- Email: [email protected]
Comparison of the induction of apoptosis in human leukemic cell lines by 2′,2′-difluorodeoxycytidine (gemcitabine) and cytosine arabinoside
Leukemra Research Vol. 19, No. 11. pp. 849-856, 1995. Coovrieht D 1995 Elsevier Science Ltd Printed in &eat Britain. All rights reserved 0145-2126195 $9.50 + 0.00
Pergamon 01452126(95)00067-4
COMPARISON OF THE INDUCTION OF APOPTOSIS IN HUMAN LEUKEMIC CELL LINES BY 2’,2’-DIFLUORODEOXYCYTIDINE (GEMCITABINE) AND CYTOSINE ARABINOSIDE David Y. Bouffard* and Richard L. Momparlert *Department of Pharmacology,UniversitC de Montreal, Montreal, QuCbec,Canada;and TPediatric Research Centre, Ste-JustineHospital, Montreal, QuCbecCanada (Received 17 November 1994.Accepted 7 April 1995) Abstract-The induction of DNA fragmentation by cytosine arabinoside (araC) and 2’,2’difluorodeoxycytidine (dFdC, gemcitabine) was compared in human leukemic cell lines. For both araC and dFdC this process was time- and concentration-dependent and resulted in loss of clonogenic survival of HL-60 myeloid leukemic cells. There was a marked difference in the potency between these two analogs in inducing apoptosis. A 6 h exposure to 5 PM araC was required to produce DNA laddering in HL-60 cells, whereas dFdC at a concentration IOO-fold less (0.05 PM) was sufficient to produce similar results. Pre-incubation of HL-60 cells with staurosporine, a non-specific protein kinase C inhibitor, increased the level of apoptosis induced by a 3 h exposure to araC or dFdC, suggesting the possible involvement of this family of enzymes in this process. Also, dFdC was able to increase the expression of both c-jun and cfos in Molt-3 leukemic cells with a concentration known to induce apoptosis in this cell line. Key words:
Gemcitabine,
apoptosis,
protein
kinase C, c-jun, c-fos.
Introduction
[lo, 111. A series of proteins that could be implicated in the regulation of apoptosis is the family of the protein kinase C (PKC). Forbes et al. [12] have shown that the induction of apoptosis in chronic lymphocytic leukemia cells could be prevented by phorbol esters (PKC modulators). Although the PKC seems to be involved in preventing apoptosis, its mechanism of action in apoptosis remains to be clarified. In some instances the non-specific PKC inhibitor staurosporine has been shown to prevent [13] and to increase [14] the level of apoptosis in cancer cells. Cytosine arabinoside (araC), one of the most effective drugs for the treatment of acute leukemia, has been shown to induce apoptosis in myeloid leukemia cells [15,16]. There is also evidence that certain oncogenes could be implicated in the process of apoptosis. The cjun and c-fos proto-oncogenes have been shown to be increased following a treatment with araC suggesting that these genes could be involved in the induction of apoptosis [17-191. 2’,2’-difluorodeoxycytidine (dFdC, gemcitabine) is a new and very interesting antimetabolite of deoxycytidine in which the two hydrogen atoms in the 2’ position of the deoxyribose sugar have been replaced by two fluorine atoms 1201. Several reports have shown that dFdC is active against many murine solid tumors and
Apoptosis or programmed cell death (PCD) is characterized by cell shrinkage and chromatin condensation with maintenance of normal organelle structure and the appearance of oligonucleosomal DNA fragmentation at multiples of approximately 200 base pairs [l, 21. PCD results from the activation of an intrinsic program after responding to specific stimuli [3]. Apoptosis can be induced in many types of cancer cells by a variety of chemotherapeutic drugs [4-71. Little is known about the cascade of events that leads to PCD. The role of protein synthesis in this process is controversial. In certain cases inhibition of protein synthesis does not prevent apoptosis [8], whereas in some instances this process depends on de novo protein synthesis [9]. In addition, in some reports inhibition of protein synthesis has been shown to trigger apoptosis Abbreviations: dFdC, 2’,2’-difluorodeoxycytidine; dFdCMP, 2’,2’-difluorodeoxycytidine S-monophosphate;dFdCTP, 2’,2’difluorodeoxycytidine S-triphosphate; araC, cytosine arabinoside; araCMP, cytosine arabinoside S-monophosphate; araCTP, cytosine arabinoside S-triphosphate; PCD, programmed cell death; PKC, protein kinase C; stauro, staurosporine. Correspondence to: R. L. Momparler, Pediatric Research Centre, Ste-JustineHospital, MontrCal, QuCbec,Canada. 849
D. Y. Bouffard and R. L. Momparler
850
ara-C (PM)
(A)
c
W
1
5
dFdC (PM) 10
c
bp
0.01
0.05
2072 2072
600 600
(B)
dFdC (0.05p.M)
ara-C (10 pM) C
3h
6h
12h
C
3h
Fig. 1. Concentration- and time-dependent DNA fragmentation in HL-60 myeloid leukemic cells produced by araC or dFdC. The cells were exposed to various concentrations of araC or dFdC and assayed for DNA fragmentation as described in Materials and Methods. The experiments were repeated three times with comparable results obtained each time. (A) Six hour exposure to different concentrations of araC and dFdC; (B) time-dependent effect of araC and dFdC.
6h
12h
0.1
Induction of apoptosisby
leukemic cell lines [21,22] and is presently undergoing phase II clinical trials [23,24]. We have shown in previous experiments that dFdC is one hundred times more cytotoxic than araC in different kinds of leukemia cell lines [25]. The mechanism of action of dFdC appears to be related to its incorporation into RNA [26] and DNA [27] and also to its inhibition of the enzymes ribonucleotide reductase [28] and deoxycytidine monophosphate deaminase [29]. dFdC has also been shown to induce apoptosis in human T-lymphoblastoid CEM cells [30]. In this report we have observed that dFdC is a much more potent inducer of apoptosis than araC, indicating that the kinetics of induction of this process for these two antimetabolites are different. We have also observed that staurosporine, a PKC inhibitor, could enhance the apoptotic activity of both araC and dFdC in leukemic cells.
gemcitabine
851
(LMP), 0.25% (w/v) bromophenol blue and 40% (w/v) sucrose were added.The sampleswere then loaded into the wells of a dry 2% agarosegel and electrophoresedat 90-100 V for 3-4 h in 1xTBE pH 8 containing 0.5 @ml ethidium bromide at 4°C. DNA laddering was observed under UV illumination. All
experiments were performed three times with similar results obtained each time. Northern blot analysis
Total cellular RNA was isolated at appropriate times from Molt-3 leukemic cells with the Trizol solution from Canadian Life Technologies Inc. (Mississauga,Canada).Electrophoresis of 20 pg total RNA was performed in a 1.1% agarose-2.6% formaldehyde gel and followed by northern blotting on to a nylon membrane (Amersham Hybond-N). The membranes were prehybridized at 42°C for 4-6 h in a buffer containing 50% formamide, 5 x SSC, 0.1% SDS, 5 x Denhardt’s solution, 100 mg/ml dextran sulphate, and 200 pg/ml shearedsalmon spermDNA. The blots were then hybridized overnight at 42°C with 2~10~ cpm of 32P-labelled probe/ml of hybridization buffer (same as prehybridization buffer but no dextran sulphate
Materials and Methods Materials
The antimetabolite, araC, was obtained from the Upjohn Company of Canada. dFdC was provided by Lilly Research Laboratories (Indianapolis, IN, U.S.A.). Staurosporine was purchased from Sigma Chemical Company (St. Louis, MO, U.S.A.). Cell culture
Human HL-60 myeloid leukemic cells were obtained from Dr R. Gallo, National Cancer Institute, Bethesda,MD, U.S.A. The human Molt-3 T-lymphoid leukemic cell line was purchasedfrom the American Type Tissue Culture Collection (Rockville, MD, U.S.A.). The cell lines were maintained in suspension culture in minimal essential medium containing non-essential amino acids (Gibco, Grand Island, NY, U.S.A.) and 10% heat-inactivated fetal calf serum (Flow Laboratories, Mississauga,Canada).The doubling time for the cell lines was between 20 and 22 h. Clonogenic assay
The proliferative viability of the leukemic cells after exposure to the various drugs was determined by cloning in soft agar. At the termination of drug exposure, the cells were
centrifuged and suspendedin drug-free medium. An aliquot of 150-200 cells was placed in 2 ml 0.3% soft agar medium containing 15% serum. After incubation for 14-16 days at 37°C in a 5% CO* incubator, the number of colonies (~500 cells) were counted. The cloning efficiency of the control was in the range of 40-60%. All experimentswere performedthree
times in triplicate. DNA fragmentation analysis After exposure to the different drugs for various exposure
times, the cells (5x10’ cells) were pelleted and suspendedin 20 pl 10 mM EDTA, 50 mM Tris-HCl pH 8.0 containing 0.5% SDS (w/v) and 0.5 mgi’ml proteinase K (Sigma). After an
incubation of 1 h at 50°C 10 pl RNaseA, DNase-free(0.5 mg/ ml; Boehringer Mannheim, Laval, Canada) was added and incubation was continued for another hour at 50°C. The
samples were then heated to 70°C and then 10 l.1110 mM EDTA pH 8.0 containing 1% (w/v) low melting point agarose
present). After hybridization, the blots were washed twice in 2xSSC, 0.1% SDS at 55°C for 30 min and then washedtwice in 0.1 xSSC, 0.1% SDS at 55°C for 30 min. The blots were then exposed to X-ray films with double intensifying screens at
-70°C. DNA probes were purified by preparative gel electrophoresis and random-primed (Boehringer Mannheim, Laval, QuCbec, Canada) to specific radioactivities. The 2.6 kb EcoRI insert of the mouse c-jun gene was purified from the
JAC.l plasmid from American Type Culture Collection (Rockville, MD, U.S.A.). The 770 bp chicken fi-actin gene insert and the 1 kb mousev-fos gene insert were obtained from Oncor (Gaithersburg, MD, U.S.A.). All experiments were performed three times with similar results obtained each time.
Results Eflects of araC and dFdC on the induction of DNA fragmentation in HL-60 cells Figure 1A shows the effect of an increase in drug concentration of araC and dFdC on the induction of DNA oligonucleosomal fragmentation in HL-60 myeloid leukemic cells for a 6 h exposure. The induction of DNA laddering was observed only with a concentration of araC over 5 PM. For the same conditions, a loo-fold lower concentration of dFdC (0.05 FM) was needed to induce apoptosis in this cell line. We have also observed that concentrations up to 100 pM araC were unable to cause DNA laddering in this cell line for a 3 h exposure, whereas 0.5 ~.IM dFdC was sufficient to induce DNA fragmentation as visualized using conventional agarose gel electrophoresis (data not shown). Even when a higher number of cells (2~10~) was used, the same differences between araC and dFdC was observed. The kinetics of induction of apoptosis was also compared between araC and dFdC in a time-dependent manner in HL-60 cells. The cells were exposed to a fixed concentration of the drugs and at appropriate times DNA laddering was analyzed by agarose gel electrophoresis as shown in Fig. 1B. In this experiment, both araC and
D. Y. Bouffard and R. L. Momparler
852
araC (yM) Stauro (nM)
dFdC (PM) Stauro (nM)
C 10
25
--
C
-
25
-
50
c
10 10 25
25
C
25
50
50
25
C 0.1 0.25 -
-
C 0.1 0.1 0.25 0.25
C
50
C
-
-
25
25
50
25
50
Fig. 2. Effect of staurosporine on the induction of DNA fragmentation in HL-60 leukemic cells prior to an exposure to araC or dFdC. The cells were pre-incubated for 3 h with 2.5 or 50 nM staurosporine and then exposed to 10 and 2.5 pM araC or 0.1 and 0.25 pM dFdC for 3 additional hours. The cells were then analyzed for DNA fragmentation. The experiments were repeated three times and similar results were obtained in each case.
dFdC demonstrated a time-dependent effect on the induction of apoptosis in HL-60 cells. For both drugs DNA laddering was apparent at 6 h and continued up to 12 h. These results also show that much less dFdC (0.05 yM) was needed to achieve the same degree of DNA fragmentation as compared to araC (10 f.tM). The use of higher concentrations of cells have resulted in similar results. As shown in Table 1, all the concentrations and time of exposure of araC and dFdC used on HL-60 cells resulted in a loss of clonogenicity as determined by the
cloning of the cells in soft agar. Similar kinetics of DNA laddering were observed with Molt-3 T-lymphoid leukemic cells (data not shown) showing that the action of araC and dFdC is not limited to the myeloid phenotype. Effect of staurosporine on araC- and dFdC-induced apoptosis The effect of staurosporine, a non-specific protein kinase C inhibitor that acts at the catalytic domain of the
Induction of apoptosis by gemcitabine
Table 1. Effect of araC and dFdC on the clonogenicity of myeloid HL-60 leukemic cell line after a treatment of 2 and 24 h Drug araC dFdC
Concentration Wf) 0.01 1.0 10.0 0.001 0.10 1.00
Table 2. Effect of staurosporine 3 h pre-incubation on the loss of clonogenic survival of myeloid HL-60 leukemic cell line for a 3 h exposure to araC and dFdC
-
Relative cell kill (%) 24 hr 2 hr ND 30f9 47fll ND 57+14 68f9
853
Drug
21+7* 49+ 10 ND 58_+ 12 >99 ND
* Mean + SE. (n = 3, in triplicate). ND, not determined. Values are expressed as % relative to control.
enzyme, was also studied in HL-60 leukemic cells. Figure 2 shows the effect of a pre-incubation (3 h) of 25 and 50 nM staurosporine on apoptosis in HL-60 prior to an exposure of 3 h to araC (10 and 25 PM) and dFdC (0.1 and 0.25 PM). The concentrations of staurosporine
araC araC dFdC dFdC Stauro Stauro araC + stauro araC t stauro araC t stauro araC + stauro dFdC + stauro dFdC t stauro dFdC t stauro dFdC + stauro
Concentration
Relative cell kill (%)
10 uM 25 uM 0.1 pM 0.25 uM 25 nM 50 nM 10 uM t 25 nM lOuMt50nM 25 uM t 25 nM 25uMt50nM 0.1 uM t 25 nM 0.1 uM t 50 nM 0.25 uM t 25 nM 0.25 pM t 50 nM
34f2’ 54fll 48+5 61+4 13*3 26k2 51+9 65+2 68+4 78k2 69+4 77+5 73+3 87&3
3h
6h
I
* Mean + SE. (rt = 3 in triplicate). Values are expressed as % relative to control.
dFdC (O.OSpM) C
.
12h
c-jun 2.7 kb
Fig. 3. Time-dependent effect of dFdC on c-jun and c-fos mRNA expression in Molt-3 cells. The cells were exposed to 0.05 uM dFdC and at an appropriate time total RNA was extracted. Northern blots were performed on 20 ug total RNA using DNA probes for c-jun and c-f&. A /I-actin DNA probe was used to standardize the amount of RNA loaded. The experiment was performed in triplicate with similar results.
854
D. Y. Bouffard and R. L. Momparler
used are known to inhibit PKC activity in vitro, and exposure to higher concentrations and longer incubation times resulted in extensive DNA fragmentation in these cell lines (data not shown). For all the combinations used between staurosporine and araC or dFdC we observed a much greater effect on the DNA fragmentation of HL-60 cells than when each drug was used alone. These results demonstrate that an inhibitor of PKC can cooperate with antimetabolites to increase the level of DNA laddering in leukemic cells. Table 2 shows that the combination of staurosporine with dFdC or araC resulted in an additive loss of clonogenicity in this cell line. The incubation of HL-60 cells for 6 h to 25 nM staurosporine resulted in a 13% loss of clonogenicity whereas the exposure for 3 h to 10 uM araC and 0.1 pM dFdC produced a loss of clonogenicity of 34 and 48%, respectively. When the cells were pre-incubated for 3 h with 25 nM staurosporine and then co-incubated for an additional 3 h with 10 pM araC or 0.1 pM dFdC the loss of clonogenicity with these combinations was 51 and 69%, respectively, showing a much greater effect than when the drugs are used alone. The same results were observed with other combinations of staurosporine with araC or dFdC. A Student’s t test showed a P < 0.05 for the combination of dFdC plus staurosporine as compared to dFdC or staurosporine alone. The combination of araC plus staurosporine showed a P2 0.1 as compared to araC or staurosporine alone. Analysis of c-fos and c-jun expression following treatment with dFdC Induction of apoptosis in leukemic cells by araC is associated with increased expression of the protooncogenes c-jun and c-fos [16]. We have investigated by northern blot analysis the expression of these protooncogenes following exposure of Molt-3 lymphoid leukemic cells to dFdC. For these studies we used the Molt-3 cell line since it showed the same sensitivity to the induction of apoptosis by dFdC as HL-60 cells (data not shown). Also HL-60 cells do not express ~53 [31], which means it will be possible to use the Molt-3 cell line in further investigations to evaluate the effect of dFdC treatment on the expression of this gene. Figure 3 shows the expression of c-&n and c-fos mRNAs in Molt-3 cells in a time-dependent manner following exposure to a fixed concentration of dFdC (0.05 uM). The expression of jun and fos increased until the sixth hour and then remained steady up to 12 h. These results demonstrate that molecular changes occur following exposure of the leukemic cells to this antimetabolite and that these modifications are timedependent. Changes in the expression of these genes were also observed in a concentration-dependent fashion
in Molt-3 cells with concentrations of dFdC known to induce apoptosis in this cell line (data not shown). Discussion In the present study, we demonstrated that both araC and dFdC induced DNA fragmentation in HL-60 leukemic cells and their action was time- and concentration-dependent (Fig. lA, B). These studies were performed in order to determine which analog was the more potent inducer of apoptosis. dFdC can induce DNA laddering in leukemic cells at concentrations lOOfold less than araC. Previous studies have demonstrated that dFdC was more cytotoxic to tumor cells than araC [21,22]. We reported that dFdC was loo-fold more cytotoxic against different leukemic cell lines than araC [25]. The results of this study indicate that the better induction of apoptosis by dFdC on leukemic cells is related to its better cytotoxic activity when compared to araC. This in vitro effect on apoptosis suggests that dFdC may be a more potent antineoplastic agent on leukemic cell lines than araC. The differences in the antileukemic activity of these deoxycytidine analogs may be explained by the differences in their molecular mechanism of action. Huang et al. [27] have demonstrated, with a DNA primer extension assay, that greater than 90% of the dFdCMP was incorporated at internal positions and could be extended by different DNA polymerases. In contrast, they noted that araCMP incorporation into DNA was predominantly at the 3’ terminal position of the elongating strand and caused chain termination as first reported by Momparler [32]. Furthermore, DNA polymerase epsilon which possesses 3’->5’ exonuclease activity was shown to efficiently remove araCMP, but not dFdCMP, from the 3’-terminus of DNA primers [27]. We have reported a greater incorporation of dFdC into DNA than araC [25]. This higher accumulation of dFdC into DNA could produce a change in the DNA configuration and be a signal for the cells to undergo apoptosis. In support of this hypothesis are the experiments of Richardson et al. [33] who demonstrated that synthetic oligodeoxynucleotides containing dFdC produced a more destabilizing effect than araC with respect to endonuclease digestion. It is important to note that, under these conditions, we did not detect DNA fragmentation induced by dFdC in different human tumor cell lines (data not shown) indicating that the sensitivity to apoptosis produced by this fluoro analog depends on the phenotype of the target cell. The molecular events responsible for the induction of apoptosis remain to be elucidated. Kufe and co-workers [16,19] demonstrated that araC induced the expression of the proto-oncogene c-jun, and proposed that the activation of this gene may be important in the induction
Induction of apoptosisby gemcitabine
of apoptosis. In our experiments we have observed that dFdC increased c-jun expression (Fig. 3) at concentrations much lower than araC reported by these investigators. We also demonstrated that dFdC increased the expression of the proto-oncogene c-fos (Fig. 3). The gene products of c-jun and c-fos have been shown to form heterodimers in the activation of transcription factor AP-1 [34]. The induction of c-jun expression by araC occurs even in the presence of cycloheximide, an inhibitor of protein synthesis [18] suggesting a mechanism of post-translational modification. We have also observed that cycloheximide did not block dFdCinduced apoptosis (data not shown). Our data suggest that dFdC acts in the same manner as araC in the control of c-jun expression, but it can induce these molecular changes at lower concentrations which correlates with its greater in vitro antileukemic activity than araC. The role of the protein kinase C family on the induction of apoptosis in leukemic cells remains to be clarified. Jarvis et al. [14] demonstrated that different inhibitors of PKC could induce apoptotic DNA fragmentation in leukemic cells. In accordance with this report is our observation that inhibition of the PKC by staurosporine resulted in a better induction of DNA fragmentation produced by araC and dFdC and enhanced the antileukemic activity of these analogs (Fig. 2, Table 2). In contrast to these observations are the reports that bryostatin, an activator of PKC, potentiates the araC induction of apoptosis in leukemic cells [35, 361. Further investigations are required to understand the pleiotropic effects of different agents on PKC. Although the role of PKC is still controversial in PCD, our results suggest that the PKC pathway might act as a suppressor of apoptosis in leukemia cells. It is of interest to note that one of the substrates of PKC is the Bcl-2 protein [37], an oncogene that has been implicated in the suppression of apoptosis 138,391. The inhibition of PKC activity by staurosporine could decrease the level of phosphorylation of the Bcl-2 protein and impair its suppressing activity on apoptosis. Other proteins which are substrates of PKC could also be implicated in the process and inhibition of their phosphorylation could play an important role in the induction of apoptosis in cells. In summary, we have demonstrated that dFdC is more potent than araC in the induction of apoptosis in leukemic cells. In addition, dFdC was more potent than araC in the activation of expression of c-jun and c-fos, genes that could be involved in the signal transduction pathway of programmed cell death. The inhibition of PKC by staurosporine has enhanced the antineoplastic activity of dFdC in HL-60 leukemic cells. More specific PKC inhibitors may have the potential for clinical use if they can enhance the antileukemic activity of deoxycytidine analogs.
855
Acknonjledgement-This work was supported by grant 2773 from the National Cancer Institute of Canada.
References 1. Wyllie A. H., Kerr J. F. & Currie A. R. (1980) Cell death: the significance of apoptosis. ht. Rev. Cytol. 68, 251. 2. Arends M. J., Morris R. G. & Wyllie A. H. (1990) Apoptosis: the role of the endonuclease. Am. J. Pathof. 136, 593. 3. Kerr 3. F. R., Wyllie A. H. & Currie A. H. (1972) Apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics. Br. J. Cancer 26, 239. 4. Kaufmann S. H. (1989) Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. Cancer Res. 49, 5870. 5. Barry M. A., Behnke C. A. & Eastman A. (1990) Activation of program cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem. Pharrnac. 40, 2353. 6. Robertson L. E., Chubb S., Meyn R. E., Story M., Ford R., Hittelman W. N. & Plunkett W. (1993) Induction of apoptosis by 2-chloro-2’-deoxyadenosine and 9-fl-o-arabinosyl-2-fluoroadenine. Blood 81, 143. 7. Gorczyca W., Gong J., Ardelt B., Traganos F. & Darzynkiewicz Z. (1993) The cell cycle related differences in susceptibility of HL-60 to apoptosis induced by various antitumor agents. Cancer Res. 53, 3186. 8. Takano Y. S., Harmon B. V. & Kerr J. F. R. (1991) Apoptosis induced by mild hyperthermia in human and murine tumour cell lines: a study using electron microscopy and DNA gel electrophoresis. J. Pathol. 163, 329. 9. Wyllie A. H., Morris R. G., Smith A. L. & Dunlop D. (1984) Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol. 142, 67. 10. Collins R. J., Harmon B. V., Souvlis T., Pope J. H. & Kerr J. F. R. (1991) Effects of cycloheximide on B-chronic lymphocytic leukaemic and normal lymphocytes in vitro: induction of apoptosis. Br. J. Cancer 64, 518. 11. Martin S. J., Lennon S. V., Bonham A. M. & Cotter T. G. (1990) Induction of apoptosis (programmed cell death) in human leukemic HL-60 cells by inhibition of RNA or protein synthesis. J. Immunol. 145, 1859. 12. Forbes I. J., Zalewski P. D., Grannakis C. & Cowled P. A. (1992) Induction of apoptosis in chronic lymphocytic leukemia cells and its prevention by phorbol diester. Exp. Cell Res. 198, 367. 13. Cotter T. G., Lennon S. V., Clynn J. M. & Green D. R. (1992) Microfilament-disrupting agents prevent the formation of apoptotic bodies in tumour cells undergoing apoptosis. Cancer Res. 52, 997. 14. Jarvis W. D., Turner A. J., Povirk L. F., Traylor R. S. & Grant S. (1994) Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C. Cancer Res. 54, 1707. 15. Fram R. J. & Kufe D. (1982) DNA strand breaks caused by inhibitors of DNA synthesis: l-fi-o-arabinofuranosylcytosine and aphidicolin. Cancer Res. 42, 4050. 16. Gunji H., Kharbanda S. & Kufe D. (1991) Induction of internucleosomal DNA fragmentation in human myeloid
856
D. Y. Bouffard and R. L. Momparler
leukemia cells by I-B-o-arabinofuranosylcytosine. Cancer Res. 51, 741. 17. Mitchell T., Sariban E. & Kufe D. (1986) Effects of l-p-oarabinofuranosylcytosine on proto-oncogene expression in human U-937 cells. Mol. Pharmac. 30, 398. 18. Kharbanda S. M., Sherman M. L. & Kufe D. W. (1990) Transcriptional regulation of c-jun gene expression by arabinofuranosylcytosine in human myeloid leukemia cells. J. clin. Invest. 86, 1517. 19. Brach M. A., Herrmann F. & Kufe D. W. (1992) Activation of the AP-1 transcription factor by arabinofuranosylcytosine in myeloid leukemia cells. Blood 79, 728. 20. Hertel L. W., Kroin .I. S., Misner J. W. & Tustin J. M. (1988) Synthesis of 2-deoxy-2,2-difluoro-o-ribose and 2deoxy-2,2-difluoro-o-ribofuranosyl nucleosides. J. Org. Chem. 53, 2406. 21. Hertel L. W., Boder G. B., Kroin J. S., Rinzel S. M., Poore G. A., Todd G. C. & Grindey G. B. (1990) Evaluation of the antitumor activity of Gemcitabine (2’,2’-difluorodeoxycytidine). Cancer Res. 50, 4417. 22. Hanauske A. R., Degen D., Marshall M. H., Hilsenbeck S. G., Grindey G. B. & Von Hoff D. D. (1992) Activity of 2’,2’-difluorodeoxycytidine (Gemcitabine) against human tumor colony forming units. Anti-Cancer Drugs 3, 143. 23. Mertens W. C., Eisenhauer E. A., Moore M., Venner P., Stewart D., Muldal A. & Wong D. (1993) Gemcitabine in advanced renal cell carcinoma. A phase II study of the National Cancer Institute of Canada Clinical Trials Group. Annals Oncol. 4, 331. 24. Christman K., Kelsen D., Saltz L. & Tarassoff P. G. (1994) Phase I1 trial of gemcitabine in patients with advanced gastric cancer. Cancer 73, 5. 25. Bouffard D. Y., Momparler L. F. & Momparler R. L. (1991) Comparison of the antineoplastic activity of 2’,2’difluorodeoxycytidine and cytosine arabinoside against human myeloid and lymphoid leukemic cells. Anti-Cancer Drugs 2,49. 26. Ruiz van Haperen V. W., Veerman G., Vermorken J. B. & Peters G. J. (1993) 2’,2’-Difluoro-deoxycytidine (gemcitabine) incorporation into RNA and DNA of tumour cell lines. Biochem. Pharmac. 46, 762. 27. Huang P., Chubb S., Hertel L. W., Grindey G. B. & Plunkett W. (1991) Action of 2’,2’-difluorodeoxycytidine on DNA synthesis. Cancer Res. 51, 6110. 28. Heinemann V., Xu Y. Z., Chubb S., Sen A., Hertel L. W.,
Grindey G. B. & Plunkett W. (1990) Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2’,2’difluorodeoxycytidine. Mol. Pharmac. 38, 567. 29. Heinemann V., Xu Y. Z., Chubb S., Sen A., Hertel L. W., Grindey G. B. & Plunkett W. (1992) Cellular elimination of 2’,2’-difluorodeoxycytidine 5’-triphosphate: a mechanism of self-potentiation. Cancer Res. 52, 533. 30. Huang P. & Plunkett W. (1992) A quantitative assay for fragmented DNA in apoptotic cells. Anal. Biochem. 207, 163. 31. Wolf D. & Rotter V. (1985) Major deletions in the gene encoding the ~53 tumor antigen cause lack of ~53 expression in HL-60 cells. Proc. natn. Acad. Sci. USA 82, 790. 32. Momparler R. L. (1969) Effect of cytosine arabinoside 5’triphosphate on mammalian DNA polymerase. Biochem. Biophys. Res. Commun. 34, 465. 33. Richardson F. C., Richardson K. K., Kroin J. S. & Hertel L. W. (1992) Synthesis and restriction enzyme analysis of oligodeoxynucleotides containing the anti-cancer drug 2’,2’-difluorodeoxycytidine. Nucleic Acid Res. 20, 1763. 34. Angel P., Hattori K., Smeal T. & Karin M. (1988) The jun proto-oncogene is positively autoregulated by its product, JUNIAP-1. Cell 55, 875. 35. Grant S., Jarvis W. D., Swerdlow P. S., Turner A. J., Traylor R. S., Wallace H. J., Lin P.-S., Pettit G. R. & Gewirtz D. A. (1992) Potentiation of the activity of l-j-oarabinofuranosyl-cytosine by the protein kinase C activator bryostatin 1 in HL-60 cells: association with enhanced fragmentation of mature DNA. Cnncer Res. 52, 6270. 36. Jarvis W. D., Povirk L. F., Turner A. J., Traylor R. S., Gewirtz D. A., Pettit G. R. & Grant S. (1994) Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[fi-o-arabinofuranosyl] cytosineinduced apoptosis in HL-60 human promyelocytic leukemia cells. Biochem. Pharmac. 47, 839. 37. May W. S., Tyler P. G., Armstrong D. K. & Davidson N. E. (1993) Role for serine phosphorylation of Bcl-2 in an anti-apoptotic signalling pathway triggered by IL-3, erythropoietin, and bryostatin 1. Blood 82 (suppl. l), 1738. 38. Korsmeyer S. J. (1992) Bcl-2 initiates a new category of oncogenes: regulators of cell death. Blood 80, 879. 39. Miyashita T. & Reed J. C. (1993) Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood 81, 151.
Pergamon 01452126(95)00067-4
COMPARISON OF THE INDUCTION OF APOPTOSIS IN HUMAN LEUKEMIC CELL LINES BY 2’,2’-DIFLUORODEOXYCYTIDINE (GEMCITABINE) AND CYTOSINE ARABINOSIDE David Y. Bouffard* and Richard L. Momparlert *Department of Pharmacology,UniversitC de Montreal, Montreal, QuCbec,Canada;and TPediatric Research Centre, Ste-JustineHospital, Montreal, QuCbecCanada (Received 17 November 1994.Accepted 7 April 1995) Abstract-The induction of DNA fragmentation by cytosine arabinoside (araC) and 2’,2’difluorodeoxycytidine (dFdC, gemcitabine) was compared in human leukemic cell lines. For both araC and dFdC this process was time- and concentration-dependent and resulted in loss of clonogenic survival of HL-60 myeloid leukemic cells. There was a marked difference in the potency between these two analogs in inducing apoptosis. A 6 h exposure to 5 PM araC was required to produce DNA laddering in HL-60 cells, whereas dFdC at a concentration IOO-fold less (0.05 PM) was sufficient to produce similar results. Pre-incubation of HL-60 cells with staurosporine, a non-specific protein kinase C inhibitor, increased the level of apoptosis induced by a 3 h exposure to araC or dFdC, suggesting the possible involvement of this family of enzymes in this process. Also, dFdC was able to increase the expression of both c-jun and cfos in Molt-3 leukemic cells with a concentration known to induce apoptosis in this cell line. Key words:
Gemcitabine,
apoptosis,
protein
kinase C, c-jun, c-fos.
Introduction
[lo, 111. A series of proteins that could be implicated in the regulation of apoptosis is the family of the protein kinase C (PKC). Forbes et al. [12] have shown that the induction of apoptosis in chronic lymphocytic leukemia cells could be prevented by phorbol esters (PKC modulators). Although the PKC seems to be involved in preventing apoptosis, its mechanism of action in apoptosis remains to be clarified. In some instances the non-specific PKC inhibitor staurosporine has been shown to prevent [13] and to increase [14] the level of apoptosis in cancer cells. Cytosine arabinoside (araC), one of the most effective drugs for the treatment of acute leukemia, has been shown to induce apoptosis in myeloid leukemia cells [15,16]. There is also evidence that certain oncogenes could be implicated in the process of apoptosis. The cjun and c-fos proto-oncogenes have been shown to be increased following a treatment with araC suggesting that these genes could be involved in the induction of apoptosis [17-191. 2’,2’-difluorodeoxycytidine (dFdC, gemcitabine) is a new and very interesting antimetabolite of deoxycytidine in which the two hydrogen atoms in the 2’ position of the deoxyribose sugar have been replaced by two fluorine atoms 1201. Several reports have shown that dFdC is active against many murine solid tumors and
Apoptosis or programmed cell death (PCD) is characterized by cell shrinkage and chromatin condensation with maintenance of normal organelle structure and the appearance of oligonucleosomal DNA fragmentation at multiples of approximately 200 base pairs [l, 21. PCD results from the activation of an intrinsic program after responding to specific stimuli [3]. Apoptosis can be induced in many types of cancer cells by a variety of chemotherapeutic drugs [4-71. Little is known about the cascade of events that leads to PCD. The role of protein synthesis in this process is controversial. In certain cases inhibition of protein synthesis does not prevent apoptosis [8], whereas in some instances this process depends on de novo protein synthesis [9]. In addition, in some reports inhibition of protein synthesis has been shown to trigger apoptosis Abbreviations: dFdC, 2’,2’-difluorodeoxycytidine; dFdCMP, 2’,2’-difluorodeoxycytidine S-monophosphate;dFdCTP, 2’,2’difluorodeoxycytidine S-triphosphate; araC, cytosine arabinoside; araCMP, cytosine arabinoside S-monophosphate; araCTP, cytosine arabinoside S-triphosphate; PCD, programmed cell death; PKC, protein kinase C; stauro, staurosporine. Correspondence to: R. L. Momparler, Pediatric Research Centre, Ste-JustineHospital, MontrCal, QuCbec,Canada. 849
D. Y. Bouffard and R. L. Momparler
850
ara-C (PM)
(A)
c
W
1
5
dFdC (PM) 10
c
bp
0.01
0.05
2072 2072
600 600
(B)
dFdC (0.05p.M)
ara-C (10 pM) C
3h
6h
12h
C
3h
Fig. 1. Concentration- and time-dependent DNA fragmentation in HL-60 myeloid leukemic cells produced by araC or dFdC. The cells were exposed to various concentrations of araC or dFdC and assayed for DNA fragmentation as described in Materials and Methods. The experiments were repeated three times with comparable results obtained each time. (A) Six hour exposure to different concentrations of araC and dFdC; (B) time-dependent effect of araC and dFdC.
6h
12h
0.1
Induction of apoptosisby
leukemic cell lines [21,22] and is presently undergoing phase II clinical trials [23,24]. We have shown in previous experiments that dFdC is one hundred times more cytotoxic than araC in different kinds of leukemia cell lines [25]. The mechanism of action of dFdC appears to be related to its incorporation into RNA [26] and DNA [27] and also to its inhibition of the enzymes ribonucleotide reductase [28] and deoxycytidine monophosphate deaminase [29]. dFdC has also been shown to induce apoptosis in human T-lymphoblastoid CEM cells [30]. In this report we have observed that dFdC is a much more potent inducer of apoptosis than araC, indicating that the kinetics of induction of this process for these two antimetabolites are different. We have also observed that staurosporine, a PKC inhibitor, could enhance the apoptotic activity of both araC and dFdC in leukemic cells.
gemcitabine
851
(LMP), 0.25% (w/v) bromophenol blue and 40% (w/v) sucrose were added.The sampleswere then loaded into the wells of a dry 2% agarosegel and electrophoresedat 90-100 V for 3-4 h in 1xTBE pH 8 containing 0.5 @ml ethidium bromide at 4°C. DNA laddering was observed under UV illumination. All
experiments were performed three times with similar results obtained each time. Northern blot analysis
Total cellular RNA was isolated at appropriate times from Molt-3 leukemic cells with the Trizol solution from Canadian Life Technologies Inc. (Mississauga,Canada).Electrophoresis of 20 pg total RNA was performed in a 1.1% agarose-2.6% formaldehyde gel and followed by northern blotting on to a nylon membrane (Amersham Hybond-N). The membranes were prehybridized at 42°C for 4-6 h in a buffer containing 50% formamide, 5 x SSC, 0.1% SDS, 5 x Denhardt’s solution, 100 mg/ml dextran sulphate, and 200 pg/ml shearedsalmon spermDNA. The blots were then hybridized overnight at 42°C with 2~10~ cpm of 32P-labelled probe/ml of hybridization buffer (same as prehybridization buffer but no dextran sulphate
Materials and Methods Materials
The antimetabolite, araC, was obtained from the Upjohn Company of Canada. dFdC was provided by Lilly Research Laboratories (Indianapolis, IN, U.S.A.). Staurosporine was purchased from Sigma Chemical Company (St. Louis, MO, U.S.A.). Cell culture
Human HL-60 myeloid leukemic cells were obtained from Dr R. Gallo, National Cancer Institute, Bethesda,MD, U.S.A. The human Molt-3 T-lymphoid leukemic cell line was purchasedfrom the American Type Tissue Culture Collection (Rockville, MD, U.S.A.). The cell lines were maintained in suspension culture in minimal essential medium containing non-essential amino acids (Gibco, Grand Island, NY, U.S.A.) and 10% heat-inactivated fetal calf serum (Flow Laboratories, Mississauga,Canada).The doubling time for the cell lines was between 20 and 22 h. Clonogenic assay
The proliferative viability of the leukemic cells after exposure to the various drugs was determined by cloning in soft agar. At the termination of drug exposure, the cells were
centrifuged and suspendedin drug-free medium. An aliquot of 150-200 cells was placed in 2 ml 0.3% soft agar medium containing 15% serum. After incubation for 14-16 days at 37°C in a 5% CO* incubator, the number of colonies (~500 cells) were counted. The cloning efficiency of the control was in the range of 40-60%. All experimentswere performedthree
times in triplicate. DNA fragmentation analysis After exposure to the different drugs for various exposure
times, the cells (5x10’ cells) were pelleted and suspendedin 20 pl 10 mM EDTA, 50 mM Tris-HCl pH 8.0 containing 0.5% SDS (w/v) and 0.5 mgi’ml proteinase K (Sigma). After an
incubation of 1 h at 50°C 10 pl RNaseA, DNase-free(0.5 mg/ ml; Boehringer Mannheim, Laval, Canada) was added and incubation was continued for another hour at 50°C. The
samples were then heated to 70°C and then 10 l.1110 mM EDTA pH 8.0 containing 1% (w/v) low melting point agarose
present). After hybridization, the blots were washed twice in 2xSSC, 0.1% SDS at 55°C for 30 min and then washedtwice in 0.1 xSSC, 0.1% SDS at 55°C for 30 min. The blots were then exposed to X-ray films with double intensifying screens at
-70°C. DNA probes were purified by preparative gel electrophoresis and random-primed (Boehringer Mannheim, Laval, QuCbec, Canada) to specific radioactivities. The 2.6 kb EcoRI insert of the mouse c-jun gene was purified from the
JAC.l plasmid from American Type Culture Collection (Rockville, MD, U.S.A.). The 770 bp chicken fi-actin gene insert and the 1 kb mousev-fos gene insert were obtained from Oncor (Gaithersburg, MD, U.S.A.). All experiments were performed three times with similar results obtained each time.
Results Eflects of araC and dFdC on the induction of DNA fragmentation in HL-60 cells Figure 1A shows the effect of an increase in drug concentration of araC and dFdC on the induction of DNA oligonucleosomal fragmentation in HL-60 myeloid leukemic cells for a 6 h exposure. The induction of DNA laddering was observed only with a concentration of araC over 5 PM. For the same conditions, a loo-fold lower concentration of dFdC (0.05 FM) was needed to induce apoptosis in this cell line. We have also observed that concentrations up to 100 pM araC were unable to cause DNA laddering in this cell line for a 3 h exposure, whereas 0.5 ~.IM dFdC was sufficient to induce DNA fragmentation as visualized using conventional agarose gel electrophoresis (data not shown). Even when a higher number of cells (2~10~) was used, the same differences between araC and dFdC was observed. The kinetics of induction of apoptosis was also compared between araC and dFdC in a time-dependent manner in HL-60 cells. The cells were exposed to a fixed concentration of the drugs and at appropriate times DNA laddering was analyzed by agarose gel electrophoresis as shown in Fig. 1B. In this experiment, both araC and
D. Y. Bouffard and R. L. Momparler
852
araC (yM) Stauro (nM)
dFdC (PM) Stauro (nM)
C 10
25
--
C
-
25
-
50
c
10 10 25
25
C
25
50
50
25
C 0.1 0.25 -
-
C 0.1 0.1 0.25 0.25
C
50
C
-
-
25
25
50
25
50
Fig. 2. Effect of staurosporine on the induction of DNA fragmentation in HL-60 leukemic cells prior to an exposure to araC or dFdC. The cells were pre-incubated for 3 h with 2.5 or 50 nM staurosporine and then exposed to 10 and 2.5 pM araC or 0.1 and 0.25 pM dFdC for 3 additional hours. The cells were then analyzed for DNA fragmentation. The experiments were repeated three times and similar results were obtained in each case.
dFdC demonstrated a time-dependent effect on the induction of apoptosis in HL-60 cells. For both drugs DNA laddering was apparent at 6 h and continued up to 12 h. These results also show that much less dFdC (0.05 yM) was needed to achieve the same degree of DNA fragmentation as compared to araC (10 f.tM). The use of higher concentrations of cells have resulted in similar results. As shown in Table 1, all the concentrations and time of exposure of araC and dFdC used on HL-60 cells resulted in a loss of clonogenicity as determined by the
cloning of the cells in soft agar. Similar kinetics of DNA laddering were observed with Molt-3 T-lymphoid leukemic cells (data not shown) showing that the action of araC and dFdC is not limited to the myeloid phenotype. Effect of staurosporine on araC- and dFdC-induced apoptosis The effect of staurosporine, a non-specific protein kinase C inhibitor that acts at the catalytic domain of the
Induction of apoptosis by gemcitabine
Table 1. Effect of araC and dFdC on the clonogenicity of myeloid HL-60 leukemic cell line after a treatment of 2 and 24 h Drug araC dFdC
Concentration Wf) 0.01 1.0 10.0 0.001 0.10 1.00
Table 2. Effect of staurosporine 3 h pre-incubation on the loss of clonogenic survival of myeloid HL-60 leukemic cell line for a 3 h exposure to araC and dFdC
-
Relative cell kill (%) 24 hr 2 hr ND 30f9 47fll ND 57+14 68f9
853
Drug
21+7* 49+ 10 ND 58_+ 12 >99 ND
* Mean + SE. (n = 3, in triplicate). ND, not determined. Values are expressed as % relative to control.
enzyme, was also studied in HL-60 leukemic cells. Figure 2 shows the effect of a pre-incubation (3 h) of 25 and 50 nM staurosporine on apoptosis in HL-60 prior to an exposure of 3 h to araC (10 and 25 PM) and dFdC (0.1 and 0.25 PM). The concentrations of staurosporine
araC araC dFdC dFdC Stauro Stauro araC + stauro araC t stauro araC t stauro araC + stauro dFdC + stauro dFdC t stauro dFdC t stauro dFdC + stauro
Concentration
Relative cell kill (%)
10 uM 25 uM 0.1 pM 0.25 uM 25 nM 50 nM 10 uM t 25 nM lOuMt50nM 25 uM t 25 nM 25uMt50nM 0.1 uM t 25 nM 0.1 uM t 50 nM 0.25 uM t 25 nM 0.25 pM t 50 nM
34f2’ 54fll 48+5 61+4 13*3 26k2 51+9 65+2 68+4 78k2 69+4 77+5 73+3 87&3
3h
6h
I
* Mean + SE. (rt = 3 in triplicate). Values are expressed as % relative to control.
dFdC (O.OSpM) C
.
12h
c-jun 2.7 kb
Fig. 3. Time-dependent effect of dFdC on c-jun and c-fos mRNA expression in Molt-3 cells. The cells were exposed to 0.05 uM dFdC and at an appropriate time total RNA was extracted. Northern blots were performed on 20 ug total RNA using DNA probes for c-jun and c-f&. A /I-actin DNA probe was used to standardize the amount of RNA loaded. The experiment was performed in triplicate with similar results.
854
D. Y. Bouffard and R. L. Momparler
used are known to inhibit PKC activity in vitro, and exposure to higher concentrations and longer incubation times resulted in extensive DNA fragmentation in these cell lines (data not shown). For all the combinations used between staurosporine and araC or dFdC we observed a much greater effect on the DNA fragmentation of HL-60 cells than when each drug was used alone. These results demonstrate that an inhibitor of PKC can cooperate with antimetabolites to increase the level of DNA laddering in leukemic cells. Table 2 shows that the combination of staurosporine with dFdC or araC resulted in an additive loss of clonogenicity in this cell line. The incubation of HL-60 cells for 6 h to 25 nM staurosporine resulted in a 13% loss of clonogenicity whereas the exposure for 3 h to 10 uM araC and 0.1 pM dFdC produced a loss of clonogenicity of 34 and 48%, respectively. When the cells were pre-incubated for 3 h with 25 nM staurosporine and then co-incubated for an additional 3 h with 10 pM araC or 0.1 pM dFdC the loss of clonogenicity with these combinations was 51 and 69%, respectively, showing a much greater effect than when the drugs are used alone. The same results were observed with other combinations of staurosporine with araC or dFdC. A Student’s t test showed a P < 0.05 for the combination of dFdC plus staurosporine as compared to dFdC or staurosporine alone. The combination of araC plus staurosporine showed a P2 0.1 as compared to araC or staurosporine alone. Analysis of c-fos and c-jun expression following treatment with dFdC Induction of apoptosis in leukemic cells by araC is associated with increased expression of the protooncogenes c-jun and c-fos [16]. We have investigated by northern blot analysis the expression of these protooncogenes following exposure of Molt-3 lymphoid leukemic cells to dFdC. For these studies we used the Molt-3 cell line since it showed the same sensitivity to the induction of apoptosis by dFdC as HL-60 cells (data not shown). Also HL-60 cells do not express ~53 [31], which means it will be possible to use the Molt-3 cell line in further investigations to evaluate the effect of dFdC treatment on the expression of this gene. Figure 3 shows the expression of c-&n and c-fos mRNAs in Molt-3 cells in a time-dependent manner following exposure to a fixed concentration of dFdC (0.05 uM). The expression of jun and fos increased until the sixth hour and then remained steady up to 12 h. These results demonstrate that molecular changes occur following exposure of the leukemic cells to this antimetabolite and that these modifications are timedependent. Changes in the expression of these genes were also observed in a concentration-dependent fashion
in Molt-3 cells with concentrations of dFdC known to induce apoptosis in this cell line (data not shown). Discussion In the present study, we demonstrated that both araC and dFdC induced DNA fragmentation in HL-60 leukemic cells and their action was time- and concentration-dependent (Fig. lA, B). These studies were performed in order to determine which analog was the more potent inducer of apoptosis. dFdC can induce DNA laddering in leukemic cells at concentrations lOOfold less than araC. Previous studies have demonstrated that dFdC was more cytotoxic to tumor cells than araC [21,22]. We reported that dFdC was loo-fold more cytotoxic against different leukemic cell lines than araC [25]. The results of this study indicate that the better induction of apoptosis by dFdC on leukemic cells is related to its better cytotoxic activity when compared to araC. This in vitro effect on apoptosis suggests that dFdC may be a more potent antineoplastic agent on leukemic cell lines than araC. The differences in the antileukemic activity of these deoxycytidine analogs may be explained by the differences in their molecular mechanism of action. Huang et al. [27] have demonstrated, with a DNA primer extension assay, that greater than 90% of the dFdCMP was incorporated at internal positions and could be extended by different DNA polymerases. In contrast, they noted that araCMP incorporation into DNA was predominantly at the 3’ terminal position of the elongating strand and caused chain termination as first reported by Momparler [32]. Furthermore, DNA polymerase epsilon which possesses 3’->5’ exonuclease activity was shown to efficiently remove araCMP, but not dFdCMP, from the 3’-terminus of DNA primers [27]. We have reported a greater incorporation of dFdC into DNA than araC [25]. This higher accumulation of dFdC into DNA could produce a change in the DNA configuration and be a signal for the cells to undergo apoptosis. In support of this hypothesis are the experiments of Richardson et al. [33] who demonstrated that synthetic oligodeoxynucleotides containing dFdC produced a more destabilizing effect than araC with respect to endonuclease digestion. It is important to note that, under these conditions, we did not detect DNA fragmentation induced by dFdC in different human tumor cell lines (data not shown) indicating that the sensitivity to apoptosis produced by this fluoro analog depends on the phenotype of the target cell. The molecular events responsible for the induction of apoptosis remain to be elucidated. Kufe and co-workers [16,19] demonstrated that araC induced the expression of the proto-oncogene c-jun, and proposed that the activation of this gene may be important in the induction
Induction of apoptosisby gemcitabine
of apoptosis. In our experiments we have observed that dFdC increased c-jun expression (Fig. 3) at concentrations much lower than araC reported by these investigators. We also demonstrated that dFdC increased the expression of the proto-oncogene c-fos (Fig. 3). The gene products of c-jun and c-fos have been shown to form heterodimers in the activation of transcription factor AP-1 [34]. The induction of c-jun expression by araC occurs even in the presence of cycloheximide, an inhibitor of protein synthesis [18] suggesting a mechanism of post-translational modification. We have also observed that cycloheximide did not block dFdCinduced apoptosis (data not shown). Our data suggest that dFdC acts in the same manner as araC in the control of c-jun expression, but it can induce these molecular changes at lower concentrations which correlates with its greater in vitro antileukemic activity than araC. The role of the protein kinase C family on the induction of apoptosis in leukemic cells remains to be clarified. Jarvis et al. [14] demonstrated that different inhibitors of PKC could induce apoptotic DNA fragmentation in leukemic cells. In accordance with this report is our observation that inhibition of the PKC by staurosporine resulted in a better induction of DNA fragmentation produced by araC and dFdC and enhanced the antileukemic activity of these analogs (Fig. 2, Table 2). In contrast to these observations are the reports that bryostatin, an activator of PKC, potentiates the araC induction of apoptosis in leukemic cells [35, 361. Further investigations are required to understand the pleiotropic effects of different agents on PKC. Although the role of PKC is still controversial in PCD, our results suggest that the PKC pathway might act as a suppressor of apoptosis in leukemia cells. It is of interest to note that one of the substrates of PKC is the Bcl-2 protein [37], an oncogene that has been implicated in the suppression of apoptosis 138,391. The inhibition of PKC activity by staurosporine could decrease the level of phosphorylation of the Bcl-2 protein and impair its suppressing activity on apoptosis. Other proteins which are substrates of PKC could also be implicated in the process and inhibition of their phosphorylation could play an important role in the induction of apoptosis in cells. In summary, we have demonstrated that dFdC is more potent than araC in the induction of apoptosis in leukemic cells. In addition, dFdC was more potent than araC in the activation of expression of c-jun and c-fos, genes that could be involved in the signal transduction pathway of programmed cell death. The inhibition of PKC by staurosporine has enhanced the antineoplastic activity of dFdC in HL-60 leukemic cells. More specific PKC inhibitors may have the potential for clinical use if they can enhance the antileukemic activity of deoxycytidine analogs.
855
Acknonjledgement-This work was supported by grant 2773 from the National Cancer Institute of Canada.
References 1. Wyllie A. H., Kerr J. F. & Currie A. R. (1980) Cell death: the significance of apoptosis. ht. Rev. Cytol. 68, 251. 2. Arends M. J., Morris R. G. & Wyllie A. H. (1990) Apoptosis: the role of the endonuclease. Am. J. Pathof. 136, 593. 3. Kerr 3. F. R., Wyllie A. H. & Currie A. H. (1972) Apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics. Br. J. Cancer 26, 239. 4. Kaufmann S. H. (1989) Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. Cancer Res. 49, 5870. 5. Barry M. A., Behnke C. A. & Eastman A. (1990) Activation of program cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem. Pharrnac. 40, 2353. 6. Robertson L. E., Chubb S., Meyn R. E., Story M., Ford R., Hittelman W. N. & Plunkett W. (1993) Induction of apoptosis by 2-chloro-2’-deoxyadenosine and 9-fl-o-arabinosyl-2-fluoroadenine. Blood 81, 143. 7. Gorczyca W., Gong J., Ardelt B., Traganos F. & Darzynkiewicz Z. (1993) The cell cycle related differences in susceptibility of HL-60 to apoptosis induced by various antitumor agents. Cancer Res. 53, 3186. 8. Takano Y. S., Harmon B. V. & Kerr J. F. R. (1991) Apoptosis induced by mild hyperthermia in human and murine tumour cell lines: a study using electron microscopy and DNA gel electrophoresis. J. Pathol. 163, 329. 9. Wyllie A. H., Morris R. G., Smith A. L. & Dunlop D. (1984) Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol. 142, 67. 10. Collins R. J., Harmon B. V., Souvlis T., Pope J. H. & Kerr J. F. R. (1991) Effects of cycloheximide on B-chronic lymphocytic leukaemic and normal lymphocytes in vitro: induction of apoptosis. Br. J. Cancer 64, 518. 11. Martin S. J., Lennon S. V., Bonham A. M. & Cotter T. G. (1990) Induction of apoptosis (programmed cell death) in human leukemic HL-60 cells by inhibition of RNA or protein synthesis. J. Immunol. 145, 1859. 12. Forbes I. J., Zalewski P. D., Grannakis C. & Cowled P. A. (1992) Induction of apoptosis in chronic lymphocytic leukemia cells and its prevention by phorbol diester. Exp. Cell Res. 198, 367. 13. Cotter T. G., Lennon S. V., Clynn J. M. & Green D. R. (1992) Microfilament-disrupting agents prevent the formation of apoptotic bodies in tumour cells undergoing apoptosis. Cancer Res. 52, 997. 14. Jarvis W. D., Turner A. J., Povirk L. F., Traylor R. S. & Grant S. (1994) Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C. Cancer Res. 54, 1707. 15. Fram R. J. & Kufe D. (1982) DNA strand breaks caused by inhibitors of DNA synthesis: l-fi-o-arabinofuranosylcytosine and aphidicolin. Cancer Res. 42, 4050. 16. Gunji H., Kharbanda S. & Kufe D. (1991) Induction of internucleosomal DNA fragmentation in human myeloid
856
D. Y. Bouffard and R. L. Momparler
leukemia cells by I-B-o-arabinofuranosylcytosine. Cancer Res. 51, 741. 17. Mitchell T., Sariban E. & Kufe D. (1986) Effects of l-p-oarabinofuranosylcytosine on proto-oncogene expression in human U-937 cells. Mol. Pharmac. 30, 398. 18. Kharbanda S. M., Sherman M. L. & Kufe D. W. (1990) Transcriptional regulation of c-jun gene expression by arabinofuranosylcytosine in human myeloid leukemia cells. J. clin. Invest. 86, 1517. 19. Brach M. A., Herrmann F. & Kufe D. W. (1992) Activation of the AP-1 transcription factor by arabinofuranosylcytosine in myeloid leukemia cells. Blood 79, 728. 20. Hertel L. W., Kroin .I. S., Misner J. W. & Tustin J. M. (1988) Synthesis of 2-deoxy-2,2-difluoro-o-ribose and 2deoxy-2,2-difluoro-o-ribofuranosyl nucleosides. J. Org. Chem. 53, 2406. 21. Hertel L. W., Boder G. B., Kroin J. S., Rinzel S. M., Poore G. A., Todd G. C. & Grindey G. B. (1990) Evaluation of the antitumor activity of Gemcitabine (2’,2’-difluorodeoxycytidine). Cancer Res. 50, 4417. 22. Hanauske A. R., Degen D., Marshall M. H., Hilsenbeck S. G., Grindey G. B. & Von Hoff D. D. (1992) Activity of 2’,2’-difluorodeoxycytidine (Gemcitabine) against human tumor colony forming units. Anti-Cancer Drugs 3, 143. 23. Mertens W. C., Eisenhauer E. A., Moore M., Venner P., Stewart D., Muldal A. & Wong D. (1993) Gemcitabine in advanced renal cell carcinoma. A phase II study of the National Cancer Institute of Canada Clinical Trials Group. Annals Oncol. 4, 331. 24. Christman K., Kelsen D., Saltz L. & Tarassoff P. G. (1994) Phase I1 trial of gemcitabine in patients with advanced gastric cancer. Cancer 73, 5. 25. Bouffard D. Y., Momparler L. F. & Momparler R. L. (1991) Comparison of the antineoplastic activity of 2’,2’difluorodeoxycytidine and cytosine arabinoside against human myeloid and lymphoid leukemic cells. Anti-Cancer Drugs 2,49. 26. Ruiz van Haperen V. W., Veerman G., Vermorken J. B. & Peters G. J. (1993) 2’,2’-Difluoro-deoxycytidine (gemcitabine) incorporation into RNA and DNA of tumour cell lines. Biochem. Pharmac. 46, 762. 27. Huang P., Chubb S., Hertel L. W., Grindey G. B. & Plunkett W. (1991) Action of 2’,2’-difluorodeoxycytidine on DNA synthesis. Cancer Res. 51, 6110. 28. Heinemann V., Xu Y. Z., Chubb S., Sen A., Hertel L. W.,
Grindey G. B. & Plunkett W. (1990) Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2’,2’difluorodeoxycytidine. Mol. Pharmac. 38, 567. 29. Heinemann V., Xu Y. Z., Chubb S., Sen A., Hertel L. W., Grindey G. B. & Plunkett W. (1992) Cellular elimination of 2’,2’-difluorodeoxycytidine 5’-triphosphate: a mechanism of self-potentiation. Cancer Res. 52, 533. 30. Huang P. & Plunkett W. (1992) A quantitative assay for fragmented DNA in apoptotic cells. Anal. Biochem. 207, 163. 31. Wolf D. & Rotter V. (1985) Major deletions in the gene encoding the ~53 tumor antigen cause lack of ~53 expression in HL-60 cells. Proc. natn. Acad. Sci. USA 82, 790. 32. Momparler R. L. (1969) Effect of cytosine arabinoside 5’triphosphate on mammalian DNA polymerase. Biochem. Biophys. Res. Commun. 34, 465. 33. Richardson F. C., Richardson K. K., Kroin J. S. & Hertel L. W. (1992) Synthesis and restriction enzyme analysis of oligodeoxynucleotides containing the anti-cancer drug 2’,2’-difluorodeoxycytidine. Nucleic Acid Res. 20, 1763. 34. Angel P., Hattori K., Smeal T. & Karin M. (1988) The jun proto-oncogene is positively autoregulated by its product, JUNIAP-1. Cell 55, 875. 35. Grant S., Jarvis W. D., Swerdlow P. S., Turner A. J., Traylor R. S., Wallace H. J., Lin P.-S., Pettit G. R. & Gewirtz D. A. (1992) Potentiation of the activity of l-j-oarabinofuranosyl-cytosine by the protein kinase C activator bryostatin 1 in HL-60 cells: association with enhanced fragmentation of mature DNA. Cnncer Res. 52, 6270. 36. Jarvis W. D., Povirk L. F., Turner A. J., Traylor R. S., Gewirtz D. A., Pettit G. R. & Grant S. (1994) Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[fi-o-arabinofuranosyl] cytosineinduced apoptosis in HL-60 human promyelocytic leukemia cells. Biochem. Pharmac. 47, 839. 37. May W. S., Tyler P. G., Armstrong D. K. & Davidson N. E. (1993) Role for serine phosphorylation of Bcl-2 in an anti-apoptotic signalling pathway triggered by IL-3, erythropoietin, and bryostatin 1. Blood 82 (suppl. l), 1738. 38. Korsmeyer S. J. (1992) Bcl-2 initiates a new category of oncogenes: regulators of cell death. Blood 80, 879. 39. Miyashita T. & Reed J. C. (1993) Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood 81, 151.