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Effects of Keratinocyte Growth Factor on the Proliferation and Radiation Survival of Human Squamous Cell Carcinoma Cell Lines In Vitro and In Vivo
Int. J. Radiation Oncology Biol. Phys., Vol. 40, No. 1, pp. 177–187, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0360-3016/98 $19.00 1 .00
PII S0360-3016(97)00561-0
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Biology Contribution EFFECTS OF KERATINOCYTE GROWTH FACTOR ON THE PROLIFERATION AND RADIATION SURVIVAL OF HUMAN SQUAMOUS CELL CARCINOMA CELL LINES IN VITRO AND IN VIVO SHOUCHENG NING, M.D., PH.D.,* CHAOXIANG SHUI, M.D.,* WAQQAR B. KHAN, B.S.,* WILLIAM BENSON, B.S.,† DAVID L. LACEY, M.D.† AND SUSAN J. KNOX, M.D., PH.D.* *Department of Radiation Oncology, Stanford University Medical Center, Stanford, CA 94305-5105, and †Amgen Inc., 1840 DeHavilland Drive, Thousand Oaks, CA 91320 Purpose: Keratinocyte growth factor (KGF) has potent mitogenic activity on normal epithelial cells and has been found to enhance intestinal crypt cell survival in irradiated mice and to prevent radiation and chemotherapyinduced mucositis in animal models. The purpose of the study reported here is to investigate the effect of recombinant human KGF on the proliferation and survival of human squamous carcinoma cell lines following irradiation. Methods and Materials: The level of KGF receptor (KGFR) mRNA in normal Balb/Mk cell line and human head and neck squamous carcinoma cell lines was assessed using a RNase protection assay. The clonogenic assay and MTT assay were used to study the proliferative effects of KGF on human tumor cell lines and Balb/MK cell line in vitro. Effects of KGF on in vivo tumor growth and radiosensitivity were studied in three KGFR-positive human squamous cell carcinoma xenografts (FaDu, Detroit 562 and A431) in nude mice, and a murine KGFR-negative melanoma tumor (B16) in Balb/c mice. Results: Seven of 10 tumor cell lines studied expressed KGFR mRNA. None of these tumor cell lines showed enhanced proliferation when exposed to KGF for 2 days or less. Prolonged exposure to KGF for 7 days or longer resulted in low level stimulation of proliferation in both clonogenic and MTT assays in four of seven KGFRpositive cell lines. Two KGFR-negative cell lines also had a low proliferative response to KGF in a clonogenic assay, but not in the MTT assay. Normal keratinocyte Balb/MK cells, which expressed a moderate level of KGFR mRNA, had a strongly proliferative response to KGF. Its KGF enhancement ratio (KER) of plating efficiency was 24 –70 times higher than that of the tumor cells studied (p < 0.001). The KGF-stimulated tumor cell growth was almost completely inhibited by heparin or epidermal growth factor (EGF). There were no significant differences (p > 0.05) in the survival of any of tumor cell lines in the presence or absence of KGF (100 ng/ml) irradiated with doses of 0 –15 Gy, and no significant differences (p > 0.05) between the radiobiological parameters D0, Dq, and n number from the SHMT model, a, b, and a/b ratio from the LQ model and SF2 for radiation survival curves for cell lines irradiated in the presence or absence of KGF. Three KGFR-positive human squamous cell carcinoma xenografts in nude mice, and a murine KGFR-negative melanoma tumor in Balb/c mice treated with 1.0 mg/kg of KGF for 3 days grew at the same rate as in untreated mice. Conclusion: The recombinant human KGF resulted in little or no stimulation of the proliferation of human head and neck squamous tumor cell lines and did not affect the radiosensitivity of these cell lines in vitro and in vivo. Therefore, KGF may be of clinical value in preventing radiation-induced mucositis and may have the potential to increase the therapeutic index of radiotherapy for treatment of cancers. © 1998 Elsevier Science Inc. Keratinocyte growth factor, KGF, KGFR, EGF, Radiosensitivity, Squamous carcinoma cells, Human tumor xenograft.
INTRODUCTION Keratinocyte growth factor (KGF), a member of the heparin-binding fibroblast growth factor (FGF) family (FGF-7), was originally purified from conditioned medium of a human embryonic lung fibroblast line, M426 (20). In addition to lung fibroblasts, KGF is also produced by mesenchymal cells from a variety of other tissues, including the human
gastrointestinal tract, bladder, prostate, mammary gland and skin in vitro (21). KGF has potent mitogenic activity on epithelial cell types (2, 11, 18, 24, 25, 27), but has no detectable effects on fibroblasts, endothelial cells, and other nonepithelial cells that respond to other FGF family members (1, 10), suggesting that KGF may be a specific paracrine mediator for normal epithelial growth and differentiation (10). The KGF receptor (KGFR) is a transmembrane
Reprint requests to: Susan J. Knox, Ph.D., M.D., Stanford University Medical Center, Department of Radiation Oncology (A-093), 300 Pasteur Dr., Stanford, CA 94305-5105. Acknowledgments—This work was supported in part by the NIH
Grant CA56464, a grant from Amgen Inc., and a Lazard Faculty Scholarship. We thank Sheila Scully of Amgen for her excellent help in the preparation of the manuscript. Accepted for publication 3 July 1997. 177
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tyrosine kinase that is a splice variant of the FGFR-2/BEK gene (16). The KGFR is expressed only in epithelial cells and binds KGF and aFGF with high affinity and bFGF at a lower affinity. KGFR mRNA has been detected in normal skin keratinocytes, epithelial cells of the oral cavity and entire gastrointestinal tract (11, 15), mammary epithelium (24), type II pneumocytes (25), and hepatocytes (11). In addition to its mitogenic effects on normal epithelial cell proliferation and differentiation, KGF has been reported to stimulate DNA synthesis in esophageal cancer cells in vitro (12), induce carcinoembryonic antigen (CEA) production by colon carcinoma cells (11), and activate the androgen receptor in prostatic carcinoma cell lines (9). KGFR mRNA has also been detected in about 50% of human epithelial carcinoma cell lines in vitro (22, 23). This includes tumor cell lines from human breast, lung, esophagus, stomach, colon, pancreas, liver, prostate, kidney, bladder, and ovary (9, 12, 15, 22, 23, 26). Expression of KGFR on tumor cells may correlate inversely with the malignant potential of Dunning R3327PAP prostatic cancer cells (26). These in vitro data suggest that the epithelial-derived tumor cells may respond to paracrine sources of KGF in vivo, and exogenous KGF could potentially affect responses of epithelial tumor cells to cytotoxic therapy. However, little is known about the effects of exogenous KGF on the proliferation of the epithelial tumor cells and on the survival of irradiated tumor cells. Recently, KGF has been found to enhance intestinal crypt cell survival in irradiated mice (Radiation Research, in press) and to prevent radiation and chemotherapy-induced mucositis in animal models (David Lacey, Amgen Inc., unpublished data). In the study reported here, we used a variety of human head and neck squamous cell carcinoma cell lines that expressed KGFR mRNA and studied the effects of recombinant human KGF on the proliferation and survival of these cell lines in vitro and in vivo following irradiation. METHODS AND MATERIALS Growth factors The recombinant human KGF and recombinant mouse epidermal growth factor (EGF) were prepared and provided by Amgen Inc., (Thousand Oaks, CA). Both factors were stored at 270°C, and diluted in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NY) with 10% fetal calf serum before use unless otherwise specified. Cell lines The human tumor cell lines studied included four lingual squamous cell carcinoma cell lines, SCC-4, -9, -15, and -25; a pharyngeal squamous cell carcinoma cell line, FaDu; a pharyngeal epidermoid cell carcinoma cell line, Detroit 562; a laryngeal epidermoid cell carcinoma cell line, HEp-2; a nasal squamous cell carcinoma cell line, RPMI 2650; an oral epidermoid cell carcinoma cell line, KB, and a submaxillary gland epidermoid carcinoma cell line, A253. A normal keratinocyte cell line, Balb/MK, was used as a
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positive control (20). All the cell lines were purchased from American Type Culture Collection (Rockville, MD). The lingual carcinoma cell lines were maintained in DMEM and Ham’s F12 (1:1) mixed media (Gibco, Grand Island, NY) supplemented with 10% FCS and 0.4 mg/ml hydrocortisone (Sigma, St. Louis, MO) and split weekly. The other tumor cell lines were maintained in DMEM plus 10% FCS. In some experiments, tumor cells were cultured in the media containing only 1% FCS. The Balb/MK cell line was maintained in DMEM plus 10% FCS, 5 ng/ml EGF. Additionally, 100 U/ml penicillin and 0.5 mg/ml streptomycin (Gibco, Grand Island, NY) were added to all the media. All cultures were incubated at 37°C in 95% air and 5% CO2.
RNase protection assay for KGFR mRNA expression Total RNA was isolated from tumor cells and Balb/MK cells using the RNA STAT-60 kit (TEL-TEST ‘‘B,’’ Inc., Friendswood, TX). A 129-bp fragment corresponding to nucleotides 1358 –1487 of the human KGFR sequence (GenBank accession #M80634) was generated by PCR and cloned into pSP72 (Promega, Madison, WI) and a 104-bp fragment of the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence was cloned into pGEM3Z (Promega, Madison, WI). After verification by sequencing, the plasmids were linearized and radiolabeled antisense riboprobes were transcribed using the Riboprobe system (Promega, Madison, WI) and 32P-UTP (Amersham, Arlington Heights, IL). Full length probes were purified by electrophoretic separation on a 6% polyacrylamide/7 M urea gel. The RNase protection assay was performed using the RPA II kit (Ambion, Inc., Austin, TX). Briefly, 50 mg of total RNA from each sample was hybridized overnight at 45°C with 105 cpm each of the KGFR and GAPDH probes. Following RNase digestion, the protected fragments were separated on a 6% polyacrylamide/7 M urea gel. The gel was exposed to a phosphor screen and the signal volumes were integrated on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The levels of KGFR mRNA was expressed as the ratio of the KGFR and GAPDH signals.
MTT assay Normal Balb/MK cells and tumor cells, at a density of 5,000 cells/ml, were cultured in 96-well plates (Costar, Cambridge, MA) in 200 ml of DMEM media plus 10% FCS and KGF at a final concentration ranging from 0 to 400 ng/ml. After incubation for a desired period at 37°C, 10 ml of 5 mg/ml MTT was added to each well. Cells were incubated for another 3 h and supernatant was removed. The crystal formazan product was dissolved by adding 200 ml of DMSO (Sigma, St. Louis, MO) and shaking thoroughly for 10 min. The optical density (O.D.) at 562 nm and 650 nm (reference) was measured using a computer-assisted Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA).
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Radiation survival curves Radiation survival curves were generated for each cell line using a clonogenic assay with or without KGF. Tumor cells were cultured in 60-mm Petri dishes in complete DMEM (or 1:1 mixed with Ham’s F12) and 100 ng/ml KGF. After incubation for 48 h at 37°C, dishes were irradiated with 0 –15 Gy at a dose rate of 4.30 Gy/min using a 137 Cs irradiator at room temperature. Cells were immediately trypsinized, diluted with growth media supplemented with 10% FCS, and plated in Petri dishes. After incubation for 10 –12 days in the presence or absence of 100 ng/ml of KGF, dishes were stained and colonies containing $50 cells were counted and used to calculate the surviving fraction. The parameters of D0, Dq, and n number from a single-hit multitarget model (SHMT) and a, b, and a/b ratio from a linear-quadratic model (LQ) of cell survival curves were calculated using software FIT 2.03 program (generously provided by Norman W. Albright, University of California at San Francisco, CA) (3). Experiments were repeated at least twice.
Fig. 1. Relative levels of KGFR mRNA expression in normal Balb/MK cells and human squamous tumor cells. (A) Cells were incubated in growth media and total RNA was collected using the RNA STAT-60 kit. The expression of KGFR mRNA was measured by the RNase protection assays on triplicate 50 mg RNA samples using human KGFR and GAPDH probes. (B) The level of KGFR mRNA in Balb/MK and tumor cells was expressed as the ratio of the KGFR and GAPDH signals. Data is shown as the mean 6 SD.
Clonogenic assay Exponentially growing cells were detached with 0.05% trypsin, counted and plated in 60-mm Petri dishes (Becton Dickinson Labware, Franklin Lakes, NJ) at appropriate dilutions in complete DMEM (or 1:1 mixed with Ham’s F12) plus various doses of KGF ranging from 0 to 100 ng/ml. Three to five dishes were plated per dose point. To compare the proliferation-stimulating activity of KGF with EGF, tumor cells were cultured with 10 ng/ml EGF alone, 10 ng/ml KGF alone, or both EGF and KGF. Following exposure to growth factors, the media were removed, and dishes were rinsed twice with Hank’s solution and filled with fresh growth media. After incubation for 10 –12 days, cells were stained with 0.25% crystal violet. Colonies, defined as $50 cells, were counted. For some cell lines, the number of large colonies with a diameter $1.0 mm was also recorded. The plating efficiency (PE) was calculated as the percentage of cells plated that grow into colonies.
Mouse tumor models and tumor growth delay study Female nude mice were used for KGFR-positive human squamous cell carcinoma xenografts (FaDu, Detroit 562 and A431), and female Balb/c mice were used for KGFR-negative murine melanoma B16 tumors. All mice were 10 to 12 weeks old and ranged in weight between 25 and 30 g. Mice were obtained from the Stanford University Research Animal Facility. The mice were normally bred and maintained
Fig. 2. Effect of KGF on proliferation of Balb/MK cells measured by MTT assay showing O.D. as a function of KGF dose (0.1– 400 ng/ml). Cells were incubated in DMEM media plus KGF at 37°C for 4 days. Data are presented as the mean 6 SD of four replicate cultures. Experiments were repeated twice.
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Fig. 3. Effect of KGF on proliferation of tumor cells and Balb/MK cells in a clonogenic assay. Cells were incubated with KGF at 37°C for 10 days. Colonies containing more than 50 cells were counted. (A) Cloning efficiency (as the percent of control cultures without KGF) of tumor cell lines as a function of KGF concentration. (B) Comparison of the cloning efficiency of Balb/MK and tumor cells as a function of KGF concentration. The scale for the Y axis (cloning efficiency) ranges from 0 –500% for A and 0 –5000% for B. Data represent the mean 6 SD from two independent experiments.
under specific pathogen-free conditions, and sterilized food and water were available ad lib. Animals were injected intradermally (i.d.) in the left flank with 5 3 106 tumor cells in a suspension volume of 50 ml. One tumor per animal was implanted. Groups of mice with an average tumor size of 100 mm3 were treated with 1) NaCl solution (0.9%) as a control; 2) KGF 1.0 mg/kg s.c. for 3 consecutive days; 3) fractionated x-ray irradiation with 2.50 Gy daily for 5 consecutive days; 4) KGF 1.0 mg/kg s.c. for 3 consecutive days before irradiation and irradiation with 2.50 Gy daily for 5 consecutive days; and 5) fractionated irradiation for 5 days as above plus KGF for 3 consecutive days after radiation. Six to eight animals were used in each group. Mice were irradiated in a mouse back jig with a Philips RT-250 200 kVp X-ray unit (12.5 mA; Half Value Layer, 1.0-mm Cu). The length, width, and height (in mm) of the tumors were measured with calipers three times a week. Measurements were continued until the tumor volume reached four times (4 3) the original pretreatment volume. Tumor volume (mm3) was calculated according to the formula: tumor volume 5 p/6 3 length 3 width 3 height. The data are expressed as the mean tumor volume-quadrupling time (days) 6 standard deviation (SD), or as percent (%) of the pretreatment volume on day 0. Statistics Data were statistically analyzed using a two-tailed Student’s t-test.
RESULTS KGFR mRNA expression by human head and neck squamous carcinoma cell lines To determine whether human head and neck squamous tumor cells expressed KGFR mRNA, a RNase protection assay (RPA) was performed to quantitate the KGFR mRNA level in the cell lines studied. As shown in Fig. 1, KGFR mRNA expression was detected by RPA assay in 7 out of the 10 tumor cell lines. The level of KGFR mRNA was expressed as the ratio of KGFR and GAPDH signals and was in a range of 0.3–12.3 in these seven tumor cell lines. The KGFR/GAPDH signal ratio of KGFR mRNA in normal Balb/MK cells was 3.0. KGFR mRNA levels in HEp-2, SCC-15, and KB tumor cell lines were not detectable in the RPA assay. Effects of KGF on proliferation of Balb/MK cells Normal Balb/MK keratinocytes were cultured with concentrations of KGF ranging from 0.1 to 400 ng/ml for 4 days. Cell proliferation was measured by MTT and clonogenic assays. The MTT assay utilizes a colorimetric reaction secondary to succinate dehydrogenase activity in mitochondria, which is an indirect measure of cell viability and proliferation. Figure 2 shows the O.D. as a function of KGF concentration for Balb/MK cells. There was a linear-log relationship between O.D. and KGF dose at concentrations ranging from 0.4 ng/ml to 20 ng/ml, with the maximal effect of KGF occurring at concentrations of $20 ng/ml. In the
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Table 1. Characteristics of normal Balb/MK keratinocytes and epithelial tumor cell lines Cell lines
KGFR mRNA*
PEcontrol†
PEKGF‡
KER§
Balb/MK Detroit 562 FaDu SCC-9 SCC-25 SCC-4 A253 RPMI 2650 SCC-15 HEp-2 KB
1 1 1 1 1 1 1 1 2 2 2
0.9 6 0.1 6.4 6 0.8 21.5 6 1.3 20.0 6 1.8 17.5 6 4.9 19.1 6 1.2 23.3 6 0.7 17.7 6 2.6 6.0 6 0.6 19.4 6 5.9 57.1 6 1.3
33.6 6 3.9 15.2 6 0.8 34.5 6 17.1 33.5 6 3.3 29.1 6 10.6 21.2 6 4.8 22.7 6 1.2 19.2 6 2.5 15.2 6 2.5 29.5 6 19.9 58.2 6 1.1
36.30 1.38 0.60 0.68 0.66 0.11 20.03 0.08 1.53 0.52 0.02
* KGFR mRNA was measured by a RNase protection assay. Plating efficiency (%) of control cells: (number of colonies/ number of cells plated) 3 100. ‡ Plating efficiency (%) at KGF dose of 100 ng/ml for 10 days. § KGF enhancement ratio of PE at KGF dose of 100 ng/ml for 10 days: (PEKGF2PEcontrol)/PEcontrol. †
clonogenic assay, the plating efficiency (PE) was stimulated by adding KGF in growth media at a dose as low as 0.1 ng/ml (Fig. 3B) and the maximal stimulatory effect of KGF occurred at doses of $100 ng/ml. The KGF enhancement ratio (KER) of PE was 36.3 at a KGF dose of 100 ng/ml (Table 1). Proliferative effect of KGF on tumor cells The MTT and clonogenic assays were used to study the proliferation-stimulating effects of KGF on human head and neck squamous cell carcinoma cell lines. In the clonogenic assays, tumor cells were incubated with various concentrations of KGF ranging from 0 –1000 ng/ml. All the tumor cell lines studied showed no stimulated proliferation in response to KGF exposure of 2 days or less. However, exposure to KGF for 7 or more days did increase PE by KGFR-positive cell lines Detroit 562, FaDu, SCC-9, and SCC-25 in a dose-dependent manner (Fig. 3A). The KERs were 1.38 for Detroit 562, and 0.60 – 0.68 for FaDu, SCC-9, and SCC-25 in the presence of 100 ng/ml KGF for 10 days (Table 1). There was no further enhancement of PE by KGF at concentrations up to 1,000 ng/ml in these tumor cell lines. The KGFR-negative cell lines SCC-15 and HEp-2 also showed an increased PE when exposed to 100 ng/ml KGF for 10 –12 days (Table 1). However, the enhancement of PE by KGF on tumor cell lines was significantly less than that observed with normal Balb/MK cells (p , 0.001) (Fig. 3B). The PE of Balb/MK cells was increased 10-fold at the low concentration of KGF (0.1 ng/ml) and to 36-fold at the high concentrations of KGF (.100 ng/ml). KGF also increased the size of colonies in two (FaDu and SCC-25) of seven KGFR-positive cell lines studied. The number of large colonies (with a diameter of $1.0 mm) was increased from 26% in control cultures to 63% in cultures containing 10 ng/ml KGF for the FaDu cell line, and from 13% in control cultures to 42% in cultures containing 100 ng/ml KGF for
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the SCC-25 cell line, representing a 2.4 and 3.2 fold increase, respectively. KGFR-positive cell lines SCC-4, A253 and RPMI2650, and KGFR-negative cell line KB showed no stimulated proliferation in response to KGF treatment in both the clonogenic assay and MTT assay. Interestingly, KGF in media containing only 1% FCS supported the sustained growth and colony formation of tumor cell lines (such as KB cell line) that did not show any stimulated proliferation (KER, 0.02) in response to KGF in normal growth media containing 10% FCS (Fig. 4). The plating efficiency of KB cells in 1% FCS media without KGF decreased to 4.8% 6 1.0 from 57.1% 6 1.3 in media containing 10% FCS. These cells died quickly in the cultures with 1% FCS in the absence of KGF, suggesting that KGF is able to partially replace the serum requirement and support the sustained growth of these cell lines in vitro. In MTT assays, tumor cells were cultured with or without 20 ng/ml KGF for 4 days. The KGFR-positive Detroit 562, FaDu, SCC-9, and SCC-25 cell lines showed significantly stimulated proliferation in response to exogenously added KGF. The KGFR-negative HEp-2 and SCC-15 cell lines, which showed increased PE after stimulation with KGF for 10 –12 days, did not show a reproducible stimulatory response to a 4-day exposure to KGF in the MTT assay. Other KGFR-positive cell lines SCC-4, A253, and RPMI 2650 and KGFR-negative cell lines KB and HEp-2 failed to show
Fig. 4. Effect of KGF on the cloning efficiency of KB cells in 10 and 1% fetal calf serum. Cells were cultured in media containing 10 or 1% FCS with KGF at concentrations of 0.1–1000 ng/ml at 37°C for 10 days. Data are presented as the cloning efficiency (as the percent of control cultures without KGF). The plating efficiency was 57.1% 6 1.3 in normal media containing 10% FCS and 4.8% 6 1.0 in media with 1% FCS. Data represent the mean 6 SD from two independent experiments.
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Fig. 5. Inhibitory effect of heparin on KGF-induced tumor cell proliferation measured in a MTT assay. Cells were grown in normal media without KGF and heparin (open circles, E), with 20 ng/ml KGF (close diamonds, r), or with 20 ng/ml KGF and 0.5 U/ml heparin (close squares, ■). O.D. is shown as a function of exposure time (1–7 days) for SCC-9, SCC-25, FaDu, and Detroit 562 cell lines. Data are presented as mean 6 SD of four replicate cultures.
significant and consistent responses to KGF in similar MTT assays (data not shown). Because KGF is a heparin-binding molecule (20) and heparin has an inhibitory effect on the KGF-induced mitogenic activity on Balb/MK cells (19), we also tested the interaction of KGF and heparin on the tumor cell lines. In these studies, four KGFR-positive tumor cell
lines Detroit 562, FaDu, SCC-9, and SCC-25 were incubated with 20 ng/ml KGF plus 0.5 U/ml heparin for 1–7 days. Heparin alone did not show any effect on the tumor cell growth. When simultaneously used with KGF, heparin almost completely blocked the KGF-stimulated cell growth of these KGFR-positive tumor cell lines (Fig. 5).
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KGF overlapped each other. The surviving fractions at 2 Gy (SF2) for these four cell lines ranged from 0.35 to 0.66 (Table 2). There were no significant differences in the survival of any of the tumor cell lines studied that were incubated in the presence or absence of KGF at any of the radiation doses utilized. When compared, the radiobiological parameters of survival curves analyzed using the LQ model (a, b, and a/b ratio) and the SHMT model (D0, Dq, and n number), there were also no significant differences (p . 0.05) between any of these parameters for most of the cell lines irradiated and cultured in the presence or absence of KGF (Table 2). The a/b ratios for SCC-25 and Detroit 562 cell lines increased following exposed to 100 ng/ml KGF for 12 days compared to radiation alone, while the a/b ratios for SCC-9 and HEp-2 cell lines decreased. However, there were no significant differences (p . 0.05) between any other parameters for these cell lines irradiated in the presence or absence of KGF for 12 days.
Fig. 6. Differential effects of KGF and EGF on colony formation of normal Balb/MK and tumor cells. Cells were cultured in media with or without KGF and EGF at a dose of 10 ng/ml at 37°C for 10 days. Data are presented as mean 6 SD from five replicate cultures.
Differential effects of KGF and EGF on colony formation Because epidermal growth factor (EGF) also stimulates epithelial cell growth (7), the potentially synergetic action of KGF and EGF was studied in a clonogenic assay in the normal Balb/MK cell line and the KGFR-positive (Detroit 562, A253, SCC-25) and KGFR-negative (KB and HEp-2) tumor cell lines. Cells were incubated for 10 days in media with or without 10 ng/ml KGF alone, 10 ng/ml EGF alone, or with the combination of the both growth factors. As shown in Fig. 6, KGF at 10 ng/ml significantly increased the colony formation by Detroit 562 and SCC-25 tumor cells (p 5 0.002), but did not affect colony formation by A253, HEp-2, and KB tumor cell lines (p . 0.05). EGF significantly inhibited colony formation by these tumor cell lines (p # 0.002), even in the presence of KGF. Normal Balb/MK keratinocytes showed a strong response to EGF as well as to KGF in the colony-forming assay. But the combined use of KGF and EGF was not able to produce the synergetic stimulation of the colony formation. Effects of KGF on the radiation survival of tumor cells The effect of KGF on the survival of human head and neck squamous cell carcinoma cell lines following irradiation was assessed using clonogenic assays. Cells were plated in culture media with or without 100 ng/ml of KGF and irradiated with 0 –15 Gy using a 137Cs source. Figure 7 shows the radiation survival curves for four KGFR-positive tumor cell lines that responded to KGF stimulation in the absence of irradiation (Fig. 2 and Table 1). As can be seen, the radiation survival curves in the absence and presence of
Effects of KGF on in vivo tumor growth and radiosensitivity Three KGFR-positive human squamous cell carcinoma xenografts (FaDu, Detroit 562, and A431) in nude mice, and a murine KGFR-negative melanoma tumor (B16) in Balb/c mice were studied. Mice were treated with KGF for 3 consecutive days alone, fractionated x-ray irradiation for 5 consecutive days alone, or in combination with KGF either before or after radiation. This treatment regimen was utilized because our previous study demonstrated that administration of 1.0 mg/kg KGF for 3 consecutive days before irradiation was effective in stimulating the intestinal stem cell proliferation (Radiation Research, in press). A representative tumor growth curve for FaDu tumor xenografts is shown in Fig. 8. There were no statistically significant differences in tumor growth between untreated mice and mice treated with KGF alone, or between groups of mice treated with radiation alone and radiation with KGF (p . 0.05). The tumor regrowth delay data were analyzed in terms of tumor volume-quadrupling time (TVQD), and again, KGF did not significantly affect the TVQD of either irradiated or unirradiated tumors (p . 0.05). Similar results were obtained for the other three cell lines studied in vivo (Table 3). DISCUSSION The primary objective of the experiments reported here was to study the potential effect of recombinant human KGF on the proliferation and survival of irradiated human squamous cell carcinoma cell lines. KGF had a minimal proliferation-stimulating effect of 7 out of 10 tumor cell lines studied in vitro when incubated with KGF for $7 days, but no significant stimulatory effect on cell lines exposed to KGF for #2 days, even on cell lines that expressed a relatively high level of KGFR. To compare the radiosensitivity of human tumor cell lines in the absence and presence of KGF, we generated the radiation survival
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Fig. 7. Effect of KGF on the survival of tumor cell lines following gamma irradiation in a clonogenic assay. The survival fraction is shown as a function of radiation dose (Gy). Cells were irradiated in the absence of KGF (open circles, E), preincubated with 100 ng/ml KGF for 2 days and irradiated (close squares, ■), or preincubated with 100 ng/ml KGF for 2 days, irradiated and continued in culture for another 10 days in the presence of KGF (close diamonds, r). Colonies were counted on day 10. Data are from representative experiments. The radiobiological parameters calculated using the SHMT and LQ models are shown in Table 2.
curves using a clonogenic assay and analyzed these survival curves with two mathematical models, the LQ and SHMT models. All of the survival curves were well fitted by these two models. In general, the radiobiological parameters of the tumor cell lines did not change significantly in the presence of KGF. However, following exposure to KGF for
12 days, the a/b ratios were increased for Detroit 562 and SCC-25 and decreased for SCC-9 and HEp-2 compared to cells similarly irradiated but incubated without KGF. Nevertheless, there were not any other significant differences between any of the other radiobiological parameters studied. Therefore, when all of the data is considered, it appears
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Table 2. Radiobiological parameters of survival curves for tumor cells irradiated in presence or absence of KGF LQ†
SHMT* Cell line SCC-4 SCC-9 SCC-15 SCC-25 FaDu Detroit562 A253 HEp-2 RPMI 2650 KB
Treatment Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF
2d 1 Rad§ 12d 1 Rad¶ 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad
D0
Dq
n
a
b
a/b
SF2‡
1.02 1.01 1.05 1.41 1.41 1.39 0.90 1.17 1.00 1.05 1.08 1.08 1.40 1.37 1.31 1.37 1.36 1.43 1.46 1.49 1.46 1.08 1.09 1.11 1.47 1.44 1.44 1.36 1.45 1.36
2.08 2.14 1.86 2.34 2.30 2.60 1.84 1.45 1.99 1.61 1.45 1.62 2.82 2.38 2.56 2.64 2.53 1.90 2.47 2.36 2.33 1.51 1.61 1.92 1.04 1.19 1.13 2.17 1.68 1.94
7.69 8.41 5.86 5.23 5.10 6.45 7.74 3.47 7.30 4.64 3.81 4.51 7.50 5.65 7.09 6.93 6.41 3.75 5.44 4.89 4.92 4.05 4.40 5.66 2.04 2.29 2.19 4.94 3.20 4.16
0.30 0.29 0.34 0.30 0.32 0.20 0.03 0.17 0.02 0.37 0.48 0.42 0.13 0.16 0.20 0.26 0.29 0.34 0.24 0.24 0.26 0.44 0.39 0.30 0.51 0.48 0.51 0.31 0.35 0.34
0.05 0.05 0.05 0.02 0.02 0.03 0.14 0.09 0.12 0.05 0.03 0.04 0.04 0.04 0.04 0.03 0.03 0.02 0.03 0.03 0.03 0.04 0.04 0.05 0.01 0.01 0.01 0.03 0.02 0.03
6.0 5.8 6.8 15.0 16.0 6.7 0.21 1.9 0.17 7.4 16.0 10.5 3.3 4.0 5.0 8.7 9.7 15.1 8.0 8.0 8.7 11.0 9.8 6.0 51.0 48.0 51.0 10.3 14.8 11.3
0.45 0.46 0.41 0.51 0.49 0.59 0.54 0.50 0.59 0.39 0.34 0.37 0.66 0.62 0.57 0.53 0.50 0.57 0.55 0.55 0.53 0.35 0.39 0.45 0.35 0.37 0.35 0.48 0.46 0.45
* The single-hit multitarget model. D0 is the dose (Gy) required to reduce the fraction of surviving cells to 37% of its previous value in the exponential region of the survival curves. Dq is the quasi-threshold dose (Gy) to measure the shoulder width of survival curves. † The linear quadratic model. ‡ Surviving fraction at 2 Gy. § Cells were exposed to KGF at a dose of 100 ng/ml for 2 days before radiation. ¶ Cells were exposed to KGF for 2 days before radiation and continued KGF exposure for 10 days postradiation. The KGF concentration was 100 ng/ml in DMEM supplemented with 10% FCS.
that the KGF did not alter the in vitro radiosensitivity of the human tumor cell lines studied. Importantly, in vivo KGF neither stimulated tumor growth nor changed the radiation sensitivity of KGFR-positive human tumor xenografts in nude mice. At the same concentration of KGF, the normal epithelial cell line Balb/MK had a KGF enhancement ratio of PE 24 –70 times that of the tumor cells (p , 0.001). The differential effect of KGF on the proliferation of normal epithelial cells and tumor cells may simply be due to the high affinity of receptors on Balb/MK cells (1, 5). Alternatively, it is also possible that there are functional differences between KGFRs and/or associated signal transduction pathways in normal and malignant cells. Heparin at a nontoxic level of 0.5 U/ml completely blocked the stimulatory effect of KGF on tumor cell proliferation, presumably because of partially overlapping binding sites of heparin and KGFR on KGF molecules (6). Interestingly, a subset of KGFR mRNA-positive cell lines did not show the stimulated proliferation in response to
Fig. 8. Effects of KGF on FaDu tumor growth in nude mice. Tumorbearing nude mice (eight mice per group) were treated with normal saline (open circles, E); KGF 1 mg/kg for 3 days alone (open squares, h); radiation 2.50 Gy daily for 5 days alone (close circles, F); KGF for 3 days followed by 5 days radiation (close diamonds, }); or radiation for 5 days followed by KGF for 3 days (close squares, ■). Radiation was given from days 0–4. The data are expressed as the mean percent (%) of the pretreatment volume on day 0 6 SD.
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Table 3. Effects of KGF on tumor growth and tumor regrowth delay following irradiation of KGFR-positive and KGFR-negative tumors Tumor volume-quadrupling time (days, mean 6 SD) Treatment
FaDu
A431
Detroit 562
B16
Untreated control KGF alone Radiation alone KGF 1 Radiation* Radiation 1 KGF†
15.2 6 1.7 14.2 6 1.2 47.9 6 3.9 42.8 6 1.6 47.1 6 3.7
9.0 6 0.4 20.0 6 1.0 23.0 6 0.6 22.0 6 0.8 25.0 6 0.5
13.9 6 3.8 13.7 6 5.1 44.7 6 12.4 44.0 6 13.2 52.4 6 8.6
17.0 6 0.4 19.0 6 0.8 35.0 6 1.1 34.0 6 1.2 36.0 6 0.9
* Mice were given 1.0 mg/kg KGF 2 days pre-, 1 day pre- and 2 h preradiation. Mice were given 1.0 mg/kg KGF at 2 h, 1 day, and 2 days after last fraction of irradiation.
†
KGF, whereas there was some enhanced proliferation of KGFR mRNA-negative cell lines (HEp-2, SCC-15 and KB) following addition of KGF to the media. Presumably, these three tumor cell lines did express KGFR mRNA but at a level too low to be detected by the RNase protection assay. Unfortunately, it was not possible to quantitate KGFR expression using a Western blot analysis due to the currently unavailability of the specific antibodies directed to the KGFR, so it was not possible to correlate levels of KGFR expression and proliferative responsiveness to KGF. The apparent discrepancies between KGFR mRNA expression and KGF-stimulated proliferative responses might be explained by a difference in receptor affinity or function, or by a poor correlation between mRNA and receptor levels. Epidermal growth factor (EGF) is a potent mitogen for epithelial cell growth and acts via its receptor (EGFR), an intrinsic membrane glycoprotein with an extracellular binding domain. It is widely distributed in human normal tissues and epithelial-derived tumor cells. EGF and its receptor are believed to play an important role in the development and growth of some tumor cells (7). In this study, EGF consistently inhibited colony formation in all the tumor cell lines tested, whereas it stimulated the clonogenic efficiency of
normal Balb/MK cells. When combined with KGF, EGF abolished the stimulatory effect of KGF on tumor cell proliferation. The reason for this is unclear. However, EGF has been reported to enhance the sensitivity of human epithelial tumor cells to irradiation (14) as well as to some chemotherapeutic agents in vitro (8, 13) and in vivo (4, 17). Radiation-induced mucositis is common during and after local irradiation of the head and neck and gastrointestinal tract, and can be a dose limiting toxicity. We have recently reported that KGF enhances the survival of irradiated intestinal crypt cells in mice (Radiation Research, in press). KGF has also been reported by others to prevent or minimize radiation and chemotherapy-induced mucositis in a variety of animal models (Amgen, Inc., unpublished data). We have reported here that recombinant human KGF resulted in little or no stimulation of the proliferation of human head and neck squamous tumor cell lines and did not affect the radiosensitivity of these cell lines in vitro and in vivo. Therefore, KGF may be of clinical value in preventing radiation-induced mucositis and may have the potential to increase the therapeutic index of radiotherapy for treatment of head and neck, esophageal, abdominal, and pelvic cancers.
REFERENCES 1. Aaronson, S. A.; Bottara, D. P.; Miki, T.; Ron, D.; Finch, P. W.; Fleming, T. P.; Ahn, J.; Taylor, W. G.; Rubin, J. S. Keratinocyte growth factor: Fibroblast growth factor family member with unusual target cell specificity. Ann. NY Acad. Sci. 638:62–77; 1991. 2. Alarid, E. T.; Rubin, J. S.; Young, P.; Chedid, M.; Aaronson, S. A.; Cunha, G. R. Keratinocyte growth factor functions in epithelial induction during seminal vesicle development. Proc. Natl. Acad. Sci. USA 91:1074 –1078; 1994. 3. Albright, N. W. Computer programs for the analysis of cellular survival data. Radiat. Res. 112:337–340; 1987. 4. Amagase, H.; Kakimoto, M.; Hashimoto, K.; Fuwa, T.; Tsukagoshi, S. Epidermal growth factor receptor-mediated selective cytotoxicity of antitumor agents toward human xenografts and murine syngeneic solid tumor. Jpn. J. Cancer Res. 80:670 – 678; 1989. 5. Bottaro, D. P.; Rubin, J. S.; Ron, D.; Finch, P. W.; Florio, C.; Aaronson, S. A. Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptor. J. Biol. Chem. 265:12767–12770; 1990.
6. Bottaro, D. P.; Fortney, E.; Rubin, J. S.; Aaronson, S. A. A keratinocyte growth factor receptor-derived peptide antagonist identifies part of the ligand binding site. J. Biol. Chem. 268: 9180 –9183; 1993. 7. Carpenter, G.; Cohen, S. Epidermal growth factor. J. Biol. Chem. 265:7709 –7712; 1990. 8. Christen, R. D.; Hom, D. K.; Porter, D. C.; Andrews, P. A.; Macleod, C. L.; Howell, S. B. Epidermal growth factor regulates the in vitro sensitivity of human ovarian carcinoma cells to cisplatin. J. Clin. Invest. 86:1632–1640; 1990. 9. Culig, Z.; Hobisch, A.; Cronauer, M. V.; Radmayr, C.; Trapman, J.; Hittmair, A.; Bartsch, G.; Klocker, H. Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor—I, keratinocyte growth factor, and epidermal growth factor. Cancer Res. 54:5474 –5478; 1994. 10. Finch, P. W.; Rubin, J. S.; Miki, T.; Ron, D.; Aaronson, S. A. Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science 245:752–755; 1989. 11. Housley, R. M.; Morris, C. F.; Boyle, W.; Ring, B.; Biltz, R.; Tarpley, J. E.; Aukerman, S. L.; Devine, P. L.; Whitehead,
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R. H.; Pierce, G. F. Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract. J. Clin. Invest. 94:1767–1777; 1994. Ishii, H.; Hattori, Y.; Itoh, H.; Kishi, T.; Yoshida, T.; Sakamoto, H.; Oh, H.; Yoshida, S.; Sugimura, T.; Terada, M. Preferential expression of the third immunoglobulin-like domain of K-sam product provides keratinocyte growth factordependent growth in carcinoma cell lines. Cancer Res. 54: 518 –522; 1994. Kroning, R.; Jones, J. A.; Hom, D. K.; Chuang, C. C.; Sanga, R.; Los, G.; Howell, S. B.; Christen, R. D. Enhancement of drug sensitivity of human malignancies by epidermal growth factor. Br. J. Cancer 72:615– 619; 1995. Kwok, T. T.; Sutherland, R. M. Enhancement of sensitivity of human squamous carcinoma cells to radiation by epidermal growth factor. J. Natl. Cancer Inst. 81:1020 –1024; 1989. LaRochelle, W.; Dirsch, O. R.; Finch, P. W.; Cheon, H. G.; May, M.; Marchese, C.; Pierce, J. H.; Aaronson, S. A. Specific receptor detection by a functional keratinocyte growth factorimmunoglobulin chimera. J. Cell Biol. 129:357–366; 1995. Miki, T.; Fleming, T. P.; Bottaro, D. P.; Rubin, J. S.; Ron, D.; Aaronson, S. A. Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. Science 251:72–75; 1991. Murayama, Y. Growth-inhibitory effects of epidermal growth factor on human breast cancer and carcinoma of the esophagus transplantal into nude mice. Ann. Surg. 211:263–268; 1990. Pierce, G. F.; Yanagihara, D.; Klopchin, K.; Danilenko, D. M.; Hsu, E.; Kenney, W. C.; Morris, C. F. Stimulation of all epithelial elements during skin regeneration by keratinocyte growth factor. J. Exp. Med. 179:831– 840; 1994. Ron, D.; Bottaro, D. P.; Finch, P. W.; Morris, D.; Rubin, J. S.; Aaronson, S. A. Expression of biologically active recombinant keratinocyte growth factor. Structure/function analysis of amino-terminal truncation mutants. J. Biol. Chem. 268:2984 – 2988; 1993.
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20. Rubin, J. S.; Osada, H.; Finch, P. W.; Taylor, W. G.; Rudikoff, S.; Aaronson, S. A. Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc. Natl. Acad. Sci. USA 86:802– 806; 1989. 21. Rubin, J. S.; Bottaro, D. P.; Chedid, M.; Miki, T.; Ron, D.; Cheon, H. G.; Taylor, W. G.; Fortney, E.; Sakata, H.; Finch, P. W.; LaRochelle, W. J. Keratinocyte growth factor. Cell Biol. Int. 19:399 – 410; 1995. 22. Rubin, J. S.; Bottaro, D. P.; Chedid, M.; Miki, T.; Ron, D.; Cunha, G. R.; Finch, P. W. Keratinocyte growth factor as a cytokine that mediates mesenchymal– epithelial interaction. Exs. 74:191–214; 1995. 23. Siddiqi, I.; Funatomi, H.; Kobrin, M. S.; Friess, H.; Buchler, M. W.; Korc, M. Increased expression of keratinocyte growth factor in human pancreatic cancer. Biochem. Biophys. Res. Commun. 215:309 –315; 1995. 24. Ulich, T. R.; Yi, E. S.; Cardiff, R.; Yin, S.; Bikhazi, N.; Biltz, R.; Morris, C. F.; Pierce, G. F. Keratinocyte growth factor is a growth factor for mammary epithelium in vivo. The mammary epithelium of lactating rats is resistant to the proliferative action of keratinocyte growth factor. Am. J. Pathol. 144: 862– 868; 1994. 25. Ulich, T. R.; Yi, E. S.; Longmuir, K.; Yin, S.; Biltz, R.; Morris, C. F.; Housley, R. M.; Pierce, G. F. Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo. J. Clin. Invest. 93:1298 –1306; 1994. 26. Yan, G.; Fukabori, Y.; Nikolaropoulos, S.; Wang, F.; Mckeehan, W. L. Heparin-binding keratinocyte growth factor is a candidate stromal to epithelial cell andromedin. Mol. Endocrinol. 6:2123–2128; 1992. 27. Yi, E. S.; Yin, S.; Harclerode, D. L.; Bedoya, A.; Bikhazi, N. B.; Housley, R. M.; Aukerman, S. L.; Morris, C. F.; Pierce, G. F.; Ulich, T. R. Keratinocyte growth factor induces pancreatic ductal epithelial proliferation. Am. J. Pathol. 145:88 – 85; 1994.
PII S0360-3016(97)00561-0
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Biology Contribution EFFECTS OF KERATINOCYTE GROWTH FACTOR ON THE PROLIFERATION AND RADIATION SURVIVAL OF HUMAN SQUAMOUS CELL CARCINOMA CELL LINES IN VITRO AND IN VIVO SHOUCHENG NING, M.D., PH.D.,* CHAOXIANG SHUI, M.D.,* WAQQAR B. KHAN, B.S.,* WILLIAM BENSON, B.S.,† DAVID L. LACEY, M.D.† AND SUSAN J. KNOX, M.D., PH.D.* *Department of Radiation Oncology, Stanford University Medical Center, Stanford, CA 94305-5105, and †Amgen Inc., 1840 DeHavilland Drive, Thousand Oaks, CA 91320 Purpose: Keratinocyte growth factor (KGF) has potent mitogenic activity on normal epithelial cells and has been found to enhance intestinal crypt cell survival in irradiated mice and to prevent radiation and chemotherapyinduced mucositis in animal models. The purpose of the study reported here is to investigate the effect of recombinant human KGF on the proliferation and survival of human squamous carcinoma cell lines following irradiation. Methods and Materials: The level of KGF receptor (KGFR) mRNA in normal Balb/Mk cell line and human head and neck squamous carcinoma cell lines was assessed using a RNase protection assay. The clonogenic assay and MTT assay were used to study the proliferative effects of KGF on human tumor cell lines and Balb/MK cell line in vitro. Effects of KGF on in vivo tumor growth and radiosensitivity were studied in three KGFR-positive human squamous cell carcinoma xenografts (FaDu, Detroit 562 and A431) in nude mice, and a murine KGFR-negative melanoma tumor (B16) in Balb/c mice. Results: Seven of 10 tumor cell lines studied expressed KGFR mRNA. None of these tumor cell lines showed enhanced proliferation when exposed to KGF for 2 days or less. Prolonged exposure to KGF for 7 days or longer resulted in low level stimulation of proliferation in both clonogenic and MTT assays in four of seven KGFRpositive cell lines. Two KGFR-negative cell lines also had a low proliferative response to KGF in a clonogenic assay, but not in the MTT assay. Normal keratinocyte Balb/MK cells, which expressed a moderate level of KGFR mRNA, had a strongly proliferative response to KGF. Its KGF enhancement ratio (KER) of plating efficiency was 24 –70 times higher than that of the tumor cells studied (p < 0.001). The KGF-stimulated tumor cell growth was almost completely inhibited by heparin or epidermal growth factor (EGF). There were no significant differences (p > 0.05) in the survival of any of tumor cell lines in the presence or absence of KGF (100 ng/ml) irradiated with doses of 0 –15 Gy, and no significant differences (p > 0.05) between the radiobiological parameters D0, Dq, and n number from the SHMT model, a, b, and a/b ratio from the LQ model and SF2 for radiation survival curves for cell lines irradiated in the presence or absence of KGF. Three KGFR-positive human squamous cell carcinoma xenografts in nude mice, and a murine KGFR-negative melanoma tumor in Balb/c mice treated with 1.0 mg/kg of KGF for 3 days grew at the same rate as in untreated mice. Conclusion: The recombinant human KGF resulted in little or no stimulation of the proliferation of human head and neck squamous tumor cell lines and did not affect the radiosensitivity of these cell lines in vitro and in vivo. Therefore, KGF may be of clinical value in preventing radiation-induced mucositis and may have the potential to increase the therapeutic index of radiotherapy for treatment of cancers. © 1998 Elsevier Science Inc. Keratinocyte growth factor, KGF, KGFR, EGF, Radiosensitivity, Squamous carcinoma cells, Human tumor xenograft.
INTRODUCTION Keratinocyte growth factor (KGF), a member of the heparin-binding fibroblast growth factor (FGF) family (FGF-7), was originally purified from conditioned medium of a human embryonic lung fibroblast line, M426 (20). In addition to lung fibroblasts, KGF is also produced by mesenchymal cells from a variety of other tissues, including the human
gastrointestinal tract, bladder, prostate, mammary gland and skin in vitro (21). KGF has potent mitogenic activity on epithelial cell types (2, 11, 18, 24, 25, 27), but has no detectable effects on fibroblasts, endothelial cells, and other nonepithelial cells that respond to other FGF family members (1, 10), suggesting that KGF may be a specific paracrine mediator for normal epithelial growth and differentiation (10). The KGF receptor (KGFR) is a transmembrane
Reprint requests to: Susan J. Knox, Ph.D., M.D., Stanford University Medical Center, Department of Radiation Oncology (A-093), 300 Pasteur Dr., Stanford, CA 94305-5105. Acknowledgments—This work was supported in part by the NIH
Grant CA56464, a grant from Amgen Inc., and a Lazard Faculty Scholarship. We thank Sheila Scully of Amgen for her excellent help in the preparation of the manuscript. Accepted for publication 3 July 1997. 177
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tyrosine kinase that is a splice variant of the FGFR-2/BEK gene (16). The KGFR is expressed only in epithelial cells and binds KGF and aFGF with high affinity and bFGF at a lower affinity. KGFR mRNA has been detected in normal skin keratinocytes, epithelial cells of the oral cavity and entire gastrointestinal tract (11, 15), mammary epithelium (24), type II pneumocytes (25), and hepatocytes (11). In addition to its mitogenic effects on normal epithelial cell proliferation and differentiation, KGF has been reported to stimulate DNA synthesis in esophageal cancer cells in vitro (12), induce carcinoembryonic antigen (CEA) production by colon carcinoma cells (11), and activate the androgen receptor in prostatic carcinoma cell lines (9). KGFR mRNA has also been detected in about 50% of human epithelial carcinoma cell lines in vitro (22, 23). This includes tumor cell lines from human breast, lung, esophagus, stomach, colon, pancreas, liver, prostate, kidney, bladder, and ovary (9, 12, 15, 22, 23, 26). Expression of KGFR on tumor cells may correlate inversely with the malignant potential of Dunning R3327PAP prostatic cancer cells (26). These in vitro data suggest that the epithelial-derived tumor cells may respond to paracrine sources of KGF in vivo, and exogenous KGF could potentially affect responses of epithelial tumor cells to cytotoxic therapy. However, little is known about the effects of exogenous KGF on the proliferation of the epithelial tumor cells and on the survival of irradiated tumor cells. Recently, KGF has been found to enhance intestinal crypt cell survival in irradiated mice (Radiation Research, in press) and to prevent radiation and chemotherapy-induced mucositis in animal models (David Lacey, Amgen Inc., unpublished data). In the study reported here, we used a variety of human head and neck squamous cell carcinoma cell lines that expressed KGFR mRNA and studied the effects of recombinant human KGF on the proliferation and survival of these cell lines in vitro and in vivo following irradiation. METHODS AND MATERIALS Growth factors The recombinant human KGF and recombinant mouse epidermal growth factor (EGF) were prepared and provided by Amgen Inc., (Thousand Oaks, CA). Both factors were stored at 270°C, and diluted in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NY) with 10% fetal calf serum before use unless otherwise specified. Cell lines The human tumor cell lines studied included four lingual squamous cell carcinoma cell lines, SCC-4, -9, -15, and -25; a pharyngeal squamous cell carcinoma cell line, FaDu; a pharyngeal epidermoid cell carcinoma cell line, Detroit 562; a laryngeal epidermoid cell carcinoma cell line, HEp-2; a nasal squamous cell carcinoma cell line, RPMI 2650; an oral epidermoid cell carcinoma cell line, KB, and a submaxillary gland epidermoid carcinoma cell line, A253. A normal keratinocyte cell line, Balb/MK, was used as a
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positive control (20). All the cell lines were purchased from American Type Culture Collection (Rockville, MD). The lingual carcinoma cell lines were maintained in DMEM and Ham’s F12 (1:1) mixed media (Gibco, Grand Island, NY) supplemented with 10% FCS and 0.4 mg/ml hydrocortisone (Sigma, St. Louis, MO) and split weekly. The other tumor cell lines were maintained in DMEM plus 10% FCS. In some experiments, tumor cells were cultured in the media containing only 1% FCS. The Balb/MK cell line was maintained in DMEM plus 10% FCS, 5 ng/ml EGF. Additionally, 100 U/ml penicillin and 0.5 mg/ml streptomycin (Gibco, Grand Island, NY) were added to all the media. All cultures were incubated at 37°C in 95% air and 5% CO2.
RNase protection assay for KGFR mRNA expression Total RNA was isolated from tumor cells and Balb/MK cells using the RNA STAT-60 kit (TEL-TEST ‘‘B,’’ Inc., Friendswood, TX). A 129-bp fragment corresponding to nucleotides 1358 –1487 of the human KGFR sequence (GenBank accession #M80634) was generated by PCR and cloned into pSP72 (Promega, Madison, WI) and a 104-bp fragment of the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence was cloned into pGEM3Z (Promega, Madison, WI). After verification by sequencing, the plasmids were linearized and radiolabeled antisense riboprobes were transcribed using the Riboprobe system (Promega, Madison, WI) and 32P-UTP (Amersham, Arlington Heights, IL). Full length probes were purified by electrophoretic separation on a 6% polyacrylamide/7 M urea gel. The RNase protection assay was performed using the RPA II kit (Ambion, Inc., Austin, TX). Briefly, 50 mg of total RNA from each sample was hybridized overnight at 45°C with 105 cpm each of the KGFR and GAPDH probes. Following RNase digestion, the protected fragments were separated on a 6% polyacrylamide/7 M urea gel. The gel was exposed to a phosphor screen and the signal volumes were integrated on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The levels of KGFR mRNA was expressed as the ratio of the KGFR and GAPDH signals.
MTT assay Normal Balb/MK cells and tumor cells, at a density of 5,000 cells/ml, were cultured in 96-well plates (Costar, Cambridge, MA) in 200 ml of DMEM media plus 10% FCS and KGF at a final concentration ranging from 0 to 400 ng/ml. After incubation for a desired period at 37°C, 10 ml of 5 mg/ml MTT was added to each well. Cells were incubated for another 3 h and supernatant was removed. The crystal formazan product was dissolved by adding 200 ml of DMSO (Sigma, St. Louis, MO) and shaking thoroughly for 10 min. The optical density (O.D.) at 562 nm and 650 nm (reference) was measured using a computer-assisted Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA).
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Radiation survival curves Radiation survival curves were generated for each cell line using a clonogenic assay with or without KGF. Tumor cells were cultured in 60-mm Petri dishes in complete DMEM (or 1:1 mixed with Ham’s F12) and 100 ng/ml KGF. After incubation for 48 h at 37°C, dishes were irradiated with 0 –15 Gy at a dose rate of 4.30 Gy/min using a 137 Cs irradiator at room temperature. Cells were immediately trypsinized, diluted with growth media supplemented with 10% FCS, and plated in Petri dishes. After incubation for 10 –12 days in the presence or absence of 100 ng/ml of KGF, dishes were stained and colonies containing $50 cells were counted and used to calculate the surviving fraction. The parameters of D0, Dq, and n number from a single-hit multitarget model (SHMT) and a, b, and a/b ratio from a linear-quadratic model (LQ) of cell survival curves were calculated using software FIT 2.03 program (generously provided by Norman W. Albright, University of California at San Francisco, CA) (3). Experiments were repeated at least twice.
Fig. 1. Relative levels of KGFR mRNA expression in normal Balb/MK cells and human squamous tumor cells. (A) Cells were incubated in growth media and total RNA was collected using the RNA STAT-60 kit. The expression of KGFR mRNA was measured by the RNase protection assays on triplicate 50 mg RNA samples using human KGFR and GAPDH probes. (B) The level of KGFR mRNA in Balb/MK and tumor cells was expressed as the ratio of the KGFR and GAPDH signals. Data is shown as the mean 6 SD.
Clonogenic assay Exponentially growing cells were detached with 0.05% trypsin, counted and plated in 60-mm Petri dishes (Becton Dickinson Labware, Franklin Lakes, NJ) at appropriate dilutions in complete DMEM (or 1:1 mixed with Ham’s F12) plus various doses of KGF ranging from 0 to 100 ng/ml. Three to five dishes were plated per dose point. To compare the proliferation-stimulating activity of KGF with EGF, tumor cells were cultured with 10 ng/ml EGF alone, 10 ng/ml KGF alone, or both EGF and KGF. Following exposure to growth factors, the media were removed, and dishes were rinsed twice with Hank’s solution and filled with fresh growth media. After incubation for 10 –12 days, cells were stained with 0.25% crystal violet. Colonies, defined as $50 cells, were counted. For some cell lines, the number of large colonies with a diameter $1.0 mm was also recorded. The plating efficiency (PE) was calculated as the percentage of cells plated that grow into colonies.
Mouse tumor models and tumor growth delay study Female nude mice were used for KGFR-positive human squamous cell carcinoma xenografts (FaDu, Detroit 562 and A431), and female Balb/c mice were used for KGFR-negative murine melanoma B16 tumors. All mice were 10 to 12 weeks old and ranged in weight between 25 and 30 g. Mice were obtained from the Stanford University Research Animal Facility. The mice were normally bred and maintained
Fig. 2. Effect of KGF on proliferation of Balb/MK cells measured by MTT assay showing O.D. as a function of KGF dose (0.1– 400 ng/ml). Cells were incubated in DMEM media plus KGF at 37°C for 4 days. Data are presented as the mean 6 SD of four replicate cultures. Experiments were repeated twice.
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Fig. 3. Effect of KGF on proliferation of tumor cells and Balb/MK cells in a clonogenic assay. Cells were incubated with KGF at 37°C for 10 days. Colonies containing more than 50 cells were counted. (A) Cloning efficiency (as the percent of control cultures without KGF) of tumor cell lines as a function of KGF concentration. (B) Comparison of the cloning efficiency of Balb/MK and tumor cells as a function of KGF concentration. The scale for the Y axis (cloning efficiency) ranges from 0 –500% for A and 0 –5000% for B. Data represent the mean 6 SD from two independent experiments.
under specific pathogen-free conditions, and sterilized food and water were available ad lib. Animals were injected intradermally (i.d.) in the left flank with 5 3 106 tumor cells in a suspension volume of 50 ml. One tumor per animal was implanted. Groups of mice with an average tumor size of 100 mm3 were treated with 1) NaCl solution (0.9%) as a control; 2) KGF 1.0 mg/kg s.c. for 3 consecutive days; 3) fractionated x-ray irradiation with 2.50 Gy daily for 5 consecutive days; 4) KGF 1.0 mg/kg s.c. for 3 consecutive days before irradiation and irradiation with 2.50 Gy daily for 5 consecutive days; and 5) fractionated irradiation for 5 days as above plus KGF for 3 consecutive days after radiation. Six to eight animals were used in each group. Mice were irradiated in a mouse back jig with a Philips RT-250 200 kVp X-ray unit (12.5 mA; Half Value Layer, 1.0-mm Cu). The length, width, and height (in mm) of the tumors were measured with calipers three times a week. Measurements were continued until the tumor volume reached four times (4 3) the original pretreatment volume. Tumor volume (mm3) was calculated according to the formula: tumor volume 5 p/6 3 length 3 width 3 height. The data are expressed as the mean tumor volume-quadrupling time (days) 6 standard deviation (SD), or as percent (%) of the pretreatment volume on day 0. Statistics Data were statistically analyzed using a two-tailed Student’s t-test.
RESULTS KGFR mRNA expression by human head and neck squamous carcinoma cell lines To determine whether human head and neck squamous tumor cells expressed KGFR mRNA, a RNase protection assay (RPA) was performed to quantitate the KGFR mRNA level in the cell lines studied. As shown in Fig. 1, KGFR mRNA expression was detected by RPA assay in 7 out of the 10 tumor cell lines. The level of KGFR mRNA was expressed as the ratio of KGFR and GAPDH signals and was in a range of 0.3–12.3 in these seven tumor cell lines. The KGFR/GAPDH signal ratio of KGFR mRNA in normal Balb/MK cells was 3.0. KGFR mRNA levels in HEp-2, SCC-15, and KB tumor cell lines were not detectable in the RPA assay. Effects of KGF on proliferation of Balb/MK cells Normal Balb/MK keratinocytes were cultured with concentrations of KGF ranging from 0.1 to 400 ng/ml for 4 days. Cell proliferation was measured by MTT and clonogenic assays. The MTT assay utilizes a colorimetric reaction secondary to succinate dehydrogenase activity in mitochondria, which is an indirect measure of cell viability and proliferation. Figure 2 shows the O.D. as a function of KGF concentration for Balb/MK cells. There was a linear-log relationship between O.D. and KGF dose at concentrations ranging from 0.4 ng/ml to 20 ng/ml, with the maximal effect of KGF occurring at concentrations of $20 ng/ml. In the
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Table 1. Characteristics of normal Balb/MK keratinocytes and epithelial tumor cell lines Cell lines
KGFR mRNA*
PEcontrol†
PEKGF‡
KER§
Balb/MK Detroit 562 FaDu SCC-9 SCC-25 SCC-4 A253 RPMI 2650 SCC-15 HEp-2 KB
1 1 1 1 1 1 1 1 2 2 2
0.9 6 0.1 6.4 6 0.8 21.5 6 1.3 20.0 6 1.8 17.5 6 4.9 19.1 6 1.2 23.3 6 0.7 17.7 6 2.6 6.0 6 0.6 19.4 6 5.9 57.1 6 1.3
33.6 6 3.9 15.2 6 0.8 34.5 6 17.1 33.5 6 3.3 29.1 6 10.6 21.2 6 4.8 22.7 6 1.2 19.2 6 2.5 15.2 6 2.5 29.5 6 19.9 58.2 6 1.1
36.30 1.38 0.60 0.68 0.66 0.11 20.03 0.08 1.53 0.52 0.02
* KGFR mRNA was measured by a RNase protection assay. Plating efficiency (%) of control cells: (number of colonies/ number of cells plated) 3 100. ‡ Plating efficiency (%) at KGF dose of 100 ng/ml for 10 days. § KGF enhancement ratio of PE at KGF dose of 100 ng/ml for 10 days: (PEKGF2PEcontrol)/PEcontrol. †
clonogenic assay, the plating efficiency (PE) was stimulated by adding KGF in growth media at a dose as low as 0.1 ng/ml (Fig. 3B) and the maximal stimulatory effect of KGF occurred at doses of $100 ng/ml. The KGF enhancement ratio (KER) of PE was 36.3 at a KGF dose of 100 ng/ml (Table 1). Proliferative effect of KGF on tumor cells The MTT and clonogenic assays were used to study the proliferation-stimulating effects of KGF on human head and neck squamous cell carcinoma cell lines. In the clonogenic assays, tumor cells were incubated with various concentrations of KGF ranging from 0 –1000 ng/ml. All the tumor cell lines studied showed no stimulated proliferation in response to KGF exposure of 2 days or less. However, exposure to KGF for 7 or more days did increase PE by KGFR-positive cell lines Detroit 562, FaDu, SCC-9, and SCC-25 in a dose-dependent manner (Fig. 3A). The KERs were 1.38 for Detroit 562, and 0.60 – 0.68 for FaDu, SCC-9, and SCC-25 in the presence of 100 ng/ml KGF for 10 days (Table 1). There was no further enhancement of PE by KGF at concentrations up to 1,000 ng/ml in these tumor cell lines. The KGFR-negative cell lines SCC-15 and HEp-2 also showed an increased PE when exposed to 100 ng/ml KGF for 10 –12 days (Table 1). However, the enhancement of PE by KGF on tumor cell lines was significantly less than that observed with normal Balb/MK cells (p , 0.001) (Fig. 3B). The PE of Balb/MK cells was increased 10-fold at the low concentration of KGF (0.1 ng/ml) and to 36-fold at the high concentrations of KGF (.100 ng/ml). KGF also increased the size of colonies in two (FaDu and SCC-25) of seven KGFR-positive cell lines studied. The number of large colonies (with a diameter of $1.0 mm) was increased from 26% in control cultures to 63% in cultures containing 10 ng/ml KGF for the FaDu cell line, and from 13% in control cultures to 42% in cultures containing 100 ng/ml KGF for
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the SCC-25 cell line, representing a 2.4 and 3.2 fold increase, respectively. KGFR-positive cell lines SCC-4, A253 and RPMI2650, and KGFR-negative cell line KB showed no stimulated proliferation in response to KGF treatment in both the clonogenic assay and MTT assay. Interestingly, KGF in media containing only 1% FCS supported the sustained growth and colony formation of tumor cell lines (such as KB cell line) that did not show any stimulated proliferation (KER, 0.02) in response to KGF in normal growth media containing 10% FCS (Fig. 4). The plating efficiency of KB cells in 1% FCS media without KGF decreased to 4.8% 6 1.0 from 57.1% 6 1.3 in media containing 10% FCS. These cells died quickly in the cultures with 1% FCS in the absence of KGF, suggesting that KGF is able to partially replace the serum requirement and support the sustained growth of these cell lines in vitro. In MTT assays, tumor cells were cultured with or without 20 ng/ml KGF for 4 days. The KGFR-positive Detroit 562, FaDu, SCC-9, and SCC-25 cell lines showed significantly stimulated proliferation in response to exogenously added KGF. The KGFR-negative HEp-2 and SCC-15 cell lines, which showed increased PE after stimulation with KGF for 10 –12 days, did not show a reproducible stimulatory response to a 4-day exposure to KGF in the MTT assay. Other KGFR-positive cell lines SCC-4, A253, and RPMI 2650 and KGFR-negative cell lines KB and HEp-2 failed to show
Fig. 4. Effect of KGF on the cloning efficiency of KB cells in 10 and 1% fetal calf serum. Cells were cultured in media containing 10 or 1% FCS with KGF at concentrations of 0.1–1000 ng/ml at 37°C for 10 days. Data are presented as the cloning efficiency (as the percent of control cultures without KGF). The plating efficiency was 57.1% 6 1.3 in normal media containing 10% FCS and 4.8% 6 1.0 in media with 1% FCS. Data represent the mean 6 SD from two independent experiments.
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Fig. 5. Inhibitory effect of heparin on KGF-induced tumor cell proliferation measured in a MTT assay. Cells were grown in normal media without KGF and heparin (open circles, E), with 20 ng/ml KGF (close diamonds, r), or with 20 ng/ml KGF and 0.5 U/ml heparin (close squares, ■). O.D. is shown as a function of exposure time (1–7 days) for SCC-9, SCC-25, FaDu, and Detroit 562 cell lines. Data are presented as mean 6 SD of four replicate cultures.
significant and consistent responses to KGF in similar MTT assays (data not shown). Because KGF is a heparin-binding molecule (20) and heparin has an inhibitory effect on the KGF-induced mitogenic activity on Balb/MK cells (19), we also tested the interaction of KGF and heparin on the tumor cell lines. In these studies, four KGFR-positive tumor cell
lines Detroit 562, FaDu, SCC-9, and SCC-25 were incubated with 20 ng/ml KGF plus 0.5 U/ml heparin for 1–7 days. Heparin alone did not show any effect on the tumor cell growth. When simultaneously used with KGF, heparin almost completely blocked the KGF-stimulated cell growth of these KGFR-positive tumor cell lines (Fig. 5).
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KGF overlapped each other. The surviving fractions at 2 Gy (SF2) for these four cell lines ranged from 0.35 to 0.66 (Table 2). There were no significant differences in the survival of any of the tumor cell lines studied that were incubated in the presence or absence of KGF at any of the radiation doses utilized. When compared, the radiobiological parameters of survival curves analyzed using the LQ model (a, b, and a/b ratio) and the SHMT model (D0, Dq, and n number), there were also no significant differences (p . 0.05) between any of these parameters for most of the cell lines irradiated and cultured in the presence or absence of KGF (Table 2). The a/b ratios for SCC-25 and Detroit 562 cell lines increased following exposed to 100 ng/ml KGF for 12 days compared to radiation alone, while the a/b ratios for SCC-9 and HEp-2 cell lines decreased. However, there were no significant differences (p . 0.05) between any other parameters for these cell lines irradiated in the presence or absence of KGF for 12 days.
Fig. 6. Differential effects of KGF and EGF on colony formation of normal Balb/MK and tumor cells. Cells were cultured in media with or without KGF and EGF at a dose of 10 ng/ml at 37°C for 10 days. Data are presented as mean 6 SD from five replicate cultures.
Differential effects of KGF and EGF on colony formation Because epidermal growth factor (EGF) also stimulates epithelial cell growth (7), the potentially synergetic action of KGF and EGF was studied in a clonogenic assay in the normal Balb/MK cell line and the KGFR-positive (Detroit 562, A253, SCC-25) and KGFR-negative (KB and HEp-2) tumor cell lines. Cells were incubated for 10 days in media with or without 10 ng/ml KGF alone, 10 ng/ml EGF alone, or with the combination of the both growth factors. As shown in Fig. 6, KGF at 10 ng/ml significantly increased the colony formation by Detroit 562 and SCC-25 tumor cells (p 5 0.002), but did not affect colony formation by A253, HEp-2, and KB tumor cell lines (p . 0.05). EGF significantly inhibited colony formation by these tumor cell lines (p # 0.002), even in the presence of KGF. Normal Balb/MK keratinocytes showed a strong response to EGF as well as to KGF in the colony-forming assay. But the combined use of KGF and EGF was not able to produce the synergetic stimulation of the colony formation. Effects of KGF on the radiation survival of tumor cells The effect of KGF on the survival of human head and neck squamous cell carcinoma cell lines following irradiation was assessed using clonogenic assays. Cells were plated in culture media with or without 100 ng/ml of KGF and irradiated with 0 –15 Gy using a 137Cs source. Figure 7 shows the radiation survival curves for four KGFR-positive tumor cell lines that responded to KGF stimulation in the absence of irradiation (Fig. 2 and Table 1). As can be seen, the radiation survival curves in the absence and presence of
Effects of KGF on in vivo tumor growth and radiosensitivity Three KGFR-positive human squamous cell carcinoma xenografts (FaDu, Detroit 562, and A431) in nude mice, and a murine KGFR-negative melanoma tumor (B16) in Balb/c mice were studied. Mice were treated with KGF for 3 consecutive days alone, fractionated x-ray irradiation for 5 consecutive days alone, or in combination with KGF either before or after radiation. This treatment regimen was utilized because our previous study demonstrated that administration of 1.0 mg/kg KGF for 3 consecutive days before irradiation was effective in stimulating the intestinal stem cell proliferation (Radiation Research, in press). A representative tumor growth curve for FaDu tumor xenografts is shown in Fig. 8. There were no statistically significant differences in tumor growth between untreated mice and mice treated with KGF alone, or between groups of mice treated with radiation alone and radiation with KGF (p . 0.05). The tumor regrowth delay data were analyzed in terms of tumor volume-quadrupling time (TVQD), and again, KGF did not significantly affect the TVQD of either irradiated or unirradiated tumors (p . 0.05). Similar results were obtained for the other three cell lines studied in vivo (Table 3). DISCUSSION The primary objective of the experiments reported here was to study the potential effect of recombinant human KGF on the proliferation and survival of irradiated human squamous cell carcinoma cell lines. KGF had a minimal proliferation-stimulating effect of 7 out of 10 tumor cell lines studied in vitro when incubated with KGF for $7 days, but no significant stimulatory effect on cell lines exposed to KGF for #2 days, even on cell lines that expressed a relatively high level of KGFR. To compare the radiosensitivity of human tumor cell lines in the absence and presence of KGF, we generated the radiation survival
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Fig. 7. Effect of KGF on the survival of tumor cell lines following gamma irradiation in a clonogenic assay. The survival fraction is shown as a function of radiation dose (Gy). Cells were irradiated in the absence of KGF (open circles, E), preincubated with 100 ng/ml KGF for 2 days and irradiated (close squares, ■), or preincubated with 100 ng/ml KGF for 2 days, irradiated and continued in culture for another 10 days in the presence of KGF (close diamonds, r). Colonies were counted on day 10. Data are from representative experiments. The radiobiological parameters calculated using the SHMT and LQ models are shown in Table 2.
curves using a clonogenic assay and analyzed these survival curves with two mathematical models, the LQ and SHMT models. All of the survival curves were well fitted by these two models. In general, the radiobiological parameters of the tumor cell lines did not change significantly in the presence of KGF. However, following exposure to KGF for
12 days, the a/b ratios were increased for Detroit 562 and SCC-25 and decreased for SCC-9 and HEp-2 compared to cells similarly irradiated but incubated without KGF. Nevertheless, there were not any other significant differences between any of the other radiobiological parameters studied. Therefore, when all of the data is considered, it appears
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Table 2. Radiobiological parameters of survival curves for tumor cells irradiated in presence or absence of KGF LQ†
SHMT* Cell line SCC-4 SCC-9 SCC-15 SCC-25 FaDu Detroit562 A253 HEp-2 RPMI 2650 KB
Treatment Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF Rad KGF KGF
2d 1 Rad§ 12d 1 Rad¶ 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad 2d 1 Rad 12d 1 Rad
D0
Dq
n
a
b
a/b
SF2‡
1.02 1.01 1.05 1.41 1.41 1.39 0.90 1.17 1.00 1.05 1.08 1.08 1.40 1.37 1.31 1.37 1.36 1.43 1.46 1.49 1.46 1.08 1.09 1.11 1.47 1.44 1.44 1.36 1.45 1.36
2.08 2.14 1.86 2.34 2.30 2.60 1.84 1.45 1.99 1.61 1.45 1.62 2.82 2.38 2.56 2.64 2.53 1.90 2.47 2.36 2.33 1.51 1.61 1.92 1.04 1.19 1.13 2.17 1.68 1.94
7.69 8.41 5.86 5.23 5.10 6.45 7.74 3.47 7.30 4.64 3.81 4.51 7.50 5.65 7.09 6.93 6.41 3.75 5.44 4.89 4.92 4.05 4.40 5.66 2.04 2.29 2.19 4.94 3.20 4.16
0.30 0.29 0.34 0.30 0.32 0.20 0.03 0.17 0.02 0.37 0.48 0.42 0.13 0.16 0.20 0.26 0.29 0.34 0.24 0.24 0.26 0.44 0.39 0.30 0.51 0.48 0.51 0.31 0.35 0.34
0.05 0.05 0.05 0.02 0.02 0.03 0.14 0.09 0.12 0.05 0.03 0.04 0.04 0.04 0.04 0.03 0.03 0.02 0.03 0.03 0.03 0.04 0.04 0.05 0.01 0.01 0.01 0.03 0.02 0.03
6.0 5.8 6.8 15.0 16.0 6.7 0.21 1.9 0.17 7.4 16.0 10.5 3.3 4.0 5.0 8.7 9.7 15.1 8.0 8.0 8.7 11.0 9.8 6.0 51.0 48.0 51.0 10.3 14.8 11.3
0.45 0.46 0.41 0.51 0.49 0.59 0.54 0.50 0.59 0.39 0.34 0.37 0.66 0.62 0.57 0.53 0.50 0.57 0.55 0.55 0.53 0.35 0.39 0.45 0.35 0.37 0.35 0.48 0.46 0.45
* The single-hit multitarget model. D0 is the dose (Gy) required to reduce the fraction of surviving cells to 37% of its previous value in the exponential region of the survival curves. Dq is the quasi-threshold dose (Gy) to measure the shoulder width of survival curves. † The linear quadratic model. ‡ Surviving fraction at 2 Gy. § Cells were exposed to KGF at a dose of 100 ng/ml for 2 days before radiation. ¶ Cells were exposed to KGF for 2 days before radiation and continued KGF exposure for 10 days postradiation. The KGF concentration was 100 ng/ml in DMEM supplemented with 10% FCS.
that the KGF did not alter the in vitro radiosensitivity of the human tumor cell lines studied. Importantly, in vivo KGF neither stimulated tumor growth nor changed the radiation sensitivity of KGFR-positive human tumor xenografts in nude mice. At the same concentration of KGF, the normal epithelial cell line Balb/MK had a KGF enhancement ratio of PE 24 –70 times that of the tumor cells (p , 0.001). The differential effect of KGF on the proliferation of normal epithelial cells and tumor cells may simply be due to the high affinity of receptors on Balb/MK cells (1, 5). Alternatively, it is also possible that there are functional differences between KGFRs and/or associated signal transduction pathways in normal and malignant cells. Heparin at a nontoxic level of 0.5 U/ml completely blocked the stimulatory effect of KGF on tumor cell proliferation, presumably because of partially overlapping binding sites of heparin and KGFR on KGF molecules (6). Interestingly, a subset of KGFR mRNA-positive cell lines did not show the stimulated proliferation in response to
Fig. 8. Effects of KGF on FaDu tumor growth in nude mice. Tumorbearing nude mice (eight mice per group) were treated with normal saline (open circles, E); KGF 1 mg/kg for 3 days alone (open squares, h); radiation 2.50 Gy daily for 5 days alone (close circles, F); KGF for 3 days followed by 5 days radiation (close diamonds, }); or radiation for 5 days followed by KGF for 3 days (close squares, ■). Radiation was given from days 0–4. The data are expressed as the mean percent (%) of the pretreatment volume on day 0 6 SD.
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Table 3. Effects of KGF on tumor growth and tumor regrowth delay following irradiation of KGFR-positive and KGFR-negative tumors Tumor volume-quadrupling time (days, mean 6 SD) Treatment
FaDu
A431
Detroit 562
B16
Untreated control KGF alone Radiation alone KGF 1 Radiation* Radiation 1 KGF†
15.2 6 1.7 14.2 6 1.2 47.9 6 3.9 42.8 6 1.6 47.1 6 3.7
9.0 6 0.4 20.0 6 1.0 23.0 6 0.6 22.0 6 0.8 25.0 6 0.5
13.9 6 3.8 13.7 6 5.1 44.7 6 12.4 44.0 6 13.2 52.4 6 8.6
17.0 6 0.4 19.0 6 0.8 35.0 6 1.1 34.0 6 1.2 36.0 6 0.9
* Mice were given 1.0 mg/kg KGF 2 days pre-, 1 day pre- and 2 h preradiation. Mice were given 1.0 mg/kg KGF at 2 h, 1 day, and 2 days after last fraction of irradiation.
†
KGF, whereas there was some enhanced proliferation of KGFR mRNA-negative cell lines (HEp-2, SCC-15 and KB) following addition of KGF to the media. Presumably, these three tumor cell lines did express KGFR mRNA but at a level too low to be detected by the RNase protection assay. Unfortunately, it was not possible to quantitate KGFR expression using a Western blot analysis due to the currently unavailability of the specific antibodies directed to the KGFR, so it was not possible to correlate levels of KGFR expression and proliferative responsiveness to KGF. The apparent discrepancies between KGFR mRNA expression and KGF-stimulated proliferative responses might be explained by a difference in receptor affinity or function, or by a poor correlation between mRNA and receptor levels. Epidermal growth factor (EGF) is a potent mitogen for epithelial cell growth and acts via its receptor (EGFR), an intrinsic membrane glycoprotein with an extracellular binding domain. It is widely distributed in human normal tissues and epithelial-derived tumor cells. EGF and its receptor are believed to play an important role in the development and growth of some tumor cells (7). In this study, EGF consistently inhibited colony formation in all the tumor cell lines tested, whereas it stimulated the clonogenic efficiency of
normal Balb/MK cells. When combined with KGF, EGF abolished the stimulatory effect of KGF on tumor cell proliferation. The reason for this is unclear. However, EGF has been reported to enhance the sensitivity of human epithelial tumor cells to irradiation (14) as well as to some chemotherapeutic agents in vitro (8, 13) and in vivo (4, 17). Radiation-induced mucositis is common during and after local irradiation of the head and neck and gastrointestinal tract, and can be a dose limiting toxicity. We have recently reported that KGF enhances the survival of irradiated intestinal crypt cells in mice (Radiation Research, in press). KGF has also been reported by others to prevent or minimize radiation and chemotherapy-induced mucositis in a variety of animal models (Amgen, Inc., unpublished data). We have reported here that recombinant human KGF resulted in little or no stimulation of the proliferation of human head and neck squamous tumor cell lines and did not affect the radiosensitivity of these cell lines in vitro and in vivo. Therefore, KGF may be of clinical value in preventing radiation-induced mucositis and may have the potential to increase the therapeutic index of radiotherapy for treatment of head and neck, esophageal, abdominal, and pelvic cancers.
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