Vol.

176,

No.

May

15,

1991

3, 1991

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Pages

1345-1350

MYELOPEROXIDASE IS A PRIMARY RESPONSE GENE IN HL60 CELLS, DIRECTLY REGULATED DURING HEMATOPOIETIC R.W.

Meier*,

*Laboratory Berne,

T.

of Clinical Tiefenau

Chen*,

R.R.

DIFFERENTIATION

Friis",

and A. Tobler*'

and Experimental Hospital CH 3004

Research, University Berne, Switzerland

*+ Central Hematology Inselspital CH 3010 Berne,

Laboratory

of

Switzerland

Received March 27, 1991 suMr4ARY: The expression of myeloperoxidase was studied in three human myeloid leukemic cell lines. The myeloperoxidase transcript was strongly expressed in promyelocytic HL 60 cells, whereas much lower levels were detected in immature monocytic U 937 cells. Phorbol-12-myristate-13-acetate induction resulted in inhibition of myeloperoxidase expression within 24hrs. This regulatory event could not be blocked by cycloheximide. Furthermore, cycloheximide did not superinduce myeloperoxidase mRNA levels in KG 1, HL 60 and U 937 cells, arguing against the existence of a negative gene regulator for myeloperoxidase. Therefore, the myeloperoxidase gene can be classified as a primary response gene. 0 1991 Academic Press, WC.

The

process

differentiation

specialized

cell

types

individual lines

cell

tiation pathway dissecting the induced a variety abnormal

cells

with

cell

to

diminish at

proliferation.

pass through different stages of from unipotent pluripotent to their differentiation, which ramifies

successively

different

stages

(1).

in

Myeloid

their

as well

as the

types, for

leukemic

respective

allow the study of in vitro genetic responses of these

HL 60 cells

their

cells

terminates in different mature blood cell rate and the number of cell cycles

blocked

stimuli.

eukaryotic

interrelated

is

Early hemopoietic progenitor intermediate differentiation During precursor cells (1). and finally proliferation

of

monocytic

the each cell

differen-

differentiation by cells to external U 937 cells

can be

to differentiate of external growth

along the monocytic pathway in response to stimuli (2,3,4,5,6). These inducers control From the growing body of and suppress malignancy.

Abbreviations: CHX, cycloheximide; phorbol-12myristate-13acetate.

MPO,

myeloperoxidase;

PMA,

0006-291X/91$1.50 1345

Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol.

176,

in

No.

vitro

induction

concluded evoke of or

BIOCHEMICAL

3, 1991

that

the

experiments

different

it possible to study myeloid differentiation. is

we show preceded

jun,

although

decrease

of

gene activity 937 cells. MATERIALS

that by

MPO mRNA levels. not

leukemic For

COMMUNICATIONS

cells

it

pathways

example,

of MPO mRNA levels the

proto-oncogenes

is apparently The constitutive

be reversed

not

by cycloheximide

be

depression

the inducing stimulus of the MPO cDNA (9) in more detail regulation

of

can

ultimately the

whether Cloning

depression induction

RESEARCH

signal

(7,B).

gene

induction

their could

with

event

its

the the

BIOPHYSICAL

intracellular

same regulatory

MPO occurs in HL 60 cells I,25 dihydroxyvitamin D3.

Here

AND

in

is PMA has made during

HL 60 cells c-fos

necessary depression in

and for of

KG

1

C-

the MPO and U

AND METHODS

All cell lines were cultivated in enriched McCoy's Cell culture: supplemented with 10% fetal calf serum. To 11 medium (Gibco BRL), McCoy's 5A medium IOml MEM sodium pyruvate (IOOmM), 4ml MEM vitamin solution 100x, 8ml MEM amino acids 50x, 4ml non-essential amino acids 100x, 4ml glutamine 100x (200mM) were added; all these 4ml L-serine Zlmg/ml, I, 6 ml reagents were provided by Gibco BRL; L-asparagine lOmg/ml, 6ml 7,5% (w/v) sodium hydrogen carbonate, 8ml 1M Hepes and penicillin/streptomycin were included. The pH was adjusted to 7.2 by sodium hydroxide. Cells were grown to a titer of about 1 -1 O6 cells/ml. 12-15hrs before PMA induction cells were harvested and resuspended in fresh medium with a titer of about 0.8*106

cells/ml.

RNA extraction and Northern blot analysis: Total RNA was extracted as described (IO). To inhibit RNA degradation 0.5% SDS was included in TE buffer or water. ?Oug total RNA was electrophoretically separated as described (11) with 1.2mg/ml ethidium bromide in the sample buffer (12). Blotting and hybridisation was done as described previously (13) using the EcoR I/ Hind III fragment of the pMPO-2 plasmid (9). For the detection of the c-fos transcript a IOOObp PstI fragment of pfos-1 was used; c-d mRNA was identified with an EcoR I/Xba I fragment derived from v-d cloned in pGEM-4 (14). The hybridisation temperature was raised to 48'C and washing of the blot was done in O.lxSSC, 0.1% SDS at 55’C or at 65'C for 30min. RESULTS

AND DISCUSSION

Myeloperoxidase is highly expressed in promyelocytes and to a lesser extent in myelocytes, monocytes and granulocytes. In the more immature myeloblasts its expression is undetectable as judged by histochemical methods (15). In agreement with these previous studies the myeloblastic cell line KG 1 did not express detectable levels of MPO transcript as compared to the more mature promyelocytic HL 60 cells (Fig.1). MPO mRNA was easily detected in these cells by Northern blot analysis of IOpg total RNA, whereas in the monocytic U 937 cells the level was drastically reduced but still

I346

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Fig-l. KG 1 and U937 cells were incubated with CHX. At the indicated time points total RNA was analysed for MPO RNA levels. correspond to 30, 20, IOug RNA without Lane 1,2,3 CHX in the culture medium. Lane 4,5,6,7,8: 101.19 RNA extracted at 1, 2, 4, 12hrs and 24hrs after start of CHX treatment.1Opg total RNA from HL 60 cells were analysed after 0, 2, 4, 12, 24hrs CHX treatment corres1,2,3,4,5. ponding to lanes In none of the cell lines could the 3.3kb MPO transcript be induced during this period of time.

detectable with a random-primed cDNA probe (Fig.1 ). This result suggested the possible expression of a negatively acting effector gene in U 937 and in KG 1 cells. Therefore, the three cell lines were treated up to 24hrs with cycloheximide. No superinduction of the MPO transcript was observed in either of these cells, arguing against the existence of a constitutively expressed repressor (Fig-l). Studies

on the

mRNA levels

in

depressive HL 60

cells

regulatory induced

mechanisms with

PMA

to

determining

monocytic

MPO

differen-

tiation agreed with the above observations. HL 60 cells were treated with 60ngml-' PMA for 24hrs. At different times, total RNA was extracted and analysed for MPO mRNA levels. At 12hrs the level of MPO transcript was clearly reduced and became undetectable at 24hrs (Fig.2B). This decrease of MPO expression was preceded by a simultaneous induction of mRNAs for the proto-oncogenes c-fos and c-j (Fig.3A) and a rapid depression of c-myc mRNA (data not shown). Whereas c-fos was only transiently induced, under the control of an autoregulatory mechanism (16,17), the c-u transcript was still detectable at a low level even after 24hrs of PMA exposure. As monocytic differentiation is not linked to the expression of c-a, this gene can no longer be considered as a trigger molecule for monocytic differentiation (18). The upregulation of c-i expression occurring in parallel to the decrease processes

in

MPO mRNA levels

protein

synthesis

response

genes

argues

strongly

for

specific

regulatory

being initiated. For the down-regulation of the MPO transcript by PMA, the expression of these two gene products is probably unnecessary, since complete inhibition of de novo protein synthesis by 40pgml-' cycloheximide (data not shown) also resulted in MPO mRNA depression (Fig.3B). This independence from ongoing makes

(19).

the

MPO

Our studies

gene

with 1347

a

member

HL 60,

of

the

primary

KG 1 and U 937 cells

Vol.

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AND

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RESEARCH

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MPO

123456

MPO I--

“’

*

2 3 4

- 23!.b

*’

5

6

7

9

9

B

MPO

0

I)+

1

0

g’l)cc-l)“cll

2 ,iactin 3 123456789 123456 2. Depression of MPO mRNAafter induction of HL 60 cells with times after addition of PMA, At different %$?i. phorbolester. total RNA was tested for its steady-state level of MPO transcript. (A) HL 60 cells were kept in culture for three months under conditions of maximal cell proliferation. (B) HL 60 cells were cultured for only three weeks. Under both conditions the MPO transcript was regulated. Lanes 1,2,3,4,5,6, correspond to (C) HL 60 cells were treated with 0,3,6,9,12,24hrs PMA treatment. PMA pulses ranging from 10 to 40 min. After washing the cells with incubated 2 x 1 culture volumes of fresh medium, they were further for 24hrs. Lane 1: continuous presence of PMA for 24hrs; lane 2: 10min pulse; 20min pulse: adherent (lane 3), non-adherent cells (lane 4); 40min pulse: adherent (lane 5), non-adherent cells (lane 6).

Fig. 3. Induction of c-fos and c-m are not necessary for the depression of MPO mRNA. (A) c-fos and c-u transcripts are both induced after addition of PMA. c-fos is detectable from 0.5 to 2hrs after induction (lane 2: 10min; lane 3: 30min; lane 4: 60min; lane 5: 90min; lane 6: Zhrs), whereas increased c-u levels could still be observed 6hrs, 12hrs and 24hrs (lanes 7,8,9) after addition of PMA. Lane 1: uninduced control cells. (B) HL 60 cells were cultivated with 40ug/ml CHX with or without PMA and the level of MPOwas detected with a MPO-2 cDNA probe using 1Opg total RNA on Northern. Lane 1: control without CHX or PMA. Lanes 2, 3: 3hrs +CHX; 3hrs +CHX+PMA; lanes 4, 5: 9hrs +CHX; 9hrs +CHX+PMA; lanes 6, 7 and lanes 8, 9 "-"PMA, "+"PMA for 12, respectively 16hrs, always in the presence of CHX. The down-regulation of MPO also took place with CHX in the culture medium.

argue against the expressed negatively transcription unit.

existence acting

of an inducible gene modulator

or constitutively controlling the

MPO

In the in vitro differentiation experiments described above the induction always occurred in the continuous presence of the inducer. Therefore, we compared the results after a brief, temporary exposure of the HL 60 cells to PMA with those where PMA treatment continued for the entire 24hrs of the experiment. Pulses of 10 to 40min showed the same effect as did the permanent addition of the inducer (Fig.2C). The extent of this regulatory event is, therefore, not correlated with the exposure time to the inducer in the culture medium. The initiation of the differentiation cascade was 1348

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also morphologically evident by cell adhesion to plastic dishes after stimuli for at least 20 and 40 min. Rapidly adhering cells as well as those floating after 10, 20 and 40 min showed the same potency to down-regulate the MPO transcript within 24hrs. Furthermore, HL 60 cells kept in culture for three months under conditions of maximal cell proliferation still showed the same capability to down-regulate MPO mRNA as rapidly as those cells cultivated only for 2 to 3 weeks (Fig.ZA,B). To our knowledge this is the first report about a cycloheximide insensitive gene permanently down-regulated during myeloid cell differentiation. Therefore, the MPO gene can be considered a primary response gene. So far these early or primary response genes have been defined loosely as a set of genes which are directly induced during in vitro cell differentiation, like c-fos and c-a. We are probably justified in hoping that at least some primary response genes initiate the cascade of cell differentiation which terminates finally in a mature cell type. Thus, the goal now in view is to attempt to overcome the differentiation block in leukemic cells by artificial induction of trigger molecules for differentiation, or conceivably also by inhibition of particular gene activities, such as for example those of c-myc (20) or myeloblastin (21). Myeloperoxidase belongs to this set of genes, specifically down-regulated during myeloid differentiation, since its expression specifically declines while the steady-state levels of other transcripts increase or remain constant. ACKNOWLEDGMENTS

We thank G. Niklaus for critical reading of the kindly provided v-f+ grants from the SWISS A.T. and the Bernische

technical assistance and Dr. S. Mathews for manuscript. Drs. R. Klemenz and W. Conca and v-m DNA probes. Work was funded by National Science Foundation 31.9141.87 to Krebsliga.

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