THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 51, Issue of December 23, pp. 321553216], 1994 Printed in U.S.A.

Isolation and Characterization of HL-60 Cells Resistant to ~itroprusside-in~uced ~i~erentiation*

(Received for publication, July 27, 1994)

Renate B. Pilz$§, Marjaneh BerjisS, Soha D. Idrisst, Jurgen S. Scheelen, Modem SuhasiniS, Lin Gaol, Immo E. Schefflerll, and Gerry R. Boss$** From the Devartments of Wedicine, IBiolom. and llChemistry, University of California at San Diego, La Jolla, California 92093-0652

, .. -“,

Sodium nitroprusside and sodium nitrite, which gen- erate nitric oxide and increase the intracellular cGMP concentration, and 8-bromo-cGMP, a membrane-perme- able cGMP analog, induce myelomon~ytic differentia- tion of HL-60 cells (Boss, G. R. (1989) Proc. NutZ. Acad. Sci. U. S. A. 86,7174-7178). We have selected HL-60 cells resistant to nitroprusside-induced differentiation as as- sessed by acquisition of the O m - 1 antigen, reduction of nitro blue tetrazolium, and morphologic maturation. The variant cells were also resistant to differentiation induced by sodium nitrite and two cGMP analogs but still differentiated in response to other inducing agents such as dimethyl sulfoxide and cAMP analogs and showed the same changes in c-myc and c-fos expression in response to the latter drugs as occurred in parental cells. We studied the early steps of the NOicGMP signal transduction pathway in the variant cells and found that basal and n i t ~ p r u s s i d e - s t i m ~ a ~ guanylate cy- clase activity was similar in parental and variant cell extracts and that nitroprusside increased the intracel- lular cGMP concentration to the same extent in parental and variant cells. As part of these studies we found that HL-60 cells expressed only as and guanylate cyclase mRNA; the abundance of these two mRNA species was similar in parental and variant cells. Neither nitroprus- side nor 8-bromo-cGMP changed the intracellular cal- cium concentration in parental or variant cells. The data suggest that the defect in the variant cells is after guanylate cyclase activation in the NOlcGMP transduc- tion pathway and that the cGMP and CAMP transduc- tion pathways operate independently in inducing differ- entiation of HL-60 cells.

The study of myeloid cell differentiation has been aided greatly by the establishment of cell lines from leukemic patients that can be induced to differentiate into morphologically and functionally mature cells (1). Probably the best studied system is the promyelocytic cell line HL-60 which ceases growth and differentiates along the myelomonocytic lineage in response to a number of agents including CAMP analogs and drugs which increase the intracellular cAMP concentration (2 , 3).

Previously, we showed that sodium nitroprusside and sodium nitrite induce myelomonoc~ic differentiation of HL-60 cells (4).

* This work was supported in part by National Institutes of Health Grants KO8 CA01548 (to R. B. Rj, RO1 GM18835 (to I. E. S.) , and ROI. GM49360 (to G. R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Tel.: 619-534-8805; Fax: 619-534-1421. I To whom correspondence and reprint requests should be addressed.

** Henry J. Kaiser Family Foundation Scholar during part of this work and supported by the Stern Foundation.

.”

These two agents increase the intracellular cGMP concentra- tion by generating nitric oxide (NO), a potent activator of cy- tosolic guanylate cyclase (5). The differentiation of HL-60 cells induced by sodium nitroprusside and sodium nitrite was aug- mented markedly by the phosphodiesterase inhibitor theophyl- line and was directly proportional to increases in the intracel- lular cGMP concentration (4). Moreover, 8-bromo-cGMP, a membrane-permeable cGMP analog, also induced HL-60 cells to differentiate with the degree of differentiation increasing significantly when theophylline was added (4). Thus, it appears that myelomonocytic differentiation of RL-60 cells can be in- duced by increasing either the intracellular cAMP or cGMP concentration.

To examine whether the CAMP- and cGMP-dependent signal transduction pathways operate independently during differen- tiation of HL-60 cells, we selected HL-60 cells resistant to growth i ~ i b i t i o n a n d differentiation induced by n i t rop~ss ide . We found that t h e n i t r o p ~ s s i d e - r e s ~ s ~ n t cells differentiated normally in response to dimethyl sulfoxide, sodium butyrate, and CAMP analogs but differentiated poorly in response to so- dium nitrite and membrane-permeable cGMP analogs; more- over, the variant cells exhibited the same change in oncogene expression as parental cells when exposed to dimethyl sulfox- ide and cAMP analogs. The defect in the nitroprusside-resis- tant cells was not secondary to decreased guanylate cyclase mRNA expression, decreased basal or nitric oxide-stimulated guanylate cyclase activity, decreased intracellular production of cGMP in response to nitroprusside, or changes in calcium metabolism. It appears that the cGMP and CAMP signal trans- duction pathways operate independently in inducing differen- tiation of HL-60 cells.

EXPERIMENTAL PROCEDURES Materials

Sodium nitroprusside and sodium nitrite were from Sigma and were made fresh prior to use; NO was >99.9% pure and was dissolved in oxygen-free 500 mM triethanolamine, pH 7.8, to a saturating concen- tration of 1.91 m. 8-Bromo-cGMP and 8-bromo-CAMP were from Boehringer Mannheim; A@,Z’-O-dibutyryl cAMP (dibutyryl CAMP), ni- tro blue tetrazolium (NBT),’ and phorbol-12-myristate-13-acetate were from Sigma. 8-para-Chlorophenylthio-cGMP (8-pCF”I-cGMP) was gen- erously provided by Drs. S. Lchmann and u. Walter. Stability of nitro- prusside was assessed by monitoring its absorbance spectrum between 350 and 700 nm; stability of 8-bromo-cGMP was assessed by monitoring its absorbance at 254 nm after separation from breakdown products by high performance liquid chromato~aphy on a strong anion exchange column (6).

[O-~~PIGTP (specific activity 1 mCiimmol) was from DuPont NEN. The radioimmunoassay kit used to measure cGMP was from Amer- sham, and Indo 1/AM and ionomycin were from Calbiochem. The fol-

The abbreviations used are: NBT, nitro blue tetrazolium; IMDM, Iscove’s modified Dulbecco’s medium; FBS, fetal bovine serum; kb, ki- lobase(s); 8-pCPT-cGMP, 8-para-chlorophenylthio-cGMP.

32155

32156 Nitroprusside-resistant HA-60 Cells lowing soluble guanylate cyclase cDNAs were generously provided: rat lung a1 and p1 subunits by Dr. M. Nakane (Abbott Laboratories, Abbott Park, IL), human fetal brain a, and rat brain pz subunits by Dr. D. Koesfing (Free University of Berlin, Germany), and human adult brain cy3 and p3 subunits by Dr. G. Guellaen (Unite de Recherche Inserm, Creteil, France).

Methods Selection of Nitroprusside-resistant HL-60 Cells-HL-60 cells were

cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin, and 50 pg/ml streptomycin at 37 "C in an atmosphere of 95% air, 5% CO,. To several cultures of 1 x 10' cells, 1 mM nitroprusside was added, and every 3-4 days the cells were centrifuged and resuspended in fresh media con- taining 1 mM nitroprusside. When cell viability decreased to <1%, cells were resuspended in media without nitroprusside and allowed to grow for several days before nitroprusside was readded. This i n ~ r m i t ~ n t exposure to nitroprusside was maintained for several months until cultures could be kept in 1 mM nitroprusside indefinitely with a viability of >99%. When cells were removed from the selective media for as long as 3 months, re-exposure to nitroprusside did not result in terminal differentiation.

The nitroprusside-resistant cells were subcloned by limiting dilution, and five independently derived clones were studied. In this manuscript we present the results of studies on one of these clones, but similar results were obtained in the other four clones.

Assessment of Cell Differentiation-Cultures were initiated at a den- sity of 0.5-1.5 x lo5 cells/ml in IMDM supplemented with 10% heat- inactivated dialyzed horse serum in the presence of the inducing agents indicated in the figures and tables. After 6 days of culture, cells were assessed for their degree of differentiation by three different methods: (i) acquisition of the OKM-1 antigen, the complement receptor CR,, (CD llb), which is present on mature myelomonocytic cells and expressed by <lo% of undifferentiated HL-60 cells (7); tiii NBT reduction after mem- brane perturbation with phorbol 12-myristate 13-acetate which meas- ures activity of a membrane-bound NADPH oxidase present in mature myelomonocytic cells (8); and (iii) Wright-Giemsa staining to assess morphologic differentiation.

The number of cells staining positive for the OK"1 antigen was determined by flow cytometry after incubation with O K " 1 antibody and fluorescein-conjugated goat anti-mouse IgG (4). The number of cells that reduced NBT was determined by incubating cells for 30 min in phosphate-buffered saline containing 1 mg/ml NBT, 100 ng/ml phorbol 12-myristate 13-acetate, and 20% FBS; 200 cells were evaluated and the number of cells with >3 black formazan precipitates was recorded. Wright-Giemsa staining was performed as described previously (4) with the experimental conditions unknown to the observer. Only cultures with >85% cell viability were included in all three analyses.

Northern Blot Analysis of e-Myc and c-Fos mRNA Expression-Cells were cultured as described under assessment of cell differentiation but harvested 6 h after the inducing agent was added. Total cytoplasmic RNA was isolated from 10-15 x lo6 cells using acidic guanidium iso- thiocyanate, and 10 pg were loaded on a 1% denaturing agarose-form- aldehyde gel (9); after electrophoresis, the RNA was blotted onto nylon membranes and hybridized to probes which were radioactively labeled using the random hexamer priming method (10). The c-myc and c-fos probes were a 1.4-kb ClaIIEcoRI fragment and a 2.7-kbXhoUNco1 frag- ment of human genomic clones, respectively (11, 12).

Northern Blot Analysis of Guanylate Cyclase mRNA Expression- Total cytoplasmic RNA was isolated as described above and enriched for poly(A)' RNA by batch absorption to oligo(dT)-bo~d latex particles. Northern blots were prepared as described above with 10 pg of poly(A)+- enriched RNA per lane; 5 pg of total eytoplasmic RNA from insect (SF9) cells expressing either rat alpl or human a3p3 guanylate cyclase served as positive controls for blots hybridized with al/& or adpa, probes, re- spectively. The probes for soluble guanylate cyclase subunits were: a 0.9-kb BglII fragment of rat lung a1 cDNA and a 0.9-kb PstI fragment of rat lung p1 cDNA(13); a 2.1-kb Sac11 fragment of human fetal brain a, cDNA (14) and an 0.8-kb HindIIIIBamHI fragment of rat pz cDNA (15); a 413-base pair Sac1 fragment of human brain a3 cDNA and a 407-base pair PuuIIISphI fragment of human brain p3 cDNA (16). Blots were washed in 2 x SSC (0.3 M NaCl, 30 mM sodium citrate, pH 7.0),0.1% SDS a t 42 "C (low stringency) and in 0.1 x SSC, 0.1% SDS at 55 "C (high stringency).

Measurement of Guanytate Cyclase Activity-Cells were extracted at a density of 25 x lo6 cells/ml by Dounce homogenization in 25 mM triethanolamine, pH 7.8, 1 mM EDTA, 1 m~ EGTA, 5 mM dithiothreitol, 25 mM ~-me~aptoethanol, and 0.5 m~ phenylmethylsulfonyl fluoride.

The extracts were centrifuged for 5 min at 800 x g to remove nuclei, and part of the s u p e m a ~ n t was used to measure total cellular guanylate cyclase activity; the remainder of the supernatant was centrifuged for 1 h at 100,000 x g, and the supernatant from this second centrifugation was used to measure cytosolic guanylate cyclase activity. Approximately 10 pg of cellular or cytosolic protein were incubated for 10 rnin at 37 "C in a final volume of 20 pl containing 25 mM triethanolamine, pH 7.8, 5 mM MnCI,, 0.5 mM isobutylmethylxanthine, 300 p~ GTP, 0.1 pCi of [a-32PlGTP, 3 units of creatine phosphokinase, 30 mM creatine pbos- phate, and 1 mM P-glycerol phosphate. In experiments where NO acti- vation of guanylate cyclase was measured, 3 mM MgCl, replaced the MnCl, and NO dissolved in deoxygenated buffer was added to a final concentration of 2 p ~ . The reaction was started by adding the substrate and was terminated by adding 5 pl of 8 M HCOOH. Substrate and product were separated by thin layer chromato~aphy in a butanol/ aceton~water/acetic acidlammonium hydroxide (35:25:22.5:15:2.5) sol- vent system (17). For each sample, the radioactivity recovered in CGMP was divided by the sum of radioactivity recovered in GMP, GDP, GTP, and G M P to calculate a conversion factor; a reagent blank (obtained by replacing enzyme with extract buffer) was subtracted from each sample. Enzyme activity was linear with time to at least 15 min and with protein from 2.5-20 pg; the data are expressed as nanomoles of GTP converted to cGMP per min per mg of protein.

Measurement of Intracellular cGMP Concentration-Approximately 25 x lo6 cells were incubated in the absence or presence of 1 mM theo- phylline for 1 h at a density of 1 x 106/ml in IMDM supplemented with 10% FBS. To appropriate cultures, 1 m~ nitroprusside was added; after 20 min, 2 volumes of ice-cold phosphate-buffered saline were added and the cells were centrifuged for 3 min at 4 "C. The media were aspirated and the cells were sonicated in 2 ml of ice-cold 70% ethanol; after 20 min on ice, the tubes were centrifuged at 1500 x g for 5 min and the super- natants were concentrated under reduced pressure. The residue was resuspended in 200 pl of 100 mM sodium acetate buffer, pH 5.5, and the cGMP concentration was measured by radioimmunoassay using IZsI- labeled cGMP according to the manufacturer's protocol. The values obtained for the intracellular cGMP concentration are less than those reported previously (4) because the antibody used in the previous stud- ies was subsequently found by the manufacturer to cross-react with some other cellular constituent.

Measurement of Intracellular Ionized Calcium Concentration-The intracellular concentration of ionized calcium was measured by flow cytometry using the calcium chelator Indo 1 (18). Approximately 4 x lo6 cells were incubated at 37 "C in IMDM supplemented with 10% heat- inactivated dialyzed horse serum for 1 h with a 10 p~ concentration of the acetoqmethyl ester of Indo 1 (Indo l/AM). The cells were washed once with phosphate-buffered saline and resuspended in fresh media. They were then incubated at 37 "C from 1 min to 1 h with various agents including the calcium ionophore ionomycin and immediately analyzed by flow cytometry.

RESULTS

Effect ofNitroprusside on the Growth of Parental and Variant HL-60 Cells

Parental HL-60 cells had a doubling time of 24 h in the absence of nitroprusside, but, in the presence of 1 m~ nitro- prusside, the cells ceased to proliferate after approximately two cell doublings (Fig. 1). The variant cells exhibited logarithmic growth in the absence or presence of 1 mM nitroprusside with a doubling time of approximately 32 h (Fig. 1).

EEect of Inducing Agents on ~~ f f e ren t~a t ion o f Parental and ~ i t r o p r u s s ~ ~ e - r e s ~ s t a ~ t HL-60 Cells

In the absence of an inducing agent, <5% of parental and variant cells were O m - 1 antigen-positive and reduced NBT (Fig. 2, A and B) . Dimethyl sulfoxide and sodium butyrate are potent inducers of myelomonocytic differentiation of HL-60 cells (3) and induced the same percentage of variant and pa- rental cells to acquire the O K " 1 antigen and reduce NBT (Fig. 2, A and B; dimethyl sulfoxide is shown in Fig. 2A and sodium butyrate in Fig. 2B).

As previously reported, 1 mM nitroprusside induced about 65% of parental cells to acquire the O K " 1 antigen and about 30% of parental cells to acquire membrane-bound NADPH OX-

Nitroprusside-resistant HL-60 CeZZs 32157

A Open Bars: Parental HL-60 Cells Hatched Bars: SNP-Resistant HL-60 Cells

100 t 1 20.0

2 10.0 .

;; 5.0

2 2.0

5 1.0 .

E

0 I

u x ...‘ .-

0

0,5 0 - -

1 2 3 4 5 6 7

Time [dl

FIG. 1. Effect of sodium nitroprusside on the growth of paren- tal and variant HL-60 cells. Cultures of parental (circles) and nitro- p~sside-resistant HL-60 cells (triangZes) were initiated at a density of 5 x lo* celldml in I ~ D M supplemented with 10% heat-inactivated FBS to triplicate cultures, 1 mM sodium nitroprusside (SNP) was added (closed circles and dosed triangles). Cell density was assessed daily for 1 week.

idase activity (4); in marked contrast, nitroprusside induced only 8% of the variant cells to test positive for the OKM-1 antigen and ~ 3 % to reduce NBT (Fig. 2, A and B; similar dif- ferences between parental and variant cells were observed for sodium nitrite). A s we found previously (4), 8-bromo-cG~P was a less potent inducer of the O K “ 1 antigen in parental cells than nitroprusside (Fig. 2 A ) , but the two agents exhibited sim- ilar potency in inducing the cells to reduce NBT (Fig. 2Z3 1. This may be because induction of membrane antigens such as OKM-1 requires a more sustained exposure to the inducing agent than induction of functional markers of maturation such as NADPH oxidase (19), and we found that 8-bromo-cGMP, even in the presence of theophylline, was less stable than ni- troprusside at 37” C in culture medium containing heat-inac- tivated serum. The variant cells showed minimal differentia- tion in response to 8-bromo-cGMP, both when measured by acquisition of the O K ” 1 antigen (Fig. 2A 1 and by NBT reduc- tion (Fig. 2B); the small degree of NBT reduction in the variant cells was attributable to the theophylline (Fig. 2 3 ) . Another membrane-permeable cGMP analog, 8-pCPT-cGMP, also in- duced differentiation of parental HL-60 cells but not of the variant cells (data not shown). Interestingly, the variant cells differentiated to the same extent as parental cells in response to either 3 my 8-bromo-cAh4P or 0.5 mM dibutyryl CAlVfP (Fig. 2, A and B) .

When differentiation of HL-60 cells was assessed by morpho- logic ~a tura t ion , similar results were obtained in contrast to parental cells, only a small percentage of the variant cells dif- ferentiated into myelocytes, and none of the variant cells dif- ferentiated into metamyelocytes on exposure to nitroprusside or 8-bromo-cGMP plus theophylline (Table I). The variant cells showed the same degree of m o ~ h o ~ o ~ c a ~ di~erentiation as parental cells in response to dimethyl sulfoxide (Table I) and 8-bromo-CAMP (data not shown).

Efeect of ~ n d u c ~ n ~ Agents on c-myc and c-fos ~ R N A Expression in Parental and Ni~raprussid~-resistant

HL-60 Cells As observed with other drugs that induce myelomonocytic

differentiation of HL-60 cells (20, 211, 1 mM nit~prusside sig- nificantly decreased the amount of c-myc mRNA in parental HL-60 cells (Fig. 3 A , compare lanes 1 and 3, parental cells cultured in the absence and presence of 1 mM nitroprusside, respectively). En the variant HL-60 cells, nitroprusside had no effect on c-myc mRNA expression (Fig. 3 A , lanes 1 and 3, ni- troprusside-resistant cells incubated in the absence and pres-

m 5 80 0 BI *s .- 60 m a

I 2

r 40

:: 20 8?

0 Control 1.2 % 1rnM 3mM 1rnM 3rnM 0.5rnM

DMSO SNP 8Br- The0 BBr- dibutyryl cGMP cAMP cAMP

+ 1 mM Theo

Open Bars: Parental HL-60 Cells Hotched Bars: SNP Resistant HL-60 Cells

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f50uM 1rnM 3mM 1mM 3mM 0.5mM butyrate SNP 8Br- The0 8Br- dibutyry-

cCMP +1 mM The0

cAMP cAMP

FIG. 2. Differentiation of parental and nitroprusside-resistant HL-60 cells. Cultures were initiated at a density of 0.5-1.5 x lo5 celldml in IMDM supplemented with 10% heat-inactivated dialyzed horse serum with the indicated drugs added to duplicate cultures. After 6 days, cells were assessed for their degree of differentiation as de- scribed under “Methods.” Data represent the mean * s. D. of at least three independent experiments. A, the percent O K ” 1 positive cells is shown for each condition. B, the percent of cells capable of reducing NBT is shown for each condition. ~ b b r e ~ a t i o n s are: DMSO, dimethyl sulfoxide; SNP, sodium nitroprusside; 8-Br-cGMP, 8-bromo-cGMP; 8-Br-ulMP, 8-bromo-CAMP; Theo, theophylline. COR^^ = untreated cells.

ence of 1 m~ nitroprusside, respectively). Dimethyl sulfoxide and dibutyryl CAMP on the other hand markedly decreased the amount of c-myc mRNA in both parental and nitroprusside- resistant HL-60 cells, although the residual amount of c-myc mRNA appeared to be slightly higher in the nitroprusside- resistant cells (Fig. 3A, compare lanes 2 and 4, cells incubated with 1.2% dimethyl sulfoxide and I mM dibutyryl CAMP, respec- tively, to lanes 1 ) .

Previous workers have found that c-fos mRNAincreases when HL-60 cells are treated with phorbol esters or C A M P analogs (22-24). We found that dibutyryl CAMP induced c-fos mRNA to a similar extent in the parental and variant cells but that ni- troprusside had no significant effect on c-fos expression in either cell type (Fig. 3B; compare lanes 2 and 3, cells incubated in the presence of 1 m~ nitroprusside and 1 mM dibutyryl CAMP, re- spectively, to lanes I , cells incubated in the absence of drugs).

Examination of the Mechanism of Nitroprusside Resistance in the Variant Cells

Most of the biological effects of nitroprusside are mediated through release of NO which is a potent activator of heme-

32158 Nitroprusside-resistant HI,-60 Cells

Nitroprusside

A Parental Resistant 1 2 3 4 1 2 3 4

c-myc

p-actin . ~.

Nitroprusside

1 2 3 1 2 3 B Parental Resistant

c-fos

p-actin

Flo. 3. Northern blot xnralysis of c-3lyc :and c-1‘0s m1tNA. Pa- rental or n i t r o p r u s s i d ~ ~ - l . ~ ~ s i ~ t : ~ ~ ~ ~ ~1.11s w w ( 1 ru l tund ns d ~ w r ~ l ~ r d in the legrnd of Fig. 2 hut harvrs td (; h aftrr addlng druCs. ’Total cytoplasmic RNA was isolatrtl, c~l~~ctrophorc~srrl. :Ind b l ~ l t t c d onto nylon nwmhrancn as clescrihrtl undrr “Mrthotls.“ A, hyhridimtion of R S A to a human c-myc prohr. Cells wcrr culturrd in the ahsrnrr of drugs f lanes I ) or in the prcwncr of 1.Y; dimrthyl sulfoxide Ilonrs 21, 1 nl\l nitroprusside (1onv.s 3 1. or 1 mhr dihutyryl CAMP flnnrs 4 ). 13, hybridization o f RNA to a human c-fi)s prohr. Crlls werr culturrd in thr ahsrnce of drugs t lnnw I ) or in the prrsrncc o f 1 mxf nitroprusside ( lonm 2 I or 1 mxr tllhutyryl CAMP (lotws 3 ). In the lowrr panel ofA and H , thr hlots wrrr rrprohed with a p-actin prohr to drmonstrntr that thr ohservrd diffcrrncrs in the amounts of c-mvr and c-fos mRNA wrrr not secondary to unequal grl loading.

containing guanylate cyclase and increases the intracellular cGMP concentration in many cell systems ( 5 , 25). Cyclic GMP activates a specific protein kinase and regulates the activity of at least two different CAMP phosphodiesterases (26, 27). In neurosensory cells and in renal and gut epithelial cells, cGMP can modulate ion channels, and, in HL-60 cells, changes in the intracellular calcium concentration can augment differentia- tion (26, 28, 29). To examine the mechanism of nitroprusside resistance in the variant cells we measured: 1) guanylate cy- clase mRNA expression; 2) basal and NO-stimulated guanylate cyclase activity in whole cell extracts and cytosolic extracts; 3) the intracellular cGMP concentration under hasal conditions and in nitroprusside-treated cells; and 4 ) the ionized calcium

~ ”. . .

Vnrinnt Pnrrntnl \‘:Inant l’:Brc,ntal \‘:trl:tnt

78 3 :1 < I C l

36 46 41 80

21 4X 9 t i

23 C l

I :> x1 1 6 12 < I

84 79 X 1 r, < I

89 10 I < I < I

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Concentration in nontreated and nitroprussidr-trcntcd crlls. We have previously shown that neither nitroprusside nor 8-bromo-cGMP significantly change the intracr.llular cAXlP concentration in HL-60 cells suggrsting thcse ct.1Is contain little cGMP-regulated phosphodicsterasc activity 1 . 1 ).

Gunnvlntr Cvc1n.w mNNA Exprrssion i n I’orrntnl n n r l .Vitro- prusside-rrsistanf IT.“ Cvlls-Uv cDNA cloning:. thrw typrs of cr and p subunits of cytosolic guanylate cycl:~sc. have hc.m identified, although the and (3., suhunits cloned from human brain may represent the human homologs of thv ( x I and (3, suhunits cloned from rat and hovine lung f 14-16. 301. Flowvcv-, r r 2 cloned from human fetal brain and 8, cloned from rat kidnry are clearly distinct and appear to diffrr in their tissur distri- bution from a, and P I f 14, 15).

We assessed by Northern hlotting \vhich cytosolic kT,.u:lnylntr cyclase subunits are expressed in HL-60 crlls. \Vhrn Northorn blots containing 10 pg of HL-60 polytA)’-rnrichcd RNA t v r r r hybridized with human (r . , and (31 cDNA prohrs or with rat c r l

and p, cDNA probes, no signal was ohtainrd. evrn unrlrr con- ditions of reduced stringency and under conditions where 5 t ~ g oftotal cytoplasmic RNA from SF9 cells rxprrssinf: human ~ r , / < ! or r a t olpl guanylate cyclase producrd a s t r o n g s i c a l ftiata n o t shown). However, a human (1, cDNA prohe and :I rat p , ! cT)SA probe hybridized to mRNAs of ahout 4 kt) and 2.2 kt). rc.sprc- tivelv, which were of equal abundance in parental and nitro- prusside-resistant HL-60 cells (Fig. 4 ). Exprrssion of (1. guanyl - a te cyclase mRNA of similar size has hccn previously rrported in a human eythroleukemia cell line 141: /3? mRSA of ahout 2.5 kb is expressed in rat liver and kidney, hut hvmatnpnirtic tissues were not examined (151. On Northern hlots of‘ various rat tissues, differential expression of ra t pi and rat /j2 mRS.4 and lack of cross-hybridization hctween /jI and (3 , c.I)NA prnhes have been demonstrated f 15).

Gunn.vlntr Cvc1n.w Actir-ity in I’nrrntol a n d .Vrtropru.widl~- rrsistnnt HI,-60 Cells-The total amount of guanylate cyclasr activity was similar in whole cell extracts (800 x g supern:~t :~nt I

and cytosolic extracts ( 100.000 x g suprrn:ltant) prrp:lrrtl from parental HL-60 cells f0.79 2 0.10 and 0.82 * 0.12 nmol’minlml of cell extract, respectively; similar valurs w t v ohtainrd in extracts of variant HI,-60 cells~. This suggested that I l I A X ) cells have predominantly cytosolic guanylntr cycl:tsr activity with little or no memhrane-bound manylatch cycl:tscb act ivi ty: membrane-hound (receptor) guanylate cyclases arc. rxprc-wd in a tissue-specific manner f31-33). r ,g . human prripheral blood monocytes contain only soluhlc mlanylate cyclasr activity with no detectable rrceptor g-uanylate cyclasf~ activity ~341.

When either the 800 x g or 100,000 x suprmnt:mts of cc.11 extracts were treated with NO. thrrc was a n approxim:ltc. 2-fold increase in guanylate cyclase activity in parrntal and variant HL-60 cells (Table 11; the sprcific activity of k-tanyl:ltr cyclase in the 100,000 x g supernatant \vas approximately 1.H- fold higher than in the 800 x g supernatant hcc:lrlsch of remov:Il

Nitroprusside-resistant HL-60 CdIs 32159

1 2 1 2 Kb

7.5-

4.4-

2.4-

1.4-

FIG. 4. Northern blot analysis of guanylate cyclase mRNA. Pa- rental Ilnnr l ) or nitroprusside-resistant ~lnnc. 2 1 HL-60 cells were harvested during logarithmic growth; Northern blots were prepared with poly(A)'-enrichrd RNA and hybridized to either rr2 ( p o n d A I or p, ( p n n d 13) guanylate cyclase cDNA probes as described under "Meth- ods." Sizes of RNA molecular weight markers in kilohases are shown on the /I$.

TAIIIX I1 Grcnnylotr r.vr1n.w netiuit~y in pnrrntol nnd nitrr~prtrssidr-rpnislnnf

H I A O rd l extracts Guanvlate cyclase activity was measured in a 800 x g and a 100.000

x g supernatant of parental or var iant HL-60 cell extracts in the pres- ence o f 5 mhl MgCI, as described under "Methods." NO was added to some of the samples at a final concentration of 2 p . 1 as described under "Methods." The data are the mean t S.D. of at least three independent experiments performed in duplicate.

Gunnylntr cylasr nctivits ~ " - . - . -

Crll l i n r s -NO +NO

snn X g 1on.ooo X g sno x g 1nn.nnn x g ~~~ .

- ~~

nrnolinrin /mg protrin Parental 0.51 0.08 0.89 -c 0.10 1.03 -c 0.15 1.78 -c 0.21 Nitroprusside- 0.54 t 0.09 0.87 -c 0.11 1.07 T 0.19 1.85 2 0.23

resistant

of particulate protein). This degree of enz-me stimulation by NO is considerably less than that observed for rat or bovine lung cytosolic guanylate cyclase (5 , 35), but is similar to what we found for the human placental enzyme (17) and other work- ers found in rat spleen, rat adrenal glands, and human plate- lets (5 , 36 , 37). The low degree of NO stimulation of the HL-60 and human placental enzymes was not secondary to the assay method because we have observed a >25-fold stimulation of the purified bovine lung enzyme (17) and of the rat lung enzyme expressed in insect (SF91 cells.'

Intracdlular cGMP Concentration in Pnwntol and Nitro- prusside-rfsistant HI,-60 Cells-Under basal conditions, the intracellular cGMP concentration was approximately the same in parental and variant HL-60 cells (Table 111) and was similar to values reported in HL-60 cells by others (38). When parental and variant HL-60 cells were treated with 1 mh1 nitroprusside plus 1 mxr theophylline, the cGMP concentration increased more than 2-fold in both cell types (Table 111). These data sug- gest there was no defect in transport of nitroprussideN0 into the variant cells and are consistent with the above described observation that NO stimulated guanylate cyclase activity in the parental and variant cells to the same degree. The rela- tively modest increase in the intracellular cGMP concentration induced by nitroprusside that we found in HL-60 cells has also been found in nitroprusside-treated human peripheral blood

R. B. Pilz, L. Gao, and G . R. Ross, unpuhlished observations.

T.\III.K I l l Intmrvllulnr &.MI' cnnrrntmtron rn pr~rrntnl r r n r l

n i trol)runsir lc-r~~.~r.~tn~~t I I I A O c d 1 . v

Approximately 25 x 10" cells were incuhated for 20 min in 1\11)\1 supplrmented with lor; FRS under th r intlicatvtl contlitilms. Thr. crlls were extracted. and thr cC\lP concrntratinn \vas mcasurc.d hy :I radio- immunoassay using ""I-laheled cC\lP as drsrr ihrd u n c h '~\!vthnds." The da ta are thr mean -c S.D. from thrw ~nt ic~prnt lmt r~uprr lmontn performed in tluplicatc-.

~~ ~~ ~~~ ~ ~

c(;\If' c o n c ~ n t r n t ~ n n

1':Irmt.d S~lrrlpnl,~l[l , , -rp*l. i l; lnt Additions to r u l t u r ( . ~~~ ~

H I . - c ; f l cr.ll* ! f I . . f X l CI.ll. ~~~ ~

/hI<,/ I / / W / / <

None 1.23 t 0.4 I .:il e 0 . 3

1 mlr Nitroprusside 2.7X 2 0.6 . 3 . 5 1 2 I 1 r,

1 mL1 Theophylline 1.16 t n.3 I . 4 A 2 f l . 4 + 1 mzc theophylline

~~~~ ~ ~

monocytes (39) and nitroprussidr- and SO-trrntrd human hepatocellular fHep3R) cells (40) .

As shown previously, 1 msr theophylline did not incrcvlsc t h r intracellular cGMP concentration (Tablr 111 and Krf. 41: t h r combination of theophylline and nitroprussidr has no rffrct on the intracellular CAMP concentration (4 I.

Intracellular Concpntration n f Ionizrd Colcitrnt in PnrPntnl and Nitroprussidf,-rcsistant HIA-60 CdIs-Thrre was no changr in the intracellular concentration of ionizrd calcium in rither parental or variant cells treated with 1 msr nitroprussidr p l u s 1 m.\r theophylline, 3 m11 8-bromo-cC.MP plus 1 n11r throphyl- line, or 3 mxl A-bromo-CAMP plus 1 mv throphyllinr (data not shown). There was, however, more than a IO-fold incrrasr in the ionized calcium concentration whrn cithrr crll linr was treated with 3 p11 ionomycin.

l~Is~rsslos

We have selected HL-60 cells that p"0w rxponentially in the presence of 1 mv nitroprusside and are rrsistnnt to nitroprus- side-induced differentiation. Even aftrr growing thrsr crlls in the absence of nitroprusside for srveral months. rr-rxpnsing them to nitroprusside did not induce differrntiation. Thr crlls were also resistant to differentiation inducrd by sodium nitritc. 8-bromo-cGMP, and 8-pCPT-cGblP, hut thry diffrrrntintrtf nor- mally in response to polar planar compounds and cA3lP ann- logs. Therefore, the nitroprusside-rrsistant crlls did not show a generalized block in differentiation, hut rather a drfrct that appeared specific to the NO/cG!LfP simal transductic~n path- way. I t is possible that nitroprusside induces differrntiation of HL-60 cells through some mechanism othrr than thr cG>lP signal transduction pathway. However, t h r following ohsrma- tions suggest that nitroprusside inducrs differentiation of HL-60 cells by increasing the intracellular cGMP concrntra- tion: ( i ) theophylline a u p e n t e d t h e effrct of nitroprussidr (Ta- ble I and Ref. 4); ( i i ) the degree ofdifferentiation is proportional to increases in the intracrllular cGMP concrntration ( 4 ) : xnd ( i i i ) the nitroprusside-resistant HL-60 cells wrrr resistant to differentiation induced by 8-bromo-cGMP and R-pCIT-cC;3TP (Fig. 2 and Table I).

We found no difference hetween parrntal and nitroprussidr- resistant HL-60 cells in basal or NO-stimulated ~ a n y l a t r cy- clase activity (Table 11). Basal enzyme activity in hoth cell typrs was from 5 to 30 times higher than found in srvrral non-human tissues (41-44). hut similar to what \vr found prcviously in human placental extracts (17) and two diffrrmt k~nup.; o f workers have found in human platrlrt extracts ( 8 7 , 4.51. ( h a - nylate cyclase activity was stimulntrd only 1.8-fold hy SO in HL-60 cell extracts, and. consistent with this modrst incrrasp in enzyme activity, the intracrllular cC3lP concrntrntion in-

32160 Nitroprusside-resistant HL-60 Cells

creased only 2-fold in HL-60 cells treated with nitroprusside (Table 111). It appears that the degree of NO stimulation of cytosolic guanylate cyclase activity is tissue-specific since other workers have found NO-induced increases in guanylate cyclase activity ranging from 1.7-30-fold depending on the tissue stud- ied (5, 36). This large range of variation in NO stimulation of guanylate cyclase activity may relate to the presence of at least two different a: and two different p subunits of cytosolic gua- nylate cyclase which can yield four different isozymes (14-16, 30,461. Since we found only 1y2 and pz guanylate cyclase mFWA expressed in HL-60 cells, it is possible that the aZp2 enzyme, which has not yet been studied in detail, is less NO-responsive than is the more widely expressed alpl enzyme (47). Consistent with this hypothesis we found that the human erythroleukemic cell line HEL, which expresses cy2 guanylate cyclase mRNA (14), shows only a 2- to %fold stimulation of cytosolic guanylate cyclase activity by nitroprusside: similar to human platelets (37,451 and HL-60 cells; moreover, we have found that human placenta also expresses a2 guanylate cyclase mRNA? and this may explain the modest 2- to &-fold stimulation of human pla- cental cytosolic guanylate activity by NO and nitroprusside (17).

Since CAMP-dependent protein kinase can be activated by cGMP, the question arises whether some of the consequences of an increase in the intracellular cGMP concentration can be attributed to cross-activation of CAMP-dependent protein ki- nase (48-50). Our results suggest that in HL-60 cells an in- crease in the intracellular cGMP concentration does not lead to activation of CAMP-dependent protein kinase since the variant cells did not differentiate in response to an increase in the intracellular cGMP concentration but did differentia^ in re- sponse to CAMP analogs (Fig. 2) and to a forskolin-induced increase in the intracellular CAMP concentration (data not shown). Similar conclusions were reached recently by Eigenthaler et al. (51) who showed that in human platelets deficient in cGMP-dependent protein kinase activity an in- crease in the cGMP concentration did not lead to phosphoryl- ation of vasodilator-stimulated phosphoprotein, a shared sub- strate of the two cyclic nucleotide-dependent protein kinases. Thus, at least in HL-60 cells and human platelets the cGMP and CAMP signal transduction pathways appear to operate in- dependently of each other.

There are at least four cGMP receptor proteins in mamma- lian cells: a cG~P-activated CAMP phosphodiesterase, a cGMP- inhibited CAMP phosphodiesterase, a cGMP-gated ion channel protein, and a cGMP-dependent protein kinase (26-28). We previously found no change in the intracellular CAMP concen- tration in HL-60 cells treated with nitroprusside or 8-bromo- cGMP suggesting these two agents induce differentiation inde- pendently of a change in CAMP phosphodiesterase activity (4); moreover, 8 - p C ~ - c G ~ P also induced differentiation of paren- tal HL-60 cells, and this membrane-permeable cGMP analog does not activate either of the two known cGMP-regulated phosphodiesterases (52). Present evidence indicates that cGMP-gated ion channels are limited to neurosensory and re- nal and gut epithelial cells (26,28). Thus, it seems most likely that nitroprusside and cGMP analogs induce differentiation of HL-60 cells by activating the c G ~ - d e ~ n d e n t protein kinase. Consistent with this hypothesis, we have identified by two- dimensional gel electrophoresis several cGMP-dependent pro- tein kinase substrates in HL-60 cells; at least one of these proteins shows increased phosphorylation in parental cells, but not in variant cells, after exposure to nitroprusside or 8-bromo- cGMP.3

3 G. R. Boss, J. S. Scheele, and R. B. Pilz, submitted for publication.

In conclusion, it appears that the defect in the nitroprusside- resistant cells is distal to guanylate cyclase activation in the NO/cGMP signal transduction pathway and may be at the level of protein phospho~lation by cG~P-dependent protein kinase.

Acknowtedgments-We thank Dr. M. Adler for the omyc and e-fos probes, Drs. G. GuellaGn, D. Koesling, and M. Nakane for the guanylate cyclase cDNA probes, and S. Shanks and S. Wancewicz for help in preparing the manuscript.

2. 1.

3.

4. 5.

6. 7.

8.

10. 9.

11.

12.

13.

14.

15.

17. 16.

18.

19.

21. 20.

22.

23.

24.

25.

26.

27.

29. 28.

30.

31. 32.

33.

34.

35

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