Aryl hydrocarbon hydroxylase and tyrosine aminotransferase activities in somatic-cell hybrids derived from hepatoma tissue culture HTC (rat) cells and 3T3 (mouse) benzo[a]pyrene-resistant cells

Vol. 56, No. 3, 1974

BIOCHEMICAI

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ARYL HYDROCARBON HYDROXYLASE AND TYROSINE AMINOTRANSFERASE ACTIVITIES SOMATIC-CELL

HYBRIDS DERIVED FROM HEPATOMA TISSUE CULTURE HTC (RAT) AND 3T3 (MOUSE) BENZO[a]PYRENE-RESISTANT

E. Brad +

*

Laboratory Health,

+

Thompson,

of Biochemistry, Bethesda, Maryland

Division of Hematology, California 90027

+I-

W. F. Benedict,*

Ida

National 20014

Children's

Section on Developmental Pharmacology, Human Development, National Institutes

Received

December

+t

Institute,

Hospital

CELLS

CELLS

S. Owens,

Cancer

IN

+t

and D. W. Nebert

National

of Los Angeles,

Institutes

of

Los Angeles,

National Institute of Child Health and of Health, Bethesda, Maryland 20014

11,1973

ABSTRACT. Somatic-cell hybrids were formed between a 3T3 (mouse) benzo[a]pyrene-resistant subline having very low basal or inducible aryl hydrocarbon hydroxylase and tyrosine aminotransferase activities and hepatoma tissue culture (rat) cells which lack the hydroxylase activity but contain the steroid-inducible aminotransferase. The benz[a]anthracene-inducible hydroxylase activity was absent or very low in all the hybrids. As has been the case in other hybrids from various parental lines, the aminotransferase was no longer inducible. INTRODUCT-CON. fibroblasts level

Aryl

this

Conversely,

enzyme tyrosine

dexametha:sone, zyme exist the

parent

in

(BA),

detectable

only

specific

In hybrids

ranged

studies

is which

activity.

Is

activity

between

the

high

formed

inherent

not

cell

line

3) to more

hydroxylase hydroxylase

0 1974 by Academic Press, Inc. of reproduction in my form reserved.

of

the

source

of the

ranged than these

of hepatoma

cell

that

of

the

3T3 The

somatic-cell by these

enhanced increased

origin

lines

greater.

BA-treated

3T3 parent

en-

two parent

lo-fold

the

by

of this

from

unanswered of

(HTC) cells.

levels

of these

3T3

or induced

in HTC cells

inducible)

One question is

culture

inducible

by fusion

in mouse

basal

tissue

activity

605 Copyright All rights

is

in the majority

6 and 40 (1).

parental

hepatic

(i.e. --

inducible

no detectable

activity

of about

is

hepatoma

hydroxylase

activity

specific

hybrids

in rat

low basal

of BA-inducible

activity

whereas

aminotransferase

3T3 cells.

level

hydroxylase

is

whereas

(i.e. --

potentially

hydroxylase

by benz[a]anthracene

of

(l),

hydrocarbon

being

prior

hydroxylase or is

the

expressed?

Vol. 56, No. 3, 1974

In the to the

BIOCHEMICAL

present

toxic

report,

effects

we have

(2-4)

demonstrate

cytotoxicity,

cells

enhanced

weeks'

cant

amounts

have

little

nique

time of

should

a cell

magnitudes

of induction

activities.

These

our

cells

possess

line

(1)

a dominant

of

allow

the

hybrids

hydroxylase

is

suggest

that

which

growth

of

medium

have of cells

low basal

over

that tech-

or inducible

between

negligible

the

3T3

signifi-

by means of this

and/or

prevents

activity

Exposure

survival

hybrids

It

and phenolic

which

and tyrosine

of 3T3 origin

factor

cells

very

showed

medium.

of epoxy

in the

fused-cell

therefore

is resistant

hydroxylase

induced.

death

contained

studied

of both

study

because

As predicted,

which

These

results

previous

cause

activity.

3T3 subline.

inducible

enzyme being

and should

We then

activity.

the

of benzo[a]pyrene

therefore

which

per ml of growth

likely

by the

the hydroxylase

and this

from

formed

RESEARCH COMMUNICATIONS

a 3T3 subline

having

most

or no hydroxylase

hydroxylase

cells

concentrations

we developed

parent

that

or products

to increasing

several

selected

of 10 ~g of benzo[a]pyrene

has been established

intermediates

AND BIOPHYSICAL

the

HTC

or only

small

aminotransferase hydroxylase

that

the

expression

in hybrids

present

3T3 variant

of hydroxylase

acti-

vity. MATERIALS AND METHODS The enzyme protein

for

determinations,

as previously is

assays

and the

described

defined

as that

hydroxylated

(1).

amount

product

of enzyme

Tyrosine

of p-hydroxyphenylpyruvic

protein.

HMT medium

uM methotrexate, Cell

rived

from

lines. wild-type

is

of aryl

equivalent

specific

culture

medium

is

the of

activity

formation 1 pmole

expressed

specific

formed

exactly

hydroxylase

to that

activity

the

performed

per min at 37"

aminotransferase

Eagle's

were

hydrocarbon

catalyzing

acid

activities,

preparations

fluorescence

as nmoles

0.4

chromosomal

Hydroxylase

protein.

and aminotransferase

--One unit

causing

hydroxybenzo[a]pyrene. mg of cellular

hydroxylase

of of 3-

as units

activity

is

per

expressed

per min per mg of cellular (1)

containing

100 PM hypoxanthine,

and 16 uM thymidine. HTC (ARl, HTC cells

TAT+, (5)

AHH-)

is

and is

606

a subline resistant

of HTC cells to

that

6-mercaptopurine

was deat con-

Vol. 56, No. 3, 1974

centrations

as high

bosyltransferase possesses aryl

BIOCHEMICAL

to us: by Dr.

bridge

(6).

pyrene

in the 1.0

The 3T3 cells

were

The most

the

clone

which

aminotransferase

We have

also

trations

cells

found

the

lack

basal

Cell ---

Eusion.

thymidine

inactivated

Sendai

the

previously

methods

putative

hybrid

each were

cover

which

contained

obtained

from

slips;

it

only such

therefore a single

a glass event.

before

of

tissue

chip

the

signs

of

After were

and designated

of

later

The resultant

enzyme and chromosomal

carried

trypsin,

colony

analyses

were

many

contained

cover

slip below

represents grown

by

and 200 cells

reported

were

out

medium,

The dishes

probably

clones

were

to remove

Each hybrid

having

B-propiolactone-

in selective

with

concen-

clones

by addition

dishes.

and therefore

to increasing

activities.

treated

low

in HMT medium

develop

hybrids

tyro-

are very

hydroxylase

one month

culture

of toxicity

survive

exposed

the

thereafter.

and both

eventually

enhanced

second

and 10 pig/ml

activities

cultures

was possible colony.

week,

Cam-

of benzo[aJ-

pg/ml

and therefore

selection (1).

0.5

hydroxylase

hydrocarbon

in one flask

in a series

hybridization

in HMT medium

described

Technology,

benzo[a]pyrene-resistant

that

process,

and the

colonies

plated

broken

virus,

week,

will

aryl

of

no morphological

kinase

data)

The fusion

lacks

generously

per ml was selected

hydrocarbon

and inducible

3T3 line

fourth

showed

subline:

(unpublished

but

concentrations

first

the

of 7,12-dimethylbenz[a]anthracene

negligible

ent

AHH-)

HTC subline

(TAT+)

Institute

10 pg benzo[a]pyrene

and aryl

These

pg/ml

growing

TAT-,

phsophori-

This

aminotransferase

in increasing

5 pg/ml

(BPR,

or absent.

grown

week,

of

in HMT medium.

Massachusetts

0.1

hypoxanthine-guanine

kinase-deficient

third

levels

3T3-4(B)

sine

the

actively

to continuous

medium:

RESEARCH COMMUNICATIONS

(AHH-).

is a thymidine Green,

pg/ml

survive

activity

Howard

growth

lack

tyrosine

hydroxylase clone

cells

cannot

glucocorticoid-inducible

The 31'3-4(E)

week,

These

and therefore

hydrocarbon

given

as 10m4M.

AND BIOPHYSICAL

pieces was

an independ-

an additional

month

performed.

RESULTS AND DISCUSSION The 3T3-4(E) of 68 to 69 (Table

line I).

had only

telocentric

The 3T3-4(E)

chromosomes

(BPR, TAT-,

607

AHH-)

(1) variant

and a narrow contained

range 59 to

line

Cell

sublines

10 11 12 13 15 ABl Al32 AB3 AB5 AB7 AB8 AB4 AB6

to

Table

I

110-137 115-123 97-109 103-111 90-101 100-105 98-109 119-129 92-101 103-105 101-112 95-111 91-115 57-59

59-69 58-12qa

range

24-31 28-31 29-34 24-29 22-24 23-27 24-27 26-29 24-26 25-28 26-29 23-26 20-26 12-19

0 29-5ga

0

percent Bi-armed

induc'ble (BP',

Aryl

and

co.2 co.2 co.2 co.2 co.2 0.3 0.4 0.4 co.2 0.2 0.4 1.1 0.6

0.3 co.2

3.2

enzyme

clones.

in

--et the

al.

25% were 1).

0.8 0.3 0.6 0.2 1.4 1.4 co.1 1.9

8.4

0.8

Basal

clustered

Tyrosine aminotransferase

activity

activitities 13 hybrid

and about (CT. ref.

Benz[a]anthracenetreated

Specific

aminotransferase sublines,

58 to 67 chromosomes, metaphase were bi-armed

co.2 co.2 co.2 co.2 co.2 <0.2 <0.2 <0.2 co.2 <0.2 0.2 co.2 1.2

o.2b co.2

0.2

Basal

hydrocarbon hydroxylase

hydroxylase and TAT , AHH - ) parent

AHH-) metaphases contained aFifty percent of HTC (ARl, TAT+, and 129. Approximately one-half of the chromosomes in each b Below the limits of sensitivity for the enzyme assay.

Footnotes

Clone

Hybrids

)

68-69

Total

Chromosomes

Chromosomal complqment and maximally the HTC (ARl, TAT , AHH-) and 3T3-4(E)

3~3-~(E)(BP~,+TAT-,-MIH-) HTC (ARl, TAT , AHH

Parental

3T3-4(E)

I.

Table

Thompson

line,

between


16.3

0.8

121

Dexamethasonetreated

3T3-4(E)

5

Vol. 56, No. 3, 1974

69 chromosomes 62 and 67. centric.

with

All

no strong

As noted

about

by differential

(1))

and by survival (BPR,

in the

process tissue

nies,

obtained

grown were

ducible to

six

tions

of

continuous

known

to

induce

cells--failed

to

differences

from

hydroxylase,

element

bimodal

distribution

hybrid

The basal

enzyme

of exposure In the

(1,

7-10).

the

activity

Cells

AHH-),

(1);

is

through

then

with

in the

20 putative

Thirteen

hybrid

other

cells

clone

3T3-4(E)

the

13 hybrids,

clones.

some negative Clone

than

or decreased

AHH-)

slightly

AB6 had a

with

extreme

TAT-,

interesting

in BA-inducible

consistent

was higher

one

in 3T3-4(E)

showed

of

in-

concentra.

extent

rise

showing

(BPR,

for

13 PM BA--the

and loss

colo-

in detail

fullest

some cells

other

was unchanged

to those

each of

a significant

segregation

with

or the

equal

and the

Two clones

AB4 showed

in this

lines

a mock fusion

examined

to its

activity.

and with

chromosome

survived

In 11 of

cultures

activity

chromosomes,

line

were

the

present

deter-

parental

in numbers

chips.

activity.

with

still

slip

karyotype,

Clone

of about

therefore

of the

none

telo-

one month.

on cover

of

number were

between

were

a 3T3 marker

separately

after

enzyme

activity

a 1s + 1s

segregation.

that

in either

parent

the

subline,

upon one to six

days

to BA. 8 clones

zyme was uniformly consistent

TAT+,

medium

hydroxylase

segregated

cell

Hybrids

by following

carried

treatment

of

3T3-4(E)

and the

of

consistent

hydroxylase

original

basis

rest.

which

partially

were

colonies

stimulate the

a modal

HMT medium.

discarded

the

a result

control

counts,

or aminotransferase

days

contains

32 ranged subline

was done on 5 to 10 metaphases

on the

hydroxylase

parent

are bi-armed.

in selective

as single

identified

HTC line

RESEARCH COMMUNICATIONS

counted,

mouse-derived

and HTC (ARl,

Chromomsomal analysis hybrids

the

experiments,

plates

40 metaphases

selective

AHH-)

were

of

of which

in the

AND BIOPHYSICAL

this

chromosome

fusion

culture

(l),

half

TAT-,

and then

from

before

mined

3T3-4(E)

mode;

chromosomes

62 chromosomes,

used

BIOCHEMICAL

with

examined

for

low and not results

A recent

from study

tyrosine

inducible several

(11)

aminotransferase by dexamethasone.

previous

involving

studies

mouse-mouse

609

of

activity, This fused-cell

the

en-

finding

is

hybrids

and human-hamster

hybrids

Vol.

56,

No.

3, 1974

demonstrated on the

BIOCHEMICAL

varying

parental

degrees

lines

hybrids

specific

activity

between

activity

of about

3.0

paper, the

hybrids

formed

seen

in the

protein,

might

process

have

inducible

3T3-4(E)

that

the

(1)

the

created

some dominant

hydroxylase

activity.

with

is

of aryl caused

We cannot,

hydroxylase

cross

described

3T3-4(E) factor

(BPR, which

hydrocarbon

has the

it

these

this lacked results induction

in 3T3-specific

rule

AHH-)

in

hydroxylase

by an increase

TAT-,

specific

because that

x (i.e. --

induced

We believe

however,

depended

3T3-4(E)(mouse)

a maximally

In the

enhancement.

activity.

by which

(l),

and a 3T3 variant--selected

enhancement

x HTC hybrids

that

of BA-inducible

parent.

COMMUNICATIONS

activity

report

and 62) compared

by HTC cells

and hydroxylase the

enhancement

to show this

the view

in 3T3-4(E)

2.5

RESEARCH

hydroxylase

In our previous

a marked

hydroxylase--failed

reinforce

that

showed

BIOPHYSICAL

of BA-induced

involved.

HTC(rat)

AND

out

variant

capacity

the

mRNA.

possibility

was selected to repress

BA-

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Benedict, W. F., Nebert, D. W., and Thompson, E. B. (1972) Proc. Nat. Acad. Sci. U.S.A. 69, 2179-2183. Grover, P. L., Sims, P., Huberman, E., Marquardt, H., Kuroki, T., and Heidelberger, C. (1971) Proc. Nat. Acad. Sci. U.S.A. 68, 1098-1101. Benedict, W. F., Gielen, J. E., and Nebert, D. W. (1972) Int. .I. Cancer 2, 435-451. Gelboin, H. V., Huberman, E., and Sachs, L. (1969) Proc. Nat. Acad. Sci. U.S.A. 64, 1188-1194. Levisohn, S. R., and Thompson, E. B. (1973) J. Cell Physiol. 81, 225-232. Matsuya, Y., and Green, H. (1969) Science 163, 697-698. Thompson, E. B., and Gelehrter, T. D. (1971) Proc. Nat. Acad. Sci. U.S.A. 68, 2589-2593. Schneider, J. A., and Weiss, M. C. (1971) Proc. Nat. Acad. Sci. U.S.A. 68, 127-131. Weiss, M. C., and Chaplain, M. (1971) Proc. Nat. Acad. Sci. U.S.A. 68, 3026-3030. Weibel, F. J., Gelboin, H. V., and Coon, H. G. (1972) Proc. Nat. Acad. Sci. U.S.A. fi, 3580-3584.

610