Biosynthesis of proteins, nucleic acids and glycosphingolipids by synchronized KB cells

BIOCHEMICAL

Vol. 54, No. 2,1973

BIOSYNTHESIS

OF PROTEINS,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NUCLEIC ACIDS AND GLYCOSPHINGOLIPIDS

BY SYNCHRONIZED KB CELLS Subroto Chatterjee, Charles Departments of Biochemistry, Michigan State University,

Received

July

C. Sweeley, Microbiology East Lansing,

and Leland F. Velicer and Public Health Michigan 48823

27, 1973

Summary The biosynthesis of glycosphingolipids and various types of proteins and nucleic acids at specific periods of the cell cycle was studied by using synchronized KB cells. Maximum incorporation of radioactive galactose, leucine and thymidine into several proteins and nucleic acids occurred as has been reported previously (6,ll). Maximum incorporation of k-l[14C] galactose into glycosphingolipids was observed during the M and G-l phases. There was a 5 fold increase in the levels of gangliosides and combined neutral glycosphingolipids during the M and G-l phases. Thus, regulated biosynthesis of glycosphingolipids and macromolecules might be important in the cyclic expression of some of the functional properties which are characteristic of these compounds. Introduction Synchronized events for

of mammalian

example,

growth with

that

(3) this

exposed

normal

is

and rate

of the

of glycosphingolipids biosynthesis

do not that

cells

such a phenomenon. for

cells,

demonstrated

of Forssman

cells.

hapten

plant

Parallel

agglutinins

whereas

are

such sites

an increase

are

in both

glycosphingolipid

decrease Polyoma

such a phenomenon

of glycosphingolipids

of

(4). (5)

regulatory

inhibition

sites

in normal

biochemical

It has been demonstrated,

exhibit

and a significant

in cell

of various

(l-3).

binding

phase

above findings,

study

density-dependent

and Kijimoto

NIL hamster

not exhibit

Because

cells

of synthesis cells,

culture

exhibit

in neoplastic

Hakomori

NIL hamster

did

cells

the mitotic

exposed

contact-inhibited cells

in tissue

the proposal

during

Recently, level

cells

neoplastic

finding only

have been used in the

normal

while

permanently

the

cells

virus

upon attaining

of this

activity

transformed high

in

NIL hamster

cell

density.

and the possibility

of the

processes,

we have investigated

and various

macromolecules

in

involvement

during

the discrete

Vol.54,No.

phases from

in the

growth

such studies

cellular

cycle

might

control

Materials

provide

of 2,250

block

terminated

insight

procedure

suspension

were

using

Suitable 2 hr time

and temporal

(6).

Hopefully,

relationships

changes

information

between

of these

various

compounds.

as "zero"

withdrawn,

aliquots

of the for

a period

Q-l[14C]galactose

(45-55

mCi/mmole),

2[14C]thymidine

(50 mCi/mmole)

by adding

20 vol

and was followed

were washed

were

twice

each

and then

30 sec.

was

of the counts

were

and the

of the solubilized

samples

pellets

in a Beckman LS-50

D-1[14C]galactose

liquid

with

10% CC13COOH.

labeled

cell

pellets

The acid

was extracted

and centrifuged.

and was saline The

analyses.

mixed

above.

with

5 ml of ice

cold

The pellet

the cell

was extracted

One ml of 1 N NaOH was added

contents

solubilized withdrawn

The pellets

overnight

in one ml of 1 N NaOH; radioactivity

described

above.

cell

material with

to the

Suitable was

spectrometer.

labeled

insoluble 3 times

overnight. and radioactivity

scintillation

586

of

buffer-isotonic 5 min.

at for

30 min at room temperature

and C-U[14C]leucine

3 times

labeled

The reaction

phosphate

were

After

were

1 &i

withdrawn

(270 mCi/mmole),

appropriate

as indicated

material

containing

at 480 xg for

for

were

was pulse

Nuclear).

O.lM

saved

cell for

sample

C-U[*'+C]leucine

5% CC13COOH and centrifuged.

by volume)

and cell

suspensions

in a medium

of ice-cold

centrifuged

precipitable

determined

block

aliquots

blue,

cell

by centrifugation

5% CC13COOH and vortexed

aliquots

by a double

thymidine

Triplicate

trypan

(New England

2[1"C]Thymidine-labeled

once more with

with

of 24 hr;

stirring

suspensions

(to).

synchronized

constant

(pH 7.4),

when the second

time

mixed

was prepared

a hemocytometer.

intervals

terminated

KB cells

The time

1 hr at 37°C with

(2:1,

into

ml of synchronized

was considered

performed

acid

KB cells.

and Methods

thymidine

cells

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of synchronized

mechanisms

A culture

cell

BIOCHEMICAL

2, 1973

5 vol were

air

pellets

were

extracted

from T&1[14C]galactose of chloroform-methanol dried

was measured

and solubilized subsequently

as

BIOCHEMICAL

Vol.54,No.2,1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

of 2[l'+C]thymidine by KB cells synchronized with Fig. 1. Incorporation A culture of 2,250 ml of synchronized KB cells double thymidine (2mM) block. At indicated intervals 2 ml samples was prepared by the method of Bello (6). from the culture were removed and pulse labeled with 1 &i of 2[14C]thymidine for 1 hr. After the incubation period the cell pellets were extracted twice with 5 ml of ice cold 5% TCA, and specific activity determined (-+-). Three 0.5 ml aliquots of the cell suspension were withdrawn, and cell counts (-o-) made at the indicated times. The shaded zone represents the G2-M-G, phases. Fig. 2. Incorporation of I-U[14C]leucine into TCA soluble (-o--) and insoluble fractions (-a-) of KB cell5 synchronized with double thymidine (2mM) block. At indicated time intervals 2 ml samples were withdrawn and incubated for 1 hr with 1 &i of L-U[14C]leucine. Following the incubation period cells were extracted three times with 10% TCA, and specific activity determined. Glycosphingolipids labeled cell

cell

pellets

suspensions (7).

lipids

was determined lipids

from

samples

at to,

Radioactivity

phase

isolated

and control

withdrawn

Suzuki

lower

were

another

collected

were

from 400 ml of unlabeled

fraction

48 hr of dialysis

dried

of JZJ-l[14C]galactose

4, 8, 10 and 12 hr intervals

in the nondialyzable after

series

under

from

against

a stream

according

ice

of nitrogen

cold

upper

to

phase

water.

The

and radioactivity

measured. Gangliosides by the procedure the lower

phase

and quantitated determined

in the

unlabeled

of Svennerholm lipids

of Folch

(8).

of Lowry

phase

Neutral

extracts

by the procedures

by the method

upper

described et aZ.

the standard. 587

lipid

samples

were

glycosphingolipids

quantitated derived

of unlabeled

cell

samples

were

elsewhere

(9).

Protein

was

(10)

using

bovine

serum albumin

from isolated

as

BIOCHEMICAL

Vol.54,No.2,1973

LA-,2 4 6

0

8

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IO I2 I4 16 I8 20 22 24 Time fhrl

Fig. 3. Incorporation of D-[14C]galactose into TCA insoluble, chloroform-methanol (2:1, by vol) extracted pefiets (-o-) of KB cells synchronized with double thymidine (2mM) block. At indicated intervals 5 ml samples were removed and incubated for 1 hr with 2.5 UCi of &-1[14C]galactose. Specific activity was determined as described in Materials and Methods. Fig. 4. Incorporation of D-1[14C]galactose into upper phase (-o-) and lower phase (-o-) lipid fractions of KB cells synchronized with double thymidine (2mM) block. At indicated intervals 10 ml samples were withdrawn and incubated with 5 &i of D-1[14C]galactose. Glycosphingolipids were isolated from the labeled ceT1 pellets and specific activities determined as described.

Results In Fig. total

cell

removal

1, the rate

count

cell

Cell

count S phase

counts Figs.

began

C-U[l%]leucine

doubled

after

unchanged

acid

during

the

throughout

phases

insoluble

period

started

to divide between

of cells

from

S phases

of proteins, of the protein

588

cell (Fig.

as indicated

6-12

hrs.

The

the thymidine

of the cell

block.

cycle. and

Incorporation

2) paralleled

for

and

rapidly

glycoproteins

cycle.

after

was maintained

period

the

with

Immediately

the S phase

This

the cells

activities

at various into

entered

12 hr of removal

2-4 show specific

glycosphingolipids

cells

is compared

KB cells.

of 2[1%]thymidine.

was almost

remained

block,

Subsequently,

7 hrs.

incorporation

of synchronized

thymidine

incorporation

approximately

second

two cycles

of the second

by increased

the

over

of 2[14C]thymidine

of

the pattern

BIOCHEMICAL

Vol. 54, No. 2, 1973

observed

for

precursor that

total

cell

occurred

the

rate

relatively

count

during

the

unchanged

chloroform

approximately

methanol

from

occurred

Later

(2:1,

late

cell

cycle

activities

the

were

the

finding

soluble

released

from

fraction

of both

the upper

was

Figs.

after

at

thymidine

block.

and was

1 and 3).

phase

and lower

incubation

same level

material

occurred

cycle

of labeled

the

insoluble

the second

of the cell (see

incorporation

acid

(glycoprotein)

to another

to approximately

with

acid

into

washing

the M and G, phases

cycle

dropped

by vol)

cells

of this

cycle.

in the S phase

one cell

during

in the

compounds

into

the cell

the

Maximum specific lipids

of L-U[l%]leucine

of LI-1[14C]galactose

4 hr after

reproducible

Maximum incorporation as compared

throughout

The peak was observed

1).

M and G, phase,

of incorporation

Maximum incorporation after

(Fig.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

galactose as that

phase

&1[14C]galactose into

these

observed

during

the

S phase. The concentrations recovered

from cells

in Table

1.

occurred

during

of purified at to,

gangliosides

S, G2, M and G, phases

The maximum increase

in the

the M and G, phases,

glycosphingolipid

content

following

of cells

release

and neutral

which

level

resulting

of the cell

of these

the second

are given

increase

five-fold

thymidine

cycle

glycosphingolipids

in an overall

was approximately

from

glycosphingolipids

within

in lo-12

hrs

block.

Discussion The results that the

several

presented

kinds

ccl 1 cycle

same is

true

in this

communication

of macromolecules

of synchronized

are

mammalian

of glycosphingolipids,

synthesized cells

which

confirm

are

previous

reports

at discrete

(l-3).

phases

of

We can now add that

synthesized

mainly

during

the

M and G1 phases. Although extent

the synthesis

throughout

into

glycoprotein

with

those

reported

of protein

the cell

cycle,

occurred

late

for

synchronized

and glycoprotein

maximum incorporation in the

S phase.

HeLa cells

These (ll),

was evident

to some

of L&l[lV]galactose results

are

and synchronized

consistent

the

BIOCHEMICAL

Vol.54,No.2,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Glycosphingolipid Different Time

Levels

Stages

(hrs)

I

of Human KB Cells

of the Cell

Total

the

Cycle* Total Neutral Glycosphingolipids***

Gangliosides**

tO

During

0.58

0.90

4

(S phase)

0.63

1.05

8

(G2 phase)

0.90

1.54

10

(M phase)

2.38

5.26

12

(Gl phase)

2.72

5.54

*Human KB cells were synchronized by the method of Bello (6). At O(t,), 4, 8, 10, and 12 hr 400 ml of cell suspension were withdrawn (these cells were derived from the same batch of synchronised cells as utilized for the radioactivity incorporation studies) and pelletized. Glycosphingolipids were subsequently isolated and analyzed as described (9). The data represent averages of duplicate experiments. **Specific activity per mg protein. ***Specific

activity

mouse L cells

These

(12).

is

expressed

as nmoles

lipid

is

expressed

as nmoles

sphingosine

investigators

D-[14C]glucosamine =I

and LJ1'+C]fucose

S phase.

our

cells

However,

(13),

results

in which

it

glucosamine

occurred

during

discrepancy

is

The most finding

that

lipids

dramatic the

do not

was observed

agree that

with

acid

per mg protein.

maximum incorporation

glycoprotein

the M phase.

result

highest

rate

occurred

by studying was further

gangliosides

into

that

sialic

occurred

similar

late

studies

of

in the

with

KB

maximum incorporation

of E-[*4C]-

At present

for

the reason

this

not clear.

glycosphingolipids obtained

found

bound

and combined

in the

current

of incorporation during

the rate evaluated

obtained

the M and Gl phases.

by quantitative

analysis

into

The qualitative

of labeled

glycosphingolipids.

590

was the

of TI-l[14C]galactose

of incorporation

neutral

investigation

ga!actose

of the actual The data

pattern into levels

obtained

these of from

BIOCHEMICAL

Vol. 54, No. 2, 1973

these

analyses

are

lipid

synthesis

occurred

Our results small

amounts

specific during

throughout

translated synthesis

observation proteins,

that

very early

functioning

in glycosyl

glycosphingolipid on the

basis

These

might

also

during

the cyclic

expression

experimental

stages

for

to thank

Mrs.

that

The finding

might

glycosyl

transferases

preferentially

of the cell

cycle

enzymes

in small

of cell

that

membrane

the evaluation

are

that

glycosphingo-

to the are glycoprior

of some kinds could

to of

be explained

amounts.

glycosphingolipids

will

of the cellular

are Perhaps

biosynthesis.

biosynthesis

of

synthesis

at a time

stages

hypothesis

in

the synthesis

be related

Synthesis

the

synthesized

at maximum rates

glycoprotein

cycle.

of glycosphingolipid

approach

probably

is clear

for

of preexisting

support

is

reactions.

remaining

final

maximum glycosphingo-

and occurs

cycle

glycolipid

of the presence results

cell

be synthesized

in the

incorporated

class

in the

transfer

it

regulated

in the cell

most of the

and hence

but

The messages

late

that

material

cycle,

carefully

phases.

occurs

above finding

most cellular

the cell is

cell

the

RESEARCH COMMUNICATIONS

the M and G, phases.

that

macromolecules

perhaps

with

during

suggest

specific

lipid

compatible

AND BIOPHYSICAL

provide

function

an

of this

of lipids.

Acknowledgements We wish

work was supported Health

Service,

in part

Alice

Swanson

by Grants

and by a grant

from

for

expert

technical

AM 12434 and 12101 the Elsa

U. Pardee

from

assistance.

This

the U. S. Public

Foundation.

References :: i: 2: 7. 8. 9.

Mitchson, J. M. (1969) Science 165, 657-663. Rueckert, R. R., and Mueller, G. C. (1960) Cancer Res. 20, 1584-1591. Abercrombie, M., and Ambrose, E. J. (1962) Cancer Res..2& 525-548. Fox, T. O., Sheppard, J. R., and Burger, M. M. (1971) Proc. Nat. Acad. Sci. U. S. 68: 244-247. Hakomori, S, I., and Kljimoto, S. (1972) Nature 239, 87-88. Bello, L. J. (1969) Biochim. Biophys. Acta.179, 204-213. Suzuki, K. (1965) J. Neurochem. 12, 629-638. Svennerholm, L. (1957) Biochim. Bzphys. Acta. 24, 604-611. Chatterjee, S. and Sweeley, C. C. (1973) manuscrFt submitted to these communications.

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Vol. 54, No. 2, 1973

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BIOCHEMICAL

AND BIOPHYSICAL

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RESEARCH COMMUNICATIONS

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