Human C-myc and N-ras expression during induction of HL-60 cellular differentiation

Vol.

126,

No. 3, 1985

February

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

15, 1985

999-l

005

HUMAN C-MYC AND N-RAS EXPRESSION DURING INDUCTION OF HL-60 CELLULAR DIFFERENTIATION Tetsu

Watanabe,

Laboratory

Received

Eric

Sari ban,

Thomas Mitchell,

and Donald

of Clinical Pharmacology, Dana-Farber Cancer and Harvard Medical School, Boston, MA

December

20,

Kufe

Institute

1984

The genome of the human HL-60 promyelocytic leukemia cell contains The present amplified c-myc sequences and the transforming N-ras oncogene. study has monitored c-myc and N-ras expression in ~~-60 cells during induction of myeloid and monocytic differentiation with dimethyl sulfoxide, hexamethylene bisacetamide, 12-O-tetradecanoylphorbol-lj’-acetate and 1 ,25 dihydroxy-vitam,in D$. The results demonstrate that induction of HL-60 differentiation is as oclated with decreases in c-myc RNA, while there is little if any effect on expression of the N-ras gene. Although the diminution in c-myc expression occurred as an early event in the induction of HL-60 differentiation, the rate of decrease in c-myc transcripts varied with respect to cessation of proliferation. Thus, the appearance of the mature phenotype and loss of proliferative capacity are associated with declines in c-myc RNA, while these events appear to occur in the absence of B 1985 Academic Press, Inc. significant alterations in N-ras expression.

The

human

expression mature with

a monocytic HL-60

HL-60 loss The

transforming with

induction

of cellular

These

3).

pathway

cells

cells

to either

of proliferative

capacity

genome

N-ras

reduction

contains

oncogene

(8).

can also

be induced

TPA (4).

c-myc

Abbreviations used are: bisacetamide: HMBA; 1 ,25 tetradecanoylphorbol-13-acetate: tetrazolium reduction: NBT.

HL-60 after

gene cells

treatment

to differentiate

Similarly,

pathway

granulocytes

used to study (1).

granulocytes

with

(5).

or monocytes

1 ,25(OH)

D

23

The

induction

is

associated

(Z-5). amplified The

in a reduction in

has been

toward

a monocyte-macrophage

differentiation

HL-60

line

differentiation

when treated

along

DMSO has resulted

A similar

cell

and functionally

DMSO or HMBA (2,

induces

with

during

promyelocytic

morphologically

along

of

HL-60

c-myc

induction

sequences of HL-60

in the level

RNA has

been

observed

(6,

7) and the

differentiation

of c-myc expression during

(9).

1,25(OH)2D3-

dimethyl sulfoxide: DMSO; hexamethylene dihydroxy-vitamin D : 1 ,25(OH) D 12-0TPA; non-specific3esterase: NSZ; hi troblue

0006-291X/85 999

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Copyright 0 1985 rights of reproduction

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by Academic Press. Inc. in any form reserved.

Vol.

126,

and

No. 3, 1985

BIOCHEMICAL

TPA-induced

HL-60

monocytic

diminution

in c-myc

expression

maturation

sequence

(10).

slowing

of HL-60 The

HL-60

cells

genes

(8).

The

expression

in

HL-60

decline

in in

cells

result the

differentiation,

during

c-myc

demonstrate

expression

of

but

of decrease

as an early

both

multiple

N-ras event

in c-myc RNA varied

precedes

oncogene

involving c-myc and

is

of HL-60 if

Although

in induction with

both

monocytic

little

gene.

in

and N-ras

inducers

there

the

phenotype.

myeloid

that

the in

thus

mechanism

of the transforming occurred

expression

monitored

c-myc RNA,

event

and the N-ras

transforming thus

Furthermore,

of the mature

induction

in decreased

the rate

in

11).

COMMUNICATIONS

be an early

c-myc sequences

has

results

c-myc expression

to

and appearance

study

The

diminution

decline

a multi-step

present

differentiation. differentiation

The

RESEARCH

(10,

has been found

of amplified

has suggested

BIOPHYSICAL

differentiation

proliferation

coexistence

AND

the

any the

of HL-60 different

inducers.

MATERIALS

AND METHODS

CELL CULTURE HL-60 cells were maintained in RPMI-1640 medium (Flow Laboratories, 1% penicillin/streptcmycin, 1.0 mM McLean, VA) containing 4 mM L-glutamine, sodium pyruvate in 20$5heat-inactivated fetal bovine serum at a density ranging from 1 to 2 x 10 /ml in a 5% CO2 atmosphere. The HL-60 cells were grown in suspension culture in the presence of 1.25% DMSO (Fisher St. Louis, MO), 3.3 Cytocentrifuge smears of cr-napthyl acetate Adherence was determined by cellular esterase (NSE) and NBT reduction (12). The percentage of positive cells attachment to the tissue culture flask. Viable cells were was determined by counting 200 cells in duplicate. determined by trypan blue exclusion. NORTHERN BLOT ANALYSIS HL-60 total cellular RNA was purified by the guanidine thiocyanatecesium chloride method (13). The RNA was dissolved in 40% formamide, 2.2 M formaldehyde, 40 mM morpholino-propanesulfonic acid (MOPS), 10 mM sodium acetate and 1 mM EDTA. Samples (15 ug) were heated to 55OC for 15 min. and then quickly chilled at O°C. Gel electrophoresis was performed in 1% agarose containing 2.2 M formaldehyde, 40 mM MOPS, 10 mM sodium acetate, 1 The gel was then mM EDTA and 0.1% ethidium bromide at 40V for 16 hours. washed and transferred onto nitrocellulose filters (14). The filters were prehybridized at 42OC for 8-12 hours in buffer consisting of 50% formamide, 5x SSC (SSC: 0.15 M sodium chloride, 0.015 sodium citrate), 0.1% SDS, lx Denhardt's solution and salmon sperm DNA (200 ug/ml). gThe RNA qeya;;',; then hybridized at 42°C for 24 hours with 0.5 to 1 x 10 cpm of buffer. The oncogene fragments were cDNA probe per ml. of hybridization I 000

Vol.

126,

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No. 3. 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

purified from vector sequences by avrose 4 el electrophoresis and nicktranslated to specific activities of 10 to 10 cpm/pg cDNA. The 1.4 kb Cla I/Eco RI fragment of the human c-myc 3’-exon was purified from the pMC41-3RC plasmid (15). The 1 .5 kb p52C- Eco RI fragment of the HL-60 N-ras gene was Filters were then washed purified from a reconstructed pBR322 plasmid (5). twice with 2x SSC, 1.0% SDS at room temperature and then twice with 0.1x Filters were exposed to x-ray film for 24 (c-myc SSC, 0.1% SDS at 50°C. hybrids) or 168 (N-ras hybrids) hours at -7OOC using an intensifying screen.

RESULTS We have treated DMSO, 4 mM HMBA, resulted

in

treated

with

before

achieving

in Figure treated

DMSO,

HL-60 inducer

cells

is

in

( Lanes

8-l 3).

logarithmic

HL-60

phase

growth

than with

delayed

(2-5).

at

times

harvested c-myc

varying

expression.

Figure

cells

with

treated

In each case c-myc expression of inducer.

to

grow

curves

are

after

48 hours

illustrated for

using

the

DMSO (Lanes

decreases

.25$

cells

TPA-

TPA and

addition

2 illustrates

was nearly

In contrast,

for

of growth

investigations

differentiation

1

while

cessation

previous

with

TPA treatment

proliferation,

Representative rather

of HL-60

for

of

continued

c-myc RNA from HL-60

addition

growth

1,25(OHj2D3

in concert

were

to monitor

after

cessation

HMBA and

inducers

in

x lOWaM TPA and 5 x 10d7M 1,25(OH)2D3.

cytostasis.

cells

decline

hours

cells

The immediate

1.

other

3.3

an immediate

HL-60

these

HL-60

of

a rapid 2-7)

and HMBA

undetectable

by 1 .5

in c-myc expression

20

0 24

48

72

96

HOWS

Flgure 1: Effects of DMSO, HMBA, TPA and 1,25(3H) D on HL-60 Cell Growth. HL-60 cells in logari hmic growth phase we e trea e Cell mM HMBA 0 , 3 . 3 x IO-‘M TPA 0 or 5 x IO-‘, 1 ,25%?Lh ^ _) A. “**’ DMSO density ” ’ was monitored for each group was over 95% for each group.

and

for

untreated

controlcc~lls

0.

Viability

Vol. 126, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL

1 2

RESEARCH COMMUNICATIONS

3 4 5 6 7 8 9 10111213141516

28s -

* 18s Effects of DMSO, HMBA and TPA on HL-60 c-myc Expression. The 7.4 kb Cla I/Eco RI fragment of the human c-myc 3'-exon was from HL-60 cells treated for varying hybridized to RNA (15 kg) obtained int_%rvals with 1.25% DMSO (Lanes 2-7), 4 mM HMBA (Lanes 8-13) and 3.3 x 10 M TPA (Lanes 14-16). Lane 1: control; Lane 2: DMSO, 0.5 hr.; Lane 3: DMSO, 1.0 hr.; Lane 4: DMSO, 1.5 hr.; Lane 5: DMSO, 2.0 hr.; Lane 6: DMSO, 3.0 hr.; Lane 7: DMSO, 6.0 hrs.; Lane 8: HMBA, 0.5 hr.; Lane 9: HMBA, 1.0 hr.; Lane 10: HMBA, 1.5 nr.; Lane 11: HMBA, 2.0 hr.; Lane 12: HMBA, 3.0 hr.: Lane 13: HMBA, 5.0 hr; Lane 14: TPA, 6.0 hr.: Lane 15: TPA. 24 hr.: and Lane 16: TPA, 48 hr.

were

not

as pronounced

c-myc

RNA was still

hours

of exposure Although

if

any

decrease

HMBA or TPA.

in the TPA-treated

detectable to this

associated in the Figure

at six

inducer with level

3 shows

cells

hours

resulted

(Fig.

after

2; Lanes

addition

in nearly

in c-myc expression,

of N-ras

RNA in HL-60

the hybridizations

of TPA,

undetectable

declines

cells

performed

with

while

24

levels.

there treated

The

14-16).

was little with

DMSO,

RNA obtained

12345678910 28S-:

,-

A

12345678910 28s

18s Figure 3: Effects of DMSO, HMBA and TPA on HL-60 c-myc and N-ras Expression. The “'P- abeled 1.4 kb Cla I/Eco RI fragment of the human c-myc 32 3'-exon (A) and the P-labeled p52C- Eco RI DNA fragment of the HL-60 N-ras gene (B) were hybridized to RNA (15 ug) obtained from HL-60 cells treated for varyjRg intervals with 1.25% DMSO (Lanes 2-II), 4 mM HMBA (Lanes 5-71, or Lane 1: Control; Lane 2: DMSO, 24 hr.; Lane 3.3 x 10 M TPA (Lanes 8-10). 3: DMSO, 48 hr.; Lane 4: DMSO, 72 hr.; Lane 5: HMBA, 24 hr.; Lane 6: HMBA, 40 hr.; Lane 7: HMBA, 72 hr.; Lane 8: TPA, 24 hr.: Lane 9: TPA. 48 hr.; and Lane 10: TPA, 72 hr. Uninduced HL-60 cells (96 hrs): 6.7 + 1.3% NBT positive (mean + S.D. of two determinations); DMSO-treated cells (96 hr): 87.5 ? 0.5% NBT positive; and HMBA-treated cells (96 hrl: 95.3 + 0.8% NBT positive. Uninduced HL-60 cells (48 hrl: no adherence; TPA-treated cells (48 hr): 84.1% adherence (mean of two determinations). 1002

Vol.

126,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1234 A.

28s -

18s -

1234 B.

28s . 185 -

4: Effect of 1,25(OH) D on HL-60 c-myc and N-ras Expression. The P-.,L$beled 1.5 kb Cla/Eco 32 fragment of the human c-myc 3’-exon (A) and the JLP-labeled p52C- Eco RI fragment of the HL-60 N-ras gene (6) were hybridized to RNA (15 ug) obtained fran HL-60 cell? treated for 24 hr (Lane 2), 48 hr (Lane 31, and 72 hr (Lane 4) with 5 x 10 M 1,25(OH) D . RNA fran control HL-60 cells (c-myc and N-ras expression identical 2af r untreated cells at 0 and 72 hr.). 1,25(OH) D -treated cells (day 3): 95.1 f 0.6% NBT positive. Control HL-60 ce?ld (day 31 : 21.25 +_ 2.5% NSE positive; 1,25(0H)2D3-treated cells (day 3): 87.2 k 2.3% NSE positive. g;gure

from HL-60

cells

treated

c-myc expression alteration mature

remained

in

the

phenotype

percent

effects

(OH12D3-treated change

in

were

myc transcripts

are

90% of the

cells.

included

N-ras

in the

(Fig.

despite

of a

The histochemical

staining

and

similar

after

the

was less

rapid

after

addition

to Figure

3.

observed

were

Although

there

treated

with

the growth

was no significant appearance

legend

cells

While

the

was detectable, although

48 and 72 hours. there

expression

4).

RNA in

expression

24,

undetectable, expression

of

Thus,

for

N-ras

cells

DMSO or HMBA.

1,25(OHj2D3

of

HL-60

c-myc

inducers

on c-myc and N-ras

in the level

decline with

level

data

these

essentially

in nearly

adherence

Similar

with

1,25

was no significant 1 ,25(OHj2D3,

but ,slower

than

patterns

with

of inducer,

the

1 ,25(OHJ2D3

in the

that

the observed

DMSO, HMBA and diminution

in c-

treatment.

DISCUSSION

Previous in

the

oncogene

DNA of

studies

have demonstrated

HL-60

cells

has suggested

that

(6,

7).

multiple

that

c-myc sequences

The detection proto-oncogenes 1003

are

of an active

amplified HL-60

may be activated

N-ras during

Vol. 126, No. 3, 1985

the

transformation

levels

of

both

the

the myeloid

induction

declines that

the

c-myc

phenotype

are

of HL-60

associated

necessarily

with

dependent

These

and the

declines

in c-myc

upon significant

monitored

to differentiate

with

that

significant

findings

would

appearance

of

RNA, while

these

alterations

the

demonstrate

associated

RNA levels.

proliferation

induced

The results is

N-ras

We have

cells

lineages.

differentiation not

(8).

in HL-60

and monocytic

but

cessation

cell

expression

of HL-60

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a hematopoietic

of c-myc and N-ras

along

not

BIOCHEMICAL

suggest

the

mature

events

in the level

are

of N-ras

expression. A decrease treated

with

between

the

in the level DMSO,

with

the

of events

However,

cessation

of

c-myc

Fur thermor

These

myc expression

is

differentiation

(16).

terminal early

decreased

induction

(17).

observed

with

not required

for

the

moderately

of c-myc and

Similarly,

(18).

would

final

cell

murine

there

was

Ha-ras

while

The

induction

therefore

appear 1004

has

no significant

F9

to primarily

and

c-myc that

c-

terminal

resulted

in a

expression change

differentiation of

of

before

Ki-ras

was

several

suggestion

divisions

expression

cell

the

levels

DMSO and HMBA.

levels

F9 cells

expression,

Thus,

for

undetectable with

and

the

proliferated

be consistent

differentiation in

or

and monocyte

inducers.

than

cells

this

expression

growth,

low

would

of mouse teratocarcinoma

proto-oncogenes differentiation

of

findings

decrease

only

expression

presence

c-myc

of HL-60

and HMBA-induced

the

support

myeloid

different

that

of

has been observed would

of both

the

slowing

cessation

e , the in

rapidly

data

between

with

less

transcripts.

similar

varied

RNA declined

divisions

The

inducers

in an immediate

DMSO-

Our

(10).

the relationship

proliferation

TPA resulted

phenotype

a mature

cells

relationship

of

multiple

HL-60

A sequential

in c-myc expression,

cells

when using

differentiation.

although

HL-60

for

a decrease

appearance

1 ,25(0H)2D3-treated

RNA has been reported

and TPA (9-11).

of inducer,

and

sequence

of

1 ,25(OH),D3

addition

proliferation

of c-myc

in Ha-ras

unchanged

during

by transfer

of c-fos

HL-60

involve

cellular alterations

in

Vol.

126,

c-myc expression whether

BIOCHEMICAL

No. 3, 1985

rather

the diminution

than that

rate

differentiation

may be associated

than the level

of c-ras

in c-myc

transcriptional

rather

AND

genes.

expression

is

of c-myc RNA.

or instability

of c-ras

BIOPHYSICAL

with

alterations

RESEARCH

COMMUNICATIONS

It is not clear, related

however,

to decreases

in

Furthermore,

induction

of

of the c-ras

gene product

RNA.

ACKNOWLEDGEMENTS This investigation was supported by PHS Grant #CA 19589 awarded by the National Cancer Institute, DHHS and an American Cancer Society Faculty Research Award (DWK). Reprint requests should be addressed to D.W. Kufe at the Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. The pMC41-3RC plasmid containing the human c-myc 3'-exon was provided by Dr. F. Won&-Staal, National Cancer Institute, Bethesda, MD. Dr. James Cunningham, Harvard Medical School, provided the pBR322 plasmid containing the p52Cfragment of the HL-60 N-ras gene. The 1,25(OHj2D3 was a gift from Dr. M. Uskokovic, Hoffman-La Roche, Nutley, NJ.

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Collins, S., Gallo R. and Gallagher, R. (1977) Nature 270,347-349. Collins, S., Ruscetti, F., Gallagher, R. and Gallo R. (1978) Proc. Natl. Acad. Sci. 75,2458-2462. Collins, S., Bodner, A., Ting, R. and Gallo R. (1980) Int. J. Cancer 25,213-218.

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Li, C. and Crosby, W. (1971) Am. J. Clin. Path. 55.283-290. L., 13. Chirgwin, J., Przybyla, A., MacDonald, R. and Rutter, W. (1979) Biochemistry 18,5294-5299. 14. Thomas, P. (1980) Proc. Natl. Acad. Sci. 77.5201-5205. 15. Dalla-Favera, R., Gelmann, E., Martinotti, S., Franchini, G., Papas, T., Gallo, R. and Wong-Staal, F. (1982) Proc. Natl. Acad. Sci. 79,6497-6502. 16. Stewart, T., Bellve, A., Leder, P. (1984) Science 226.707-710. 17. Campisi, J., Gray, H., Pardee, A., Dean, M. and Sonnenshein, G. (1984) Cell 36,241-247. 18. Muller, H. and Wagner, E. (1984) Nature 311,438-442. 12.

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