Tyrphostin ErbB2 Inhibitors AG825 and AG879 Have Non-specific Suppressive Effects on gp130/ STAT3 Signaling

Although the interaction between gp130 and the ErbB family has frequently been shown in cancer cells, the mechanism of this interaction remains unclear and controversial. In the present study, we found that specific tyrphostin inhibitors of ErbB2 (AG825 and AG879), but not ErbB1 inhibitor (AG1478), suppressed IL-6-induced tyrosine phosphorylation of STAT3 in schwannoma cells. However, biochemical evidence for transactivation of ErbB2 by IL-6 was not observed. Additionally, the inhibition of ErbB2 expression, with either a specific RNAi or transfection of an ErbB2 mutant lacking the intracellular domain did not inhibit the IL-6-induced tyrosine phosphorylation of STAT3. Thus, it seems that tyrphostins, which are known as specific inhibitors of the ErbB2 kinase, may have non-specific suppressive effects on the IL-6/STAT3 pathway.

The functional role of the CARM1-SNF5 complex and its associated HMT activity in transcriptional activation by thyroid hormone receptor

We have investigated the function and mechanisms of the CARM1-SNF5 complex in T3-dependent transcriptional activation. Using specific small interfering RNAs (siRNA) to knock down coactivators in HeLa alpha2 cells, we found that coactivator associated arginine methyltransferase 1 (CARM1) and SWI/SNF complex component 5 (SNF5) are important for T3-dependent transcriptional activation. The CARM1- SWI/SNF chromatin remodeling complex serves as a mechanism for the rapid reversal of H3-K9 methylation. Importantly, siRNA treatment against CARM1 and/or SNF5 increased the recruitment of HMTase G9a to the type 1 deiodinase (D1) promoter even with T3. Knocking- down either CARM1 or SNF5 also inhibited the down- regulation of histone macroH2A, which is correlated with transcriptional activation. Finally, knocking down CARM1 and SNF5 by siRNA impaired the association of these coactivators to the D1 promoter, suggesting functional importance of CARM1- SNF5 complex in T3-dependent transcriptional activation.

Development of ligand-dependent regulatory system and its application to gene therapy of insulin-dependent diabetes mellitus

To develop an inducible expression system, the enhanced artificial nuclear receptors and target reporters were constructed. Artificial nuclear receptors were generated by fusing three domains, consisting of DNA-binding domain (DBD) of GAL4, ligand binding domain (LBD) of progesterone or estrogen receptor, and activation domain (AD) of VP16, sterol regulatory element binding protein (SREBP)-1a, or SREBP-2. The activation domain of SREBP-1a showed most potent transcriptional activity. The maximal level of target reporter gene expression was extremely elevated by the usage of ATP citrate-lyase (ACL) minimal promoter -60/+67 in place of artificial TATA promoter, while the SV40 enhancer severely increased the basal transcription in the absence of ligand. The induction system, developed in the present study, was applied to cell therapy, resulting in successful induction of single-chain insulin analogue (SIA) gene expression to correct the hyperglycemia in diabetic animals. By means of subcutaneous cell therapy, the SIA gene expression rapidly occurred after the local topical application of ligand. These results suggest that our system represents a powerful tool for transcriptional regulation of target gene that can be used for diverse applications, ranging from basic research to gene therapy.

Anti-HER2/neu Peptide Producing Vector System for Biologic Therapy - Is It Possible to Mass-produce the Peptide?

A humanized monoclonal antibody against HER2 has been using in a clinical setting and has been shown to possess therapeutic properties. A mimetic peptide against HER2 was also reported to bind to the HER2 receptor with some therapeutic potential. Based on a previous report and the sequence of Herceptin, we designed oligonucleotides of anti-HER2 mimetic peptides, named V2 and V3 peptides, in order to develop a peptide- producing vector system for biologic therapy against HER2- overexpressing cancers. We also adopted the sequence of a previously reported mimetic peptide, V1 (Park BW et al. Nat. Biotechnol, 2000, 18: 194-198), as a reference peptide. We examined the effects of the V2 and V3 peptides against the HER2-overexpressing cell lines, SK-BR-3 and T6-17. Transient transfection of the construct expressing V1 and V2 inhibited cell proliferation in HER2-overexpressing cell lines by 20 - 30%, but had no effect on the HER2-negative NIH3T3 cells. The proliferation inhibition shown by V2 was slightly better than that shown by V1. Recombinant peptides V2 and V3 were produced on a large scale in an E. coli system, and the V2 peptide showed anti-HER2-specific tumor cell proliferation inhibition of 10% to 30%. Current results suggest that anti-HER2 mimetic peptides, overexpressed by a constitutive promoter or produced in an E. coli system, could specifically inhibit the proliferation of HER2-expressing cancer cells. Further efforts to augment the biologic specificity and efficacy and to develop new technologies for the purification of the peptide from the E coli system are needed.

Acetyl-CoA carboxylase beta expression mediated by MyoD and muscle regulatory factor 4 is differentially affected by retinoic acid receptor and retinoid X receptor

Mammals have two major isoforms of acetyl-CoA carboxyase (ACC). The 275 kDa beta-form (ACC beta) is predominantly in heart and skeletal muscle while the 265 kDa alpha-form (ACC alpha) is the major isoform in lipogenic tissues such as liver and adipose tissue. ACC alpha is thought to control fatty acid oxidation by means of the ability of malonyl-CoA to inhibit carnitine palmitoyl-CoA transferase-1 (CPT-1), which is a rate-limiting enzyme of fatty acid oxidation in mitochondria. Previously, it was reported that MyoD and other muscle regulating factors (MRFs) up-regulate the expression of ACC beta by interactions between these factors and several cis-elements of ACC beta promoter. We described here that ACC beta expression mediated by MRFs is regulated by retinoic acids. Endogenous expression of ACCb in differentiated H9C2 myotube was significantly increased by retinoic acid treatment. However, on transient transfection assay in H9C2 myoblast, ACC beta promoter activity was suppressed by RXRa and more severely by RAR alpha. These effects on ACCb expression in myoblasts and myotubes by RXR alpha and RAR alpha seem to be mediated by their interactions with MRFs because no consensus sequence for RXR alpha and RAR alpha has been found in ACC beta promoter and retinoic acid receptors did not affect this promoter activities by itself. In transient transfection in NIH3T3 fibroblast, the activation of ACC beta promoter by MyoD, main MRF in myoblast, was significantly suppressed by RAR alpha and to a less extent by RXR alpha while the RXR alpha drastically augmented the activation by MRF4, major MRF in myotube. These results explained that retinoic acids differentially affected the action of MRFs according to their types and RXR alpha specially elevates the expression of muscle specific genes by stimulating the action of MRF4.

The Mechanisms of Antitumor Effect of Anti-p185HER2/neu Monoclonal Antibody and Peptide Mimetic

PURPOSE: Anti-p185HER2/neu monoclonal antibody (mAb) that can induce phenotypic reversion and monoclonal antibodies specific for the p185HER2/neu growth factor receptor that are able to diminish its kinase-signaling properties represent a specific advance in the therapy for p185HER2/neu-expressing human cancers. With mAb treatment, down-regulation of p185HER2/neu surface receptors and attenuation of the kinase-signaling properties have been observed and regarded as a basic phenomenon; however, the mechanisms for mAb-induced phenotypic reversion are not clear. METHODS: We used human tumor-cell lines of SK-BR-3, T6-17, and U373MG. With immunoprecipitation and Western blotting, we investigated the changes in p185HER2/neu receptor phosphorylation and the expression of signal-regulatory proteins (SIRPs) after mAb treatment. To identify the proteins interacting with Tat-binding protein-1 (TBP1), we used the Clonotech Gal4 matchmaker two-hybrid system. RESULTS: Minimal to moderate reduction in phosphotyrosine (pTyr) content was observed in SK-BR-3 and T6-17 cells with short-term (10-30 minutes) incubation after mAb treatment, but that did not alter total p185HER2/neu receptor density. SIRPs phosphorylation after peptide treatment was increased. With mAb treatment, three proteins were shown to interact with TBP1, and all of the interacting proteins are subunits of proteasome 26S. Collectively, anti- p185HER2/neu mAb or peptide down-regulates the surface receptors and attenuates the kinase signaling, which then both induces higher proteasome activity through increased TBP1 and increases SIRPs expres sion. CONCLUSION: Increased proteasomal activity may degrade abnormal proteins and increased SIRPs may regulate signal transduction toward the norm. Therefore, activation of a protein-degradation pathway and induction of signal-regulatory proteins may be possible mechanisms for the ultimate anti-tumor effects of the anti-p185HER2/neu mAb or peptide.

The Study on the Mechanism Regulating the Production of Extracellular Matrix in Renal Tubular Epithelial Cells Cultured in High Glucose Concentration / 대한신장학회잡지

Thickening of tubular basement membrane and progressive tubulointerstitial fibrosis has been reported as important components of diabetic nephropathy, In order to investigate the mechanisms of tubulinterstitial changes in diabetic nephropathy, we evaluated the effects of a high concentration of glucose(25mM; 450mg/dL) on glucose transporter GLUT1 level, fibronectin production and tissue inhibitors of metalloproteinases (TIMP)-1 concentration in renal tubular(LLC-PK1) cells. As the effect of high glucose-induced alteration in LLC-PK1 cells, the expression of facilitative glueose transporter, GLUT1 was decreased after longer than 24-hours exposure to 25mM glucose, compared to control(5.6mM). The administration of protein kinase C (PKC) inhibitor GF109203X(10 microM) did not show significant effect on high glucose-induced decrease of GLUT1 level. On western blot analysis of fibronectin production, The exposure of LLC-PK cells to 25mM glucose for 48 hours significantly increasc4 fibro- nectin production, dose-dependently. The addition of GF102903X at the concentration of 10pM induced the significant increase of fibronectin level in LLC-PK1cells under glucose-free condition, whereas there was no significant effect on the high glucose-induced increase of fibronectin production. The addition of anti-TGF-beta antibody at 30 microgram/mL partly inhibited the high glucose-induced increase of fibronectin production. Concerning the changes of tissue inhibitor of metallo-proteinase(TIMP)-1 levels in the presence of high glucose, the exposure to high glucose for 24 and 43 hours increased TIMP-1 levels in culture supernatant of LLC-PK1 cells, dose-dependently. The TIMP-1 levels of 48-hour exposure to 15 and 25mM glucose were also significantly higher than those of 24-hourexposure. The treatment with 10 microM GF102903X or 30 microgram/mL anti-TGF-Beta antibody had no significant effects on TIMP-1 levels measured under the high glucose culture condition. In conclusion, the expression of facilitative glucose transporter, GLUT1 is inhibited and the production of fibronectin is increased in renal tubular cells cultured in the presence of high concentration of glucose, which is partly mediated by TGF-beta. The TIMP-1 level is also increased under high glucose culture condition. The enhanced productions of fibronectin and TIMP-1 of renal tubular cells under high glucose concentration may contribute to tubulointerstitial fibrosis that occurs in diabetic nephropathy.

One Year result of Dermatophagoides farinae antigen - speciffic autologous antibody complex immunotherapy in respiratory allergic patients sensitized to house dust mite / 천식및알레르기

BACKGROUND: Although good clinical effects have been reported, immunotherapy with house dust mite ( HDM ) antigen - autologous specific antibody complex ( IC - IT ) is not yet accepted as an effective immunomodulating tool in HDM allergic diseases. We aimed to prove the clinical effect of IC - IT in HDM sensitized respiratory allergic subjects. Method : Six HDM sensitized respiratory allergic subjects were enrolled. Autologous D. farinae specific IgG was purified with DEAE ion exchange and affinity chromatography. After one year of IC - IT treatment the clinical effects were analyzed with symptom scores, methacholine PC20, ELISA assay of D. farinae specific antibodies and intradermal skin reactivity. Result : The rhinitis symptom score significantly improved after a one - year administration of IC - IT ( 1.23 +/- 0.30 vs. 0.37 +/- 0.15, p< 0.05), but no significant differences were found in asthma symptom score, intradermal skin reactivity to D. farinae and ELISA optic absorbances of D. farinae specific IgE, IgG, and IgG subclasses. Methacholine PC20 values improved in all 6 patients who were administered with IC - IT ( 0.35 vs. 1.66 mg/ml, p< 0.05 ). CONCLUSION: IC - IT may be efficient for management of HDM atopic asthma. Further studies are needed before clinical application of IC - IT in house dust mite atopic subjects.

Purification of the major allergens from Korean Dermatophagoides pteronyssinus and production of the recombinant antigens / 천식및알레르기

BACKGROUND: Purified major allergens of house dust mite are essential for evaluation of the allergic mechanism in molecular basis and development of new modalities of immunemodulation. OBJECTIVE: In this study, we aimed to purif group 1 and group 2 allergens from Dermatophagoides pteronyssinus (Dp). In addition, cDNAs corresponding to Der pI and II in Korean Dp were isolated and recombinant Der p1 and Der pII were synthesized. MATERIALS AND and METHOD: Der pI allergen was purified by ammonium sulfate precipitation, anion -exchange column chromatography, and gel filtrat,ion chromatography. Der pII allergen was purified by anion exchange chromatography, gel filtration chromatography, and a preparative isoelectric focusing method. RESULTS: Eight hundred ug of Der pI and 50 ug of Der pII were obtained from 100 g of culture medium and 1 g of mite bodies, respectively. The purities of these allergens were confirmed by SDS PAGE and the strong reactivity to the patient sera was identified. In order to produce a recombinant allergens, poly(A) RNA from house dust mites were isolated and used for cDNA synthesis by RT PCR. The cDNA was inserted into prokaryotic expression vector and the vectors were transformed into E. coli. A little amount of recombinant Der pI protein was produced due to the low solubility, and 1.2 mg of recombinant Der pII was produced from 1 L of E. coli culture medium. The antigenicity of Der pI was relatively weak, however, Der pII showed a strong antigenicity. Amino acid sequence of the amplified cDNA deduced from DNA sequences of Der pII showed 6 different variants. The variation of amino acid sequences suggests the possibility of high incidence of mutation of Der pII protein. CONCLUSION: A simplified method for the purification of Der pI and Der pII was developed. Recombinant allergens will be useful for the diagnosis and treatment of allergy with lower costs.

The Study on the Mechanism of Cyclosporine A Induced Apoptosis in Renal Tubular Epithelial Cells / 대한이식학회지

A major limiting factor in the use of cyclosporine A (CsA) is nephrotoxicity, but the mechanisms of nephrotoxicity are not fully understood. In order to elucidate the pathogenesis of CsA tubulotoxicity, we examined mechanisms (DNA synthesis, necrosis and apoptosis) of cellular injury induced by CsA in cultured LLC-PK1 renal tubular cell line. The possible role of Fas antigen in the mediation of CsA-induced cell death and the hypothesis that CsA-mediated injury activates the glucose transporter GLUT1, a stress response gene in renal tubular cells were also investigated. CsA treatment for 24 hours in LLC-PK1 cells showed significantly decreased 3H-thymidine uptake in a dose dependent manner (0.1 microgram/ml to 1 mg/ml), indicating that DNA damage is a sensitive indicator of CsA induced nephrotoxicity. A dose of 10 microgramml CsA caused a significant increase in LDH release (M+/-S.D., 11.0+/-3.0% vs 27.0+/-9.8, p<0.05). On flow cytometric analysis, 9.9 4.2% of control cells appeared in a region of decreased forward light scatter and increased side scatter, respectively. Both indices representing characteristics of apoptotic cell death. Compared to control, treatment with 10 ng/ml of CsA for 24 hours significantly increased the proportion of cells in apoptotic region to 38.9 13.5%. This finding was supported by electrophoretic analysis of the DNA extracted from CsA-treated cells, where a series of bands corresponding to integer multiples of 180 to 200 base pairs was visualized. CsA (0.1 microgram/ml) treatment for 24 hours was seen to cause a significant elevation in the expression of the 45 kD Fas protein by Western blot analysis. In addition, the exposure to CsA was also associated with an increase of GLUT1 protein levels up to 2.2 fold (mean) on Western blot analysis. In conclusion, CsA is directly toxic to tubular cells with inhibiting DNA synthesis and inducing cell death in the form of necrosis or apoptosis. Fas antigen-ligand system and glucose transporter GLUT1 may play roles in mediating CsA induced tubular cytotoxicity.

The Study on the Mechanism of Cyclosporine A Induced Apoptosis in Renal Tubular Epithelial Cells / 대한이식학회지

A major limiting factor in the use of cyclosporine A (CsA) is nephrotoxicity, but the mechanisms of nephrotoxicity are not fully understood. In order to elucidate the pathogenesis of CsA tubulotoxicity, we examined mechanisms (DNA synthesis, necrosis and apoptosis) of cellular injury induced by CsA in cultured LLC-PK1 renal tubular cell line. The possible role of Fas antigen in the mediation of CsA-induced cell death and the hypothesis that CsA-mediated injury activates the glucose transporter GLUT1, a stress response gene in renal tubular cells were also investigated. CsA treatment for 24 hours in LLC-PK1 cells showed significantly decreased 3H-thymidine uptake in a dose dependent manner (0.1 microgram/ml to 1 mg/ml), indicating that DNA damage is a sensitive indicator of CsA induced nephrotoxicity. A dose of 10 microgramml CsA caused a significant increase in LDH release (M+/-S.D., 11.0+/-3.0% vs 27.0+/-9.8, p<0.05). On flow cytometric analysis, 9.9 4.2% of control cells appeared in a region of decreased forward light scatter and increased side scatter, respectively. Both indices representing characteristics of apoptotic cell death. Compared to control, treatment with 10 ng/ml of CsA for 24 hours significantly increased the proportion of cells in apoptotic region to 38.9 13.5%. This finding was supported by electrophoretic analysis of the DNA extracted from CsA-treated cells, where a series of bands corresponding to integer multiples of 180 to 200 base pairs was visualized. CsA (0.1 microgram/ml) treatment for 24 hours was seen to cause a significant elevation in the expression of the 45 kD Fas protein by Western blot analysis. In addition, the exposure to CsA was also associated with an increase of GLUT1 protein levels up to 2.2 fold (mean) on Western blot analysis. In conclusion, CsA is directly toxic to tubular cells with inhibiting DNA synthesis and inducing cell death in the form of necrosis or apoptosis. Fas antigen-ligand system and glucose transporter GLUT1 may play roles in mediating CsA induced tubular cytotoxicity.

Characterization of regulatory elements on the promoter region of human ATP-citrate lyase

ATP-citrate lyase (ACL), an enzyme catalyzing the first step in biosynthesis of fatty acids, is induced during the lipogenesis and cholesterologenesis. We demonstrate that the region -213 to -128 of human ACL promoter is responsible for conferring glucose-mediated transcription. This region in the ACL promoter contains Sp1 binding sites determined by DNase I foot-printing assay. Gel retardation assay using oligonucleotides from -179 to -141 and -140 to -110 showed two specific DNA-protein complexes postulated to be formed by transcription factor Sp1. Competition gel shift and supershift assays have confirmed that these DNA-protein complexes were the result of induced Sp1 as well as another Sp1-related proteins. Western blot analysis also demonstrated that transcription factor Sp1 was slightly increased in the nuclear proteins extracted from Alexander cells following supplementation of glucose. In addition, expression of 110 kDa protein reacting with antibody against Sp3 was dramatically increased by glucose supplementation, while isoforms of Sp3, about 80 kDa in size was decreased in its amounts. Our results suggest that changes in the expression of Sp1 family proteins play an important role in activation of the ACL promoter by glucose.

Regulation of ATP-citrate lyase gene transcription

It has been suggested that glucose metabolites and insulin are the most important factors inducing ATP-citrate lyase (ACL) by a high carbohydrate diet. We have used a primary culture of rat hepatocytes to confirm the role of glucose and insulin in terms of ACL gene expression. The results showed that glucose displayed a direct effect on ACL gene expression and the insulin helps the glucose effect. The nucleotide sequences from -512 to -485 of the ACL promoter are highly homologous (70%) to the sequences surrounding the carbohydrate response element (ChoRE) of the S14 gene. The gel retardation analysis using ChoRE of the S14 gene showed that the ACL promoter which contains the ChoRE-like sequence specifically inhibited the formation of the complex by the nuclear proteins isolated from rat liver. To localize the regions which are involved in the regulation of ACL gene expression, transient expression assay using ACL promoter-CAT (chloramphenicol acetyltransferase) constructs containing various lengths of a 5' flanking region of the ACL gene were carried out. The proximal promoter region -419 to -1 containing several potential Sp1 binding sites showed the strong enhancing effect, which increases the transcription of CAT genes in the various cell lines, such as the CHO (Chinese hamster ovary) cell, the HepG2 cell, and primary cultured rat hepatocytes. In response to glucose, among the ACL promoter-CAT constructs, only pNP33-CAT (-1342 to -1) showed a 2.64 fold increase in CAT activity by a high concentration of glucose. The activation of ACL gene expression by glucose seems to be regulated in a complicated manner involving interactions between the contexts of the several sequence elements and various transacting factors, which is not a simple mechanism directed only by a short sequence element.

Induction of hepatic ATP-citrate lyase by insulin in diabetic rat--effects of insulin on the contents of enzyme and its mRNA in cytosol, and the transcriptional activity in nuclei

The effects of insulin on ATP-citrate lyase, its mRNA in cytosol, and the transcriptional activity in nuclei of diabetic rat liver were studied. Experimental diabetes was induced by an intraperitoneal injection of streptozotocin, and livers were removed from rats at 0, 1, 3, 6, 16, and 72 hours after the administration of insulin. ATP-citrate lyase began to increase at 16 hours, and continuously increased until 72 hours. The amount of mRNA encoding ATP-citrate lyase increased abruptly at 16 hours, then decreased to near basal level in 72 hours. No change in the transcription rate was observed until 3 hours after insulin administration. However, the activity increased 4-fold at 6 hours and 7-fold at 16 hours, 16-fold at 6 hours and 28-fold at 16 hours when pGACL1 and pGACL2 were used as probes, respectively, preceding the increase in the amounts of mRNA and the enzyme. It is suggested that the increase in the amount of ATP-citrate lyase by insulin is primarily due to the increase in the transcriptional activity of the gene in nuclei, which results in the subsequent increase in the amount of mRNA for the biosynthesis of ATP-citrate lyase in cytosol.

Regulation of fatty acid synthase at transcriptional and post-transcriptional levels in rat liver

The regulation of fatty acid synthase in rat liver was investigated at transcriptional and post-transcriptional levels. When rats were fasted for 3 days and refed on a high-carbohydrate diet, the amounts of FAS in liver cytosol began to increase at 12 hours and further increased until 48 hours. The amount of mRNA for FAS began to increase at 6 hours and reached to a maximum level at 12 hours, indicating that the expression of mRNA for FAS precedes the increase of FAS protein pool. After 12 hours the amounts of mRNA gradually decreased and remained at a much lowered level between 24 and 48 hours. The elevated amount of FAS mRNA reflected on the amount of FAS protein in the first 24 hours, but these two parameters were not paralleled thereafter, probably due to the changes in the translational efficiencies. The run-on transcriptional activity of FAS gene began to increase at 4 hours after refeeding a high-carbohydrate diet and further increased to reach a maximum level 25 fold of the initial level at 12 hours, followed by a 16 fold level between 24 and 48 hours. The elevation of run-on transcriptional activity of FAS gene preceded the increase of FAS mRNA in the liver cytosol by 2 hours, and a similar increasing pattern was observed until 12 hours. However, FAS mRNA concentration decreased gradually after 12 hours, while the transcriptional activity remained at a high level until 48 hours. The changes in FAS mRNA content in the cytosol of rat liver were closely related to the transcriptional activity of FAS gene in the early phase of induction, but another regulatory mechanism seems to operate in the decrease of mRNA after 12 hours.

A study on the regulation of translocation of glucose transporters during hepatocarcinogenesis induced by 3'-Me DAB

The mechanism of glucose transported (GT) expression on the plasma membranes of hepatoma cells in rats induced by 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) was studied. Cytochalasin B binding to plasma membrane fractions from control and 3'-MeDAB group in the absence of cold cytochalasin B showed 9,825 +/- 925 and 30,165 +/- 625 dpm/mg membrane protein. Scatchard plot analysis showed that the GTs present on the plasma membrane fractions in control and 3'-Me DAB groups were 5.0 and 16.0 pmol/mg membrane protein and their Kd values were 151 and 157 nM, respectively. These results suggest that the numbers of GTs in plasma membrane were increased in the 3'-Me DAB group compared to the control group. In contrast, the amounts of GTs in low density microsomal (LDM) fractions measured by a photoaffinity labeling technique using [3H]-cytochalasin B were 31,207 and 11,702 dpm/mg protein in the control and 3'-Me DAB group, respectively. These results suggest that GTs were translocated from LDM to plasma membranes during carcinogenesis. To confirm these results by an independent method 10% SDS-polyacrylamide gel electrophoresis was carried out. Gel slice No. 13 corresponding to MW of 45 kDa from plasma membrane fractions showed increased radioactivities in the 3'-Me DAB group compared to the control group. However, LDM fractions of the 3'-Me DAB group showed decreased radioactivities compared to the control group. Western blot analysis using anti-human RBC GT antibody present in the plasma membranes and LDM fractions from control and 3'-Me DAB groups did not show any significant difference, indicating low cross-reactivity between them. These results indicate that increased glucose transport seems to be more likely due to reciprocal redistribution of GTs between plasma membrane and LDM fractions.

Characterization of Tumor Specific Antigens on the Plasma Membrane Surface of Rat Hepatomas lnduced by 3'-Me DAB and ldentification of the Common Tumor Specific Antigens from Rat Hepatomas lnduced by Different Chemical Hepatocarcinogens

Three different chemical carcinogens, 2-acetylaminofluorene (AAF), diethylnitrosamine(DENA), and 3'-methyl-4dimethylaminoazobenzene (3'-Me DAB) were used to induce hepatomas in rats. Plasma membrane surface proteins of normal rat liver cells and rat hepatomas were extracted with 3M KCI. From the analysis of the proteins of normal rat liver and rat hepatoma induced by 3'-Me DAB by discontinuous polyacrylamide gel electrophoresis(Disc-PAGE), under nonreducing and nondenaturing conditions polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol (SDS-PAGE), Sephadex G-200 gel permeation chromatography, DEAE-A50 ion-exchange chromatography and two-dimensional gel electrophoresis, at least three tumor specific antigens were identified. One had a molecular weigh of 66,000 (pl=6.79) while the other two had the same molecular weight 73,000 but differed in their isoelectric points (7.58 and 7.81). For immunological analysis of tumor specific antigens, the absorbed antiserum was prepared. Plasma membrane surface proteins of rat hepatoma induced by 3'-Me DAB were used to obtain New Zealand White male rabbit antiserum. Rabbit antiserum was then reacted with the proteins isolated from the plasma membrane surface of normal rat liver and the absorbed antiserum reacting specifically with the tumor specific antigens derived by 3'-Me DAB was obtained. Using the absorbed antiserum, the immunoreactivities of plasma membrane surface proteins isolated from rat hepatomas induced by 3'-Me DAB, AAF, and DENA were compared by Ouchterlony double immunodiffusion analysis and immunoelectrophoresis. To characterize the proteins reacting to the absorbed antiserum, immunoglobulin G was separated from the absorbed antiserum and coupled to cyanogen bromide-activated Sepharose CL-4B. The isolated proteins from the plasma membrane surface proteins of 3'-Me DAB-induced hepatoma using this immunoaffinity chromatography had molecular weights of 66,000 and 73,000. The localization of these proteins on surface plasma membranes of rat hepatomas induced by 3'-Me DAB was confirmed by an immunofluorescence technique. The experimental results revealed the existence of cross-reacting common antigens on the plasma membrane surface of rat hepatomas induced by different hepatocarcinogens.