BIOCHEMICAL

Vol. 63, No. 4, 1975

INSULIN

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

INDUCTION OF TYROSINE AMINCYIRANSFERASE IN SYNCHRONIZED HEPATOMA CELLS

Janet

Rettig

Emanuel*

-I

and Thomas D. Gelehrter

Department of Human Genetics, Yale University of Human Genetics New Haven, CT 06510, and-!-Departments and Internal Medicine, University of Michigan Ann Arbor, MI 48104 Received

February

20,1975

Summary: Addition of insulin to unsynchronized rat hepatoma cells, previously incubated with dexamethasone, promotes a rapid increase in tyrosine aminotransferase activity. Mitotic cells in the presence or absence of colcemid are not inducible by either insulin or dexamethasone. Inducibility by dexamethasone is restored only after the third hour of the Gl phase, presumably related to the appearance of the putative posttranscriptional repressor (Martin, D.W. et al. (1969). Proc. Nat. Acad. Sci., 63: 842-849). In contrast, inducibility by insulin is restored immediately upOn passage of the synchronized cells into Gl. These data suggest that insulin can induce tyrosine aminotransferase in the absence of the repressor. In an unsynchronized culture the

(HTCl)

rate

anism

of

normal

of

rat

liver

(4),

insulin

(5)

or dialyzed

iously

induced

with

causes

a further

which that

is

independent

to steroid bovine

control

In an attempt

Copyright All rights

used

o 1975 by Academic Press, Inc. of reproduction in any form reserved.

(6)

amount

cycle;

all

expression further

HTC, hepatoma

825

as well

defined,

affect of

the the

tissue

enzyme, We have

synthesis Gl.

derived only

in

is

con-

Addition

of

HTC cells,

prev-

serum-free

medium,

by a mechanism shown previously aspects

same enzyme

culture;

by a mech-

of TAT'

different

differences

in

as cells

induction enzyme

tissue

increase

2.1.1.5)

to unsynchronized

induced

hepatoma

a lo-fold

G2, M and early

of

and aerum of

cell

RNA synthesis.

to elucidate

are:

the

in a chemically

in the

insulin

(2,3,4)

to steroid

during

serum

of concomitant

corticosteroids,

The abbreviations aminotransferase.

of

rat

(E.C.

HTC cells

sensitive

dexamethasone

post-transcriptional

1

(1). are

doubling

induce

aminotransferase

Gl and S phases

and insensitive

of established

hormones

tyrosine

RNA synthesis

the mid and late stitutive

corticosteroid

synthesis

requiring

from

cells,

population

of

the

(7). in the

modes of

and TAT,

tyrosine

Vol. 63, No. 4,1975

action

of

BIOCHEMICAL

these

inducers,

induction

in different

ulations

of HTC cells

ed monolayer

we have phases

were

cultures.

dexamethasone

from

obtained

We have

that

studied

of the

the

cell

RESEARCH COMMUNICATIONS

sensitivity

cycle.

of TAT to insulin

Highly

by mitotic

observed

show a cell-cycle

is different

AND BIOPHYSICAL

synchronized

selection

that

cells

phase-specific

from

colcemid-treat-

previously

sensitivity

pop-

induced to insulin

with which

to dexamethasone.

METHODS A.

Cell

growth

culture

as previously

monolayer

prior

This

allowed

normal

level

previously pM)was

cells,

and yet

cell

cycle

side

transport

least

parameters,

150 x 25 mm Falcon (Eagle)

supplemented Cultures

chloride. was replaced growing

were

and the

of mitotic

cells,

added.

Colcemid prior

divisions

of colcemid

was added

incubated

hours

to a final

to harvest.

plate

was removed

appear

of mitotic

concentration mitotic

lo-15

826

calf

measurable

to block

nucleo-

after

Twenty-four

Medium

and 0.02%

calcium

the medium

20 hours; hours

prior

half-serum

$I dexamethasone

of 0.25 were

PM at

three

harvested

per

Essential

which

and calcium-free 0.1

cells

x lo6

sera,

an additional

cells

"mitotic

to alter

necessary

at 37'C

to harvest

comparable

(9).

and 5% fetal

for

conditions

of colcemid

yield

by plating

confluent.

Detached

these

and irregular

cells

20 hours

of TAT and

was also

highest

not

24 hours

(6)

Under

in

complement, for

in 30 ml of Minimal

incubated

prior

serum

concentration

the level

established

and not

Sixteen

below

of hepatoma

5% bovine

the medium

did

and induced

induction

cells

cell

in spinner

cells

(2,S).

the

cultures

logarithmically

behavior

produced

culture

with

serum

which

were

usual

growing

The effective

line

tissue

the

(2).

and was far

cultures

half

maintained

synchronized

synchronized

multipolar

in another

were

minimal

cycle

concentration

Monolayer

hours

and cell

as that

the

this

figures";

medium

provided

reported

chosen

with

were

logarithmically

of TAT in steroid-induced

to that (0.25

over

medium

growth

medium

stocks

Cells

(7).

in growth

by incubation

to use.

HTC cell

described

cultures

conditioned

the

-and synchrony:

cells

were

to harvest growth was added.

to twelve by aspiration

aceto-orcein

as

“mitotic

figures”)

to

cells

total

B.

Assays

ation

of

1 UCi

cells

for

60

synthesis

into

10% -3 1 H]

Novelli

--RESULTS

TAT

and

the

without

ratio

of

mitotic

cells

tritium

counts

per

material

minute

after

incub-

Ci/mM;

Amersham)

with

5 x

incubation

mixture

were

directly

assayed was

by

in

the

in

mitotic

cells

in

(1-C)

show

insulin (1-D).

the

by

the

assayed

method

of

milli-units

method

as

Lowry

per

rig/ml)

mitotic

exhibit

mg

of

Mans

described

--et

al.

lo5

prev(12).

Enzyme

protein.

addition, Interphase

an

colcemid

of

even

when remaining

a response

cultures

(1-B)

show

Mitotic

cells

collected

is after

in

827

is

TAT

with

that of

contrast, two

incubation from

that

hours med-

colcemid-

unsynchronized also

seen

unsynchron

about the

colcemid

dexamethasone

above

maximal

of

in

activity

selection

to

that

concentration

In

in

mitotic

absence

than

the

inducing

(1-A).

present

similar the

in

alone

colcemid

cells

higher

a maximally

which

(2,3,4),

induced

increase

response

insulin

previously

previously

colcemid

immediate

to

significantly

and no

responsiveness reported

is cells

virtually

presence

As

cells

Mitotic of

in

cells.

HTC

presence

the

difference

HTC

cells.

(4

treated

of

discs

as

total

19

or

cells”.

precipitable

‘SA.

measured

interphase

unsynchronized

insulin

ium

acid

expressed

induced

of

after

as

aminotransferase

was

and in

cells

mitotic

aliquots

1 demonstrates

incubated

ized

“percent

quantitated

filter

The

(with

DISCUSSION

activity

and

chromatin

boundary.

thymidine

Tyrosine

mitotic

induced

as

3MM

was

Figure between

expressed

-methyl

Protein

AND

nuclear

was

Whatman

activity

clumped

visible

Duplicate

(10).

(11).

tightly

trichloroacetic

minutes. on

specific

is

DNA

precipitated

iously

no

scored

:

with

and

incorporated

and

cells

cells. show

no

response

Vol. 63, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

C

“b / /” -.A

0

I

2

0

I

2

0

1

2

0

1

2

HOURS

Figure 1: Insulin induction of TAT in mitotic and interphase cells. After exposure to colcemid for 12hours as described in Methods, cells were collected and resuspended at 5 x 105 cells/ml in conditioned half-serum growth medium containing steroid and colcemid, and maintained as spinner cultures. A, mitotic cells; B, interphase cells remaining after mitotic selection. Parallel cultures of HTC cells were incubated under identical conditions except that they were not exposed to colcemid. C, unsynchronized cells never exposed to colcemid; 2, unsynchronized cells with colcemid added at time of resuspension. Each cell population was divided and TAT activity measured on one ml aliquots of cells after 0, 1, 2 and 2 l/2 hours incubation with (0-O) or without (0 - 0) 4 rig/ml insulin. to insulin,

and Gl cells

to insulin,

further

suggesting

insulin

induction

for

lack

of

ure

of response

ent

in

in mitotic

with

lower

that

cell

cycle

putative On the

(13). repressor,

basis

of

(0.01

that

levels

Inducing

steroids

with

'8).

also

induced

the

during

the metabolic

are

828

responsive

not

Nor can

responsible the

fail-

already to

pres-

increase

TAT

protein)

18). tyrosine

of the

to antagonize

the

aminotransferase

enzyme

inducible

TAT mRNA and allowing inhibitors,

is

failed

of

steroid

thought

fully

'80 milliunits/mg

expression

the

are

of TAT activity

repressor

only

stablizing

cells

nM) of dexamethasone

is produced

studies

mitotic

added

of colcemid

as insulin

by a post-transcriptional which

presence

high

cells,

It has been proposed

mRNA (l),

has been

half-maximally

concentrations

is regulated

the

by the

mitotic cells

colcemid

in these

be explained

steroid-induced

activity

to which

specific phases the

of

the

action

of

it

to accumulate.

insulin

mediated

induc-

the

Vol. 63, No. 4, 1975

BIOCHEMICAL

0

Lx

,

2

4

AND BiOPHYSlCAL

RESEARCH COMMUNICATIONS

x 8

6

12

IO

0

14

:

16

E

HOURS

Figure 2: Insulin induction of TAT during the cell cycle. Mitotic collected after 12 hours incubation with colcemid, resuspended at ml in conditioned half-serum growth medium containing steroid but and maintained as spinner cultures. Percent mitotic cells, X-X, thymidine incorporation into DNA, m - l , were monitored as markers cycle phase as described in Methods. At 0, 2, 4, 7, 10 hours after of cells, TAT activity was measured and insulin (4 bg/ml) added to TAT activity was measured 1, 2, 2 l/2 hours after insulin addition.\@ dexamethasone only;{0 - Oldexamethasone plus insulin. r tion

of TAT synthesis,

ciency

in contrast,

of utilization

of existing

The action cycle

and thus

repressor

is

through

mitosis

to the shown into

sition

(8);

(2-4),

the high

into

the

similar cells

to the

level

are

acquired phase. tive

to induction

that

insulin

insulin

induction;

passage

into

a period

early but

of TAT is

not

of cells

in percent synthesis

Gl,

later.

829

of the

cells

parallels

cells

level

this

reported

persists

in late

Gl and S,

Mitotic to insulin

and is maintained

by dexamethasone.

mitotic

As previously

sensitivity

in Gl in which

independent

cell

passing

that

populations. but

to the

of post-transcriptional

to a lower

to

by insulin,

induction

a decline

in unsynchronized

is

relative

of TAT in mitotic

observed

upon

there

activity

with

HTC cells

by the decrease

10 hours

effi-

TAT (5,14,15).

synchrony

in RNA and protein

induced

unresponsive

Thus,

induced

is tight

about

an increased

mRNA for

or absence

There

begins

of Gl,

immediately

2.

increase

steroid part

presence

Gl as measured

DNA synthesis

early

in steroid

putative

in Figure

to represent

cytoplasmic

of insulin

21, and a sharp

(Fig.

appears

cells were 5 x 105 cells/ no colcemid, and [3H ]of cell collection subcultures. -e,.,

is

well

into

S

TAT synthesis

is

sensi-

Therefore, presence

of

it

appears

the postulated

tran

Vol. 63, No. 4,1975

BIOCHEMICAL

post-transcriptional observation

repressor.

that

"superinduced" repressor

insulin with

is

thought

basic

Two

insulin

seen

inery

of mitotic

receptors diminished

mechanisms

rate

protein

synthesis elongation

protein

is not

synthesis

and total

protein

synthesis

by insulin. The cellular

receptors

on the

response

cell

selectively

composition

of the

lectins

availability this insulin

the

both

(25,26) of the

interpretation binding

is

the

to HTC cells

of

(19-21). of the

negate

shown

on the

preliminary is

as well

the

is

cell

rate

finding

of initia-

the

rate

by specific

the magnitude

phase,

the

of the

receptors which

Since

'in mitotic

same in mitotic

TAT (5) of TAT

the

as agglutinability

that

of

stimulation

(22,23).

limiting

of total

of mRNA for the

differ-

rate

to be a glycoprotein

cycle

greatly

in mitotic

of available

germ agglutinin

receptor

in the

insulin

in the

reduced

Consequently,

has been

is

to be mediated

concentration

(24)

the

mach-

that

translated

initiation

appears

insulin

dependent

insulin

rate

such

may increase

to

membrane is

insulin

might

by wheat cell

that

to

synthetic

synthesis

TAT mRNA is

inhibition

surface

in which

of response

an increase

due to a block

Since

The receptor

recognized

plant

this

response

is a function

and of insulin.

altered.

(l&3),

protein

demonstrated is

lack

surface

is in fact

Thus,

cells

by increasing

already

D, a condition

the protein

cell

total

there

have

in mitotic

by the

in cells

the in

in the

(3).

(17)

for

alteration

Although

HTC cells,

Fan and Penman

supported

TAT activity

be proposed

alteration

of TAT synthesis

also

of actinomycin

(a)

stimulated.

cells.

tion;

(b)

is

RESEARCH COMMUNICATIONS

(16).

might

cells:

cells,

in mitotic

ential

increases

to be absent

not

conclusion

concentrations

in mitotic

are

This

further

high

AND BIOPHYSICAL

can be

glycoprotein

by it

is

possible

cells.

affinity

that

Against of

and interphase

cells

(27).

ACIUKWLEDGEMRNT This work was supported by Grants GM 17712 and GM 15419 from the National Institutes of Health. J.R.E. was supported by a training HD 00198 from the National Institutes of Health.

830

grant

Vol. 63, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

References 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22. 23. 24.

25. 26. 27.

Tomkins, G.M., Gelehrter, T.D., Granner, D.K., Martin, D.W., Samuels, H.S., Thompson, E.B. (1969) Science 166: 1474-1480. Martin, D.W., Tomkins, G.M., GGner, D. (1969) Proc. Nat. Acad. Sci. -*62. 248-55. Martin, D.W., Tomkins, G.M., Bresler, M.A. (1969) Proc. Nat. Acad. Sci. 63: 842-49. Sellers, L. and Granner, D. (1974) J. Cell Biol. 60: 337-45 Gelehrter, T.D. and Tomkins, G.M. (1970) Proc. Nat, Acad. Sci. 66: 890-97. Gelehrter, T.D. and Tomkins, G.M. (1969) Proc. Nat. Acad. Sci. E: 723-30. Gelehrter, T.D., Emanuel, J.R., Spencer, C.J. (1972) J. Biol. CGm -247: 6197-203. Emanuel, J.R. - Unpublished observations. Plageman, G.W. and Erbe, .I. (1974) Cell 2: 71-74. Mans, R.J. and Novelli, G.D. (1961) Archiv. Biochem. Biophys. 94: 48-53. Spencer, C.J. and Gelehrter, T.D. (1974) J. Biol. Chem. 249: 577-83. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.JT1951) J. Biol. Chem. -193: 265-75. Martin, D.W. and Tomkins, G.M. (1970) Proc. Nat. Acad. Sci. 65: 1064-68. Bushnell, D.E., Becker, J.E., Potter, V.R. (1974) Biochem. Bzphys. Res. Comm. 56: 815-21. Butcher, F.R., Bushnell, D.E., Becker, J.E. (1972) Exp. Cell Res. 74: 115-23. Tomkins, G.M., Martin, D.W., Stellwagen, R.H., Baxter, J.D., Mamont, P., Levinson, B.B. (1970) Cold Sprg. Hrbr. Symp. 35: 635-40. Fan, H. and Penman, S. (1970) J. Mol. Biol 507655-70. Wool, I.G., Wettenhall, R.E.H., Klein-Bremhzr, H., Abayang, N. (1972) in: Insulin Action, I.B. Fritz, ed., Academic Press, New York, pp. 415-35.P. (1973) Fed. Proc. 32: 1838-46. Cuatrecasas, Garwin, J.L. and Gelehrter, T.D. (1974) Archiv. Biochem. Biophys. -164: 5259. Neville, D.M. (1974) In Vitro 2: 445-54. P. and Tell, G. (1973) Proc. Nat. Acad. Sci. -70: 485-89. Cuatrecasas, Cuatrecasas, P. (1973) J. Biol. Chem. 248: 3528-34. Gahmberg, C.G. and Hakomori, S. (1974)Biochem. Biophys. Res. Comm. 2: 283-91. Fox, T.O., Sheppard, J.R., Burger, M.M. (1971) Proc. Nat. Acad. Sci. 68: 244-47. Smets, L.A. (1973) Nature NB 245: 113-115. c=nication. Hollenberg, M.D., personal

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