Effect of tumour-cell-derived or recombinant keratinocyte growth factor (KGF) on proliferation and radioresponse of human epithelial tumour cells (HNSCC) and normal keratinocytes in vitro

Abstract

Purpose of this work was to test the effect of tumour-cell-derived keratinocyte growth factor (KGF) or recombinant KGF (palifermin) on cell proliferation and radiation response of human HNSCC cells and normal keratinocytes in vitro. Four tumour cell cultures derived from head and neck squamous cell carcinomas, primary keratinocytes, and immortalized keratinocytes were analysed. Fibroblasts, the natural source of KGF protein, served as controls. KGF expression was observed in primary and immortalized keratinocytes, fibroblasts, and in tumour cells, while significant KGF receptor expression was only found in keratinocytes. Recombinant KGF as well as tumour-cell-derived KGF caused a significant growth stimulation and radioprotection in keratinocytes, which was abolished by a neutralizing anti-KGF antibody. This indicates that tumour-cell-derived KGF is biologically active. In the tumour cell lines, no significant growth stimulation was induced by recombinant KGF, and the neutralizing antibody did not influence tumour cell growth or radiation response. Our results indicate that the normal, paracrine KGF regulatory mechanisms, which are based on KGF receptor expression, are lost in malignant cells, with the consequence of irresponsiveness of the tumour cells to exogenous KGF. In face of the amelioration of the radiation response of normal epithelia, demonstrated in various clinical and various preclinical animal studies, recombinant KGF represents a candidate for the selective protection of normal epithelia during radio(chemo) therapy of squamous cell carcinoma.

Fig. 1

a Example of KGF expression in normal (keratinocytes, fibroblasts DF-19) and tumour (ZMK-1, GR-145, BW-225, OH-65) cells. b KGF receptor expression in keratinocytes and ZMK-1 tumour cells

Fig. 2

a Radiation-induced changes in KGF expression in normal (KTC = primary keratinocytes, HaCat = immortalized keratinocytes, DF-19 = primary fibroblasts) and tumour cells (BW-225, ZMK-1, GR-145, OH-65). Error bars represent standard deviations. Measurements were performed in duplicate and experiments were repeated two times. b Radiation-induced changes in KGF receptor expression in keratinocytes (KTC) and HaCat cells. Error bars represent standard deviations. Measurements were performed in duplicate, and experiments were repeated two times

Fig. 3

a Stimulated proliferation of keratinocytes by recombinant KGF is not concentration dependent. Data were recorded over 96 h, error bars represent ±1 standard deviation; statistically significant (P ≤ 0.05) stimulation was observed at 72 and 96 h, as marked by asterisks. Measurements were performed in triplicate, and experiments were repeated at least three times. b KGF-induced (0.2 μg/ml recombinant KGF) stimulated proliferation of irradiated keratinocytes was abrogated by the addition of anti-KGF antibody to the medium. Data shown were recorded after 96 h of incubation. Error bars represent ±1 standard deviation; statistically significant (P ≤ 0.05) inhibition of proliferation is marked by asterisks. Measurements were performed in triplicate, and experiments were repeated at least three times

Fig. 4

a Reduced proliferation of keratinocytes in media containing a specific anti-KGF antibody compared to media containing unspecific IgG antibodies. Keratinocytes were plated in supernatants derived from primary fibroblasts (DF-19), epithelial tumour cells (BW-225, ZMK-1, GR-145, OH-65), or KGF-free keratinocyte medium (KM). Data were recorded for cells irradiated at 1 Gy, 96 h after plating. Error bars represent ±1 standard deviation; statistically significant (P ≤ 0.05) inhibition of proliferation is marked by asterisks. Measurements were performed in triplicate, and experiments were repeated at least three times. b Reduced proliferation of keratinocytes in media containing a specific anti-KGF antibody compared to media containing unspecific IgG antibodies. Keratinocytes were plated in supernatants derived from primary fibroblasts (DF-19), epithelial tumour cells (BW-225, ZMK-1, GR-145, OH-65), or KGF-free keratinocyte medium. Data were recorded for cells irradiated at 4 Gy, 96 h after plating. Error bars represent ±1 standard deviation; statistically significant (P ≤ 0.05) inhibition of proliferation is marked by asterisks. Measurements were performed in triplicate and experiments were repeated at least 3 times

Fig. 5

a No stimulated proliferation by recombinant KGF added to tumour cell cultures for 72 h. Open symbols represent cells plated in KGF-free medium; closed symbols represent cells plated in medium containing 0.2 μg/ml recombinant KGF. Data were recorded over 72 h, error bars represent ±1 standard deviation. Measurements were performed in triplicate and experiments were repeated at least three times. b No effect of recombinant KGF added to tumour cell cultures for 96 h. Closed bars represent cells plated in KGF-free medium; horizontal stripes represent cells plated in medium containing 0.2 μg/ml recombinant KGF. Data were recorded over 96 h, error bars represent ±1 standard deviation. Measurements were performed in triplicate, and experiments were repeated at least three times

Fig. 6

a No effect of the neutralizing anti-KGF antibody on the proliferation of fibroblasts (DF-19) or epithelial tumour cells (BW-225, ZMK-1, GR-145, OH-65) was found. Data were recorded for non-irradiated cells, 96 h after plating. Closed bars represent cells plated in KGF-free medium; dashed bars represent cells plated in medium containing 0.2 μg/ml recombinant KGF. Error bars represent ±1 standard deviation. Measurements were performed in triplicate and experiments were repeated at least three times. b No effect of the neutralizing KGF antibody on the proliferation of fibroblasts (DF-19) or epithelial tumour cells (BW-225, ZMK-1, GR-145, OH-65) was observed. Data were recorded for cells irradiated with 1 Gy, 96 h after plating. Closed bars represent cells plated in KGF-free medium; dashed bars represent cells plated in medium containing 0.2 μg/ml recombinant KGF. Error bars represent ±1 standard deviation. Measurements were performed in triplicate and experiments were repeated at least three times. c No effect of the neutralizing KGF antibody on the proliferation of fibroblasts (DF-19) or epithelial tumour cells (BW-225, ZMK-1, GR-145, OH-65) was found. Data were recorded for cells irradiated with 4 Gy, 96 h after plating. Closed bars represent cells plated in KGF-free medium; dashed bars represent cells plated in medium containing 0.2 μg/ml recombinant KGF. Error bars represent ±1 standard deviation. Measurements were performed in triplicate and experiments were repeated at least three times