Tohoku
J. Exp.
Med., 1992,
Control
of Growth
Philadelphia Cells
168, 387-391
by
and
Differentiation
Chromosome-Positive Tyrosine
Kinase
of Leukemia
Inhibitors
YOSHIOHONMA,TAKASHIKASUKABEand MOToo HOZUMI Department of Chemotherapy, Saitama Research Institute, Saitama 362
Cancer Center
HONMA,Y., KASUKABE,T, and HOZUMI, M. Control of Growth and Differentiation of Philadelphia Chromosome-PositiveLeukemia Cells by Tyrosine Kinase Inhibitors. Tohoku J. Exp. Med., 1992, 168 (2), 387-391 Herbimycin A, a selective inhibitor of tyrosine kinase activity, induced differentiation of leukemia cells isolated from Philadelphia chromosome-positive chronic myelogenous leukemia patients. However, it did not induce differentiation of leukemia cells from acute myelogenous leukemia patients, although these cells could be induced to differentiate by treatment with appropriate compounds. A selective inhibitor of tyrosine kinase might be useful in chemotherapy of Philadelphia chromosome-positive leukemia. Philadelphia chromosome-positive leukemia ; acute myelogenous leukemia ; chronic myelogenous leukemia ; herbimycin A ; tyrosine kinase inhibitor
The neoplastic transformation of many human tumors appears to be associated with the expression of activated oncogenes. Thus inhibition of activities of the oncogene products may be a new strategy for cancer chemotherapy. Translocation of the c-abl gene from chromosome 9 to 22, resulting in the Philadelphia chromosome, occurs in more than 90% of chronic myelogenous leukemia (CML) patients. The markedly increased tyrosine kinase activity of the gene product strongly suggests that the chimeric gene is directly involved in the pathogenesis of the leukemia. If so, a selective inhibitor of tyrosine kinase activity might be useful in chemotherapy of this leukemia. MATERIALS ANDMETHODS Human leukemia cells were obtained from patients during routine diagnostic procedures, and cultured in RP-MI-1640 mediumsupplementedwith 10% heat-inactivatedfetal bovine serum at 37°Cin a humidifiedatmosphereof 5% 002 in air. The cell number was counted after 6 days treatment with herbimycin A or the same period of incubation without herbimycin A. Differentiation-associatedproperties were determinedby the methodsdescribedpreviously(Honmaet al. 1983).
Address
for reprints
: 818 Komuro,
ma-machi, 387
Saitama
362, Japan.
388
Y. Honma
et al.
RESULTSANDDISCUSSION Cells from patients with AML or CML in blast crisis were cultured with or without herbimycin A in the presence of some hematopoietic growth factors. Several leukemia cells did not grow well, so we could not evaluate the growthinhibitory effect of herbimycin A on them. However, some hematopoietic growth factors (GM-CSF, IL-2, IL-3, and/or IL-4) stimulated the growth of some, but not all, leukemia cells in primary cultures. We evaluated the growth-inhibitory effect of herbimycin A in primary cultures of leukemia cells, when the cell numbers increased more than 2-fold in 6 days (Table 1). MO cells were taken from a man with blast crisis of CML. Morphologically, most of the freshly isolated MO cells were blastic and promyelocytic. Herbimycin A induced morphological differentiation of MO cells into mature granulocytes. Under the culture conditions used, the number of viable MO cells was not significantly reduced by herbimycin A, indicating that this drug does not kill immature granulocytes selectively (Fig. 1). MO cells did not grow without added growth factors, but in the presence of GM-CSF or IL-4 their cell number increased 1.5- to 1.8-fold during culture for 6 days. GM-CSF and IL-4 did not enhance granulocytic differentiation of MO cells induced by herbimycin A, although the cells showed slight differentiation into myelocytes, but not mature granulocytes in the presence of the cytokines only. During primary culture without any added compounds, the cell number of HN cells was increased only slightly, but IL-4 or GM-CSF stimulated cell proliferation, increasing the cell number 2.5-2.7-fold during culture for 6 days. Culture of HN cells with various concentrations of herbimycin A for 6 days caused dose-dependent stimulation of lysozyme synthesis (Fig. 2). Herbimycin A inTABLE1. Effect of herbimycin A on growth and differentiation of leukemia primary culture
cells in
Control
of Philadelphia
Chromosome-Positive
Leukemia
Cells
389
Fig. 1. Effect of herbimycin A on morphological changes of MO cells in the absense and presence of GM-CSF at 50 U/ml. LI,blasts and promyelocytes ; , myelocytes ; •, metamyelocytes and mature granulocytes.
creased the naphthyl AS-D chloroacetate esterase and lysozyme activities in KA cells, and caused slight morphological differentiation of the cells into mature granulocytes (Fig. 3). The cell number of KA cells increased slightly in the control culture, but in cultures with herbimycin A at less than 15 ng/ml the viable cell number of KA cells scarcely changed. TS cells were taken from a man with CML in lymphoid crisis. During culture for 7 days, the cell number increased slightly, but the cell growth was not stimulated by IL-2, IL-3, or IL-4. Herbimycin A at over 30 ng/ml was cytotoxic to TS cells, and no significant myelomonocytic differentiation was observed in drug-treated TS cells. The effects of herbimycin A on leukemia cells from patients with AML in
Fig. 2.
Effect
of herbimycin
A on differentiation
of HN cells.
Y. Honma
390
Fig. 3.
Effect
of herbim ycin
et al.
A on differentiation
of KA cells.
primary culture were also examined. The sensitivities of the cells to growth inhibition by the antibiotic varied. Herbimycin A, even at high concentration, did not cause any differentiation of any of the AML cells tested (5 cases), although all the cells showed monocytic differentiation by 12-0-tetradecanoylphorbol-13acetate. Herbimycin A caused reversion of transformed Rous sarcoma virus-infected rat kidney cells to normal morphology. This antibiotic was also found to be effective against various cells transformed by the other tyrosine kinase oncogenes including v-abl, but did not reverse the morphology of the cells transformed by the oncogenes raf, ras, and myc (Uehara et al. 1989). Our previous (Honma et al. 1989,1992) and present results show that the antiproliferative and differentiationinducing effects of herbimycin A differed in different leukemia cells : Phl-positive (or bcr/abl gene-containing) leukemia cells appeared to be more sensitive to herbimycin A than Phl-negative leukemia cells. Thus herbimycin A and its derivatives might be useful as cancer chemotherapeutic agents against some types of leukemia with altered tyrosine kinase activities. Acknowledgments This work Education, Comprehensive
was partly
Science
supported
and Culture
10-Year
Strateegy
by
a grant
and a grant for Cancer
for cancer
from the Ministry Control,
research
from
of Health
the
Ministry
and Welfare
of
for a
Japan.
References 1) Honma, Y., Fuj ita, Y., Kasukabe, T., Hozumi, M., Sampi, K. & Sakurai, M. (1983) Induction of differentiation of human acute non-lymphocytic leukemia cells in primary culture by inducers of differentiation of human myeloid leukemia cell line HL-60. Eur. J. Cancer Clin. Oncol., 19, 251-261.
Control of Philadelphia Chromosome-Positive Leukemia Cells
391
2) Honma, Y., Okabe-Kado, J., Hozumi, M., Uehara, M. & Mizuno, S. (1989) Induction of erythroid differentiation of K562 human leukemic cells by herbimycin A, an inhibitor of tyrosine kinase activity. Cancer Res., 49, 331-334. 3) Honma, Y., Kasukabe, T., Hozumi, M., Maseki, N., Sakurai, M., Sakashita, A. & Tsuruoka, N. (1992) Herbimycin A, an inhibitor of tyrosine kinase activity, induces differentiation of human leukemic cells with a structurally altered c-abl (bcr/abl) gene. Leukemia, 6, 229-231. 4) Uehara, Y., Murakami, Y., Mizuno, S. & Kawai, S. (1989) Inhibition of transforming activity of tyrosine kinase oncogenes by herbimycin A. Virology, 164, 294-298.
J. Exp.
Med., 1992,
Control
of Growth
Philadelphia Cells
168, 387-391
by
and
Differentiation
Chromosome-Positive Tyrosine
Kinase
of Leukemia
Inhibitors
YOSHIOHONMA,TAKASHIKASUKABEand MOToo HOZUMI Department of Chemotherapy, Saitama Research Institute, Saitama 362
Cancer Center
HONMA,Y., KASUKABE,T, and HOZUMI, M. Control of Growth and Differentiation of Philadelphia Chromosome-PositiveLeukemia Cells by Tyrosine Kinase Inhibitors. Tohoku J. Exp. Med., 1992, 168 (2), 387-391 Herbimycin A, a selective inhibitor of tyrosine kinase activity, induced differentiation of leukemia cells isolated from Philadelphia chromosome-positive chronic myelogenous leukemia patients. However, it did not induce differentiation of leukemia cells from acute myelogenous leukemia patients, although these cells could be induced to differentiate by treatment with appropriate compounds. A selective inhibitor of tyrosine kinase might be useful in chemotherapy of Philadelphia chromosome-positive leukemia. Philadelphia chromosome-positive leukemia ; acute myelogenous leukemia ; chronic myelogenous leukemia ; herbimycin A ; tyrosine kinase inhibitor
The neoplastic transformation of many human tumors appears to be associated with the expression of activated oncogenes. Thus inhibition of activities of the oncogene products may be a new strategy for cancer chemotherapy. Translocation of the c-abl gene from chromosome 9 to 22, resulting in the Philadelphia chromosome, occurs in more than 90% of chronic myelogenous leukemia (CML) patients. The markedly increased tyrosine kinase activity of the gene product strongly suggests that the chimeric gene is directly involved in the pathogenesis of the leukemia. If so, a selective inhibitor of tyrosine kinase activity might be useful in chemotherapy of this leukemia. MATERIALS ANDMETHODS Human leukemia cells were obtained from patients during routine diagnostic procedures, and cultured in RP-MI-1640 mediumsupplementedwith 10% heat-inactivatedfetal bovine serum at 37°Cin a humidifiedatmosphereof 5% 002 in air. The cell number was counted after 6 days treatment with herbimycin A or the same period of incubation without herbimycin A. Differentiation-associatedproperties were determinedby the methodsdescribedpreviously(Honmaet al. 1983).
Address
for reprints
: 818 Komuro,
ma-machi, 387
Saitama
362, Japan.
388
Y. Honma
et al.
RESULTSANDDISCUSSION Cells from patients with AML or CML in blast crisis were cultured with or without herbimycin A in the presence of some hematopoietic growth factors. Several leukemia cells did not grow well, so we could not evaluate the growthinhibitory effect of herbimycin A on them. However, some hematopoietic growth factors (GM-CSF, IL-2, IL-3, and/or IL-4) stimulated the growth of some, but not all, leukemia cells in primary cultures. We evaluated the growth-inhibitory effect of herbimycin A in primary cultures of leukemia cells, when the cell numbers increased more than 2-fold in 6 days (Table 1). MO cells were taken from a man with blast crisis of CML. Morphologically, most of the freshly isolated MO cells were blastic and promyelocytic. Herbimycin A induced morphological differentiation of MO cells into mature granulocytes. Under the culture conditions used, the number of viable MO cells was not significantly reduced by herbimycin A, indicating that this drug does not kill immature granulocytes selectively (Fig. 1). MO cells did not grow without added growth factors, but in the presence of GM-CSF or IL-4 their cell number increased 1.5- to 1.8-fold during culture for 6 days. GM-CSF and IL-4 did not enhance granulocytic differentiation of MO cells induced by herbimycin A, although the cells showed slight differentiation into myelocytes, but not mature granulocytes in the presence of the cytokines only. During primary culture without any added compounds, the cell number of HN cells was increased only slightly, but IL-4 or GM-CSF stimulated cell proliferation, increasing the cell number 2.5-2.7-fold during culture for 6 days. Culture of HN cells with various concentrations of herbimycin A for 6 days caused dose-dependent stimulation of lysozyme synthesis (Fig. 2). Herbimycin A inTABLE1. Effect of herbimycin A on growth and differentiation of leukemia primary culture
cells in
Control
of Philadelphia
Chromosome-Positive
Leukemia
Cells
389
Fig. 1. Effect of herbimycin A on morphological changes of MO cells in the absense and presence of GM-CSF at 50 U/ml. LI,blasts and promyelocytes ; , myelocytes ; •, metamyelocytes and mature granulocytes.
creased the naphthyl AS-D chloroacetate esterase and lysozyme activities in KA cells, and caused slight morphological differentiation of the cells into mature granulocytes (Fig. 3). The cell number of KA cells increased slightly in the control culture, but in cultures with herbimycin A at less than 15 ng/ml the viable cell number of KA cells scarcely changed. TS cells were taken from a man with CML in lymphoid crisis. During culture for 7 days, the cell number increased slightly, but the cell growth was not stimulated by IL-2, IL-3, or IL-4. Herbimycin A at over 30 ng/ml was cytotoxic to TS cells, and no significant myelomonocytic differentiation was observed in drug-treated TS cells. The effects of herbimycin A on leukemia cells from patients with AML in
Fig. 2.
Effect
of herbimycin
A on differentiation
of HN cells.
Y. Honma
390
Fig. 3.
Effect
of herbim ycin
et al.
A on differentiation
of KA cells.
primary culture were also examined. The sensitivities of the cells to growth inhibition by the antibiotic varied. Herbimycin A, even at high concentration, did not cause any differentiation of any of the AML cells tested (5 cases), although all the cells showed monocytic differentiation by 12-0-tetradecanoylphorbol-13acetate. Herbimycin A caused reversion of transformed Rous sarcoma virus-infected rat kidney cells to normal morphology. This antibiotic was also found to be effective against various cells transformed by the other tyrosine kinase oncogenes including v-abl, but did not reverse the morphology of the cells transformed by the oncogenes raf, ras, and myc (Uehara et al. 1989). Our previous (Honma et al. 1989,1992) and present results show that the antiproliferative and differentiationinducing effects of herbimycin A differed in different leukemia cells : Phl-positive (or bcr/abl gene-containing) leukemia cells appeared to be more sensitive to herbimycin A than Phl-negative leukemia cells. Thus herbimycin A and its derivatives might be useful as cancer chemotherapeutic agents against some types of leukemia with altered tyrosine kinase activities. Acknowledgments This work Education, Comprehensive
was partly
Science
supported
and Culture
10-Year
Strateegy
by
a grant
and a grant for Cancer
for cancer
from the Ministry Control,
research
from
of Health
the
Ministry
and Welfare
of
for a
Japan.
References 1) Honma, Y., Fuj ita, Y., Kasukabe, T., Hozumi, M., Sampi, K. & Sakurai, M. (1983) Induction of differentiation of human acute non-lymphocytic leukemia cells in primary culture by inducers of differentiation of human myeloid leukemia cell line HL-60. Eur. J. Cancer Clin. Oncol., 19, 251-261.
Control of Philadelphia Chromosome-Positive Leukemia Cells
391
2) Honma, Y., Okabe-Kado, J., Hozumi, M., Uehara, M. & Mizuno, S. (1989) Induction of erythroid differentiation of K562 human leukemic cells by herbimycin A, an inhibitor of tyrosine kinase activity. Cancer Res., 49, 331-334. 3) Honma, Y., Kasukabe, T., Hozumi, M., Maseki, N., Sakurai, M., Sakashita, A. & Tsuruoka, N. (1992) Herbimycin A, an inhibitor of tyrosine kinase activity, induces differentiation of human leukemic cells with a structurally altered c-abl (bcr/abl) gene. Leukemia, 6, 229-231. 4) Uehara, Y., Murakami, Y., Mizuno, S. & Kawai, S. (1989) Inhibition of transforming activity of tyrosine kinase oncogenes by herbimycin A. Virology, 164, 294-298.
