Feb 20, 1981 - Divisions of Pharmacology, Medicine, and Pediatric Oncology, Sidney Farber Cancer Institute-Harvard Medical School, Boston, Massachusetts ...
Proc. Nati. Acad. Sci. USA
Vol. 78, No. 5, pp. 3235-3239, May 1981
Medical Sciences
Lethality of human myeloblasts correlates with the incorporation of arabinofuranosylcytosine into DNA (leukemia/cytotoxicity/nucleoside analog)
P. P. MAJOR, E. M. EGAN, G. P. BEARDSLEY, M. D. MINDEN, AND D. W. KUFE * Divisions of Pharmacology, Medicine, and Pediatric Oncology, Sidney Farber Cancer Institute-Harvard Medical School, Boston, Massachusetts 02115
Communicated by Sol Spiegelman, February 20, 1981
ABSTRACT We recently demonstrated a highly significant relationship between the incorporation.of I-,-D-arabinofuranosylcytosine (ara-C) into L1210 DNA and the loss of clonogenic survival. These studies have now been extended to the human promyeloblast (HL-60) cell line and myeloblasts from a patient with acute myelogenous leukemia. Our results demonstrate: (i) the specific internucleotide incorporation of ara-C into human myeloblast DNA; (ii) the lability of [3Hjara-C-labeled DNA to alkali which necessitates the use of nondegrading assay conditions; and (iii) a highly significant relationship (P < 0.0001) between the loss of clonogenicity of these cells and the extent of ara-C incorporation. These findings suggest that the incorporation of ara-C into DNA is one of the initial events leading to cell lethality. This method is applicable to clinical samples of bone marrow and peripheral blood as an in-vitro assay for studying the sensitivity of cell populations to this drug.
I-f3-D-Arabinofuranosylcytosine (ara-C) is the most effective agent in the treatment of acute myelogenous leukemia in man (1). However, the detailed biochemical mechanisms responsible for the anti-leukemic activity and selectivity of ara-C have not been established. The triphosphate ara-CTP inhibits DNA polymerase in vitro by competing with dCTP for binding to this enzyme (2, 3). This finding has been suggested as one mechanism responsible for inhibition of DNA replication (4-6). However, ara-CTP does not affect the incorporation of dTTP or dATP (7, 8) and therefore does not induce functional changes in DNA polymerase. It is unclear how the inhibition of DNA synthesis through the reversible displacement ofdCTP from the binding site on DNA polymerase produces cytotoxicity during brief periods of drug exposure, although the resumption of DNA synthesis after inhibition by ara-C has been characterized by reinitiation of previously replicated DNA segments (9). The inhibition of DNA synthesis could also occur through incorporation of ara-C into the DNA strand with either slowing of chain elongation or chain termination. The specific incorporation of ara-C into DNA has been demonstrated in one study (10); other studies (4, 11-13) have demonstrated the incorporation into DNA and RNA. ara-C residues have been found in DNA intermucleotide linkage (2, 10), confirming incorporation within DNA strands in vivo. Other work using purified DNA templates has suggested that ara-C functions as a chain terminator (7). The formation of faulty (ara-C) DNA might also result in lethal cellular events. Previous studies, however, failed to correlate the incorporation of ara-C into DNA with cytotoxicity
(2).
We have recently used cesium sulfate gradient centrifugation to monitor the incorporation of ara-C into L1210 cellular nucleic acids (14). This methodpermits the separation of DNA and RNA
under nondegrading conditions. Our results demonstrated the incorporation of intact ara-C into DNA and, more importantly, a highly significant relationship (P < 0.0001) was found between the degree of ara-C incorporation into DNA and the loss of clonogenic survival. These results supported the hypothesis that incorporation of ara-C in DNA is another possible mechanism of cytotoxic action. The incorporated ara-C residues into DNA could slow chain elongation or could produce faulty DNA (15). We have now extended our findings to the human promyeloblast HL-60 cell line and to myeloblasts from a patient with acute myelogenous leukemia (AML). Our present results show that the extent of incorporation of ara-C into human myeloblast DNA bears a highly significant relationship to the loss of clonogenic survival. These observations suggest that the incorporation of ara-C into human leukemic cell DNA is at least one mechanism responsible for producing lethal cellular events. They also offer a readily measurable variable, ara-C incorporation in DNA, for monitoring the effects of this drug. These methods are applicable to clinical samples of leukemic cells and should provide the means of developing tests to predict and to monitor sensitivity to ara-C. MATERIALS AND METHODS Cell Culture. The HL-60 (passage 20) cells were obtained from Robert Gallo (National Cancer Institute). These cells were maintained as a suspension culture in RPMI-1640 (GIBCO) with 10% heat-inactivated fetal calf serum, 100 units of streptomycin per ml, 100 jig of penicillin per ml, and 2 mM L-glutamine at 370C in 5% C0J95% air. Clonogenic Survival of Human- Myeloblasts. The HL-60 cells in logarithmic growth phase were washed twice in phosphate-buffered saline and resuspended in RPMI-1640 medium with 10% heat-inactivated, dialyzed fetal calf serum at a concentration of 5 x 105 cells per ml. Peripheral myeloblasts from a patient with- AML in relapse were prepared and grown in culture as described (16). ara-C (Upjohn) was added to give varying final concentrations ranging from 10-8 to 10-3 M and cells were incubated at 370C for periods varying from 1 to 24 hr. After drug exposure, the cells were collected in drug-free medium without serum. After they were counted in a model Z Coulter Counter, the HL-60 cells were plated in 0.8% methylcellulose (RPMI-1640) containing 20% fetal calf serum, 5% horse serum, and 5% PHALCM (16). The human AML blasts were plated in 0.8% methylcellulose containing Iscove special medium (GIBCO) with 20% fetal calf serum and 10% PHA-LCM (16). Viability was determined after 7-10 days by scoring colonies containing Abbreviations: ara-C, 1-,-D-arabinofuranosylcytosine; AML, acute myelogenous leukemia. * To whom reprint requests should be addressed at: Sidney Farber Cancer Institute, 44 Binney Street, Boston, MA 02115.
The publication costs ofthis article M ere defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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Proc. Natl. Acad. Sci. USA 78 (1981)
Medical Sciences: Major et al.
3236
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Fraction FIG. 1. HL-60 cells in logarithmic growth at 5 x 105 cells per ml were incubated with [3Hlara-C (13 ,uCi/ml) and, [2P]H2PO4 (5 IACi/ml) for 3(A), 12 (B), and 24 (C) hr. The total cellular nucleic acids were purified and analyzed by cesium sulfate density centrifugation. The [5Hlara-C and 32p counts banding in the RNA region (between 1.62 and 1.68 g/ml) and DNA region (between 1.42 and 1.48 g/ml) ofthe gradients were determined and used as a measure of the incorporation of ara-C into'DNA and of the relative synthetic rates of RNA and DNA.
>20 cells. Percentage colony formation was determined by the ratio ofcolonies formed by ara-C-treated cells to untreated cells. Cloning efficiency of the untreated HL-60 cells in this system
ranged from 5% to 10%. Thymidine suicide procedures were described (17). Incorporation of ara-C into Myeloblast Nucleic Acids. Myeloblasts were washed twice with phosphate-buffered saline and resuspended at 5 x 05 cells per ml in RPMI-1640 medium. Cells were incubated with 13 puCi of [3H]ara-C (26 Ci/mmol; 1 Ci = 3.7 x 1010 becquerels; New England Nuclear) per ml and 5 ACi of [32P]H2PO4 (carrier-free; New England Nuclear) per ml for 1-24 hr. These nucleic acids were then purified for analysis on cesium sulfate gradients as described (14). Digestion of [3H]ara-C-Labeled DNA. Myeloblasts were labeled with [3H]ara-C for 6 hr as described for incorporation as
-studies. The purified DNA was digested with DNase I, snake venom phosphodiesterase, and alkaline phosphatase (9, 10). After precipitation of the remaining macromolecular species with perchloric acid, the nucleosides were analyzed by highpressure liquid chromatography on a Micropak AX-10 ion exchange column (Varian) with 80% acetonitrile/20% 0.01 M KH2PO4 (pH 2.85) as eluent. The elutions were performed after the addition of appropriate markers. Fractions were collected during each elution and assayed for 3H. The digestion of [3H]ara-C labeled DNA to 3'-nucleotides was performed by the sequential action of micrococcal nuclease and spleen phosphodiesterase (18). The nucleosides and nucleotides were fractionated by high-pressure liquid chromatography on a Micropak AX-10 ion exchange column at a flow rate of 2 ml/min. 'Buffer A was 80% acetonitrile/20% 0:01 M
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Fraction FIG. 2. Incorporation of [3H]ara-C (a) and 32p (o) into AML blast nucleic acid before (A) and after (B) alkaline digestion. Peripheral myeloblasts from a patient with AML in relapse were, prepared as described (16). The erythrocyte-rosette-depleted mononuclear cells consisting of >95%.leukemic blasts were washed twice with'RPMI-1640 and resuspended at aconcentration of 5 x 105 cells per ml. Cells were incubated with 13 1ACi of [3H]araC per ml and 5 uCi of [32P]H2PO4 per ml for 6 hr. The total cellular nucleic acids were purified and analyzed by Cs2SO4 density centrifugation (A). An equal aliquot was treated with 0.4 M NaOH for 16 hr at 370C prior to analysis (B).
Proc. Natl. Acad. Sci. USA 78 (1981)
Medical Sciences: Major et al..
3237
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,uCi of [3H]ara-C per ml for 6 hr. The purified DNA was digested to nucleosides with DNase I, snake venom phosphodiesterase, and al-
kaline phosphatase. The nucleosides were purified and analyzed by high-pressure liquid chromatography on a Micropak AX-10 ion exchange column with 80% acetonitrile/20% 0.01 M KH2PO4 (pH 2.85) as eluent. Appropriate nucleoside markers were added and fractions (0.4 ml) were collected during the elution and assayed for 3H. ara-U, Arabinofuranosyluracil .
20
FIG. 4. AML blasts were labeled with [3H]ara-C as described for Fig. 2. The purified DNA was digested to 3'-nucleotides by the sequential action of micrococcal nuclease and spleen phosphodiesterase (18). The nucleotides were purified and analyzed by high-pressure liquid chromatography on a Micropak AX-10 ion exchange column. araU, Arabinofuranosyluracil.
KH2PO4 (pH 2.85); buffer B was 0.01 M KH2PO4 (pH 2.85). The elution program utilizing buffer B was as follows: (i) 0-40% for 5 min; (ii) 40% for 10 min; (iii) 40100% for 5 min; and (iv) 100% for 10 min.
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RESULTS The degree of [3H]ara-C incorporation into HL-60 nucleic acid was determined by cesium sulfate gradient centrifugation which separates RNA (banding at 1.62 to 1.68 g/ml) and DNA (banding at 1.42 to 1.48 g/ml). There was significant incorporation of [3H]ara-C in the region of the gradient; no significant 3H radioactivity was detectable in the RNA region (Fig. 1). The labeling with 32p serves as a measure of newly synthesized RNA and DNA and also demonstrates the ability to detectboth nucleic acids by this method. Similar results were obtained with the human AML blasts (Fig. 2A). Further, alkali treatment of these labeled nucleic acids (Fig. 2B) resulted in complete degradation of the RNA fraction and loss of nearly 65% of the radioactivity banding in the DNA region of the gradient. It is important to demonstrate that the 3H radioactivity detectable in the DNA region of the Cs2SO4 gradient represents [3H]ara-C. The labeled DNA was digested to nucleosides and analyzed by high-pressure liquid chromatography after the addition of appropriate markers. The nucleoside profile (Fig. 3) illustrates that the 3H radioactivity comigrated with ara-C, with 95% of the incorporated [3H]ara-C was po-
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ara-C, M FIG. 5. HL-60 cells were exposed to ara-C for 3 (A), 6 (B), 12 (C),
or 24 (D) hr at concentrations ranging from 10-8 to 10-3 M. After drug exposure, the cells were washed, resuspended in drug-free RPMI-1640 medium, and assayed for radioactivity. The cells were plated at 1000 cells per ml in 1.5% methylcellulose in RPMI-1640 containing 20% fetal calf serum, 5% horse serum, and 5% PHA-LCM (16). Clonogenic
survival was determined after 10 days-by scoring colonies greater than 50 cells. Percentage viability was determined by the ratio of colonies formed by ara-C treated cells compared to untreated cells. Dotted line, survival of cells in thymidine suicide experiments.
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Proc. Natl. Acad. Sci. USA 78 (1981)
sitioned in. internucleotide linkage rather than at the chain terminus. These findings were identical for both the HL-60 cells and the AML blasts. The relationship of ara-C incorporation into cellular DNA to drug-induced cytotoxicity was studied by comparing the amount of ara-C incorporated to the loss of clonogenic survival after drug exposure. The effect of ara-C on the clonogenic survival of HL-60 cells (Fig. 5) and the AML blasts (Fig. 6) was determined by exposure to concentrations of 10-8 M to 10-3 M for 1, 3, 6, 12, or 24 hr. The dotted lines represent the percentage of control survival for cells treated in "thymidine suicide" experiments (17) as a measure of cells in S phase during the exposure periods. The loss of clonogenic survival for both cell populations was dependent upon drug concentration and time of drug exposure. The relationship between incorporation of ara-C into DNA and loss of clonogenic survival was determined bymeasuring the amount of [3H]ara-C incorporated into DNA under conditions similar to those used for the studies monitoring clonogenic survival. Incorporation studies were performed at [3H]ara-C concentrations ranging from 10-7 to 10-4 M during incubation periods of 1 to 24 hr. The clonogenic survival at each ara-C concentration was obtained from the cloning data shown in Figs.
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5 and 6. The product of drug concentration times time of drug exposure correlated significantly, by probit analysis (19), with both pmol of ara-C incorporated into DNA (HL-60, coefficient R = 0.752, P < 0.0001; AML blasts: coefficient R = 0.594, P < 0.006), and log(% survival) (HL-60, coefficient R =-0.829, P < 0.0001; AML blasts, coefficient R = -0.718, P < 0.002). Furthermore, the relationship between percentage survival and log(pmol of ara-C incorporated into DNA) also correlated at a highly significant level (HL-60, coefficient R = -0.847, P < 0.0001; AML blasts, coefficient R = -0.885, P < 0.0001). The probit analysis (19) of this relationship for the AML blasts as determined by a computer-assisted program is illustrated in Fig. 7.
ara-C, M FIG. 6. Clonogenic survival of human myeloblasts after exposure to ara-C for 1 hr (A) 3 hr (B) 6 hr (C), or 24 hr (D). The myeloblasts were prepared as described in the legend to Fig. 2 and-were resuspended at 5 x 105 cells per ml in complete medium. ara-C was added to give final concentrations ranging from 10-7 to 10-4 M, and the cells were incubated at 370C for periods varying from 1 to 24 hr. After drug exposure,
the cells were pelleted, washed twice with 10 ml of RPMI-1640 medium, and resuspended in drug-free medium without serum. The cells were then counted in a model Z Coulter Counter and plated in 0.8% methylcellulose containing Iscove special medium with 20% fetal calf serum and 10% PHA-LCM (16). Viability was determined after 7 days by scoring colonies greater than 20 cells. Dotted lines, survival of cells in thymidine suicide experiments.
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FIG. 7. Relationship between AML blast clonogenic survival and [3H]ara-C incorporation in DNA (pmol/107 cells) at ara-C concentrations of 10-7 M (A), 10-6 M (e), 10-5 M (i), and 10-4 M (o) during periods of 1, 3, 6, 12, and 24 hr.
Medical Sciences: Major et al. DISCUSSION We previously demonstrated a highly significant relationship between the degree of ara-C incorporation into L1210 DNA and the loss of clonogenic survival (14). These studies have now been extended in human HL-60 promyeloblasts and myeloblasts from a patient with AML. The results ofthe present study demonstrate the intemucleotide incorporation of ara-C into human myeloblast DNA. We have also found a highly significant relationship between incorporation of ara-C into human myeloblast DNA and loss of clonogenic survival. Our data suggest that the incorporation of ara-C into DNA is at least one mechanism responsible for producing lethal cellular events. The specific incorporation of ara-C into cellular DNA and not RNA has been previously demonstrated by using cesium chloride gradient analysis performed under nondegrading conditions (10). Other studies have detected ara-C in both RNA and DNA (11-13) by using alkaline conditions to distinguish between these nucleic acids. Cesium sulfate distinguishes ara-C incorporation into RNA and DNA under nondegrading conditions and demonstrates that [3H]ara-C incorporation is limited to DNA. Furthermore, alkali treatment of DNA labeled with [3H]ara-C results in loss of radioactivity, thus necessitating the use of nonalkaline conditions for the separation of RNA and DNA. This finding may account for the previous inability to establish a relationship between ara-C incorporation in DNA and cytotoxicity. The further characterization and relevance of the alkali susceptibility will be considered elsewhere. Our observations provide insights into the mechanism ofaction of ara-C and they may explain its effect on differentiation (20, 21) and inhibition of self-renewal capacity (22). It will now be of interest to determine whether the extent of ara-C incorporation into human myeloblast DNA relates to clinical response. A significant correlation between ara-CTP formation and retention by acute myelocytic leukemia cells in vitro and the duration of the remission has been shown (23). However, the effects of ara-C on DNA synthesis have not correlated with cytotoxicity of clonogenic cells (24). It is now possible to use our method for monitoring incorporation ofara-C into cellular DNA as a useful clinical marker for studying the sensitivity of cell populations to this drug.
Proc. Natl. Acad. Sci. USA 78 (1981)
3239
The authors thank Ms. L. M. Grappi for her excellent secretarial assistance. This work was supported in part by Grant CA-29431-01 from the National Cancer Institute and by an American Cancer Society Junior Faculty Research Award (to D.W. K.). P. P. M. and M. D. M. are Fellows of the Medical Research Council of Canada.
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