Many growth factors play pivotal roles in cell proliferation, migration, and differentiation [14
]. Although a possible stimulating influence on leukemic cells remains questionable, most studies have reported that G-CSF is a safe agent that improves neutrophil count, thereby reducing the incidence of documented infection without regrowth of leukemic cells or other negative effects [15
]. In the present study, we evaluated the effects of 2 forms of recombinant human G-CSF (rhG-CSF) available for clinical use: filgrastim is derived from Escherichia coli and has a non-glycosylated form, whereas lenograstim is derived from Chinese hamster ovary (CHO) cells and is glycosylated. The peak serum concentrations of G-CSF after administration of a standard dose of G-CSF (5 µg/kg) is found to range from 15 to 30 ng/mL [19
]; therefore, we used a range of G-CSF concentrations spanning this concentration (0, 10, 50, and 100 ng/mL). The proliferation effect of G-CSF was prominent in Kasumi-1 cells, and the 2 forms of G-CSF showed similar effects. This might be due to the high expression of G-CSFR in AML1/ETO
+ Kasumi-1 cells, as reported in previous studies [2
]. Meanwhile, the proliferation was not stimulated in CTV-1 cells or U266 cells, which did not express G-CSFR. K562 cells with low-level expression of G-CSFR mRNA showed mild proliferation only at 100 ng/mL filgrastim after 72 h; however, this concentration cannot be applied in a clinical setting and is also much higher than the estimated serum concentrations in patients after G-CSF administration.
In addition to a proliferative effect, we noted that G-CSF induced differentiation in AML1/ETO
-positive cells with a high level of G-CSFR expression. There have been several reports on the differentiation effect of G-CSF, evidenced by morphologic changes and immunophenotypic changes [21
]. In the present study, although mild changes in CD11b expression were observed in unstimulated control cells and in stimulated CTV-1 cells, there was a prominent increase of CD11b expression in G-CSF-treated Kasumi-1 cells. However, the expression of CD66b was not affected. During the normal process of differentiation of neutrophils in bone marrow, CD66b is expressed from CD34-myeloblasts, reaching its highest level of expression at the myelocyte stage and decreasing thereafter [26
], whereas CD11b is expressed at a later stage and its expression increases during maturation [27
]. Thus, the differentiation pattern found in the present study (CD11b+/CD66b-) would not be seen in a normal process and suggests that the in vitro
differentiation induced by G-CSF was abnormal and incomplete. Induction of CD11b expression by G-CSF has been previously reported [22
]. Given that not all patients with AML1/ETO
+ AML show prominent differentiation in response to exogenous G-CSF, it is inferred that other factors such as microenvironments have to be taken into consideration for differentiation in vivo
Many studies have demonstrated the expression of G-CSFR in tumor cells or autocrine secretion of G-CSF in non-hematopoietic tumors such as colon cancer, ovarian cancer, squamous cell cancer, malignant melanoma, and sarcoma [6
]. In these reports, G-CSF was shown to stimulate proliferation and angiogenesis, and subsequently enhance malignant potential [6
]. Owing to the potential risk of stimulation of proliferation by G-CSF, information concerning G-CSFR expression in tumor cells would be helpful in the management of cancer patients. Here, we performed quantitative G-CSFR mRNA expression analyses in various solid tumor cell lines. Among the solid tumors, 13.1% expressed G-CSFR. Of note, G-CSFR expression in the hepatoblastoma cell line HepG2 was high and comparable to that in the AML1/ETO
+ Kasumi-1 cell line [2
]. However, such expression, especially when low, should be confirmed through additional testing and the statistical relevance of low expression needs to be validated.
In conclusion, G-CSFR expression and the proliferative effects of G-CSF on various malignant cells were demonstrated in the present study. Therefore, G-CSF should be used with caution in patients with hematopoietic or non-hematopoietic tumors with high G-CSFR expression. Accordingly, we suggest that screening for G-CSFR before administering G-CSF would be helpful in minimizing the risk of tumor proliferation. Expression levels of G-CSFR in primary tumor tissues should be evaluated by further study.