Recruitment of Leukemic Cells from G 0 Phase of the Cell Cycle By

CORRESPONDENCE

Recruitment of leukemic cells from G0 phase of the cell cycle by interferons results in conversion of resistance to daunorubicin

Leukemia (2003) 17, 2049–2051. doi:10.1038/sj.leu.2403085 were not affected by daunorubicin-induced cell death. To analyze whether a similar cell cycle-specific sensitivity to Ara-C TO THE EDITOR and daunorubicin was observed in other leukemic cell lines, we analyzed four acute lymphoblastic leukemia cell lines and one Although the majority of patients with acute myeloid leukemia CML blast crisis cell line, which were generated in our (AML) responds to initial treatment, relapse of the disease occurs laboratory by culturing of leukemic blasts from five different in a significant percentage of these patients.1 The cell cycle patients in serum-free medium at high cell concentrations until status of leukemic cells may play an important role in the spontaneous sustained proliferation of the leukemic cells response to treatment of leukemic cells. Especially, the broadly occurred. These cell lines cytogenetically and phenotypically used cytotoxic agent Cytarabine (Ara-C) has been demonstrated resembled the primary leukemia. Figure 1b shows the median to exert its action via intercalation into the DNA of cells cell cycle distribution within these cell lines after incubation for À6 specifically in the S phase of the cell cycle, and sensitivity to this 48 h in medium alone, or in medium containing 1 Â 10 M Ara- agent is therefore described to be specific for cells in active cell C or daunorubicin. As expected, Ara-C-induced cell death 2 cycle. The role of the cell cycle in sensitivity of leukemic cells resulted in a deletion of cells from cycling S and G2/M phases, to anthracyclins like daunorubicin remains largely unknown. resulting in relative enrichment for cells in G0 and G1 phases. In We hypothesized that a fraction of noncycling leukemic contrast, daunorubicin-induced cell death resulted in a deletion (precursor) cells residing in dormancy might be unresponsive to of cells from both activated G1 phase and cycling S and G2/M chemotherapy. To test this hypothesis, we determined in a pilot phases, resulting in a major enrichment of cells in G0 phase. study the in vivo cell cycle specificity of chemotherapeutic These data illustrated the specificity of daunorubicin not only for treatment by analysis of the cell cycle distribution of leukemic cells in active cell cycle, but also for noncycling cells in the blasts freshly isolated from the peripheral blood of three patients activated G1 phase of the cell cycle. with AML at diagnosis and at multiple consecutive days after The AML-193 cell line model was used to further investigate in vivo treatment with Ara-C, daunorubicin, and etoposide. the influence of manipulation of the cell cycle status of leukemic Detailed cell cycle analysis was performed by combined ki-67 cells on chemotherapy sensitivity. As described previously, it is staining and propidium iodide (PI) DNA staining. Ki-67 is a possible to separately manipulate the cell cycle status and the nuclear protein which is absent in G0 phase and present in all proliferative capacity of these AML-193 cells using GM-CSF and other phases of the cell cycle,3 thereby facilitating the interferon (IFN) treatment.4 Three AML-193 subcell lines were discrimination between cells in resting G0 phase, activated G1 generated by culturing of cells either in the absence of cytokines phase, and cycling S or G2/M phase. Table 1 shows the total (control), in the presence of 1000 U/ml IFN-g, or in the presence nuclear cell counts, the percentages of leukemic blasts, and the of 500 U/ml IFN-a, as described previously.4 GM-CSF was used cell cycle distribution of the cells measured in time during to induce proliferation in these cells. None of these cell cycle chemotherapy administration. Already after 1 day of treatment, manipulations did induce apoptosis. The long-term culture a decrease in the number of cycling cells was noticed. In the period in the presence of IFN-g did not affect the proliferation following days, a further deletion of all activated ki-67 positive rate of the cells in the presence or absence of GM-CSF (data not cells was observed, resulting in a profound enrichment for cells shown). However, IFN-a treatment induced a 2.5-fold decrease in the resting G0 phase of the cell cycle. This relative enrichment in both baseline proliferation and proliferation in the presence of for cells in G0 phase could in part be explained by the GM-CSF. Detailed cell cycle analysis of cells cultured in the enrichment for normal T and B cells. However, even the presence or absence of GM-CSF and interferons was performed. leukemic population in G0 phase of the cell cycle appeared to As shown in Figure 2, in control AML-193 cells cultured in the be protected from the cytotoxic effects of the treatment. absence of GM-CSF as well as interferons, most cells were in To investigate in more detail the cell cycle specificity of resting G0 phase (66%), 19% of cells were in G1 phase, and daunorubicin, we used the human myeloid leukemic cell line 15% in S or G2/M phase (n ¼ 20). Long-term interferon treatment AML-193 as a model, and studied the cell cycle distribution of of control cells induced a significant increase in activated G1 viable cells within this cell line prior to and after 40 h of cells to 31% in IFN-g- and 27% in IFN-a-treated cells chemotherapy exposure. As shown in Figure 1a, AML-193 cells (Po0.0001). Small but significant higher percentages of cells cultured in medium contained cells in resting G0 phase (lower in S or G2/M phase were observed in IFN-g-treated cells (21%) left quadrant), cells in activated G1 phase (lower right quadrant), and IFN-a-treated cells (18%), as compared to control cells and cycling cells in S or G2/M phase (upper right quadrant). As (Po0.01). These increases coincided with a decrease in the À6 expected after 40 h of incubation, 10 M Ara-C-induced number of cells in resting G0 phase to 48% in IFN-g-treated complete deletion of the cycling S or G2/M compartment was cells, and to 55% in IFN-a-treated cells. Induction of prolifera- observed, whereas noncycling cells in resting G0 phase and tion with GM-CSF resulted in increased percentages of cells in activated G1 phase of the cell cycle were protected. In contrast, activated G1 phase (45%) and in the cycling S/G2/M phase À6 after 40 h of exposure to 10 M daunorubicin, both cells from (34%). In the presence of GM-CSF, IFN treatment did not exert the cycling S/G2/M compartment and the activated G1 an additive effect on the cell cycle distribution. These results compartment were deleted. Only cells in the resting G0 phase illustrate that long-term interferon treatment of AML-193 cells Correspondence 2050 Table 1 Cell cycle distribution after in vivo chemotherapy Pt Gender/age FAB class Time point WBC (106/ml) % blasts % lymphocytes Cell cycle distribution

G0 G1 S/G2/M

1 m/29 M4 Day 0 64 30 6 30 85 9 Day 1 75 23 2 6.4 82 10 Day 2 80 18 2 1.7 58 31 Day 3 92 7 1 0.7 40 59

2 f/45 M5 Day 0 80 13 7 38.3 90 4 Day 1 54 44 2 7.9 82 12 Day 2 ND ND ND ND ND ND Day 3 94 4 2 0.5 57 21

3 m/35 M1 Day 0 52 45 3 86.2 95 1 Day 1 85 14 1 76.6 91 4 Day 2 ND ND ND ND ND ND Day 3 72 28 0 5.8 74 9 WBC ¼ white blood cell count; ND ¼ not done. The cell cycle distribution within the total nuclear cell population is listed at diagnosis and at 1, 2, and 3 days after chemotherapy administration. Owing to logistic problems, day 2 is missing in patients 2 and 3.

Figure 2 Cell cycle distribution after interferon treatment in the absence and presence of GM-CSF. Cell cycle distribution was analyzed by combined ki-67/propidium iodide DNA staining. The # percentages of cells in G0 phase (’), G1 phase ( ), S phase ( ), and Figure 1 Cell cycle specificity of daunorubicin and Ara-C. (a) Cell G /M phase (&) were determined. cycle analysis of viable AML-193 cells by PI/ki-67 analysis after 40 h of 2 À6 incubation in medium alone, or in medium containing 10 M -6 daunorubicin, or 10 M Ara-C. (b) Cell cycle analysis of viable cells of five ALL/CML cell lines after 40 h of incubation in medium alone, or À6 À6 ¼ g a in medium containing 10 M Ara-C or 10 M daunorubicin. The after 40 h (n 30). Activation of control cells by IFN- or IFN- percentages of cells in G0 phase (’), G1 phase ( ), S phase (&), and without induction of proliferation induced a two-fold increase in " G2/M phase ( ) were determined. The mean percentages of cells in median sensitivity to daunorubicin after 24 h, resulting in 48% every cell cycle fraction7s.d. are listed. lysis (n ¼ 20; Po0.0001) and 36% lysis (n ¼ 15; P ¼ 0.02) in IFN-g- and IFN-a-treated cells, respectively. The same increase in sensitivity to daunorubicin was found after induction of proliferation with GM-CSF (n ¼ 40; Po0.0001). IFN-g treatment resulted in activation of resting cells without induction of of GM-CSF-treated cells resulted in a minor additional increase proliferation, facilitating separate analysis of the effects of cell in the sensitivity to daunorubicin. Despite inhibition of cycle manipulation and proliferative status manipulation on proliferation by IFN-a treatment, no decrease in the sensitivity chemotherapy sensitivity. Flow cytometric analysis of control to daunorubicin of GM-CSF-treated cells was found (n ¼ 20; cells and IFN-g- and IFN-a- treated cells in the absence and P ¼ 0.6). After 40 h of exposure to daunorubicin, all GM-CSF- or presence of GM-CSF revealed no differences in the expression IFN-treated activated cells were equally well lysed. Only resting levels of the apoptosis-related proteins Fas, Bcl-2, Bax, p53, control cells showed a lower sensitivity to daunorubicin TNF-R1, and P-glycoprotein.4 Figure 3 illustrates the sensitivity (Po0.001). These results illustrated that the proliferation rate of AML-193 cells cultured in the six different culture conditions itself, as determined by complete progression through the cell to daunorubicin-induced apoptosis. The results of all individual cycle, did not coincide with the sensitivity to daunorubicin- experiments are plotted as percentage-specific lysis after 24 induced apoptosis, since the slowly proliferating IFN-a cell line (Figure 3a) and 40 h (Figure 3b). Medians are indicated with was equally sensitive as the GM-CSF- or IFN-g-treated cell lines. À6 bold lines. Incubation of control cells with 10 M daunorubicin In contrast, the sensitivity to Ara-C was directly related to the resulted in a median lysis of 23% after 24 h (n ¼ 40) and 49% percentages of cycling cells (data not shown).

Leukemia Correspondence 2051 Table 2 Cell cycle speciﬁcity of chemotherapy-induced apoptosis in AML-193 cells in the absence of the proliferation-inducing growth factor GM-CSF

t ¼ 0h t ¼ 48 h Medium Medium Ara-C Dauno

Control G0 6873587756768275 G1 167220783577677 S8711172572572 G2/M 8731072470773

IFN-g G0 4975447541757875 G1 287530754775876 S11721272772772 G2/M 12731372571874

IFN-a G0 5576507756757472 G1 2571317234791278 S1072971574572 G2/M 10751173570874

Percentages of cells (7s.d.) in G0,G1, S, and G2/M phases of the cell cycle after incubation for 48 h in medium alone, or after 48 h of 6 6 exposure to 10À M Ara-C or 10À M daunorubicin (n ¼ 10).

inducing leukemic cell proliferation. In the clinical studies performed until now, interferons were used as maintenance therapy after chemotherapy intervention.5 Based on our data, it may be rational to use interferon as treatment before and during chemotherapy. However, in combination with conventional untargeted chemotherapy, the interferon-induced recruitment of normal, nonleukemic cells from the resting G0 phase into active state might result in severe toxicity within normal tissues. Therefore, exploration of new agents with relative specificity for leukemic cells, but with similar ability to recruit these cells from the resting G0 phase, will be needed. Alternatively, interferon combined with leukemia-targeted chemotherapeutic agents Figure 3 The effect of cell cycle manipulation by GM-CSF and interferon treatment on sensitivity to daunorubicin. To determine the might be a feasible approach to increase the effectiveness of À6 chemotherapy sensitivity, the cells were exposed to 10 M daunor- the chemotherapy with relative specificity for the leukemic ubicin for 24 (a) and 40 h (b). The percentage-specific lysis was cells. measured in 51Cr release assays. Each experiment is plotted as a different dot. Medians are visualized as bars. I Jedema1 1Leiden University Medical Center, RMY Barge1 Department of Hematology, BA Nijmeijer1 Leiden, R Willemze1 The Netherlands To illustrate that the cell cycle specificity of daunorubicin- JHF Falkenburg1 induced cell death was independent of the cell cycle distribution of the cells at the onset of exposure, we analyzed AML-193 References cells cultured under three different culture conditions in time. Table 2 demonstrates the constant cell cycle distribution in AML-193 cells cultured in the absence of GM-CSF in time. Ara- 1 Lowenberg B, Downing JR, Burnett A. Acute myeloid leukemia. N Engl J Med 1999; 341: 1051–1062. C induced a diminishment only of cells in the cycling S/G2/M 2 Cannistra SA, Groshek P, Griffin JD. Granulocyte–macrophage compartment. After 48 h of daunorubicin-induced cell death, colony-stimulating factor enhances the cytotoxic effects of cytosine activated cells in G1, S, and G2/M phases were deleted, resulting arabinoside in acute myeloblastic leukemia and in the myeloid in relative enrichment for cells in the resting G0 phase. Similar blast crisis phase of chronic myeloid leukemia. Leukemia 1989; 3: results were obtained in the proliferating AML-193 cells cultured 328–334. in the presence of GM-CSF. 3 van Oijen MG, Medema RH, Slootweg PJ, Rijksen G. Positivity of the proliferation marker Ki-67 in noncycling cells. Am J Clin Pathol In conclusion, we demonstrated that exposure of leukemic 1998; 110: 24–31. cells to the anthracyclin daunorubicin specifically preserves 4 Jedema I, Barge RM, Willemze R, Falkenburg JH. High suscept- leukemic (precursor) cells in the resting G0 phase of the cell ibility of human leukemic cells to Fas-induced apoptosis is cycle, illustrating the relative specificity of daunorubicin for restricted to G(1) phase of the cell cycle and can be increased leukemic cells in the activated phases of the cell cycle. This is in by interferon treatment. Leukemia 2003; 17: 576–584. contrast to the clearly proliferation-dependent sensitivity for 5 Goldstone AH, Burnett AK, Wheatley K, Smith AG, Hutchinson RM, Clark RE. Attempts to improve treatment outcomes in acute Ara-C. These results may favor a therapeutic strategy using cell myeloid leukemia (AML) in older patients: the results of the United cycle modulators like interferons in an attempt to decrease the Kingdom Medical Research Council AML11 trial. Blood 2001; 98: percentage leukemic (precursor) cells in dormancy, without 1302–1311.

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