ABSTRACT
PURPOSE: Phospholipase C(PLC) isozymes play significant roles in signal transduction mechanism. PLC-gamma1 is one of the key regulatory enzymes in signal transduction for cellular proliferation and differentiation. Ras oncoprotein, EGFR, and PKC are also known to be involved in cell growth. The exact mechanisms of these signal transduction following irradiation, however, were not clearly documented. Thus, this study was planned to determine the biological significance of PLC, ras oncoprotein, EGFR, and PKC in damage and regeneration of rat intestinal mucosa following irradiation. MATERIAL AND METHOD: Sixty Sprague-Dawley rats were irradiated to entire body with a single dose of 8Gy. The rats were divided into 5 groups according to the sacrifice days after irradiation. The expression of PLC, ras oncoprotein, EGFR and PKC in each group were examined by the immunoblotting and immunohistochemistry. The histopathologic findings were observed using HandE stain, and the mitoses for the evidence of regeneration were counted using the light microscopy and PCNA kit. The phosphoinositide(PI) hydrolyzing activity assay was also done for the indirect evaluation of PLC-gamma1 activity. RESULTS: In the immunohistochemistry, the expression of PLC-beta was negative for all groups. The expression of PLC-gamma1 was highest in the group III followed by group II in the proliferative zone of mucosa. The expression of PKC-sigma1 was strongly positive in group I followed by group II in the damaged surface epithelium. The above findings were also confirmed in the immunoblotting study. In the immunoblotting study, the expressions of PLC-beta, PLC-gamma1, and PLC-sigma1 were the same as the results of immunohistochemistry. The expression of ras oncoprotein was weakly positive in groups II, III and IV. The of EGFR was the highest in the group II, III, followed by group IV and the expression of PKC was weakly positive in the group II and III. CONCLUSION: PLC-gamma1 mediated signal transduction including ras oncoprotein, EGFR, and PKC play a significant role in mucosal regeneration after irradiation. PLC-sigma1 mediated signal transduction might have an important role in mucosal damage after irradiation. Further studies will be necessary to confirm the signal transduction mediating the PLC-sigma1.
Subject(s)
Animals , Rats , Cell Proliferation , Epithelium , Hand , Immunoblotting , Immunohistochemistry , Intestinal Mucosa , Isoenzymes , Microscopy , Mitosis , Mucous Membrane , Negotiating , Phospholipases , Proliferating Cell Nuclear Antigen , Radiotherapy , Rats, Sprague-Dawley , Regeneration , Signal Transduction , Type C PhospholipasesABSTRACT
PURPOSE: Phospholipase C (PLC) isozymes ply significant roles in transmembrane signal transduction. PLC-i 1 acts as the intracellular effector in signal transduction for cellular proliferation and differentiation. Ras oncoprotein is also involved in cell growth. We determined the biological significance of PLC and ras oncoprotein in regeneration following radiation and the effect of different modes of administration of 5-FU. MATERIALS AND METHODS: To determine the effect of the administration mode of 5-FU on the regeneration of intestinal mucosa of rats following radiation, we compared the expression of PLC and ras oncoprotein in six groups. Group I had no treatment. GroupII received radiation( 8 Gy) only. Group III received radiation(8 Gy) and 5-FU(150mg/kg) continuous intravenous (iv) infusion for 12 hours. Group iv bolus injection. Group V received only 5-FU(150mg/kg) iv bolus injection. Through immunoblotting and immunohistochemistry, we examined the expression of PLC and ras oncoprotein in rat jejunum at 96 hours after radiation or 5-FU administration and at 120 hours after radiation and 5-FU administration. We also investigated the histological findings using hematoxylin and eosin stain. RESULTS: In the immunohistochemistry study, PLC-i 1 expression was the highest in group III followed by groups II and VI in that order and was weakly positive in groups V and VI. PLC-i 1 was hardly detected in the control group. The expression of ras oncoprotein wss the same as the PLC-i 1 expression for all groups. These results were confirmed by the histological findings regarding the mucosal regeneration. In the immunoblotting analysis, PLC-i 1 expression was the highest in group III followed by group IV and II in that order. This difference between the immunoblotting and immunohistochemistry study was due to the high expression of PLC-i 1 on the damaged surface epithelium rather than to its expression in the regeneration region as observed in the immunohistochemistry study for group IV. The expression of PLC- 1 was positive only in group V and VI, which received both radiation and 5-FU, and the expression of PLC-b 1was negligible for all groups. CONCLUSION: These results suggest that PLC-i 1 mediated signal transduction and ras oncoprotein may have a significant role in mucosal regeneration after radiation, and that continuous iv infusion of 5-FU may induce active regeneration in intestinal mucosa following radiation. In addition, the expression of PLC- 1 in combined group of radiation and 5-FU implies that PLC- 1 may be involved in signal transduction mediated by concerted action between radiation and 5-FU.