Ann Dtol Rhinal LaryngollOl:1992
HETEROGENEITY IN EPIDERMAL GROWTH FACTOR RESPONSIVENESS AND TUMOR GROWTH OF HUMAN MAXILLARY CANCER CELL LINES SOHTARO KOMIYAMA, MD
KATSUKO MATSUI, MS
HIROSHI MIYAZAKI, MD
FUKUOKA, JAPAN
FUKUOKA, JAPAN
FUKUOKA, JAPAN
SHOHJI KUDOH, MD
HIROMOTO MIZOGUCHI, MD
NOBUYOSHI SHIMIZU, PHD
FUKUOKA, JAPAN
Orrx, JAPAN
TOKYO, JAPAN
We have established three cell lines (IMC-2, IMC-3, and IMC-4) from a human maxillary tumor, which exhibited different sensitivities to epidermal growth factor (EGF). It was inhibitory to colony-forming abilities of IMC-3 and IMC-4 cells in culture, while it affected that of IMC-2 cells slightly if at all. The differential sensitivities to EGF among the three cell lines were reproducibly observed when several cell sublines were further established from tumors appearing in nude mice. Saturation-binding kinetics with 125I_EGF showed similar levels of EGF-binding activities among the three cell lines. However, IMC-2, IMC-3, and IMC-4 showed almost similar sensitivities to cisplatin. Autophosphorylation of EGF receptor in the presence of EGF proceeded at similar levels among the three cell lines. Tumor growth was followed in nude mice when IMC-2, IMC-3, and IMC-4 at 1 x 107 cells were inoculated. The IMC-2 tumors enlarged at much faster rates than the other two cell lines. The IMC-4 tumors showed very slow growth rates, and IMC-3 tumors enlarged at an intermediate rate. These data suggest that the maxillary tumor used comprised cell populations that differed in their growth behaviors in responseto EGF. KEY WORDS - differential growth rates, epidermal growth factor responsiveness, human maxillary cancer.
nated with Na 12S1 by the chrolamine T method as described previously. 16 An anticancer agent, cisplatin (cis-diamminedichloroplatinum), was obtained from Nihon Kayaku Company, Tokyo, Japan.
INTRODUCTION
Epidermal growth factor (EGF) is important for many kinds of cells, including epidermal and epithelial cells. 1 It facilitates cell proliferation through interaction with the membrane-anchored glycoprotein receptor of molecular weight 170,000. 2 The EGF receptor gene is often amplified in human malignancies.":" An src family-related oncogene, v-erb B, is also known to encode a truncated form of EGF receptor.v':" Overexpression of the EGF receptor gene or erb B gene causes transformation of mouse NIH3T3 cells. 9 - 11 Expression of functional EGF receptor is also required for malignant transformation by viral transforming genes: papilloma virus E5 12 and polyoma middle T antigen or v-src. 13 Both EGF and EGF receptor are expected to be closely involved in malignant transformation as well as tumor growth.
Cell Culture. Cells derived from a human solid tumor were grown in complete growth medium containing RPMI 1640 medium (Gibco Laboratory, Grand Island, New York), supplemented with 100/0 heat-inactivated fetal calf serum (Gibco), 100 U/mL of penicillin G, 100 p,g/mL of streptomycin, and 100 JLg/mL of kanamycin." Establishment of Cell Lines From Maxillary Cancer Patient. The patient was a 48-year-old man with a maxillary tumor (T3NOMO). The tumor was histologically well-differentiated squamous cell carcinoma. A tumor biopsy was performed before chemotherapy and irradiation and the specimen was cut into small pieces and subcutaneously transferred to nude mice. After 2 months, the tumor in the mice was extirpated and cultured in plastic flasks containing complete growth medium. To avoid contamination by mouse cells, the culture was performed in the presence of anti-nude mouse cell antiserum. 14.15
We have previously established tumor cell lines from a patient with maxillary cancer, and these cell lines showed various levels of drug sensitivity to anticancer agents.P:" In this study, we explore the possibility that a tumor comprises cell populations that differ in their growth rates. We established cell sublines from a tumor derived from a patient with maxillary cancer and examined the expression of EGF receptor in correlation with growth behaviors in vivo as well as in vitro in response to EGF.
To establish several purified cell lines from the cultured tumor cells, 1 x 103 to 2 X 103 cells of the tumor were inoculated into 0.30/0 soft agar, and colonies appeared after incubation for 5 to 7 weeks at 37°C. About 10 colonies were independently isolated and each colony was grown in monolayer culture in a Petri dish. An independent colony being formed was purified from each Petri dish, and final-
MATERIALS AND METHODS
Materials. The EGF was obtained from Toyobo Corporation, Osaka, Japan. The 12sI_EGF was iodi-
From the Department of Otorhinolaryngology, Faculty of Medicine. Kyushu University, Fukuoka (Korniyama, Matsui, Miyazaki, Kudoh), the Department of Biochemistry. Oita Medical School. Oita (Mizoguchi), and the Department of Molecular Biology, Keio University School of Medicine, Tokyo (Shimizu). Japan. REPRINTS - Sohtaro Komiyama, MD. Dept of Otorhinolaryngology. Faculty of Medicine, Kyushu University, Fukuoka 812, Japan.
519
Downloaded from aor.sagepub.com by guest on January 16, 2015
Komiyama et al, Epidermal Growth Factor
520
TABLE 1. MAXILLARY CANCER CELL LINES ESTABLISHED C ell Lines
Original Cell Line
IMC-2 IMC-2-1 IMC~2~3
IMC-3 IMC-3-5 IMC-4 IMC-4-5 IMC-4-6
IMC-3 IMC-4 IMC-4
80
Derivations Maxillary Tumor at Tumor in Maxillary Tumor at Maxillary Tumor at Tumor in
tumor inoculated site ascites tumor inoculated site tumor inoculated site bone marrow
ly we obtained three cell lines: IMC-2, IMC-3, and IMC-4. The three cell lines were all found to show typical human karyotypes with hyperdiploid chromosomes. Cell lines IMC-2, IMC-3, and IMC-4 showed average chromosome numbers of 73± 11, 57 ±4, and 56±5, respectively, when 29 to 40 metaphase chromosomes were analyzed. Determination of tumor growth in vivo in nude mice was carried out when all three cell lines were inoculated in nude mice according to a previous report. 17 Each tumor cell line was inoculated at 1 x 107. Binding Assays. Human cell lines were seeded in 24-well plates and grown to a near-confluent state. For the 125I-EGF binding assay, cells were washed twice with phosphate-buffered saline (PBS) and then incubated with 500 p.g of serum-free minimum essential medium (MEM) containing 20 mmol/L HEPES, 5 mg/mL bovine serum albumin, and 1251_ EGF in the presence or absence of a hundredfold excess of EGF for 2 hours at 4 0 C. 16.18 Cells were then washed with PBS three times and lysed with 1 0/0 Triton X-I00. The lysates were measured by a gamma counter. Specific 12SI_EGF binding was determined as the difference between the counts in the absence and presence of excess unlabeled EGF. For down-regulation assaysfor EGF binding, cells grown to near confluency in 24-well plates were pretreated with 10 ng/mL of EGF for 0, 4, and 24 hours, respectively; the cells were washed with PBS twice and incubated at 37°C for 2 hours in the binding buffer. After washing with binding buffer twice, 50 nmol/L of 12sI-EGF in the binding buffer was added in the presence or absence of excess competitor to assess nonspecific binding. Receptor Phosphorylation. Confluent cells in 35-mm dishes were washed twice with phosphatefree MEM, then labeled with 32P-orthophosphate (2.5 mCi/mL, Amersham Corp, Arlington Heights, Ill) in phosphate-free MEM for 1 hour. Then EGF (10 ng/mL) was directly added to the medium at 0, 15, 30, and 60 minutes before lysis. After labeling, the EGF receptors were immunoprecipitated with EGF receptor monoclonal antibody (B4G7) and pur.. ified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described before. 19 Colony Formation Assay. To assay colony forma-
E
70
"'-
HETEROGENEITY IN EPIDERMAL GROWTH FACTOR RESPONSIVENESS AND TUMOR GROWTH OF HUMAN MAXILLARY CANCER CELL LINES SOHTARO KOMIYAMA, MD
KATSUKO MATSUI, MS
HIROSHI MIYAZAKI, MD
FUKUOKA, JAPAN
FUKUOKA, JAPAN
FUKUOKA, JAPAN
SHOHJI KUDOH, MD
HIROMOTO MIZOGUCHI, MD
NOBUYOSHI SHIMIZU, PHD
FUKUOKA, JAPAN
Orrx, JAPAN
TOKYO, JAPAN
We have established three cell lines (IMC-2, IMC-3, and IMC-4) from a human maxillary tumor, which exhibited different sensitivities to epidermal growth factor (EGF). It was inhibitory to colony-forming abilities of IMC-3 and IMC-4 cells in culture, while it affected that of IMC-2 cells slightly if at all. The differential sensitivities to EGF among the three cell lines were reproducibly observed when several cell sublines were further established from tumors appearing in nude mice. Saturation-binding kinetics with 125I_EGF showed similar levels of EGF-binding activities among the three cell lines. However, IMC-2, IMC-3, and IMC-4 showed almost similar sensitivities to cisplatin. Autophosphorylation of EGF receptor in the presence of EGF proceeded at similar levels among the three cell lines. Tumor growth was followed in nude mice when IMC-2, IMC-3, and IMC-4 at 1 x 107 cells were inoculated. The IMC-2 tumors enlarged at much faster rates than the other two cell lines. The IMC-4 tumors showed very slow growth rates, and IMC-3 tumors enlarged at an intermediate rate. These data suggest that the maxillary tumor used comprised cell populations that differed in their growth behaviors in responseto EGF. KEY WORDS - differential growth rates, epidermal growth factor responsiveness, human maxillary cancer.
nated with Na 12S1 by the chrolamine T method as described previously. 16 An anticancer agent, cisplatin (cis-diamminedichloroplatinum), was obtained from Nihon Kayaku Company, Tokyo, Japan.
INTRODUCTION
Epidermal growth factor (EGF) is important for many kinds of cells, including epidermal and epithelial cells. 1 It facilitates cell proliferation through interaction with the membrane-anchored glycoprotein receptor of molecular weight 170,000. 2 The EGF receptor gene is often amplified in human malignancies.":" An src family-related oncogene, v-erb B, is also known to encode a truncated form of EGF receptor.v':" Overexpression of the EGF receptor gene or erb B gene causes transformation of mouse NIH3T3 cells. 9 - 11 Expression of functional EGF receptor is also required for malignant transformation by viral transforming genes: papilloma virus E5 12 and polyoma middle T antigen or v-src. 13 Both EGF and EGF receptor are expected to be closely involved in malignant transformation as well as tumor growth.
Cell Culture. Cells derived from a human solid tumor were grown in complete growth medium containing RPMI 1640 medium (Gibco Laboratory, Grand Island, New York), supplemented with 100/0 heat-inactivated fetal calf serum (Gibco), 100 U/mL of penicillin G, 100 p,g/mL of streptomycin, and 100 JLg/mL of kanamycin." Establishment of Cell Lines From Maxillary Cancer Patient. The patient was a 48-year-old man with a maxillary tumor (T3NOMO). The tumor was histologically well-differentiated squamous cell carcinoma. A tumor biopsy was performed before chemotherapy and irradiation and the specimen was cut into small pieces and subcutaneously transferred to nude mice. After 2 months, the tumor in the mice was extirpated and cultured in plastic flasks containing complete growth medium. To avoid contamination by mouse cells, the culture was performed in the presence of anti-nude mouse cell antiserum. 14.15
We have previously established tumor cell lines from a patient with maxillary cancer, and these cell lines showed various levels of drug sensitivity to anticancer agents.P:" In this study, we explore the possibility that a tumor comprises cell populations that differ in their growth rates. We established cell sublines from a tumor derived from a patient with maxillary cancer and examined the expression of EGF receptor in correlation with growth behaviors in vivo as well as in vitro in response to EGF.
To establish several purified cell lines from the cultured tumor cells, 1 x 103 to 2 X 103 cells of the tumor were inoculated into 0.30/0 soft agar, and colonies appeared after incubation for 5 to 7 weeks at 37°C. About 10 colonies were independently isolated and each colony was grown in monolayer culture in a Petri dish. An independent colony being formed was purified from each Petri dish, and final-
MATERIALS AND METHODS
Materials. The EGF was obtained from Toyobo Corporation, Osaka, Japan. The 12sI_EGF was iodi-
From the Department of Otorhinolaryngology, Faculty of Medicine. Kyushu University, Fukuoka (Korniyama, Matsui, Miyazaki, Kudoh), the Department of Biochemistry. Oita Medical School. Oita (Mizoguchi), and the Department of Molecular Biology, Keio University School of Medicine, Tokyo (Shimizu). Japan. REPRINTS - Sohtaro Komiyama, MD. Dept of Otorhinolaryngology. Faculty of Medicine, Kyushu University, Fukuoka 812, Japan.
519
Downloaded from aor.sagepub.com by guest on January 16, 2015
Komiyama et al, Epidermal Growth Factor
520
TABLE 1. MAXILLARY CANCER CELL LINES ESTABLISHED C ell Lines
Original Cell Line
IMC-2 IMC-2-1 IMC~2~3
IMC-3 IMC-3-5 IMC-4 IMC-4-5 IMC-4-6
IMC-3 IMC-4 IMC-4
80
Derivations Maxillary Tumor at Tumor in Maxillary Tumor at Maxillary Tumor at Tumor in
tumor inoculated site ascites tumor inoculated site tumor inoculated site bone marrow
ly we obtained three cell lines: IMC-2, IMC-3, and IMC-4. The three cell lines were all found to show typical human karyotypes with hyperdiploid chromosomes. Cell lines IMC-2, IMC-3, and IMC-4 showed average chromosome numbers of 73± 11, 57 ±4, and 56±5, respectively, when 29 to 40 metaphase chromosomes were analyzed. Determination of tumor growth in vivo in nude mice was carried out when all three cell lines were inoculated in nude mice according to a previous report. 17 Each tumor cell line was inoculated at 1 x 107. Binding Assays. Human cell lines were seeded in 24-well plates and grown to a near-confluent state. For the 125I-EGF binding assay, cells were washed twice with phosphate-buffered saline (PBS) and then incubated with 500 p.g of serum-free minimum essential medium (MEM) containing 20 mmol/L HEPES, 5 mg/mL bovine serum albumin, and 1251_ EGF in the presence or absence of a hundredfold excess of EGF for 2 hours at 4 0 C. 16.18 Cells were then washed with PBS three times and lysed with 1 0/0 Triton X-I00. The lysates were measured by a gamma counter. Specific 12SI_EGF binding was determined as the difference between the counts in the absence and presence of excess unlabeled EGF. For down-regulation assaysfor EGF binding, cells grown to near confluency in 24-well plates were pretreated with 10 ng/mL of EGF for 0, 4, and 24 hours, respectively; the cells were washed with PBS twice and incubated at 37°C for 2 hours in the binding buffer. After washing with binding buffer twice, 50 nmol/L of 12sI-EGF in the binding buffer was added in the presence or absence of excess competitor to assess nonspecific binding. Receptor Phosphorylation. Confluent cells in 35-mm dishes were washed twice with phosphatefree MEM, then labeled with 32P-orthophosphate (2.5 mCi/mL, Amersham Corp, Arlington Heights, Ill) in phosphate-free MEM for 1 hour. Then EGF (10 ng/mL) was directly added to the medium at 0, 15, 30, and 60 minutes before lysis. After labeling, the EGF receptors were immunoprecipitated with EGF receptor monoclonal antibody (B4G7) and pur.. ified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described before. 19 Colony Formation Assay. To assay colony forma-
E
70
"'-
