PURPOSE: The activation of phospholipase C (PLC) is one of the early events in various growth processes, including malignant transformation. Among PLC-isozymes, PLC-gamma is activated through direct interaction with growth factor receptor tyrosine kinase. In this study, we evaluated the patterns of PLC-gamma expression in benign and malignant tumors of the breast and compared the patterns with their normal counterpart. METHODS: Using immunoblot assay, we evaluated the patterns of expression in PLC-gamma in 20 breast cancer tissues, 13 fibroadenoma tissues, and normal tissues. The level of expression was analyzed by densitometry. RESULTS: All of 20 breast cancer tissues and 13 fibroadenoma tissues showed overexpression of PLC-gamma when compared with their normal counterparts. The level of the PLC-gamma expression was 3.9-fold and 17.3-fold higher in fibroadenomas and breastcancers, respectively, than in normal tissues. CONCLUSION: The result suggested that the level of PLC-gamma expression increases as the normal tissue undergoes progression to fibroadenoma, and finally to carcinoma. The pattern of expression of PLC-gamma in breast tissue implies that the PLC-gamma-mediated signal transduction may play a significant role in the progression of breast cancer from normal tissue.
Breast Neoplasms ; Breast* ; Densitometry ; Fibroadenoma ; Phospholipase C gamma ; Phospholipases* ; Protein-Tyrosine Kinases ; Signal Transduction ; Type C Phospholipases

In order to explore the existence and distribution of phospholipase (PLC) isozymes in the rat retina, immunohistochemical staining was applied using monoclonal antibodies against PLC isozymes (PLC beta; K92, PLC gamma; D7, F7, PLC delta; R32, S11). For immunohistochemical detection, avidin-biotin peroxidase complex (ABC) method was performed on frozed tissue sections of rat retina. Our study showed that PLC isozymes have particular distributional patterns in the retina. Namely, PLC beta is broadly distributed in the outer and inner segments of photoreceptor cell layer, nuclear layer and ganglion cell layer. PLC gamma is mainly appeared in the nerve fiber layer, ganglion cell layer and inner nuclear layer. PLC delta is confined only in the ganglion cell layer. These results clearly demonstrate the PLC isozymes may have their own role in the transduction of light pathway in the retina. However, further studies will be required to verify theirs precise role in the photoreception.
Animals ; Antibodies, Monoclonal ; Ganglion Cysts ; Immunohistochemistry ; Isoenzymes* ; Nerve Fibers ; Peroxidase ; Phospholipase C beta ; Phospholipases* ; Photoreceptor Cells ; Rats* ; Retina* ; Type C Phospholipases*

In order to explore the existence and distribution of phospholipase (PLC) isozymes in the rat retina, immunohistochemical staining was applied using monoclonal antibodies against PLC isozymes (PLC beta; K92, PLC gamma; D7, F7, PLC delta; R32, S11). For immunohistochemical detection, avidin-biotin peroxidase complex (ABC) method was performed on frozed tissue sections of rat retina. Our study showed that PLC isozymes have particular distributional patterns in the retina. Namely, PLC beta is broadly distributed in the outer and inner segments of photoreceptor cell layer, nuclear layer and ganglion cell layer. PLC gamma is mainly appeared in the nerve fiber layer, ganglion cell layer and inner nuclear layer. PLC delta is confined only in the ganglion cell layer. These results clearly demonstrate the PLC isozymes may have their own role in the transduction of light pathway in the retina. However, further studies will be required to verify theirs precise role in the photoreception.
Animals ; Antibodies, Monoclonal ; Ganglion Cysts ; Immunohistochemistry ; Isoenzymes* ; Nerve Fibers ; Peroxidase ; Phospholipase C beta ; Phospholipases* ; Photoreceptor Cells ; Rats* ; Retina* ; Type C Phospholipases*

BACKGROUND: Phospholipase C-beta4 (PLC-beta4) is known to be one of the most important signal transducing molecules; however, its biophysical and chemical characteristics are not well known due to the difficulty in purifying PLC-beta4 from bovine retina. In the present study, we used the baculovirus expression system in order to express and purify large amounts of PLC-beta4. With this system, we also tried to produce chimeric PLC-beta3/beta4 and PLC-beta4/beta3 protein in order to study the structure-activity relationship between N terminal and C terminal portion of PLC-betas. METHODS: I cloned PLC-beta4 to the baculovirus expression system by the polymerase chain reaction method and infected the PLC-beta4 to Sf9 cells. I purified recombinant PLC-beta4 proteins using sequential high performnance liquid chromatography (HPLC) by using the TSK phenyl-5PW column and the TSK heparin-5PW column. With this similar method, I was able to express chimeric PLC-beta3/beta4 and PLC-beta4/beta3 proteins. RESULTS: With the two step HPLC, I was able to purify PLC-beta4 by 30-fold; this purified PLC-beta4 contained PLC activity. I also expressed chimeric PLC-beta3/beta4 and PLC-beta4/beta3 using the baculovirus system, and their expression was confirmed by the immunoblot method. However, chimeric PLC-beta4/beta3 did not show PLC activity, while chimeric PLC-beta3/beta4 retained its PLC-activity. CONCLUSION: Expression of chimeric PLC-beta4 using the baculovirus system was an efficient method to obtain a large amount of protein. Moreover, this expression and purification method would be useful in studying the physical and chemical characteristics of this protein. In my study using chimeric PLC-beta protein by swapping the N terminal and C terminal portions of PLC-beta3 and beta4, chimeric protein lost its activity completely in PLC-beta4/beta3 chimera. This result suggested a minute change in the tertiary structure of the protein, which may significantly affect its function.
Baculoviridae ; Chimera ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Clone Cells ; Phospholipase C beta ; Phospholipases ; Polymerase Chain Reaction ; Proteins ; Retina ; Sf9 Cells ; Structure-Activity Relationship ; Type C Phospholipases

No abstract available.
Animals ; Inositol* ; Kidney* ; Liver* ; Phospholipases* ; Rats* ; Type C Phospholipases*

We previously shown that LES contraction depends on M3 receptors linked to PTX insensitive Gq protein and activation of PLC. This results in production of IP3, which mediates calcium release, and contraction through a CaM dependent pathway. In the esophagus ACh activates M2 receptors linked to PTX sensitive Gi3 protein, resulting in activation of PLD, presumably, production of DAG. We investigated the role of PLC isozymes which can be activated by Gq or Gbeta protein on ACh-induced contraction in LES and esophagus. Immunoblot analysis showed the presence of 3 types of PLC isozymes, PLC-beta1, PLC-beta3, and PLC-gamma1, but not PLC-beta2, PLC-beta4, PLC-gamma2, PLC-delta1, and PLC-delta2 from both LES and esophageal muscle. ACh produced contraction in a dose dependent manner in LES and esophageal muscle cells obtained by enzymatic digestion with collagenase. PLC-beta1 or PLC-beta3 antibody incubation reduced contraction in response to ACh in LES but not in esophageal permeabilized cells, but PLC-gamma1 antibody incubation did not have an inhibitory effect. The inhibition by PLC-beta1 or PLC-beta3 antibody on Ach-induced contraction was antibody concentration dependent. The combination with PLC-beta1 and PLC-beta3 antibody completely abolished the contraction, suggesting that PLC-beta1 and PLC-beta3 have a synergism to inhibit the contraction in LES. PLC-beta1, -beta3 or -gamma1 antibody did not reduce the contraction of LES cells in response to DAG (10 (-6) M), suggesting that this isozyme of PLC may not activate PKC. When Gq/11 antibody was incubated, the inhibitory effect of the incubation of PLC beta3, but not of PLC beta1 was additive (Fig. 6). In contrast, when Gbeta antibody was incubated, the inhibitory effect of the incubation of PLC beta1, but not of PLC beta3 was additive. This data suggest that Gq/11 or Gbeta may activate cooperatively different PLC isozyme, PLCbeta1 or PLCbeta3 respectively.
Animals ; Calcium ; Cats* ; Collagenases ; Digestion ; Esophageal Sphincter, Lower* ; Esophagus ; GTP-Binding Protein alpha Subunits, Gq-G11* ; Isoenzymes ; Muscle Cells ; Phospholipase C beta ; Type C Phospholipases

No abstract available.
Phospholipases* ; Protein Kinase C* ; Protein Kinases* ; Psychotropic Drugs* ; Type C Phospholipases*

We report two cases of gingival plasma cell granuloma in a 34-yr-old and 40-yr-old two male renal transplant recipients with cyclosporine A (CsA)-induced gingival overgrowth (GO). Histologically, these lesions were composed of mature plasma cells, showing polyclonality for both lambda and kappa light chains and fibrovascular connective tissue stroma. In addition to the fact that CsA-induced plasma cell granuloma is rare, the salient features of our cases were the secretion of interleukin-6 and overexpression of phospholipase C-gamma1 of the tumor cells, which may explain the mechanisms of CsA- induced GO.
Cyclosporine/adverse effects ; Female ; Gingival Diseases/*chemically induced/enzymology/immunology/pathology ; Granuloma, Plasma Cell/*chemically induced/enzymology/immunology/pathology ; Humans ; Immunohistochemistry ; Immunosuppressive Agents/adverse effects ; Interleukin-6/metabolism ; Kidney Transplantation ; Male ; Middle Aged ; Phospholipase C gamma ; Type C Phospholipases/metabolism

This study was conducted to investigate the effects of extremely low frequency electromagnetic fields (EMF) on signal pathway in plasma membrane of cultured cells (RAW 264.7 cells and RBL 2H3 cells), by measuring the activity of phospholipase A2 (PLA2), phospholipase C (PLC) and phospholipase D (PLD). The cells were exposed to the EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h. The basal and 0.5 microM melittin-induced arachidonic acid release was not affected by EMF in both cells. In cell-free PLA2 assay, we failed to observe the change of cPLA2 and sPLA2 activity. Also both PLC and PLD activities did not show any change in the two cell lines exposed to EMF. This study suggests that the exposure condition of EMF (60 Hz, 0.1 or 1 mT) which is 2.4 fold higher than the limit of occupational exposure does not induce phospholipases-associated signal pathway in RAW 264.7 cells and RBL 2H3 cells.
Arachidonic Acid ; Cell Line ; Cell Membrane ; Cells, Cultured ; Electromagnetic Fields ; Magnets ; Occupational Exposure ; Phospholipase D ; Phospholipases ; Phospholipases A2 ; Pyridoxal ; Signal Transduction ; Type C Phospholipases

BACKGROUND AND OBJECTIVES: Phosphoinositide specific phospholipase C (PLC) plays a pivotal role in the transmembrane signal transduction pathways by catalyzing the hydrolysis of phosphoinositide 4,5-bisphosphate (PIP2) to yield the intracellular second messengers, diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3), in response to the interaction of various ligands with the cell surface receptors. The question arises as to the physiological roles of the phosphoinositide second messenger system in the inner ear. The purpose of this study was to determine whether PLCbeta isozymes are present at the cochlea and what portion of cochlea each PLCbeta isozymes are distributed in. MAERIALS AND METHODS: Two methods, an immunohistochemical staining and western blot for PLCbeta isozymes were used in the rat cochlea. Frozen section and surface preparation were prepared for immunohistochemical staining. The PLCbeta isozymes or proteolytic digests were separated by SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose membranes. Rabbit polyclonal antibodies raised against four PLCbeta isozymes were used. RESULTS: Each PLCbeta isozymes showed differential expressions in the cochlea. PLCbeta1 immunoreactivity was observed in the inner and outer hair cells and the spiral ganglion cells; PLCbeta2 in the stria vascularis and PLCbeta3 mainly in the inner hair cells. PLCbeta4 was not observed in cochlea. In western blots of rat cochlea extracts, the PLCbeta isozymes stained several bands corresponding to the known molecular weight of PLCbeta monomers, which are probably proteolytic digests. CONCLUSION: These results suggest that differentially localized each PLCbeta isozymes in the cochlea may have specific roles in signal transduction pathway of auditory system.
Animals ; Antibodies ; Blotting, Western ; Cochlea* ; Collodion ; Ear, Inner ; Frozen Sections ; Gels ; Hair ; Hydrolysis ; Inositol 1,4,5-Trisphosphate ; Isoenzymes* ; Ligands ; Membranes ; Molecular Weight ; Phospholipase C beta ; Phospholipases* ; Rats* ; Receptors, Cell Surface ; Second Messenger Systems ; Signal Transduction ; Spiral Ganglion ; Stria Vascularis ; Type C Phospholipases