Regulation of the expression of enzymes involved in the replication of DNA in chemically-induced granulocytic differentiation of HL-60 leukemia cells

Leukemia Research 22 (1998) 687 – 695

Regulation of the expression of enzymes involved in the replication of DNA in chemically-induced granulocytic differentiation of HL-60 leukemia cells Yixiang Chen a, John A. Sokoloski a, Edward Chu b, Alan C. Sartorelli a,* a

Department of Pharmacology, Cancer Center, Yale Uni6ersity School of Medicine and VA Connecticut Healthcare System, New Ha6en, CT 06520, USA b Department of Internal Medicine and De6elopmental Therapeutics Program, Cancer Center, Yale Uni6ersity School of Medicine and VA Connecticut Healthcare System, New Ha6en, CT 06520, USA Received 14 November 1997; accepted 7 February 1998

Abstract The expression of seven enzymes involved in the biosynthesis of DNA was measured in HL-60 promyelocytic leukemia cells treated with dimethylsulfoxide (DMSO) or all-trans retinoic acid (RA) to gain information on their role in the termination of proliferation in cells undergoing granulocytic differentiation. The steady-state levels of the mRNAs for topoisomerase I, topoisomerase II, DNA polymerase-a, thymidylate synthase, thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase progressively declined from day 3 to day 7 of exposure to the polar solvent or the retinoid suggesting that the expression of these enzymes is coordinately regulated. In contrast, a pronounced difference between the two inducers of differentiation occurred in the expression of the mRNA of the M2 subunit of ribonucleotide reductase, with DMSO causing virtually complete inhibition of the expression of the M2 subunit of the enzyme from day 5 through day 7, with no change in the steady-state levels of the mRNA being produced by retinoic acid. Measurement of the enzymatic activities of two of these catalysts, thymidylate synthase and thymidine kinase, in cells exposed to the two inducers of maturation corroborated the findings at the level of the mRNAs, with corresponding decreases in the activity of these enzymes. The findings collectively demonstrate that the down-regulation of the expression of a relatively wide variety of enzymes involved in DNA replication occurs as late events in the granulocytic differentiation of HL-60 cells, ensuring that cellular replication cannot occur in terminally differentiated cells. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: HL-60 leukemia; Granulocytic differentiation; DMSO; Retinoic acid; mRNAs; DNA biosynthesis

1. Introduction There is considerable evidence that an inverse relationship exists between cellular differentiation and proliferation [1,2]. Thus, mechanisms are operative which regulate these competing events. The HL-60 promyelocytic leukemia represents an extensively studied system Abbre6iations: DMSO, dimethylsulfoxide; RA, all-trans retinoic acid; NBT, nitroblue tetrazolium; Mo-1, CD11b integrin; PBS, phosphate buffered saline; TPA, 12-O-tetradecanoylphorbol 13-acetate; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; SDS, sodium dodecylsulfate; topo II, topoisomerase II; PMSF, phenylmethylsulfonyl fluoride; topo I, topoisomerase I. * Corresponding author. Tel.: + 1 203 7854533; fax: + 1 203 7372045. 0145-2126/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved. PII: S0145-2126(98)00053-8

which is responsive to a variety of chemical and biological inducers of terminal differentiation (see for example Ref. [3]). A number of studies have provided evidence for regulatory events that may be involved in the termination of proliferation when HL-60 cells are exposed to inducers of terminal differentiation. Two exceedingly early events that have been described which are capable of regulating cellular proliferation involve the relatively rapid modulation of p21 WAF1/CIP1 [4,5] and transferrin receptor [6–9] gene expression following the chemical initiation of either myeloid or monocytic differentiation of HL-60 leukemia cells. The p21 protein, which is induced following exposure to initiators of maturation, is a general inhibitor of cyclin-dependent kinases, thereby leading to growth

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arrest [4,10,11]; the expression of this gene is increased within 1–3 h following exposure to a variety of inducers of differentiation [4]. In contrast, the expression of the transferrin receptor is down-regulated when HL-60 cells, as well as several other leukemia cell lines, are exposed to inducers of differentiation [6 – 9]. The decrease in expression of the mRNA for this receptor varied depending upon the inducer, beginning at 4 h for aclarubicin, 8 h for DMSO and at later times for retinoic acid and 12-O-tetradecanoyl phorbol 13-acetate [7]. Changes in the expression of other enzymes involved in the synthesis of DNA occurred much later, implying that their down-regulation is probably a consequence of the cessation of cell proliferation which accompanies terminal differentiation. Thus, the expression of the M1 subunit of ribonucleotide reductase [12], of thymidylate synthase and nuclear factors which interact with the 5%-upstream region of the thymidylate synthase gene [13], of thymidine kinase [14], and of topoisomerase I and II [15 – 17] were markedly depressed in HL-60 cells exposed to both myeloid and monocyte/macrophage inducers of maturation at relatively later times. The present study was designed to investigate the effects of the myeloid inducers of differentiation, DMSO and retinoic acid, on the expression of a wide variety of enzymes involved in the biosynthesis of DNA.

2. Materials and methods

2.1. Chemicals Retinoic acid, dimethylsulfoxide (DMSO), 12-O-tetradecanoylphorbol 13-acetate (TPA) and nitroblue tetrazolium (NBT) were purchased from the Sigma Chemical Co. (St. Louis, MO); [a-32P]-deoxycytidine 5%-triphosphate ([a-32P]-dCTP; 3000 Ci mmol − 1) and [2-14C]-thymidine (10 mCi mmol − 1) were obtained from Amersham Life Science Inc. (Arlington Heights, IL); [5-3H]-2%-deoxyuridine 5%-monophosphate ([3H]dUMP; 5 Ci mmol − 1) was purchased from Amersham. The molecular biology reagents, diethyl pyrocarbonate, formamide, dextran sulfate, sodium citrate, sodium acetate and salmon sperm DNA were obtained from Sigma. Prime-it II random primer labeling kit for cDNA labeling and NucTrap push columns for purification of [32P]-labeled cDNA were from Stratagene (La Jolla, CA). ECL chemiluminescent reagents were obtained from Amersham Life Science. The transfer membrane was Immobilon-P (PVDF, 0.45 mM pore size; Millipore Corp, Bedford, MA). For Northern analyses, a pBS-hTOP2 phagemid with a cloned cDNA for human topoisomerase IIa, a cDNA probe for murine thymidylate synthase (1.3 kb PstI fragment from

pBR322) and a cDNA probe for murine thymidine kinase (pMTK4) were generously provided by Dr S. Srimatkandada of the Department of Pediatrics, Yale University School of Medicine.

2.2. Cell culture HL-60 promyelocytic leukemia cells were supplied by Dr Robert C. Gallo of the University of Maryland, Baltimore, MD. Cell stocks were routinely checked for Mycoplasma contamination by the gene probe method (Gen-Probe, Inc., San Diego, CA). HL60 cells were passaged by seeding at 1.5–2× 105 cells ml − 1 in RPMI 1640 medium supplemented with 15% heat-inactivated (56°C for 30 min) fetal calf serum (Gibco Laboratories, Grand Island, NY), 100 units ml − 1 of penicillin and 100 mg ml − 1 of streptomycin. Cultures in exponential growth were passaged every 3 days. Cells were incubated at 37°C with 5% CO2 in air in a humidified incubator. The doubling time of HL-60 cells was 26–28 h. Cell stocks were re-established from cultures cryopreserved in 10% glycerol after 35–60 passages. Cell numbers were determined with a Model ZBI Coulter counter (Coulter Electronics, Hialeah, FL).

2.3. Cell differentiation Expression of Mo-1 (CD11b integrin) was employed as a measure of cellular differentiation using flow cytometry (FACS IV; Becton Dickinson, San Jose, CA). The lyophilized fluorescence-labeled Mo-1 monoclonal antibody (Coulter Immunology) was prepared by reconstitution with distilled water, followed by centrifugation at 100000× g for 10 min as recommended by the manufacturer. Then, 2× 106 HL-60 cells were harvested and washed three times with ice-cold phosphate buffered saline (PBS). Cells were then incubated with antibody on ice for 30 min, washed three times with ice-cold PBS, and the amount of antibody bound to cells was quantified. Excitation at 488 nm and emission at 530 nm were the wavelengths employed. The channel number representing the mean fluorescence intensity of 20000 cells was determined and compared to cells incubated in the absence of fluorescent-labeled antibody. The percentage of Mo-1 positive cells was determined by ascertaining the number of treated cells whose fluorescence intensity exceeded that of 95% of the cells incubated in the absence of the fluorescent-labeled antibody. The presence of dead or damaged cells was determined by pre-incubating cultures with 2.0 mg ml − 1 of propidium iodide for 5 min prior to analysis. In these experiments, less than 5% of the cells were stained with propidium iodide and these were removed from the analyses by electronic gating. After different periods of treatment with the inducers of differentiation, cell viability was also determined by dye exclusion

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using 0.1% trypan blue. The capacity of HL-60 cells to undergo functional granulocytic maturation was assessed by determination of NBT positivity. Cells (0.5– 2.0 ×106) in 1 ml of RPMI 1640 medium containing 0.1% NBT and 1 mg of TPA were incubated for 20 min at 37°C. The percentage of NBT positive cells containing blue-black formazan deposits was determined by light microscopy as previously described [18]. HL-60 cells were exposed to inducers of differentiation for 1, 3, 5 and 7 days. In both control groups (without differentiation inducers) and groups exposed to the chemical initiators of maturation, the cell seeding concentrations were adjusted for each treated and control flask such that the concentration of cells from different treatments and durations of exposure would have approximately the same number of cells (12× 105 cells ml − 1) at the time of harvesting. This approach minimized possible differences in gene expression due to cell density-dependent growth inhibition.

2.4. Northern analyses Total cellular RNA was extracted by the acid guanidinium thiocyanate-phenol-chloroform method described by Chomczynski and Sacchi [19]. In some experiments, total RNA was also extracted using the Trizol reagent from Gibco BRL (Grand Island, NY) according to the protocol provided by the company. Northern analyses were carried out by a modification of the method of Davis et al. [20]. The total RNA content of each sample was measured spectrophotometrically, and 10–15 mg of total RNA from each sample was fractionated by electrophoresis on a 1.0% agarose gel containing 2.0% formaldehyde for 3 h at 90 V. RNA was capillary-transferred to a nylon membrane (Nytran, 0.45 mm pore size; Schleicher and Schuell, Inc., Keene, NH) using 10 × SSC (1.5 M NaCl, 0.15 M sodium citrate) overnight. Following transfer, membranes were washed with water for 1 min and air dried. Samples were cross-linked to filters by brief UV irradiation at 254 nm (UV Stratalinker 1800; Stratagene). The membranes were pre-hybridized for 3 h to overnight at 42°C in 50% deionized formamide, 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS) and 0.2 mg ml − 1 of salmon sperm DNA. The [32P]-labeled cDNA probes were added to the pre-hybridization solution, and hybridization was carried out at 42°C overnight. The probe was labeled by the random priming reaction using [32P]-dCTP and the Klenow fragment of DNA polymerase I. The nylon membrane was autoradiographed using Kodak X-OMAT film at −80°C. The hybridization intensity was quantified with a laser densitometer (Personal Densitometer SI; Molecular Dynamics, Sunnyvale, CA). The cDNA probes used in the Northern blot hybridizations were derived as follows: the probe for

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topoisomerase IIa was an EcoRI fragment of 5.6 kb from the pBS-hTOP2 phagemid [21]; for thymidylate synthase, a murine cDNA of 1.3 kb cloned in the PstI site of pBR322 [22]; for thymidine kinase, a 1.16 kb murine TK1 cDNA (pMTK4) cloned in pcD [23]; for the human ribonucleotide reductase gene M2 subunit (RRM2), a BamHI/HindIII cDNA fragment of 1.17 kb from the pCRII phagemid (GenBank/EMBL/DDBJ: X59618); for the human DNA topoisomerase I, a HindIII/XmnI fragment of a cDNA from the pATH11 expression vector [24]; for the human DNA polymerase a, a PstI/HindIII fragment of a cDNA from pcD-KBpola [25]; for human hypoxanthine-guanine phosphoribosyltransferase (HGPRT), a BamHI fragment of 1.45 kb from the cDNA from the plasmid vector EBO-pCD ([26]; ATCC 59562); and a PstI 2 kb fragment from a cDNA, pA1, for b-actin, [27]. This latter probe for b-actin was used alone or hybridized simultaneously with other cDNA probes to quantify the amount of total RNA in each sample. cDNA probes were labeled using a Prime-it II random primer labeling kit from Stratagene, according to the guidelines provided by the company. Purification of the labeled probes was accomplished using the NucTrap probe purification push column (Stratagene). In some experiments, membranes were stripped of radioactive probes for rehybridization using 0.01 × SSC containing 0.1–0.6% SDS at 70– 95°C for 15–50 min until radioactivity monitored with a Geiger counter was less than 150 cpm.

2.5. Enzymatic assays Cell extracts were prepared by repeated freezing and thawing of 2× 107 cells three times in 50 mM Tris– HCl, pH 8.4, containing 1 mM b-mercaptoethanol. Cell lysates were centrifuged at 27000× g for 20 min at 4°C and the supernatant was assayed immediately for enzyme activities. Thymidylate synthase activity was measured by a modification of the method of Roberts [28]. Briefly, a 20-ml aliquot of the cell homogenate was diluted 1:1 with freshly prepared Tris–sucrose solution [final concentrations of 1 mg ml − 1 of tetrahydrofolate, 20 mM dithiothreitol, 300 mM Tris–HCl, pH 7.5, 120 mM NaF, 2.2 mM dUMP, 8.6 mM [5-3H]-dUMP (5 Ci mmol − 1; Amersham Life Science, Arlington Heights, IL), 65 mM sucrose, and 0.3% bovine serum albumin] and incubated for 1 h at 37°C. The reaction was terminated by the addition of 12 ml of 33% trichloroacetic acid. Charcoal (180 ml; 100 mg ml − 1) was added to adsorb the residual substrate. Tritiated water from the [3H]-dUMP released into the supernatant was assayed by liquid scintillation spectrometry. An unincubated control was used to correct for the presence of non-absorbable radioactivity in the substrate.

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Thymidine kinase activity was measured using [14C]thymidine as the substrate by the methodology of Cheng [29]. The assay mixture contained 50 mM Tris– HCl, pH 7.4, 10 mM MgCl2, 5 mM Na-ATP, 0.1% bovine serum albumin, 90 mM thymidine, 10 mM NaF, 0.1 mM [2-14C]-thymidine (10 mCi mmol − 1; Amersham Corp.) and 15 ml of cell supernatant. After incubation for 30 min at 37°C, an aliquot of the reaction mixture was applied to a Whatman DE-81 paper disc, washed three times with 100% alcohol, and radioactivity retained thereon quantified by scintillation spectrometry.

2.6. Western analysis of thymidylate synthase Cell extracts were prepared as described above. ECL western blot kits were obtained from Amersham Life Science. The protein content of the cellular extracts was determined by the method of Bradford [30]. The extracts were stored at − 70°C for up to 2 weeks. Protein samples were denatured with 2× SDS-sample buffer (1 ml of glycerol, 0.5 ml of b-mercaptoethanol, 3 ml of 10% SDS, 1.25 ml of 1.0 M Tris – HCl, pH 6.7, and 1.5 mg of bromophenol blue), applied to a 6% SDSminigel, and subjected to electrophoresis at a constant 110 V for 2 h. The minigel was transferred onto Millipore Immobilon-P (PVDF, 0.45 mm pore size) according to established procedures [31]. The transfer was carried out overnight at a constant 30 V. After transfer, the membrane was air dried, incubated in Blotto solution (1 × Tris buffered saline, 5% milk, 0.05% Tween 20), washed and hybridized with an anti-human thymidylate synthase polyclonal antibody as described previously [32]. Chemiluminescent detection was carried out according to the procedure described in the ECL Western blotting protocol (Amersham Life Science).

sion of thymidylate synthase mRNA in cells exposed to either the polar solvent or the retinoid increased relative to that of comparable untreated controls for up to 2 days and then progressively decreased thereafter over the 7 day period (Fig. 2). These changes corresponded to a progressive decrease in enzyme protein, as determined by Western blot analysis (Fig. 3, top panel), and a corresponding progressive decrease in enzymatic activity (Fig. 3, bottom panel). Thymidine kinase represents the initial enzyme of the salvage pathway for the formation of thymine nucleotides. In a manner analogous to that observed for thymidylate synthase, the levels of messenger RNA for thymidine kinase progressively declined from day 3 to day 7 of exposure to the inducers of differentiation (Fig. 4), and the

3. Results Exposure of HL-60 promyelocytic leukemia cells to 1.3% DMSO or 1.0 mM retinoic acid resulted in a progressive increase in the percentage of differentiated cells in the population as measured by Mo-1 positivity (Fig. 1, top panel). A similar progressive increase in NBT positivity occurred with increasing duration of treatment with DMSO or retinoic acid (data not shown). A corresponding decrease in cell number as a function of time occurred with the acquisition of the differentiated state (Fig. 1, bottom panel). Trypan blue exclusion demonstrated that cells exposed to either the polar solvent or the retinoid were viable during the differentiation into granulocyte-like cells (data not shown). The effects of 1.3% DMSO and 1 mM retinoic acid on enzymes involved in the generation of thymine nucleotides by HL-60 cells were measured. The expres-

Fig. 1. Mo-1 (CD11b) expression (top panel) and growth inhibition (bottom panel) in HL-60 cells exposed to 1.3% DMSO or 1 mM retinoic acid. HL-60 cells were seeded at 105 cells ml − 1 and treated with DMSO or retinoic acid for from 1 to 7 days. The viability of cells was verified by trypan blue exclusion. Mo-1 binding to HL-60 cells was conducted as described in Section 2 and each point represents a sample of 2×106 cells. The results are from three or more independent experiments 9S.E.

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mRNA decreased progressively beginning on day 2 of exposure to DMSO and on day 3 following treatment with the retinoid (Fig. 10).

4. Discussion

Fig. 2. Northern analysis of the time-course of the expression of thymidylate synthase in HL60 cells induced to differentiate by 1.3% DMSO or 1 mM retinoic acid. The results represent the quantitation of the time-course of the expression of thymidylate synthase mRNA in HL-60 cells induced to differentiate by DMSO and retinoic acid. The relative densities of the bands are presented as the mean value 9 S.E. derived from at least three independent experiments.

activity of the enzyme decreased beginning at day 5 (Fig. 5). Ribonucleotide reductase is the major enzyme responsible for generating cytosine, guanine and adenine deoxyribonucleotide precursors of DNA. Measurement of the expression of the mRNA of the M2 subunit of the enzyme delineated a pronounced difference between the two inducers of differentiation, with DMSO causing virtually complete inhibition of the expression of the M2 subunit of the enzyme from day 5 through 7 after an early transient increase at day 2, with no change in the steady-state levels of the message being produced by retinoic acid (Fig. 6). The steady-state levels of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) mRNA, which represents an enzyme involved in the salvage of guanine and hypoxanthine residues, after relatively large increases in expression of HGPRT mRNA at early times also declined following exposure to each of the initiators of differentiation, with the decrease beginning after two days of exposure to the polar solvent and after three days of treatment with the retinoid (Fig. 7). The polar solvent and the retinoid caused little or no change in the steady-state levels of topo II mRNA over the initial 2 days of exposure. This initial lack of effect of the inducers of differentiation was followed by a progressive decrease in the expression of the topo II gene over the 7 days of exposure (Fig. 8). An increase in the messenger RNA for topo I occurred over the initial 2 days of exposure to the inducers of differentiation, followed by a progressive decrease in the steady-state levels of topo I mRNA (Fig. 9). In a similar manner, DNA polymerase-a

To gain information on the mechanism(s) used by differentiating leukemia cells to terminate cellular replication, we have measured the expression of seven enzymes directly involved in the synthesis of DNA, including topo II, topo I, DNA polymerase-a, ribonucleotide reductase, thymidylate synthase, thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase, in HL-60 leukemia cells following treatment with the myeloid inducers of differentiation, DMSO and retinoic acid. A number of reports have documented changes in these enzymes as a result

Fig. 3. Western analysis (top panel) and activity (bottom panel) of thymidylate synthase of HL-60 cells exposed to 1.3% DMSO or 1.0 mM retinoic acid. The western blots represent the results from a representative experiment. Twenty micrograms of protein were applied to a 6% SDS-minigel and subjected to electrophoresis. Transfer and hybridizations were carried out described in Section 2. Thymidylate synthase activity data are from three independent experiments presented as a percentage of the activity of control cells in the absence of inducers 9S.E. The value for control cells was 73620 96750 cpm mg protein − 1.

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Fig. 4. Northern analysis of the time-course of the expression of thymidine kinase in HL-60 cells induced to differentiate by 1.3% DMSO or 1.0 mM retinoic acid. The results represent the quantitation of the time-course of the expression of thymidine kinase mRNA in HL-60 cells induced to differentiate by DMSO and retinoic acid. The relative densities of the bands are presented as mean values 9 S.E. derived from three independent experiments.

of the induction of differentiation in various experimental systems. Kuruto et al. [33] have shown a decrease in the level of thymidylate synthase mRNA in HL-60 cells undergoing terminal maturation. These changes may be the result of decreases in nuclear factors which interact with a sequence around the initiation codon of the thymidylate synthase gene, which have been shown to decrease in HL-60 cells undergoing monocytic differentiation following exposure to 1a,25-dihydroxyvitamin D3 [13]. The other enzyme system involved in generating thymine nucleotides, thymidine kinase, is also transcriptionally repressed in chicken myoblasts undergoing

Fig. 5. Thymidine kinase activity in HL-60 cells treated with 1.3% DMSO or 1.0 mM retinoic acid. Data are from three independent experiments presented as a percentage of the activity of control cells in the absence of inducers 9 S.E. The value for control cells was 335009 3880 cpm mg protein − 1.

Fig. 6. Northern analysis of the time-course of the expression of the ribonucleotide reductase M2 subunit in HL-60 cells induced to differentiate by 1.3% DMSO or 1.0 mM retinoic acid. The results represent the quantitation of the time-course of the expression of the ribonucleotide reductase M2 subunit gene in HL-60 cells induced to differentiate by DMSO and retinoic acid. The relative density of the bands are presented as the mean values 9 S.E. derived from three independent experiments.

terminal differentiation [34]. However, since the decrease in enzyme activity was significantly greater than the decline in the corresponding mRNA levels, a translational or post-translational mechanism was suggested as also being involved in regulating the activity of this enzyme. It is noteworthy that post-transcriptional regulation of the thymidine kinase gene was also observed in HL-60 cells undergoing monocytic differentiation

Fig. 7. Northern analyses of the time-course of the expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in HL-60 cells induced to differentiate by 1.3% DMSO or 1.0 mM retinoic acid. The results represent the quantitation of the time-course of the expression of the HGPRT gene in HL-60 cells induced to differentiate by DMSO and retinoic acid. The relative densities of the bands are presented as the mean values 9S.E. derived from at least three independent experiments.

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Fig. 8. Northern analysis of the time-course of the expression of DNA topoisomerase II in HL-60 cells induced to differentiate by 1.3% DMSO or 1 mM retinoic acid. The results represent quantitation of the time-course of the expression of the topoisomerase II gene in HL-60 cells induced to differentiate by DMSO and retinoic acid. The relative densities of the bands are presented as the mean values 9 S.E. derived from at least three independent experiments.

following treatment with the phorbol ester TPA [14]. Other studies have also shown that the expression of thymidine kinase is controlled at the transcriptional and post-transcriptional levels in other cell systems [35]. In the present report, we confirm and extend these findings, demonstrating that the steady-state levels of the mRNAs for both thymidylate synthase and thymidine kinase decline progressively as does enzyme activity in HL-60 cells undergoing myeloid differentiation and ter-

Fig. 9. Northern analysis of the time-course of the expression of topoisomerase I in HL-60 cells induced to differentiate by 1.3% DMSO or 1.0 mM retinoic acid. The results represent the quantitation of the time-course of the expression of the topoisomerase I gene in HL-60 cells induced to differentiate by DMSO and retinoic acid. The relative densities of the bands are presented as the mean values 9 S.E. derived from three independent experiments.

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Fig. 10. Northern analysis of the time-course of the expression of DNA polymerase-a in HL-60 cells induced to differentiate by 1.3% DMSO or 1.0 mM retinoic acid. The results represent the quantitation of the time-course of the expression of the DNA polymerase-a gene in HL-60 cells induced to differentiate by DMSO and retinoic acid. The relative densities of the bands are presented as the mean values 9S.E. derived from three independent experiments.

minate replication following exposure to DMSO and retinoic acid. The expression of topo I mRNA and its corresponding protein product have been shown to decrease in the slime mold Physarum polycephalum during development to dormant spherules [36]. In an analogous manner we have shown that in HL-60 leukemia cells the expression of the topo I gene decreases with time following exposure to the myeloid inducers of differentiation, DMSO and retinoic acid. A much larger body of literature exists on the regulation of topo II during leukemia cell maturation. Sahyoun et al. [37] reported that topo II was activated in vitro by protein kinase C, the target of the monocytic inducer of differentiation, TPA. The transient relaxation of DNA supercoiling and a small amount of DNA breaks, both characteristic of topo II reactions, occurred within 1–2 h of exposure to retinoic acid [38,39]. Furthermore, a decrease in TPA-induced, topo II-mediated cleavage of DNA occurred in HL-60 cells within 24 h of exposure to the phorbol ester [16,17]. This inhibition of topo II activity by TPA, as well as by the antibiotic novobiocin, has been found to be associated with the differentiation process [15]. We have reported earlier that inhibitors of topo II caused the maturation of WEHI-3B myelomonocytic leukemia cells [40]. In the present report, we demonstrate that in HL-60 cells exposed to DMSO and retinoic acid, topo II decreases progressively with time. In a similar manner the mRNAs for HGPRT and DNA polymerase-a decrease following exposure to the granulocytic inducers of differentiation. The generation of deoxyribonucleotides other than the thymine deoxyribonucleotide is catalyzed by the enzyme ribonucleotide reductase. Mann et al. [12] have

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demonstrated that the M1 subunit of ribonucleotide reductase decreases in HL-60 cells undergoing both granulocytic and monocytic differentiation induced by DMSO and TPA, respectively, concomitant with the cessation of cell division. In the present study, in contrast to the expression of the other housekeeping genes, we have shown a precipitous decline in the levels of the mRNA for the M2 subunit of ribonucleotide reductase of HL-60 cells, beginning at 5 days following their treatment with DMSO, with little or no change occurring in the steady-state levels of the mRNA for this subunit following exposure to retinoic acid, even though both of these agents caused the differentiation of these leukemia cells. These finding demonstrate that the signaling systems that are initiated by the polar solvent and the retinoid to down-regulate the expression of enzymes involved in DNA synthesis are somewhat selective. The findings, collectively, demonstrate that the down-regulation of a relatively wide variety of critical enzymes for DNA biosynthesis occurs as cells cease replication due to the induction of terminal differentiation implying that the expression of these enzymes is coordinately regulated. While it appears that these relatively late effects are not the primary events in terminating the proliferation of maturing cells, the decline of a number of enzyme systems required for DNA synthesis ensures that cellular replication remains shutdown in terminally differentiated cells. Acknowledgements This research was supported in part by United States Public Health Service Grant CA-02817 from the National Cancer Institute. References [1] Davila DG, Minoo P, Estewig DN, Kasperbauer JL, Tzen C-Y, Scott RE. Linkages in control of differentiation and proliferation in murine mesenchymal stem cells and human keratinocyte progenitor cells: the effects of carcinogenesis in mechanisms of differentiation. In: Fisher PB, editor. Model Cell Culture Systems for Studying Differentiation, vol. 1. Boca Raton: CRC Press, 1990:1. [2] Ruddon RW. Cancer Biology, 3rd ed. New York: Oxford University Press, New York, 1995:209. [3] Reiss M, Gamba-Vitalo C, Sartorelli AC. Induction of tumor cell differentiation as a therapeutic approach: preclinical models for hematopoietic and solid neoplasms. Cancer Treat Rep 1986;70:201. [4] Jiang H, Lin J, Su Z-z, Collart FR, Huberman E, Fisher PB. Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21, WAF1/CIP1 expression in the absence of pS3. Oncogene 1994;9:3397. [5] Manfredi JJ, Yang H, Waxman S. The cyclin-dependent kinase inhibitor p21 as a target for differentiation therapy. Mol Cell Differ 1996;4:33.

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