratory is directed toward understanding the origin and significance of changes in aldehyde dehydrogenase activity occurring during rat hepatocarcinogenesis [1-5]. We recently demonstrated that several rat hepatoma cell lines have an aldehyde dehydrogenase phenotype qualitatively identical to that seen in heptaotcellular carcinomas generated in vivo [6]. The phenotype is characterized by elevated NADP+-depen dent, benzaldehyde-oxidizing activity, the demonstration of new isozymes by electrophoresis and characteristic localization of aldehyde dehydro* To whom correspondence should be addressed at: Department of Biology, P.O. Box 1927, The University of Alabama, University, AL 35486, U.S.A. Abbreviation: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

genase activity detected in situ [2,3,5]. These observations suggested that rat hepatoma cell lines may be an excellent in vitro system for studying the mechanisms underlying expression of hepatoma aldehyde dehydrogenase. We have also described the major physical and functional properties of four basal and four inducible aldehyde dehydrogenases isolated from rat liver [7,8]. The four normal liver isozymes are tetramers which preferentially oxidize aliphatic aldehyde substrates using NAD + as coenzyme. However, each isozyme can be distinguished by subcellular location, substrate a n d / o r coenzyme kinetics, response to extremes of pH or heat, sensitivity to effectors, a n d / o r apparent molecular weight. The inducible rat liver aldehyde dehydrogenases are cytosolic and not detectable in normal liver. They can be separated into two distinct

0304-4165/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

181

groups [8]. The isozyme isolated from hepatocellular carcinomas and a 2,3,7,8-tetrachlorodibenzop-doxin (TCDD) -inducible isozyme are very active with benzaldehyde-like aromatic aldehydes and prefer NADP + as coenzyme. These isozymes are dimers, possess identical kinetic properties, pH profiles and responses to effectors. A phenobarbital-inducible isozyme and an aldehyde dehydrogenase appearing during the promotion phase of rat hepatocarcinogenesis appear to be identical. Both are NAD+-specific and prefer ahphatlc aldehyde substrates. They have identical pH and thermal stability profiles and responses to effectors. Their molecular weights are intermediate between the tumor-specific and TCDD-inducible isozymes and the normal liver aldehyde dehydrogenases. Development of an in vitro model for studying regulation of tumor aldehyde dehydrogenase activity is dependent on establishing that the aldehyde dehydrogenase activity found in the cell lines is identical to that found in hepatocellular carcinomas generated in vivo. This paper reports the single-step purification of aldehyde dehydrogenase from HTC rat hepatoma cells and demonstrates the identity of this isozyme with the aldehyde dehydrogenase found in tiepatocellular carcinomas. Materials and Methods

Aldehydes were from Aldrich. Propionaldehyde and benzaldehyde were redistilled before use. Enzyme grade ammonium sulfate, DEAE- and CMcellulose, NAD+, NADP+, tetraethylthiuram disulfide (disulfiram) and gentamycin were from Sigma. Reagents for polyacrylamide gel electrophoresis were from Eastman. G-200 Sephadex, 5'-AMP-Sepharose 4B, and ampholytes for isoelectric focusing were from Pharmacia Fine Chemicals. Spinner-modified Eagle's minimal essential medium, nonessential amino acids and heat-inactivated calf and fetal bovine serum were from K.C. Biological, Inc. All other chemiclas were reagent grade. New Zealand White rabbits were from a local supplier. HTC cells were carried in monolayer culture using spinner-modified Eagle's medium supplemented with nonessential amino acids 5% heat-in-

activated bovine calf serum and gentamycin (50 t~g/ml). Material for enzyme purification was obtained by placing the HTC cells in suspension culture and maintaining the concentration between 2.0.103 and 5.0.103 cells/ml. 8-10 1 cultures were harvested by centrifugation at 200 × g for 15 min. The cells were resuspended in medium, sedimented by centrifugation and stored at - 70°C in 60 mM sodium phosphate buffer (pH 8.5)/1 mM EDTA/1 mM 2-mercaptoethanol. Aldehyde dehydrogenase activity was determined at 25°C by monitoring the increase in absorbance at 340 nm caused by NADH or NADPH production during the oxidation of aldehyde substrate in a modification of the assay described previously [9]. Final substrate and coenzyme concentrations were 5 and 1 mM, respectively, unless otherwise stated. All appropriate corrections for enzyme-independent changes in absorbance were made. Activities were expressed as m l U / m g of protein (1 mlU = 1 nmol NAD(P)H produced/min). Protein concentrations were determined by the method of Lowry et al. [10] with bovine serum albumin as standard. The purification of aldehyde dehydrogenase from HTC cells was adapted from the procedures described previously for the isolation of the tumor-specific and TCDD-inducible isozymes [8]. After thawing, HTC cells were prepared as a 10% homogenate in 60 mM sodium phosphate buffer (pH 8.5)/1 mM EDTA/1 mM 2-mercaptoethanol using a loose-fitting Potter-Elvehjem homogenizer. The homogenate was centrifuged at 48 000 x g for 30 min and the supernatant dialysed against several changes of 100 mM potassium phosphate buffer (pH 7 . 5 ) / 1 mM E D T A / 1 mM 2mercaptoethanol/0.02% Triton X-100. The dialysed supernatant was applied to a 5'-AMP-Sepharose 4B affinity column equilibrated with dialysis buffer. The column was washed with 100 mM, then 450 mM potassium phosphate-containing buffer systems and finally with 25 mM sodium phosphate buffer (pH 7,5)/1 mM EDTA/1 mM 2-mercaptoethanol/0.02% Triton X-100. Aldehyde dehydrogenase was then eluted with the 25 mM sodium phosphate buffer system containing 0.25 mM NAD +. Active fractions were pooled and concentrated as necessary under N 2 using a Diaflo concentrator.

182

The tumor-specific and TCDD-inducible aldehyde dehydrogenases were purified as described previously [8]. Molecular weight estimations, pH profiles, the effect of disulfiram and various kinetic properties of HTC aldehyde dehydrogenase were determined essentially as described [7,8]. Electrophoretic analyses were performed as described [7,8]. Rabbit antiserum to HTC aldehyde dehydrogenase was generated by footpad injection of purified enzyme in Freund's complete adjuvant. At 6 weeks, and monthly thereafter, rabbits received a booster injection of enzyme in Freund's incomplete adjuvant and were bled 4 weeks later. Anti-HTC antibodies were purified from whole serum by ammonium sulfate precipitation and DEAE-CM-cellulose chromatography [11,12]. Results

Aldehyde dehydrogenase can be purified from HTC cell supernatants in a single step (Table I). All of the enzyme activity binds to 5'-AMP-Sepharose 4B and is eluted with 0.25 mM NAD +. Analysis of the affinity purified HTC aldehyde dehydrogenase preparations by analytical gel electrophoresis followed by protein and enzyme activity staining indicates such preparations are homogeneous. One major and two minor proteins, each possessing aldehyde dehydrogenase activity, are observed. No contaminating proteins were observed by either analytical or sodium dodecyl sulfate-gel electrophoresis. Gel isoelectric focusing in the pH range 5-8 produced a pI estimate of approx. 6.9-7.0 for HTC aldehyde dehydrogenase. Gel-filtration chromatography through G-200 Sephadex yields a relative molecular weight of approximately 110000 for HTC aldehyde dehy-

1.0 RNase

~e

0.8

Chymo.

0.6 Ovalburnin 0.4

Hexokinase

0.2

Aldolase

0.0'L 4.0

~5

5.5

5.0 Log MW

Fig. 1. Molecular weight estimations for HTC aldehyde dehydrogenase by G-200 gel filtration. (3, aldehyde dehydrogenases: HTC, HTC aldehyde dehydrogenase; Tumor, tumorspecific isozyme; TCDD, TCDD-inducible isozyme; PA, promotion-associated isozyme; PB, phenobarbital-inducible isozyme; Mito I and II, normal liver mitochondrial isozymes I and II; Micro I and II, normal liver rnicrosomal isozymes I and II; Chymo, chymotrypsin, e, molecular weight standards.

drogenase (Fig. 1). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that HTC aldehyde dehydrogenase is composed of a single 54 kDa MW subunit. The g m values for NAD + and NADP + are approximately 20 and 350 /~M, respectively, for HTC aldehyde dehydrogenase (Table II). The Vma~ values indicate a strong preference for NADP + as

TABLE I PURIFICATION OF ALDEHYDE DEHYDROGENASE FROM HTC CELLS Step

Vol. (ml)

Total activity a (mlU)

Total protein (mg)

Specific activity (mlU/mg)

Supernatant b 5'-AMP-Sepharose c

22 7

17519 18040

102 1

172 17910

Yield (%)

Purification (-fold)

100

104

a Average of four purifications; b supernatant from a 48000× g centrifugation of a 10% cell homogenate; c Activity elutes with a 25 mM sodium phosphate buffer system (pH 7.5) containing 0.25 mM NAD ÷.

183 TABLE II KINETIC PROPERTIES OF HTC ALDEHYDE DEHYDROGENASE Substrate K m values are in mM. Coenzyme K m values are in ~tM. K,, values were determined at 1 mM NAD ÷ for propionaldehyde, 1 mM NADP ÷ for benzaldehyde, 1 mM propionaldehyde for NAD +, and 1 mM benzaldehyde for NADP +. Relative substrate Vma~ values were determined by measuring activity at 5 mM substrate concentration compared to the activity with 5 mM propionaldehyde. Relative NADP ÷ Vma~ was determined using 1 mM NADP ÷ compared to 1 mM NAD +. --, not determined.

Formaldehyde Acetaldehyde Propionaldehyde Benzaldehyde Chlorbenzaldehyde Nitrobenzaldehyde Phenylacetaldehyde NAD

+

NADP ÷

Km

Vmax

2.9 0.3 23 343

0.01 0.07 1.00 3.61 2.22 1.34 0.03 1.00 6.90

coenzyme. H T C a l d e h y d e d e h y d r o g e n a s e clearly prefers b e n z a l d e h y d e a n d its derivatives as substrates (Km of 300 /~M vs. 3 m M for p r o p i o n a l d e h y d e ) with either N A D + or N A D P + as c o e n z y m e ( T a b l e II). Significant s u b s t r a t e inhibition was o b s e r v e d at greater than 5 m M benzaldehyde. HTC aldehyde dehydrogenase cannot oxidize p h e n y l a c e t a l d e h y d e to an a p p r e c i a b l e extent. W i t h p r o p i o n a l d e h y d e a n d N A D +, the p H velocity profile of H T C a l d e h y d e d e h y d r o g e n a s e shows increasing activity with increasing p H to p H 8.0 a n d a second s h a r p increase in activity a b o v e a p p r o , p H 8.5. W i t h b e n z a l d e h y d e a n d N A D P +, H T C a l d e h y d e d e h y d r o g e n a s e has a single s h a r p p H o p t i m u m n e a r p H 8.5 with activity declining r a p i d l y at m o r e alkaline p H s . H T C a l d e h y d e d e h y d r o g e n a s e is heat labile. Less than 10% of the N A D +- or N A D P + - d e p e n d e n t activity r e m a i n s after 2 m i n at 55°C. Disulfiram, at b o t h 25 a n d 50 ~ M , inhibits the N A D P + - d e p e n d e n t activity of H T C a l d e h y d e d e h y d r o g e n a s e 99%. N A D +-dependent e n z y m e activity is i n h i b i t e d a p p r o x i m a t e l y 60%. A n t i b o d i e s to p u r i f i e d H T C a l d e h y d e d e h y d r o genase react with c o m p l e t e i d e n t i t y to b o t h the t u m o r a n d T C D D - i n d u c i b l e isozymes (Fig. 2). In

Fig. 2. Reaction of various aldehyde dehydrogenases with anti-HTC aldehyde dehydrogenase antibodies. (A) Anti-HTC aldehyde dehydrogenase antibodies (3 mg/ml); (B) 25% normal liver homogenate supernatant; (C) 10% liver homogenate supernatant containing promotion-associated aldehyde dehydrogenase; (D) 10% liver homogenate supernatant containing phenobarbital-inducible aldehyde dehydrogenase; (E) 10% liver homogenate supernatant containing TCDD-inducible aldehyde dehydrogenase; (F) 5% HTC cell supernatant containing HTC aldehyde dehydrogenase; (G) 10% hepatocellular carcinoma homogenate supernatant containing tumor aldehyde dehydrogenase. All wells contained 15-/~1 samples. Incubation was in a humid environment for 48 h at 4°C after which slides were washed for 60 rain in running water and stained for enzyme activity with benzaldehyde and NADP ÷. An identical pattern was obtained if slides were stained for protein using Coomassie R-250.

d o u b l e diffusions, n o cross-reactivity occurs with either the p h e n o b a r b i t a l - i n d u c i b l e or p r o m o t i o n a s s o c i a t e d a l d e h y d e d e h y d r o g e n a s e n o r with norm a l liver a l d e h y d e dehydrogenases.

Discussion Based on its physical a n d functional properties, the a l d e h y d e d e h y d r o g e n a s e activity isolated from H T C rat h e p a t o m a cells is very closely related, if n o t identical, to the t u m o r a n d T C D D - i n d u c i b l e a l d e h y d e d e h y d r o g e n a s e s previously d e s c r i b e d [8] ( T a b l e III). T h e active e n z y m e f r o m all three sources is a 110 k D a d i m e r c o m p o s e d of 5 4 - k D a

184 TABLE IIl PROPERTIES OF TUMOR-RELATEDALDEHYDE DEHYDROGENASES HTC

Tumor-specific ~

TCDD-inducible ~

Molecular weight (kDa) Functional form b pl Coenzymepreference c CoenzymeKm d NAD ÷ (#M) NADP+ (~M)

110 dimer 6.9 NADP+ 20 350

110 dimer 6.8 NADP+ 70 420

110 dimer 7.05 NADP+ 70 210

Disulfiram inhibition Heat stability Substrate preference

yes yes yes labile stable stable mM Km; broad, but prefer benzaldehyde;phenylacetaldehydepoor substrate

See Ref. 8; b based on subunit of 54 kDa; c based on Km and Vmax data; d average of at least two determinations not differing by more than 10%.

subunits. They prefer N A D P + as coenzyme, preferentially oxidize benzaldehyde-like aromatic aldehydes, but cannot oxidize phenylacetaldehyde. They all have similar pH profiles and sensitivities to disulfiram. They cross-react with complete identity immunochemically. However, HTC aldehyde dehydrogenase is more thermal labile than either the tumor or TCDD-inducible aldehyde dehydrogenases and has a slightly lower K m for N A D +. The H T C - t u m o r - T C D D - i n d u c i b l e aldehyde dehydrogenase family is clearly different from the phenobarbital-inducible, promotion-associated and normal liver aldehyde dehydrogenases [7,8]. The latter enzymes all differ significantly in functional molecular weight, kinetic properties and substrate and coenzyme specificity. Moreover, none of these aldehyde dehydrogenases cross-react with antibodies to the HTC aldehyde dehydrogenase. In the absence of structural information confirming identity, the minor differences in functional properties between the HTC, tumor and TCDD-inducible aldehyde dehydrogenases observed may reflect the fact that they represent multiple molecular forms of the same enzyme. Both gel electrophoresis and isoelectric focusing studies have indicated the presence of multiple active forms of aldehyde dehydrogenase in HTC and other rat hepatoma cell lines, hepatocellular carcinomas and TCDD-treated livers [2-4,6,11]. However, we have not succeeded in separating the individual isozymic forms by the purification protocols developed to date.

The purification of aldehyde dehydrogenase from HTC cells with essentially 100% recovery in a single step makes this cell line an obvious source for the large amounts of material necessary for detailed molecular and genetic studies of the enzyme. Previously, the best yields of inducible and normal liver aldehyde dehydrogenases were 24 and 15%, respectively, using a 3-4-step purification procedure [7,8]. Having established that the aldehyde dehydrogenase found in HTC cells is very similar, if not identical, to that found in hepatocellular carcinomas developing in vivo will allow the development of an in vitro model for studying the regulation of tumor aldehyde dehydrogenase activity as previously proposed [6]. It will also greatly simplify the studies of the mechanism of expression of this enzyme during carcinogenesis, because large numbers of HTC cells can be grown in vitro under defined and easily manipulated conditions. This contrasts sharply with the time, expense, complexity and variability inherent in studying enzyme changes during carcinogenesis in vivo.

Acknowledgements This work was supported by Grant No. CA21103 to R.L. from The National Cancer Institute.

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