Induced maturation of the human promyelocytic leukemia cell line, HL-60, by 2-β-D-ribofuranosylselenazole-4-carboxamide

Vol. 115, No. 3, 1983 September 30, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS Pages 971-980

INDUCED MATURATION OF THE HUMAN PROMYELOCYTIC LEUKEMIA CELL LINE, HL-60, BY 2-8-D-RIBOFURANOSYLSELENAZOLE-4-CARBOXAMIDE

Diane L. Lucas, 1 Roland K. Robins, 2 Robert D. Knight, 1 and Daniel G. Wright I

Department of Hematology, iWalter Peed Army Institute of Pesearch, Washington, D.C. 20307 2The Cancer Research Center, Department of Chemistry, Brigham Young University, Provo, UT 84601 Received August 16, 1983

The new synthetic nucleoside analogue, 2-8-D__-ribofuranosylselenazole-4carboxamide, was evaluated for its effects upon the growth and maturation of the human promyelocytic leukemia cell line, HL-60. At a concentration of ~ 1 nm, this agent was found both to decrease HL-60 cell proliferation and to cause the cells to acquire an ability to phagocytose opsonlzed yeast and to reduce nitroblue tetrazolium dye, functions characteristic of mature myeloid cells. In addition, this agent at similar concentrations caused a marked depression of intr~cellular guanosine nucleotide pools and a reduction in the incorporaton of [14C] hypoxanthine into guanylates. These results suggested that the selenazole nucleoside caused an inhibition of inosinate monophosphate dehydrogenase, a key enzyme of guanylate biosynthesis. We therefore measured the activity of this enzyme indirectly by simultaneous-UV-radioactivity HPLC as well as by a direct radiometric method and demonstrated markedly reduced enzyme activities by both assays in drug treated cells. Dose response studies indicated that concentrations of drug which caused > 30% inhibition of IMP dehydrogenase activity induced > 50%maturation of the cells. These observations with this new nucleoside analogue provide further support for the concept that production of guanosine nucleotides and the activity of IMP dehydrogenase have a role in regulating the terminal maturation of myeloid cells.

2-B-D-Pdbofuranosylselenazole-4-carboxamide (RSC) I, a nucleoside analogue, has recently been synthesized by Srivastava and Robins (I) and has been shown to have both cytotoxic effects on P-388 and LI210 cells in vitro and oncolytic effects on lewis lung carcinoma in mice.

This compound is

structurally related to the thiazole nucleoside, 2-~-D-ribofuranosylthiazole4-carboxamide, (NSC 286193, tiazofurin) for which the oncolytic properties and

i. Abbreviations: RSC = 2-~-D-ribofuranosylselenazole-4-carboxamide; RTC : 2-~-D-ribofuranosylthiazole-4-carboxamide; IMPD : inosinate monophosphate dehydrogenase; NBT : nitroblue tetrazolium; IC50 : the concentration of drug that causes a 50% reduction in cellular proliferation; ID50 = the concentration of drug that induces maturation of 50% of the cells.

0006-291X/83 $I .50 971

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a mechanism of action have been described

(2-5).

Previous studies have shown

that tiazofurin causes inhibition of inosinate monophosphate

dehydrogenase

(IMPD) and a consequent depletion of guanosine nucleotide pools (3-5). Studies in our own laboratory have also shown that inhibition of guanosine nucleotide biosynthesis is a feature of induced maturation of the human promyelocytic leukemia cell line, HL-60 (6). selenazole nucleoside

We therefore evaluated the

for its effects both as an inducer of HL-60 cell

maturation and as an inhibitor of IMPD.

We predicted

that inhibition of IMPD

by the new compound would~be associated with induced maturation of HL-60 cells, because of our previous observations that inhibitors of IMPD (mycophenolic acid, 3-deazaguanosine,

and tiazofurin)

consistently

cause

maturation of this myeloid cell line. MATERIALS AND METHODS Cell Culture: Suspension cultures of the human promyelocytic leukemia cell line, HL-60, were maintained in RPMI 1640 medium (Microbiological Associates, Walkersville, MD) supplemented with 10% heat-inactivated (56°C for 30 min) fetal bovine serum (Reheis Chem. Co., Phoenix, AZ) under standard conditions of temperature and humidity. The cells, which were routinely passed at 4-5 day intervals, manifested stable growth characteristics with a cell doubling time of 28 hr. Cell counts were determined by hemocytometer chamber counting and the viability of cells was assessed by dye exclusion techniques using trypan blue. Cellular morphology was evaluated on cytospin preparations that had been stained with Wright-Giemsa. The effects of the selenazole and thiazole nucleosides on HL-60 cell growth and maturation were evaluated by direct addition of the drugs to cultures newly established from cells in logphase growth. Assays of HL-60 Cell Maturation Phagocytosis: The relative maturity of HL-60 cells in culture was evaluated by direct inspection of their morphology and by assessing their ability to phagocytose opsonized yeast. Heat-killed Candida albicans were opsonized by incubation with 20% human AB serum in PBS for 2 hr at 37°C aliquoted and stored at -20°C. HL-60 cells were washed twice in PBS and incubated with opsonized yeast at a yeast to cell ratio of 10:1 for 20 min at 37°C. The percentages of cells that had ingested yeast was determined by direct microscopic inspection of at least 200 cells. NBT Dye Reduction: Reduction of nitroblue tetrazolium (NBT) dye (Sigma Chem. Co., St. Louis, MO) by HL-60 cells was determined according to a previously described method (7). The cells (Ixl06) were incubated for 20 min at 37°C in RPMI 1640 medium with I00 ng/ml 12-O-tetradecanoylphorbol-13acetate (Sigma) and with 0.1% NBT. After incubation, the cells were placed onto slides by cytospin~ fixed, and stained. The percentage of cells that contained blue-black formazan deposits, the product of NBT dye reduction, was determined by microscopic examination. High Performance Liquid Chromatography: Analysis of purine nucleotide pools and their biosynthesis in HL-60 cells was achieved by adapting methods

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previously described (8). Cell suspensions were centrifuged at 4°C, 200 x g for 10 min and resuspended in a final volume of 0.5 ml to which an equal volume of 1 M perchloric acid was added. After 25 min at 4°C, the samples were centrifuged at i0,000 x g_for I0 min. The resultant supernatant was neutralized with 10 M KOH and precipitated salt was removed by centrifugation. Purine nucleotides of extracted HL-60 cells were analyzed using a Spectra Physics (Santa Clara, CA) liquid chromatography system. The mobile phase consisted of potassium phosphate buffers A, 0.08 M, pH 3.4, and B, 0.8 M, pH 4.3. UV absorbance of eluants was measured using an Altex (Berkeley, CA) dual waveiength detector and associated radioactivity measured simultaneously with a Flo-One (Radiomatic Instruments, Addison, IL) radioactive flow detector. Quantification of purine nucleotides was accomplished by using external standards (8). Inosine Monophosphate Dehydrogenase Assay: The assay for IMPD was a modification of the radiometric technique described by Cooney et al. (9). Cell samples were prepared for analysis of IMPD activity first by centrifugation to concentrate the cells followed by sonication. Cell sonicates were then centrifuged in an Eppendorf microcentrifuge for 2 min and the resulting supernatant was combined with 1 ml of 1 M sodium acetate, pH 5.0. The precipitated protein, dissolved in a buffer consisting of 0. i M TRIS, 1 mM dithiothreitol and 0.2 M KCI, served as the enzy+e source. The substrate mixture was prepared by adding 3 BI of 0. i M NAD to i00 pl of [2-3H] DiP (200 ~Ci/ml), a gift from Dr. Cooney (NCI, NIH). The assay was conducted in Eppendorf tubes which contained a solution of 50 ~i of 0 . 0 1 M uridine, 50 ~i of 2x10 -5 M allopurinol and 2 mg of KCI. To this, I00 ~i of the enzyme preparation was added and the reaction initiated by addition of 5 pl of the substrate. After a 1 hr incubation period at 37°C, the reaction was terminated by heating at 95°C for 1-2 min. The samples were then centrifuged at 12,000 x g__for 2 mfn. A i00 ~i aliquot of the reaction mixture was applied to Dowex anion exchange resin which was then washed with 1.9 ml of distilled H20. The eluant (tritiated water) was collected in scintillation vials and the radioactivity measured in Hydrofluor scintillation fluid (National Diagnostics, Somerville, NJ). RESULTS We have reported previously that inhibitors of IMPD induce HL-60 cells to mature

(6).

In the present study, we investigated the activity of a recently

synthesized nucleoside analogue,

the selenazole nucleoside,

inhibitor of IMPD and an inducer of HL-60 cell maturation. RSC is illustrated in Fig.

I.

Tiazofurin

as both an The structure of

(RTC) differs from RSC only at the

0 II

N~ S e

Ho_ HO Figure I.

OH

2- B-D-ribofuranosylselenazole-4-carboxamlde

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I00

B

P

o/ l

90 80

m

m

~

60

E~

50

"~:i

40

_~

30 2o

B

m

#

m

B

I0 I

I

I

I

I

I

9

8

7

6

5

4

DRUG CONCENTRATION(-LOG MOLARITY) Figure 2. HL-60 cells were cultured in RPMII640 medium supplemented with insulin, transferrin, and selenium to which varying amounts of selenazole or thiazole nucleosides were added. The percent inhibition of cell growth and the percentage of mature cells as assessed by reduction of NBT dye were evaluated after 6 days. Key: (0) selenazole nucleoside, and (e) thlazole nucleoside.

one position of the thiazole ring which is occupied by a sulfur rather than a selenium atom.

The activities of this compound were compared with those of

RSC in these studies.

Both RTC and RSC, when added to the culture medium of

HL-60 cells in log phase growth, decreased cellular proliferation, morphological maturation to metamyelocytes,

caused

and in a dose dependent manner

induced the cells to acquire an ability to phagocytose opsonized yeast and to reduce nitroblue tetrazolium dye, which are functions that characterize mature myeioid cells (Fig. 2).

RSC was found to be the more potent drug, with an

IC50 of ~ 50 nM compared with an IC50 of ~ 9 ~M for RTC.

With both drugs the

inducing dose 50 (ID50) was tenfold less than the IC50 (Fig. 2). Purine nucleotides were quantified by HPLC analysis of perchlorate extracts of HL-60 cells cultured with varying amounts of RSC for 72 h (Table i).

Intracellular pools of adenylates in treated cultures were similar to

those of untreated cells.

Unlike the adenylates,

however, guanosine

nucleotide pool sizes were reduced from 24 nmoles/107 cells to 8 nmols/107 cells in cultures treated with 100 nM RSC.

Depression of guanosine nucleotide

pool sizes could be detected at doses of RSC as low as 1 nM.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Table i

Incorporation of [14C] Hypoxanthlne into Purlne Nucleotides in HL-60 Cells After Treatment with Selenazole Nucleoside

Additions

IMP

XMP

GXP

AXP

1.6 75 47

3.6 56 16

24.2 943 39

87.6 3058 35

10-7 M nmoles/107 cells radioactivity (dpm x 10-2 ) specific activity

3.4 98 28

N.D. N.D. N.D.

8.1 98 3

82.7 1618 20

10-8 M nmoles/107 cells radioactivity (dpm x 10-2 ) specific activity

1.9 59 31

2.6 29 11

17.5 494 28

81.0 2013 25

10-9 M nmoles/107 cells radioactivity (dpm x 10-2 ) specific activity

1.7 40 24

3.5 49 14

19.8 669 34

83.4 2707 32

None nmoles/lO 7 cells radioactivity (dpm x 10-2 ) specific activity Selenazole Nucleoside

Titration of the effects of selenazole nucleoside on incorporation of [14C]hypoxanthine (i ~Ci/ml, 4 hr) into IMP, ILMP, GXP (ZGMP, GDP, GTP) and AXP (ZAMP, ADP, ATP) after 72 hr. Simultaneous UV-radioactivity measurements of HPLC eluants enabled quantification of nucleotide concentrations and radioactivity in neutralized I M PCA extracts. N.D., none detectable.

The conversion of IMP to )~IP is catalyzed by IMPD and inhibition of this enzyme would be expected to decrease the concentration of XMP and guanosine nucleotides as well as increase IMP pools.

An inverse correlation of the X M P

and IMP pools in cells treated with RSC is shown in Table I.

When I00 ~

RSC

P

was added to the cultures, XMP could not be detected while there was a twofold increase of IMP pools.

It is also apparent in Table 1 that the selenazole

nucleoside was very effective as an inhibitor of [14C]hypoxanthine incorporation.

Biosynthesis of both adenylates and guanylates was reduced by RSC but

it was readily apparent that the impairment of guanylate biosynthesis was much greater than that of adenylate biosynthesis.

The selective reduction in both

guanosine nucleotide pool sizes and guanylate biosynthesis is illustrated in Fig. 3 which shows profiles for purine nucleotide concentration and their associated radioactivity from the incorporation of radiolabel from [14C]Hx for untreated HL-60 cells and for cells incubated with RSC for 72 hr.

975

One should

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AF/q, 234~

/°

}

v z

8

/, 15

-.1. i

~

15

6

7

i.

9

9

4C)

PLUSRSC

RETENTIONTIME

40

{ MINUTES ) Figure 3. Chromatographic resolution of purtne nucleottdes and associated radioactivity (cpm) in HL-60 cell extracts from control (panel A) and selenazole nucleos~de-treated (panel B) cultures. HL-60 cells were cultured with the drug (i0-" M) for 72 hr. During the last 4 hr of culture, [14C} hypoxanthine, (I pCi/ml, 51.1 m Ci/mmol, New England Nuclear, Boston, MA) was added to the cultures which were then extracted with 1 M perchloric acid for 25 min at 4"C. Aliquots of neutralized extracts were applied to a Partlsil-10 SAX anlon-exchange micropartlculate column (Whatman). Nucleotide ~ peaks (OD, 254 nm) were identified by correspondence to standards. Peak numbers: (I) IMP, (2) ~IP, (3) GMP, (4) UDP, (5) ADP, (6) GDP, (7) UTP, (8) ATP, (9) GTP.

note in particular the absence of peak 2 (XMP) and the reduction in peaks 6 (GDP) and 9 (GTP) in the nucleotide profile from the RSC treated cells. levels of GMP appeared to be elevated in extracts of RSC treated cells

The (peak

3, Panel B), but this is likely due to metabolism of the drug to 2-6-Dribofuranosylselenazole-4-carboxamide

5'-phosphate which coelutes with (~P

(1). It was possible to estimate the apparent activity of IMPD from radiotracer studies by summarizing the incorporation of radiolabel into EMP and guanylates

(10).

When these calculations were made, the amount of

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Table 2 Apparent Activity of IMPD in Guanosine Nucleotide Biosynthesis from [" C]Hypoxanthine in RSC Treated HL-60 Cells Selenazole Nucleoside~

Control

10-7

Radioactivity,

999±11

(dpm x 10-2)

Percent Inhibition

98~23

-

(M)

10 -8

10-9

523±68

718±93

90

48

28

HL-60 cells were cultured with the indicated amounts of RSC for 72 hr and during the last 4 hr of culture [14C]hypoxanthine, I ~Ci/ml, was present. The results are the means of three experiments, ± S.E.M., in which the amount of radiolabel incorporated into XMP, GMP, GDP, and GTP was summarized.

radiolabel from

[14C]Hx incorporated into XMP and guanylates was markedly

reduced in cells treated with RSC (Table 2).

In cultures which contained

10 -7 M RSC there was a reduction from 99,900 dpm to 9,800 dpm (90% inhibition).

The percent inhibition of [14C]Hx incorporation into nucleotide

metabolites of IMPD in cultures containing

I ~4 RSC was 28%.

In order to determine more definitively the activity of I ~ D cells treated with RSC (10-8M) and RTC

in HL-60

(10-6M), the activity of this enzyme

was measured directly in cells which had been incubated with these agents for 72 hr.

Both RSC and RTC at these concentrations inhibited cellular

proliferation and induced maturation of the cells and, as shown in Table 3, decreased the specific activity of IMPD by 69% and 76% respectively. DISCUSSION Alterations of purine metabolism in immunodeficiency

diseases and in

malignancy have suggested that purine derivatives have a critical role in regulating the normal growth and maturation of mammalian eells. the control of purine metabolism in proliferating cells that have undergone malignant Ib~ dehydrogenase,

Studies on

cell populations and in

transformation have focused attention upon

a key rate controlling enzyme in the biosynthesis of

guanosine nucleotides. rapidly proliferating

High levels of this enzyme have been associated with cells whereas low levels have been associated with

terminally differentiated

cells

(ii).

Our current studies with the selenazole

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Table 3

IMP Dehydrogenase Activity After Cultivation of HL-60 Cells with Thiazole or Selenazole Nucleoside

% Mature Ceils

IMP Dehydrogenase Activity Addition

none

Specific Activity (picomoles/min/mg)

% Inhibition

NBT

Phagocytosis

205± 7

-

4±2

3± 1

RSC, 10-8 M

64±13

69%

65~7

47±12

RTC, 10-6 M

50* 4

76%

63±9

58~11

HL-60 cells in suspension culture were incubated for 72 hr with either RTC or RSC in RPMI 1640 supplemented with insulin, transferrin and selenium at 37°C. The cells were then washed twice in PBS, suspended in Buffer W and sonicated. A crude extract was prepared by precipitation of the sonicate with sodium acetate and IMP dehydrogenase activity was measured by the radiometric assay described in Methods. Enzyme activity is expressed as picomoles of tritated water formed/min/mg protein, ± S.E.M., n = 3.

nucleoside, which is shown to be a potent inhibitor of IMPD, support the concept that the activity of this enzyme may have role in regulating cellular maturation.

Selenazole nucleoside is structurally related to the c-

nucleosides, many of which exhibit oncolytic and antiviral properties

(12).

Exposure of HL-60 cells to selenazole nucleoside caused them to undergo morphologic maturation to the metamyeloeyte stage of granulocyte development and to acquire the differentiated functions of NBT reduction and phagocytosis. RSC was found to be an effective inducer of HL-60 cell maturation at concentrations in the nanomolar range.

In this respect it is similar to retinoic

acid, another potent inducer of HL-60 cell maturation

(13).

We have shown

previously that depression of guanosine nucleotide pool sizes, which occurs within 24 hr after exposure to inducers such as retinoic acid, dimethyl sulfoxide, dimethyl formamide, and hypoxanthine, is a consistent feature of induced HL-60 cell maturation.

Moreover, we have shown that exposure of these

cells to inhibitors of IMPD, such as mycophenolic acid and RTC, also results in decreased guanosine nucleotide pool sizes, and induced maturation of HL-60 cells (6).

reduced guanylate biosynthesis,

Like these other inhibitors of

IMPD, RSC interrupts the incorporation of radiolabeled hypoxanthine into guanosine but not adenosine nucleotides of HL-60 cells.

978

Thus, a reduction of

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guanosine nucleotide pool sizes either by inducers such as retinoic acid or by specific inhibitors of IMPD (mycophenolic acid, RTC and most strikingly RSC) is associated with maturation of these myeloid cells.

Our finding that there

is a decrease in the specific activity of IMPD in HL-60 cells induced to mature is in accord with previous reports that the specific activity of IMPD in leukemic cells is higher than that in normal blood leukocytes

(14).

The mechanism whereby the thiazole and selenazole nucleosides

inhibit

IMPD has yet to be fully defined.

However, it has been postulated that a

proximate metabolite

(3,4) and RSC (15), possibly an NAD analog,

of tiazofurin

is the principal inhibitor of IMPD.

The oncolytic properties of tiazofurin and other inhibitors of IMPD have been attributed to a reduction of guanylidate pools sufficient to cause an inhibition of DNA synthesis

(16,3,5).

addition of exogenous guanosine.

These effects can be reversed by the

However, the mechanisms by which guanylidate

pool sizes influence cellular maturation may be different from those that affect cellular proliferation.

Indeed, impaired cellular proliferation per se

is not necessarily associated with maturation, with HL-60 cells

(6,17,18).

as we and others have shown

It is of interest in this regard that the

oncolytic effects of RSC and RTC are observed at concentrations which differ from those inducing maturation.

Modest reductions

(~ 20%) in guanosine

nucleotide pool sizes are associated with cellular maturation whereas far greater reductions in guanosine nucleotide concentrations appear to be required to inhibit cell growth. approximately

The IC50 of RTC for H L - 6 ~ cells is

9 ~M; that reported for the macrophage cell line P-388 is 1 ~M

(5), indicating a similar range of susceptibility of these two cell lines to the cytotoxic effect of thiazole nucleoside.

On the other hand, the ID50 of

this compound for HL-60 cells is tenfold less.

The potency of selenazole nucleoside as an inducing agent is comparable to that of retinoic acid and two orders of magnitude greater than tiazofurin in which a sulfur atom replaces selenium.

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Structure activity correlates such

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as this may provide a basis for the rational design of new chemotherapeutic drugs. REFERENCES I.

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Cooney, D.A., Wilson, Y. and McGee, E. (1983).

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Robins, R.K. (1982) Nucleosides and Nucleotides I, 35-44.

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Becker, H.J. and Lohr, G.W. (1979) Klin. Wochenschr. 57, 1109-1115.

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