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Biological role of epidermal growth factor (EGF) receptor gene amplification in human squamous cell carcinoma
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Biological role of eidermal growth factor (EGF) receptor gene amplification in human squamous cell carcinoma II. Differential effects of EGF on the anchorage-dependent, -independent cell growth and on the cell adherence to fibronectin and collagen substrata between the parental and variant HSC-1 cells Yukiya Maruguchi, Kimio Fujii, Ken-ichi Toda and Sadao Imamura Department of Oermatology, Kyoto University In monolayer culture, EGF inhibited the anchorage-dependent growth of the parental HSC-1 cells, while it unaffected or even stimulated the growth of the variant cells. In soft agar culture, however, EGF stimulated the anchorage-independent growth of the parental cells and showed no growth stimulatory effect on the variant cells. Such growth-stimulatory effects of EGF in soft agar culture of the HSC-1 cells prompted us to examine if EGF showed any effects on the cell adherence to extracellular matrix such as fibronectin and collagen in these cells. After the cells in monolayer culture were treated with EGF. the cell adherence YIS determined by cell adhesion assay. EGF above 0.1 ng/ml reduced the cell attachment to fibronectin substrata, and the cell spreading to fibronectin and collagen substrata in the parental cells. On the other hand, EGF hardly affected the cell adherence to these matrix protein in the variant cells. Such differential zffects of EI?Fon cell adherence may be related with the difference in the effects of EGF on the anchorage-dependent and -independent growth between parental and the variant HSC-1 Cells.
A SENSITIZING EFFECTS OF HEMATOPORPHYRIN OLIGOMER AND CAFFEINE FOR THE DNA DAMADE IRRADIATED BY X-RAY ON EXPERIMENTAL SKIN TUMORS K. Ishiguro, H. Ishida, 5. Nakagawa, K. Kawahara, K. &da, N. Miyoshi" and M. Fukuda' Department of Dermatology, *Department of Pathology, Fukui Medical School, Fukui, Japan Hematoporphyrin aligomer(Hp0) was administered before X-ray lrradiation of transplantable skin tumor in C3H mouse. Caffeine was to be given the mouse after X-ray radiation. The growth of the tumor irradiated with HpO and/or caffeine was more inhibited and the necrotic area was more expanded than that of the tumor irradiated X-ray only. With the use of color image analyzer, widespread bleeding was observed in the tumor irradiated with HpO and/or caffeine. Labeling index utilizing bromodeoxyuridine(BrdU) in the viable area of the treated tumor decreased when HpO and/or caffeine were administered. Furthermore. the amount of DNA damage more increased in the tumor cells irradiated with HpO than that irradiated only. And, the repair of the damaged DNA was mow delayed in the tumor cells irradiated wth HpO and caffeine than that irradiated with the other treatments.
INTBRLBUKIN 1 ALPHA AND BETA IN SQUAWUS CELL BPITmLIoMA.
CELL CARCINOEU AND BASAL
Kava. S. Furusavs, K. Ohm*. H. Takabama. S. Watanabe and M. Mizoguchi Depgrment of Dermatology. Teikyo University School of Medicine, Tokyo and Toranom"" Hospital. Tokyo, Japan
Y.
Biological role of epidermal growth factor (EGF) receptor gene amplification in human squamous cell carcinoma I. Isolation of EGF-resistant variants from HSC-1 human squamous carcinoma cells; characterization of their EGF receptor gene and their expression Naoko Dosaka, Kimio Fujii. Yukiya Maruguchi, Kouichi Ikri and Sadao Imamura Department of Dermatology, Faculty of Medicine, KYoto University EGF shows potent growth-stimulatory effects for varieties of cells in cultwe on binding to its specific receptor. The awlification of the receptor gene and their overexpression has been reported in squamous cell carcinomas of various tissue origin and its relevance to the development of malignacy has been proposed. HOWeVer, its precise roles in oncogenesis have not been fully elucidated. HSC-1 cells are squamous carcinoma cells derived from human skin which have highly amplified EGF receptor gene. We tried to isolate variant clones which contain fewer number of EGF receptor gene copies than the parental cells so that the biological effects of EGF can be compared between the parental and the variant cells. We succeeded in developing several variants which showed much less degree of receptor gene amplification and thus, fewer number of receptor expression than the parental cells. Southern-blotting, Northern-blotting and 1251-EGF bindlng data are presented in this section.
Biological role of epidermal growth factor ~EGF) receptor gene amplification in human squamous cell carcinoma III. Role of amplified EGF receptor associated tyrosine kinase activity in the regulation of cell-extracellular matrix interaction Kimio Fujii, Vukiya Maruguchi, Naoko Dosaka and Sadao Imamura Department of Dermatology, Kyoto University EGF-induced reduction in cell adherence to fibronectin substrata was further clarified in the parental cells. The Cell adherence response to EGF was obvious as early as 2 min and came to be at the maximum at 30 min. Pretreatment of the cells with a protein synthesis inhibitor, cycloheximide, or simultaneous treatment with swine- or thiol-proteinase inhibitor did not affect the response, indicating that de now protein synthesis or activation of Proteinases is not reqzrmn the EGF-induced reduction of cell adherence. Although EGF induced the accumulation of cellular 1,2-diglyceride and the CaP+-dependent liberation of arachidonic acid into the medium in the parental HSC-1 cells, TPA and/or CaZ+ ionophore failed to mimic the effect of EGF in reducing cell adherence to fibronectin. These data suggest that the high activity of EGF receptor-associated tyrosine kinase is involved in the regulation of cell interaction with fibronectin substrata, thereby modulating the invasive and metastatic potential of squamous carcinoma ceils.
It ~89 reported that interleuki" 1 (IL-l) induced proliferaciue activity in cultured keratinocytes. To find out the function af epidermal IL-1 in squamous cell carcinoma (SCC) and basal cell epirhelioma (BCE) in viv". ski" biopsies from both tumors were examined by a" imunohistolagical technique, using polyclonal specific antibodies against human rIL-1 alpha and beta (kind gifts from Dr. Dinarello). The staining intensity with the anti-IL-1 alpha ~8s higher than that with the anti-IL-1 beta in the both tumors, rbough SCC showed higher staining i"te"sit9 than BCE. We also performed blocking of the staining with reconbinanc IL-1 (rIL-1). The staining with antiGIG alpha was strongly blocked with rIL-I alpha but that with anti-IL-1 beta ~8s on19 weakly blmked virh rIL-1 beta. Staining with the two antibodies ~a- predominantly found in the cytoplasm of both tumors. "e also examined IL-1 alpha snd beta activity of the lysate from both tumors bg neutralizing ability of anti-IL-1 alpha or beta on tbymacyte proliferation assay. Only I&l alpha activity was detected from these tumors, and the activit9 of SCC was much higher than that of BCE. However, we can't de"9 the existence of the precursor form of IL-l beta which can not be detected by the thymcyte proliferation assay. It is well know that SCC is more malignant and vigorously proliferative than BCE. however, the relationship between tumor pr"liferatio" and IGl in viva is unknown. The cytoplasmic component of SC2 is larger than that of BCE. which may be one of the reasons for the higher IL-1 activity and staining intensity in XC.
N. I&ii, H. *no. Y. Hortuchi, T. Nagatani and Ii. Narajima "f Dermatology, Yokahama City University School of Medicine, Yokohama. Japan
H. Isbii, Departolent
The mechanisms of eosinbphilia, which is associated with "eaplastlc disease and allergic or parasitic diseases. are still not clear. Recently, interleukin-5 (m-5) has bee" characterized as both a" eosinophil colony-stimularing factor and a" eosi"o@,ll differentiation factor. ‘7" the ocher hand, the ,xesence of various eosinophil chemocactic factors in serum of patients with hypereosinophilic syndrome and allergic disease has been shav". I" the present study, we demonstrated that IL-5, b"~ not IL-4 or G-CSF. has cllemotacticXtiYity for human easinophils. Furthermore, to elucidate whether there are functional differences in eosinophils between normal and eosi"o@,ilc patients, we examined eosinophil responsiveness to IL-5, eymosan-activated serun and PAT in normal volunteers, eosinophilic patients associated WiZh CUtaneDYS T cell lym@ma, eczematous dermatitis, Kimura's disease and hypereosinophilic syndrome using the modified Boyde" chamber method. Serum samples from rhese patients were i""esLigated t" have priming effects or deactivating effects on chemofactic agents. I" one patlent with cutaneous T cell lynphoma, a" extract of tumor tissue and supernaLant from cu1tvre* t"moT cells were tested for eosinopllil. chfmotaetic actiliify. The possible parent and setecrivr role of IL-5 *n human eosinopt,irsi5 discussed.
Biological role of eidermal growth factor (EGF) receptor gene amplification in human squamous cell carcinoma II. Differential effects of EGF on the anchorage-dependent, -independent cell growth and on the cell adherence to fibronectin and collagen substrata between the parental and variant HSC-1 cells Yukiya Maruguchi, Kimio Fujii, Ken-ichi Toda and Sadao Imamura Department of Oermatology, Kyoto University In monolayer culture, EGF inhibited the anchorage-dependent growth of the parental HSC-1 cells, while it unaffected or even stimulated the growth of the variant cells. In soft agar culture, however, EGF stimulated the anchorage-independent growth of the parental cells and showed no growth stimulatory effect on the variant cells. Such growth-stimulatory effects of EGF in soft agar culture of the HSC-1 cells prompted us to examine if EGF showed any effects on the cell adherence to extracellular matrix such as fibronectin and collagen in these cells. After the cells in monolayer culture were treated with EGF. the cell adherence YIS determined by cell adhesion assay. EGF above 0.1 ng/ml reduced the cell attachment to fibronectin substrata, and the cell spreading to fibronectin and collagen substrata in the parental cells. On the other hand, EGF hardly affected the cell adherence to these matrix protein in the variant cells. Such differential zffects of EI?Fon cell adherence may be related with the difference in the effects of EGF on the anchorage-dependent and -independent growth between parental and the variant HSC-1 Cells.
A SENSITIZING EFFECTS OF HEMATOPORPHYRIN OLIGOMER AND CAFFEINE FOR THE DNA DAMADE IRRADIATED BY X-RAY ON EXPERIMENTAL SKIN TUMORS K. Ishiguro, H. Ishida, 5. Nakagawa, K. Kawahara, K. &da, N. Miyoshi" and M. Fukuda' Department of Dermatology, *Department of Pathology, Fukui Medical School, Fukui, Japan Hematoporphyrin aligomer(Hp0) was administered before X-ray lrradiation of transplantable skin tumor in C3H mouse. Caffeine was to be given the mouse after X-ray radiation. The growth of the tumor irradiated with HpO and/or caffeine was more inhibited and the necrotic area was more expanded than that of the tumor irradiated X-ray only. With the use of color image analyzer, widespread bleeding was observed in the tumor irradiated with HpO and/or caffeine. Labeling index utilizing bromodeoxyuridine(BrdU) in the viable area of the treated tumor decreased when HpO and/or caffeine were administered. Furthermore. the amount of DNA damage more increased in the tumor cells irradiated with HpO than that irradiated only. And, the repair of the damaged DNA was mow delayed in the tumor cells irradiated wth HpO and caffeine than that irradiated with the other treatments.
INTBRLBUKIN 1 ALPHA AND BETA IN SQUAWUS CELL BPITmLIoMA.
CELL CARCINOEU AND BASAL
Kava. S. Furusavs, K. Ohm*. H. Takabama. S. Watanabe and M. Mizoguchi Depgrment of Dermatology. Teikyo University School of Medicine, Tokyo and Toranom"" Hospital. Tokyo, Japan
Y.
Biological role of epidermal growth factor (EGF) receptor gene amplification in human squamous cell carcinoma I. Isolation of EGF-resistant variants from HSC-1 human squamous carcinoma cells; characterization of their EGF receptor gene and their expression Naoko Dosaka, Kimio Fujii. Yukiya Maruguchi, Kouichi Ikri and Sadao Imamura Department of Dermatology, Faculty of Medicine, KYoto University EGF shows potent growth-stimulatory effects for varieties of cells in cultwe on binding to its specific receptor. The awlification of the receptor gene and their overexpression has been reported in squamous cell carcinomas of various tissue origin and its relevance to the development of malignacy has been proposed. HOWeVer, its precise roles in oncogenesis have not been fully elucidated. HSC-1 cells are squamous carcinoma cells derived from human skin which have highly amplified EGF receptor gene. We tried to isolate variant clones which contain fewer number of EGF receptor gene copies than the parental cells so that the biological effects of EGF can be compared between the parental and the variant cells. We succeeded in developing several variants which showed much less degree of receptor gene amplification and thus, fewer number of receptor expression than the parental cells. Southern-blotting, Northern-blotting and 1251-EGF bindlng data are presented in this section.
Biological role of epidermal growth factor ~EGF) receptor gene amplification in human squamous cell carcinoma III. Role of amplified EGF receptor associated tyrosine kinase activity in the regulation of cell-extracellular matrix interaction Kimio Fujii, Vukiya Maruguchi, Naoko Dosaka and Sadao Imamura Department of Dermatology, Kyoto University EGF-induced reduction in cell adherence to fibronectin substrata was further clarified in the parental cells. The Cell adherence response to EGF was obvious as early as 2 min and came to be at the maximum at 30 min. Pretreatment of the cells with a protein synthesis inhibitor, cycloheximide, or simultaneous treatment with swine- or thiol-proteinase inhibitor did not affect the response, indicating that de now protein synthesis or activation of Proteinases is not reqzrmn the EGF-induced reduction of cell adherence. Although EGF induced the accumulation of cellular 1,2-diglyceride and the CaP+-dependent liberation of arachidonic acid into the medium in the parental HSC-1 cells, TPA and/or CaZ+ ionophore failed to mimic the effect of EGF in reducing cell adherence to fibronectin. These data suggest that the high activity of EGF receptor-associated tyrosine kinase is involved in the regulation of cell interaction with fibronectin substrata, thereby modulating the invasive and metastatic potential of squamous carcinoma ceils.
It ~89 reported that interleuki" 1 (IL-l) induced proliferaciue activity in cultured keratinocytes. To find out the function af epidermal IL-1 in squamous cell carcinoma (SCC) and basal cell epirhelioma (BCE) in viv". ski" biopsies from both tumors were examined by a" imunohistolagical technique, using polyclonal specific antibodies against human rIL-1 alpha and beta (kind gifts from Dr. Dinarello). The staining intensity with the anti-IL-1 alpha ~8s higher than that with the anti-IL-1 beta in the both tumors, rbough SCC showed higher staining i"te"sit9 than BCE. We also performed blocking of the staining with reconbinanc IL-1 (rIL-1). The staining with antiGIG alpha was strongly blocked with rIL-I alpha but that with anti-IL-1 beta ~8s on19 weakly blmked virh rIL-1 beta. Staining with the two antibodies ~a- predominantly found in the cytoplasm of both tumors. "e also examined IL-1 alpha snd beta activity of the lysate from both tumors bg neutralizing ability of anti-IL-1 alpha or beta on tbymacyte proliferation assay. Only I&l alpha activity was detected from these tumors, and the activit9 of SCC was much higher than that of BCE. However, we can't de"9 the existence of the precursor form of IL-l beta which can not be detected by the thymcyte proliferation assay. It is well know that SCC is more malignant and vigorously proliferative than BCE. however, the relationship between tumor pr"liferatio" and IGl in viva is unknown. The cytoplasmic component of SC2 is larger than that of BCE. which may be one of the reasons for the higher IL-1 activity and staining intensity in XC.
N. I&ii, H. *no. Y. Hortuchi, T. Nagatani and Ii. Narajima "f Dermatology, Yokahama City University School of Medicine, Yokohama. Japan
H. Isbii, Departolent
The mechanisms of eosinbphilia, which is associated with "eaplastlc disease and allergic or parasitic diseases. are still not clear. Recently, interleukin-5 (m-5) has bee" characterized as both a" eosinophil colony-stimularing factor and a" eosi"o@,ll differentiation factor. ‘7" the ocher hand, the ,xesence of various eosinophil chemocactic factors in serum of patients with hypereosinophilic syndrome and allergic disease has been shav". I" the present study, we demonstrated that IL-5, b"~ not IL-4 or G-CSF. has cllemotacticXtiYity for human easinophils. Furthermore, to elucidate whether there are functional differences in eosinophils between normal and eosi"o@,ilc patients, we examined eosinophil responsiveness to IL-5, eymosan-activated serun and PAT in normal volunteers, eosinophilic patients associated WiZh CUtaneDYS T cell lym@ma, eczematous dermatitis, Kimura's disease and hypereosinophilic syndrome using the modified Boyde" chamber method. Serum samples from rhese patients were i""esLigated t" have priming effects or deactivating effects on chemofactic agents. I" one patlent with cutaneous T cell lynphoma, a" extract of tumor tissue and supernaLant from cu1tvre* t"moT cells were tested for eosinopllil. chfmotaetic actiliify. The possible parent and setecrivr role of IL-5 *n human eosinopt,irsi5 discussed.