Arrest of C1300 neuroblastoma cells by limiting serum or isoleucine: Implications for growth control in malignant cells

Arrest of C1300 neuroblastoma cells by limiting serum or isoleucine: Implications for growth control in malignant cells

Vol. 68, No. 4, 1976 ARREST BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS OF Cl300 NEUROBLASTOMA CELLS OR ISOLEUCINE: IMPLICATIONS MALIG...

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Vol. 68, No. 4, 1976

ARREST

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF Cl300

NEUROBLASTOMA

CELLS

OR ISOLEUCINE:

IMPLICATIONS MALIGNANT

BY LIMITING

FOR GROWTH CELLS*

Michael

E.

SERUM

CONTROL

IN

Baker

Molecular Biology Laboratory, The Salk Institute Post Office Box 1809, San Diego, California 92112 Received

December

9,1975 SUMMARY

The arrest of Cl300 neuroblastoma cells by limiting serum or isoleucine in the growth medium is described. The resumption of DNA synthesis after the return of the cells to complete medium indicates that they stop in the early G1 (or Go) phase of the cell cycle with both arrest procedures. However, the isoleucine limitation procedure also arrests about half of the cells in the G2 phase of the cell cycle. This result is used to modify a recent model for growth control of transformed cells. The has been limiting

regulation studied serum

(or Go) phase resume

insulin

studied

(2, 3),

medium

a high daily,

some

by density

the cells

being

to 105/ml. * Supported by Senior Division of the American Copyright All rights

0 1976 by Academic Press, of reproduction in any form

(5),

cells

cells

suitable

culture

(1, 2).

By

can be arrested

in G1

additions

to

approach,

made

the role

and nutrients

is not as well

of serum

(4) have

inhibition

(5 x 106/m1) released

from

fellowship Society. 1059

developed

of the cell

in synchronizing

dependent

Dernham Cancer Inc. reserved.

this

in one part

success

of cells

with

in tissue

been

synthesis.

cells

density

these

With

the situation

these

has been

in suspension,

requires

cells

(4),

nucleotides

of DNA

cells

untransformed

and then

S phase.

cyclic

of arresting there

acids

cycle

and enter

in mammalian with

or amino

transformed

difficulty

grow

(l-3)

synthesis

extensively

in the process

For

ever,

most

of the cell

growth

(l-3),

of DNA

cycle

L929 (8).

and changing

cells, This

from

(6, 7).

How-

which technique

the growth

G1 (or Go) phase

D189

due to the

by dilution

the California

Vol. 68, No. 4, 1976

This

BIOCHEMICAL

report

is concerned

neuroblastoma

cell

culture

(9-11).

been

in 1969 extensively

the process

some

limited

evidence

limitation

paper,

synthesis tumor

in the mouse

of sympathetic

properties

but there

origin

of this

has not been

Cl300

cell

much

put into line

have

emphasis

on

synthesis. properties

(12,13,15)

for

with

were

of synchrony

medium.

and the time

and a modification cells

(12-14)

these

cells

when

course

of DNA

synthesis

arrest

of Cl300

cells

for

likely

in one part

of arresting

model

investigated,

it seemed

the results

of a recent

being

of arrest

Therefore

arresting

I report

is contrasted

DNA

differentiated

as the neuronal

be developed In this

This

The

in the growth

could

with

a spontaneous

studied

of DNA

However, was

line,

AND BIOPHYSICAL RESEARCH COMMUNlCATlCNS

serum that

was procedure

of the cell

cycle.

Cl300

cells

by serum

on adding

back

serum.

by isoleucine

growth

there

control

limitation

(16)

in transformed

(7) is proposed. MATERIALS

AND

METHODS

The mouse Cl300 cell line, Clone la, was obtained from Cell cultures. Dr. David Schubert. Stock cells were cultured in suspension in plastic petri dishes (Falcon) at 37OC in a CO2 incubator in Dulbecco-Vogt’s modified Eagle’s medium (DEM) (17) plus 20% fetal calf serum. Cell doubling time was about 20 hrs. Arresting procedure. a) Serum arrest -- cells which had been in DEM t 10% calf serum for 3 days were plated in DEM plus 2% calf serum at 2 x lo6 cells/lOOmm petri dish (20 ml medium). After 3$ays the cells were spun down, washed lx with DEM and replated at 2 x 10 cells/60mm petri dish As will be shown later, most of the cells in fresh DEM for 20-24 hrs. are then in the early Gl (or Go) phase of the cell cycle. b) Isoleucine arrest -- cells which had been in DEM t 10% calf serum for 3 days were plated in DEM ( -isoleucine ) plus 10% Sephadex G50 filtered calf serum 2 x lo6 cells/lOOmm petri dish (20 ml medium) for 2 days. Flow microfluorometric analyses. Two procedures were used for staining the cells for DNA, the procedure of Tobey et al. (18) and of Crissman and gave similar patterns for DNA distributions Tobey (2 3). Both procedures in the cells. Cell blue

vialility, in d stilled

Trypan water).

blue was made Immediately

up as a stock solution (0. 2% trypan before use, 4 parts of this stock

1060

BIOCHEMICAL

Vol. 68, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cells were solution were mixed with 1 part of 42. 5 g/l solution of NaCl. immersed in this solution for 3 mins and scored in a hemacytometer for uptake of the dye. Over 100 cells were counted in each of 3 separate samples. Cell growth assay. Cells were plated at 105/60mm petri calf serum. The cell number was determined in triplicate after plating using a Coulter Counter. The variation for less than 10%.

dish in DEM t 10% at different times each time point was

RESULTS Reversible

arrest

limitation.

The

treated

with

synthesis hrs

time

is about

In Fig.

and after

of Cl300

course

the arresting

2 which

later.

before

of growth

procedure 50-fold

2 the flow

arrest

of DNA

(Fig.

neuroblastoma

cells

by serum

synthesis

after

serum

addition

is shown

in Fig.

1.

higher

than

controls,

microfluorometric

(FMF)

2a and 2b) and after

serum

A wave

begins

for of DNA

about

analysis additions

cells

12-14

of cultures (Fig.

2c

and 2d) is shown.

4

12

20 26 TIME (hours

36 1

44

Fig. 1. Initiation of DNA synthesis in Cl300 neuroblastoma cells by serum. Arrested C1300+cells (2 x 10 ) were plated in Dulbecco’s modified Eagle’s medium (DEM) - 10% calf serum in 6Omm petri dishes. Cultures were pulsed with 10 PCi [3H]-thymidine in 0. 10 ml of 1.5 x 10m4 M non-radioactive thymidine for 2-4 hr at different times after plating. Cells were collected on glass fiber filter papers, washed 2x with Tris-saline buffer, 2x with 10% TCA and lx with 957’0 ethanol. The filter papers were counted in scintillation fluid containing 4. 2% Liquifluor in a liquid scintillation counter. CPM were normalized to a 2 hr [3H]-thymidine pulse. 0 : DEM; 0 : DEM t 10% calf serum. 1061

Vol. 68, No. 4, 1976

When synthesis cell

Cl300

cells

occurs

about

doubling

arrested

BIOCHEMICAL

time

Gl

arrest. by limiting

the peak

of DNA

nutrient

to the growth

tion

than

with

arrested

to complete

cells

stop

procedure

serum

limitation.

Geimsa

stained

The over doubles

about

CHANNEL (AMOUNT

Gl

NO. OF DNA)

the cells

of DNA

1).

Since

have

been

C 1300

medium

(16).

about

10 hr after

Clone

Cla

cells In this

the return

(or Go), reveals

exclude after

figure

the

were instance,

of this

and also than

the peak

stain

trypan

of DNA

NO. OF DNA)

the return

blue

microscopic

CHANNEL (AMOUNT

are

the cell and their

synthesis

NO. OF DNA)

of the cells

incorporation

1) it is apparent

to reenter

cells

in medium

as thymidine

since

distribu-

of Cl300

2 days

0. 3% of the cells

and able

the vital

analysis

after

in Fig. in G2,

a different

after

(as well

as described

viable

CHANNEL (AMOUNT

first

at 28 and 52 hrs

less

are

and find

3, the FMF

is shown,

this

done

cells

15 hrs

(Fig.

Recently,

In Fig.

From

cells

95% of them

with

and then

shown]

arrested

that

in the growth

limitation (t=O),

in early

addition

it appears

occurred

this

[not

the peak

medium.

medium.

experiments

serum

limitation.

isoleucine

isoleucine

limitation,

(or Go).

by isoleucine

lacking

20 hrs,

synthesis

used

by serum

2.4 hr after’the

by iaoleucine

arrested

I have

arrested

is about

in early

Reversible

are

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that analysis

in mitosis. cycle cell

since number

(1.2).

CHANNEL (AMOUNT

NO OF DNA)

Fig. 2. Flow microfluorometric analysis of Cl300 cells before and after arrest by serum limitation and after the addition of 10% calf serum. (A) before arrest; (B) after arrest; (C) t=18 hr in DEM t 10% calf serum; (D) t=25 hr in DEM f 10% calf serum.

1062

these of

Vol. 68, No. 4, 1976

Single

BIOCHEMICAL

restriction

cells

which

cells

may

are

time

difficult

Usually,

Using

after

being

this

becomes

unfavorable after cells

Multiple

R,

cell

(16)

types

of transformed

ever,

in Cl300

Clone

where

the cells

arrest

for

observations R points

for for

of serum

normal

growth.

suggest cells

growth Cl300

transformed

la,

that

cells.

with

and the length

cells

are

cells

are

The

(4, 7).

called

results

the medium of normal

growth

and therefore,

of this

procedures,

of Go (Figs.

a

is limited. paper

and of

at least

restriction

growth

controls

some

point.

How-

the phase

2 and 3).

cells.

It may

property then

the same

cycle

of the R point

of limiting

separate,

syn-

experiments,

when

at a single

a general

resume

limited

other

growth

in transformed

in

will

the absence

appropriate

can be arrested the method

were

growing

when

components

at about

in the cell

a loss

cycle

Cl300

control

factor(s)

In his model,

the cell for

medium

stop

involves

untransformed

cells

of several

cells

transformed

one of several

is one point

transformation

et al.

Hypothesis

where

points

cycle,

to complete

that there

stop throughout

of the cell

untransformed

and the results

for

restriction

Revoltella

arrested

observation

point,

virus

(or Go) by limiting

nutrient

(7) has proposed

Unlike

cells.

in one part

returned

of which

restriction

these

to arrest

medium.

regardless

control

untransformed

in Gl

DNA

Pardee

for

be arrested

the growth thesizing

point

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

be that

of transformed growth

control

these cells.

If

becomes

C

6,

s

6

f-\ CHANNEL (AMOUNT

NO. OF DNA)

Fig. 3, Flow microfluorometric isoleucine limitation, (B) t=28 in DEM t 10% calf serum.

CHANNEL (AMOUNT

NO. OF DNA)

analysis hr in DEM

1063

CHANNEL (AMOUNT

NO. 0~ DNA)

of Cl300 cells (A) after arrest t 10% calf serum, and (C) t=52

by hr

BIOCHEMICAL

Vol. 68, No. 4, 1976

complex

because

nutrients

Because

of this,

those

limited

growth

an increased loss growth

down

to gradually

Cl300 to DNA

cells

clones arrest.

different

Cl300

differentiated

cells

responses of Revoltella

which

are

part

substance. another

turn

growth

off cell

to involve

DNA

of this

initiation

does

care

of the cycle

due to

not exclude

the alters

is required

the differences

is to suggest relationship

step

cycle.

the Cla

(23)

in

proper

of the cell

properties

and its

(21,221.

transformation

between that

neuronal

point

but that with

limitation

consequence

to study

more

in one part

(16) indicates

growth

of the cell This

cells

to isoleucine

known

on cell

way cell

can be arrested

One exciting clones

effects

transformed

et al.

RESEARCH COMMUNICATIONS

not at the restriction

but suggests

that for

these

are

a second

implies

different

and those

for

cells.

procedures,

synergistic

stop at a different

in normal

conditions

The

which

may

point(s)

model

choosing

cells

requirement

control This

can exert

substance

of restriction

AND BIOPHYSICAL

clone between

also

extend

the use of to the

process. ACKNOWLEDGMENT

I wish to thank Dr. David Schubert for the gift of the Cl300 cells and I am grateful to Dr. Uta Francke for her help in advice in culturing them. the microscropic analysis of Geimsa stained isoleucine arrested cells. The advice and support of Dr. Robert Holley is appreciated. I am grateful for the excellent technical assistance of Ingrid Klinger. I thank Mr. Nathan Belcher of Pfizer Inc. for his gift of mithramycin which was used in some of the FMF experiments. REFERENCES 1. 2.

3. 4. 5. 6.

Todaro, G. J., Lazar, G. K., and Green, H. (1965) J. Cell. Comp. Physiol. 66, 325-334. Temin, H. M., Pierson, R. W., Jr., and Dulak, N. C. (1972) In Growth, Nutrition and Metabolism of Cells in Culture, V. I. Cristofalo and G. Rothblat, eds. (New York:Academic Press), p. 49-81. Halley, R. W., and Kiernan, J, A. (1974) Proc. Nat. Acad. Sci. USA 71, 2908-2911. Holley, R. W., and Kiernan, J. A. (1974) Proc. Nat. Acad. Sci. USA 71, 2942-2945. Seifert, W. E., and Rudland, P. S. (1974) Nature 248, 138-139. Paul, D. (1973) Biochem. Biophys. Res. Commun. 2, 745-753.

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BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

7. Pardee, A. B. (1974) Proc. Nat. Acad. Sci. USA 5, 1286-1290. 8. Glinos, A. D., and Weerlein, R. J. (1972) J. Cell. Physiol. 2, 79-90. 9. Augusti-Tocco, G., and Sato, G. (1969) Proc. Nat. Acad. Sci. USA 64, 311-315. 10. Schubert, D., Humphreys, S., Baroni, C., and Cohn, M. (1969) Proc. Nat. Acad. Sci. USA 64, 316-323. 11. Klebe, R. J. and Ruddle, F. H. (1969) J. Cell Biol. 43, 69A. 12. Seeds, N. W., Gilman, A. G., Amano, T., and Nirenberg, M. W. (1970) Proc, Nat. Acad. Sci. USA 66, 160-167. 13. Schubert, D., Humphreys, S., devitry, F., and Jacob, F. (1971) Developmental Biol. 25, 514-546. 14. Kates, J. R., Winterton, R., and Schlessinger, K. (1971) Nature 2, 345-347. (1973) In The Role of Cycle Nucleotides 15. Furmemski, P., and Lubin, M. in Carcinogenesis, J. Schultz and H. G. Bratzner, eds. (New York: Academic Press), p. 239-261. 16. Revoltella, R. , Bertolini, L., and Pediconi, M. (1974) Exp. Cell Res. 85, 89-94. R. (1963) Proc. Nat. Acad. Sci. USA 2, 17. Vogt, M., and Dulbecco, 171-179. Crissman, H. A., and Kraemer, P. M. (1972) J. Cell 18. Tobey, R. A., Biol. 3, 638-645. M. A., Trujillo, T. T., Mullaney, P. F., and Coulter, 19. Van Dilla, J. R. (1969) ScienceE, 1213-1214. H. A., and Tobey, R. A. (1974) Science 184, 1297- 1298. 20. Crissman, (1959) Science 130, 432-437. 21. Eagle, H. R. G. (1974) In Vitro 10, 119-129. 22. Ham, X. O., and Nirezerg, M. W. (1974) Proc. Nat. Acad. 23. Breakefield, Sci. USA -71, 2530-2533.

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