Introduction

Stem cell factor (SCF, Steel-factor or kit-ligand) is a potent growth factor produced by haematopoietic and bone marrow-derived stromal cells, which is essential for normal haematopoiesis, gametogenesis, melanogenesis and mast cell differentiation (Refs 1,2,3 and reviewed in Refs 4 and 5). Binding of SCF to its receptor c-kit, leads to oligomerisation of c-kit and activation of its tyrosine kinase activity in haematopoietic progenitor cells, mast cells, interstitial cells of the Cajal and germ cells.^6,7,8,9 Mutations rendering c-kit signalling deficient in mice, through either inactivation of c-kit (White spotting/Ws^v)^10,11 or mutation of SCF (Steel/Sl^d)^12,13 lead to identical phenotypes of hypopigmentation, sterility and impaired haematopoiesis demonstrating the importance of the c-kit signalling pathway.¹⁴ This phenotype can be reverted in Sl mutant mice by the administration of recombinant SCF¹⁵ and in Ws mutant mice by functional c-kit expression.¹⁶ Interestingly, due to the lack of anti-apoptotic stimuli through the absence of functional SCF/c-kit signalling, Ws/Ws^v and Sl/Sl^d mice also demonstrate an enhanced susceptibility to treatment with ionising radiation.¹⁷ The c-kit signalling pathway has also been implicated in oncogenic transformation of several types of cancers, including germ and mast cell tumours, melanoma, acute lymphocytic leukaemia (ALL), gastrointestinal stromal tumours (GIST) and small cell lung cancer.^{8,18,19,20,21,22,23,24,25,26}

The 2-phenylamino-pyrimidine derivative imatinib (STI571, Glivec, formerly CGP57148B) is a selective inhibitor of the c-Abl tyrosine kinase and is currently used for treatment of patients with BCR/ABL positive leukaemias.²⁷ However, imatinib is also known to inhibit platelet-derived growth factor receptor (PDGFR) and c-kit, both of which belong to the receptor tyrosine kinase subclass III family.^28,29,30 Imatinib has previously been demonstrated to inhibit growth in the SCF-dependent haematopoietic cell line M-07e.³⁰ The relevance of imatinib-mediated inhibition of c-kit to cancer therapy has been demonstrated in the treatment of GIST.³¹

Imatinib represents an important new treatment strategy for chronic myeloid leukaemia (CML) and patients with c-kit-dependent malignancies. However, relapses are common, especially in patients with acute leukaemia; therefore, the combination of imatinib with chemotherapy is currently under investigation.^32,33

In this context we have investigated if administration of imatinib increases the haematopoietic toxicity of concomitant cytotoxic treatment, in vivo and in vitro, using the commonly used anthracycline idarubicin, a daunorubicin derivative that is frequently used in the treatment of several haematological malignancies including CML³⁴ and which, like other anthracyclines, exerts its cytotoxic activity by intercalating DNA and inhibiting topoisomerase.³⁵

Materials and methods

Drugs

Imatinib, provided by Novartis Pharma AG, Basel, Switzerland, was dissolved in distilled water and kept at 4°C. Idarubicin (Zavedos, Pharmacia-Upjohn, Milan, Italy) was dissolved in distilled water; aliquots were kept at -20°C and diluted before use.

Animals and treatments

Female CD-1 nu/nu mice (7–9 weeks old) were supplied by Charles River (Calco, Como, Italy) and kept under standard laboratory conditions according to the guidelines of our Institute. Animal studies were approved by the Ethics Committee for Animal Experimentation of the Istituto Nazionale Tumori, Milan, Italy. Imatinib was delivered per os (at 160 mg/kg of body weight) every 8 h for 11 days through a syringe connected to a soft plastic tube introduced into the mouse oesophagus (gavage) as previously reported.³⁶ Idarubicin was administered at 2 mg/kg i.v. once in conjunction, 4 or 8 days prior to imatinib treatment as indicated. When the two drugs were combined, idarubicin was administered 2 h after the first treatment with imatinib. Control mice received 0.9% (w/v) NaCl according to the same schedule as for imatinib treatment. Mice were observed daily for toxicity and their body weight was recorded.

In some experiments, tumour-bearing mice were used. In this case, the animals were implanted with the human BCR/ABL-positive cell line KU812 14 days before the experiment and used when the tumour reached at least 1 g, as described elsewhere.³⁷

White blood and differential cell counts

At regular intervals, tail blood was obtained for white blood cell (WBC) counts. Cells were stained with crystal violet (0.02% (w/v) in 3% acetic acid) and counted using a Burker cell counting chamber (Burker, Germany). Smears of peripheral blood were made under ambient air. Differential counts were obtained by microscopic analysis of May–Grünwald-Gemsa stained cells on glass cover slides.

Tissue analysis

At the end of the treatment period mice were killed and spleen, lung, liver, intestine and femur were excised for histological examination. Tissues were immersion-fixed in 4% paraformaldehyde prior to embedding and cutting. After haematoxylin and eosin staining, a pathologist examined the sections in a blinded manner.

Culture and treatment of murine bone marrow cells

Murine bone marrow cells were obtained by PBS-flushing of femours from female nude mice under sterile conditions. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (complemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine, 50 U/ml interleukin (IL)-6 (PeproTech EC Ltd, London, UK), 50 ng/ml murine recombinant SCF (a kind gift of Dr Carlo-Stella, National Cancer Institute, Milan, Italy) and a 1:1000 dilution of murine recombinant IL-3 from cell culture supernatant derived from CHO cells stably transfected with a murine IL-3 expression plasmid (a kind gift of Dr S Morris, St Jude's Research Hospital, Memphis, TN, USA). Cells were cultured at 37°C and in the presence of 5% CO₂. Where indicated, cells were treated with 0.01 mg/ml idarubicin for 1 h at 37°C, washed twice with DMEM and cultured in the absence or presence of imatinib. Alternatively, cells were treated with ionising radiation at 0.5 and 1.0 Gy using a cesium Cs137 source prior to culture in supplemented DMEM.

Proliferation and trypan blue exclusion assay

Murine bone marrow cells were treated with 0.01 mg/ml idarubicin for 1 h or left untreated, washed and seeded in round bottom 96 well plates at 10⁵ cells/well in a volume of 100 l in supplemented medium. Subsequently, imatinib and SCF were added either singularly or in combination to a final concentration of 3 M and 50 ng/ml, respectively, and the volume was adjusted to 200 l per well. Eight hours prior to harvesting on to glass fiber filters, 1 Ci of ³[H]-thymidine was added to each well. Proliferation was assessed by measuring the incorporation of ³[H]-thymidine using a filter scintillation counter (1430 MicroBeta, Wallac, Turku, Finland). For trypan blue exclusion assays, cells were treated similarly with or without 0.01 mg/ml idarubicin for 1 h, washed and cultured with or without 3 M Imatinib or 50 ng/ml SCF. The number of viable and dead cells was determined by staining dead cells with trypan blue every 2 days until day 12 after exposure to idarubicin.

Statistical analysis

For statistical analysis of body weight and in vitro data, the Minitab software (Minitab Inc, USA) was employed using the Mann–Whitney test. Mortality data were analysed using the Graph Pad Prism software (Graph Pad Software, Inc) employing the logrank test to calculate P values. The statistical analysis of neutrophil counts was performed using the two-tailed t-test with Welch correction (Graph Pad Software, Inc). A value of P < 0.05 was considered statistically significant.

Results

Simultaneous co-treatment with imatinib and idarubicin results in mortality and body weight loss in nude mice

To assess the toxicity of combination treatment with imatinib, idarubicin was injected either simultaneously, or 4–8 days prior to imatinib treatment.

Concomitant administration of imatinib and a single, sublethal dose of idarubicin resulted in the death of 4/16 animals, while single treatment with either imatinib (n = 16) or idarubicin (n = 16) alone did not cause any mortality (P = 0.042) (Figure 1a). Mortality was associated with body weight loss and occurred between days 6 and 8 (Figure 1b). Idarubicin or imatinib-treatment alone had very little effect on body weight. The decrease in body weight of idarubicin or imatinib vs idarubicin plus imatinib treated mice was statistically significant at day 7 of imatinib treatment (P = 0.02). When idarubicin was injected 4 or 8 days prior to imatinib, no mortality or body weight loss could be observed (Figure 1a and b).

Figure 1.

Mice treated simultaneously with imatinib and idarubicin show increased mortality and body weight loss. (a) Survival of mice treated with Imatinib (n = 16), idarubicin (n = 16) or a combination of both in which idarubicin was administered on day 0 (n = 16), 4 days (n = 6) or 8 days (n = 6) prior to imatinib. Data are expressed as percentage of viable mice at each treatment day; *P  0.05 (logrank test). (b) Body weight of mice from different treatment groups. Arrows indicate the time of idarubicin injection in each treatment group. Data presented as average body weight in gram s.e.; IDA, idarubicin; **P  0.02 (Mann–Whitney test).

Full figure and legend (27K)

The simultaneous administration of imatinib and idarubicin to tumour-bearing mice apparently caused an even higher mortality (7/8 mice), although the difference in mortality between tumour-bearing and non-tumour-bearing mice did not reach statistical significance. As in non-tumour bearing animals, mortality was associated with body weight loss and occurred between days 6 and 8 of treatment.

In a parallel experiment, sublethal whole body irradiation of mice (500 rad) in conjunction with imatinib administration led to 25% mortality (2/8), that was however not statistically significant (data not shown).

Treatment with imatinib reduces haematopoietic recovery after exposure to idarubicin in vivo

Treatment of animals with a single dose of idarubicin led to a reduction of neutrophilic granulocyte counts at day 4 after idarubicin injection followed by a recovery at days 7–10 (Figure 2a). Mice treated simultaneously with imatinib displayed a higher reduction in neutrophil counts at day 4 and a lower recovery at days 7–10 (P = 0.004, 0.04 and 0.034 between idarubicin and idarubicin/imatinib-treated groups at days 4, 7 and 10, respectively). In mice treated with imatinib alone, no significant changes in neutrophil counts could be observed (data not shown). When idarubicin was administered 4 or 8 days prior to treatment with imatinib, neutrophil counts where similar to animals treated with idarubicin alone.

Figure 2.

Treatment with Imatinib reduces haematopoietic recovery after exposure to idarubicin in vivo. (a) Neutrophil counts derived from animals of different treatment groups at different time points following idarubicin injection. Data presented as percentage of initial neutrophilic granulocyte counts prior to idarubicin treatment s.e. *P  0.05, **P  0.02 (two tail t-test). (b) Weight of spleens derived from different treatment groups measured on day 10 after imatinib treatment or injection with idarubicin (control group). Data are presented as average spleen weight in milligram s.e.; **P  0.02 (Mann–Whitney test).

Full figure and legend (19K)

After 11 days of treatment with imatinib, animals were killed and the spleens were analysed (Figure 2b). Animals treated with idarubicin alone had enlarged spleens. This effect was inhibited by the simultaneous treatment with imatinib (P = 0.019). Imatinib alone caused a minor reduction in spleen size that was not significant when compared to non-treated controls.

Co-treatment of mice with imatinib and idarubicin induces histological changes in bone marrow and spleen

Histological changes following treatment with idarubicin or idarubicin plus imatinib were observed in spleen (Figure 3, panel 1 and 2) and bone marrow (Figure 3, panel 3).

Figure 3.

Co-treatment of mice with imatinib and idarubicin induces pathology in spleen and bone marrow. (Panels 1 and 2) Haematoxilin/eosin-stained tissue sections of spleens derived from different treatment groups: (a and b) controls; (d and e) idarubicin-treated; (g and h) idarubicin and imatinib. (Panel 3) Tissue sections of bone marrow derived from different treatment groups: (c) controls; (f) idarubicin; (i) idarubicin and imatinib.

Full figure and legend (77K)

Treatment of animals with a single dose of idarubicin led to a substantial increase in myeloid cells present in the spleen coupled with lymphoid hyperplasia (Figure 3d and e) when compared to controls (Figure 3a and b). Additionally, immature granulocyte precursors were notably increased in the subcapsular splenic red pulp, and along the splenic trabeculae (not shown). Simultaneous administration of imatinib markedly suppressed these effects (Figure 3g and h). Spleens of double treated mice also showed lymphocyte depletion in the periarteriolar lymphoid sheaths, with shrinkage of both the medulla and marginal zones of secondary follicles. Bone marrow (Figure 3, panel 3) from animals treated with imatinib and idarubicin showed decreased numbers of megakaryocytes and myeloid precursor cells (Figure 3i), when compared with controls (Figure 3c) and with mice treated with idarubicin alone (Figure 3f). Imatinib administration alone did not induce histological changes in spleen or bone marrow when compared to control animals. We were unable to detect pathological changes in tissue samples prepared from intestine, liver or lung (data not shown).

SCF-induced bone marrow cell proliferation is inhibited by imatinib

We investigated if the increase in idarubicin-induced haematopoietic toxicity mediated by administration of imatinib in vivo could also be observed in murine bone marrow cells in vitro. We first assessed if the addition of imatinib to the culture medium could inhibit SCF-induced proliferation of murine bone marrow cells. As shown in Figure 4a, bone marrow cells strongly proliferated in the presence of 50 ng/ml SCF. The presence of 3 M imatinib in the culture medium led to a statistically significant reduction of bone marrow cell proliferation, as measured by ³[H]-thymidine incorporation. In the absence of SCF, baseline bone marrow cell proliferation was reduced by approximately 60% and was not affected by the addition of imatinib, indicating that the effect of imatinib on bone marrow cell proliferation was due to the inhibition of SCF signalling.

Figure 4.

Stem cell factor induced bone marrow cell proliferation is inhibited by imatinib. (a) Proliferation of murine bone marrow cells in response to SCF and/or imatinib after 48 h of culture. Cells were maintained in supplemented DMEM in the presence or absence of 50 ng/ml SCF and with or without 3 M Imatinib. (b) Proliferation after pretreatment with idarubicin (0.01 mg/ml for 1 h) or ionising radiation (0.5 or 1.0 Gy). Data represent percentage of mean ³[H]-thymidine incorporation (counts per minute) compared to control cells (left column of (a)) s.e. IDA, idarubicin. **P  0.02 (Mann–Whitney test).

Full figure and legend (22K)

The pretreatment of bone marrow cells with idarubicin or ionising radiations (Figure 4b) substantially reduced cell proliferation both in the absence and presence of SCF. However, cells cultured in the presence of SCF still displayed some proliferative response that was largely inhibited by the addition of imatinib. In the absence of SCF, treatment with idarubicin resulted in complete abrogation of proliferation. Treatment with ionising radiations (0.5 or 1.0 Gy) led to a 80% reduction of proliferation in the absence of SCF or when both SCF and imatinib were added into the culture medium. In the presence of SCF alone, treatment with ionising radiation reduced bone marrow cell proliferation by approximately 40–50%. Our results indicate that imatinib inhibits SCF-induced proliferation of bone marrow cells. Cytotoxic treatment with ionising radiations or idarubicin-reduced proliferation, which could be partially overcome by the addition of SCF, indicating that SCF may be important for bone marrow recovery after genotoxic treatment.

Treatment of bone marrow cells with imatinib reduces cell recovery after treatment with idarubicin

Survival of murine bone marrow cells cultured in the presence of SCF was assessed by trypan blue exclusion (Figure 5). To mimic the in vivo experimental procedure, bone marrow cells were cultured in the presence or absence of 3 M imatinib for a period of 12 days (Figure 5a). After an initial phase of growth, cell numbers reached a maximum at day 9 followed by a decline in cell number. The presence of imatinib significantly reduced the number of viable cells by approximately 50% at the time when the number of non-treated cells reached its maximum. However, this reduction was not due to increased cell death, but rather to inhibition of cell growth, as imatinib did not increase the number of dead cells in the culture (Figure 5b).

Figure 5.

Treatment of bone marrow cells with imatinib inhibits SCF-induced growth and reduces long-term recovery after treatment with idarubicin. Murine bone marrow cells were cultured in supplemented DMEM for 12 days in the presence of SCF. (a) Number of viable cells in the presence or absence of 3 M imatinib. Data present the number of viable cells as mean of three experiments s.e.; (b) Percentage of dead BM cells in the presence or absence of 3 M Imatinib. Data present the percentage of dead cells as mean of three experiments s.e. (c) Number of viable cells treated with 0.01 mg/ml idarubicin for 1 h and subsequently cultured in the presence or absence of 3 M Imatinib. Data presented as mean of three experiments s.e.

Full figure and legend (22K)

Similar to the in vivo experiment, we also assessed the recovery of bone marrow cell growth following a brief cytotoxic treatment with idarubicin and subsequent culture in medium supplemented with SCF in the presence or absence of imatinib (Figure 5c). Treatment of cells with 0.01 mg/ml idarubicin for 1 h resulted in an initial decline in cell numbers. In the absence of imatinib, a recovery in cell number was observed from day 7, whereas in the presence of imatinib, there was a marked delay in cell recovery. The number of viable cells did not increase until day 9 after idarubicin treatment. We conclude that imatinib inhibits SCF-induced cell growth and recovery after cytotoxic treatment without causing cell death.

Discussion

In this study, we investigated the effect of imatinib in the context of idarubicin-induced haematosuppression. Our results indicate that imatinib can prolong and aggravate idarubicin-induced haematotoxicity when administered simultaneously. The inhibitory effect of imatinib on haematopoietic recovery after idarubicin exposure was reflected by reduced neutrophil counts and spleen size of double treated animals. The in vivo effect of imatinib on idarubicin-induced cytotoxicity may derive from imatinib-mediated inhibition of c-kit signalling required for normal haematopoiesis. The ability of imatinib to induce death in BCR/ABL-expressing cells is well documented.^36,38 Also, results from several studies have shown that the pro-apoptotic effect of imatinib on BCR/ABL expressing cells is enhanced by concomitant treatment with common cytotoxic drugs in vitro.^{39,40,41,42,43,44,45} Up to now, however, limited information is available on the toxicity of imatinib in combination with cytotoxic drugs in vivo and on BCR/ABL-negative cells. In a study undertaken by Uemura and Griffin,⁴⁶ imatinib had no effect on irradiated normal bone marrow cells cultured in the absence of SCF. Our data indicate that this is also the case for idarubicin-induced cytotoxicity. However, imatinib specifically blocked SCF-induced cell proliferation. The effect of imatinib appeared to be specific for SCF-mediated proliferation since in the absence of SCF, imatinib did not cause reduction of proliferation. Furthermore, imatinib had no effect on the number of dead cells in long-term bone marrow cultures indicating that SCF provides a pro-proliferative signal that is not strictly required for the survival of murine bone marrow cells in vitro. It is also important to note that the enhanced toxicity of idarubicin in the presence of imatinib is dependent on the treatment schedule. In fact, when idarubicin was injected 4 or 8 days prior to treatment with imatinib, no toxicity was observed, indicating that haematopoietic recovery at this point was no longer influenced by imatinib. In previous studies, imatinib (100, 200 or 300 mg/kg p.o. once per day or 100 mg/kg p.o. twice per day) did not appear to dramatically worsen the tolerability of normal and immunosuppressed nude BALB/c mice towards conventional anticancer agents (cisplatinum, 5-fluorouracil, doxorubicin or Taxol R) administered at optimal doses near the MTD (Buchdunger and O'Reilly, Novartis Pharma AG, unpublished). In the present study, we have administered imatinib according to a dose and schedule previously optimized for the treatment of CML in vivo.³⁶ Continuous inhibition of BCR/ABL is an important prerequisite for the treatment of CML in experimental models. While treatment schedules administering imatinib once or twice per day are known to produce some inhibition of tumor growth, they are not sufficient to completely eradicate the tumor.³⁶ The comparatively high dose of imatinib used in the present study did not cause mortality and did not decrease WBC counts when administered alone. We used nude mice for our study since these represent a commonly used experimental model for CML. However, the observed effect may be particularly pronounced in nude mice and therefore be less prominent in wild-type animals.

A concentration of 3 M of imatinib was used in in vitro experiments. This level did not decrease proliferation rates and did not induce apoptosis in all BCR/ABL negative cells tested and it caused an almost complete inhibition of BCR/ABL autophosphorylation.⁴⁷

Treatment of animals or normal bone marrow cells with sublethal doses of ionising radiations and subsequent administration of imatinib led to similar results as observed with idarubicin. These data indicate that the inhibition of SCF-induced bone marrow cell recovery may be inhibited by imatinib following cytotoxic treatment in general.

Several studies are currently underway to evaluate the use of imatinib in combination with commonly used antileukemic therapies.^32,33 Since imatinib is generally well tolerated and very few undesired effects have been described, combination with established therapeutic strategies may prevent the development of resistance to imatinib and increase its antileukemic potential.⁴⁸

Our results do not oppose the use of imatinib in combination with generally cytotoxic treatments in principle, but elucidate the in vivo effects of imatinib on endogenous SCF signalling in an experimental model and could suggest separating the administration of imatinib and cytotoxic drugs.

The inhibitory effect of imatinib on PDGFR signalling could also account for additional haematologic stress in double-treated animals. Targeted disruption of PDGF or its receptor results in embryonic lethality^49,50 and PDGFR disruption has been associated with metabolic stress resulting from defects in placenta, heart and blood vessels in chimaeric animals.⁵¹

In summary, our results provide evidence that systemic administration of imatinib increases the sensitivity to concomitant cytotoxic treatment in a murine model. Further studies will be required to establish the effects of combining imatinib with traditional chemotherapy for the treatment of CML and c-kit-dependent malignancies.

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Acknowledgements

The authors thank Dr C Carlo-Stella, National Cancer Institute, for murine recombinant SCF and Dr P Dalerba, National Cancer Institute, for murine, recombinant IL-6. We are grateful to Dr E Buchdunger, Novartis Switzerland, for critical discussion and Dr R Gunby, National Cancer Institute, for kindly reviewing the manuscript. This work has been supported by the Italian Association for Cancer Research (AIRC), the Italian Ministry of Health (Ricerca Finalizzata) and MIUR-COFIN2001. HR is the recipient of a Marie-Curie individual fellowship (QLK3-CT-1999-51065) under the EC-5th framework program.