Morris Hepatoma Research Articles

Growth inhibition of subcutaneously transplanted hepatomas by alterations of the dietary arginine-methionine balance.

We hypothesized that alteration of the dietary arginine-methionine balance might inhibit tumor growth and suggest nutritional strategies for cancer therapy. The Morris hepatoma 3924A was subcutaneously transplanted in ACI rats. Control diets containing normal levels of arginine, methionine, and other amino acids in replacement of protein (24%), carbohydrates (59%), fat (10%), and fiber, vitamins, and minerals (7%) were fed for 28 days. Six experimental diets were adjusted to maintain amino acids at 23-25% and carbohydrates at 58-60%; these diets were 1%-2% deficient in arginine or supplemented with 1-2% arginine (expressed as percent amino acid content of diet) in combination with normal, deficient, and supplementary levels of methionine. Daily food intake was unaffected by the experimental diets. The control groups gained 26.4 +/- 2.8 g body weight, and small body weight decrements ranged from 3.5% to 8.4% in the groups fed the experimental diets. Tumor weight of controls was 8.5 +/- 1.5% of body weight. The experimental diets that produced significant tumor growth inhibition (TGI) were 1) the arginine-methionine-deficient diet, 2) the arginine-excess-methionine-deficient diet, 3) the arginine-deficient diet, and 4) the excess-arginine diet. Diets containing excess methionine failed to produce TGI. TGI resulted in tumor weights 41-46% of control values. TGI was associated with significantly lower blood urea nitrogen, plasma protein, and tumor spermidine-to-spermine ratio than in tumor-bearing controls. It is concluded that dietary alteration of a single amino acid, arginine, might be a potentially useful nutritional strategy for controlling tumor growth.

5] Membrane proteins

Publisher Summary Depending on the type of interaction, membrane proteins are either embedded into a lipid bilayer or associated with the membrane structures, in the latter case, usually by ionic interaction or by hydrogen bonds. To isolate and analyze these proteins, they must be extracted from the membrane structures— that is, solubilized. Solubilization is mostly carried out with detergents. In this chapter, to illustrate the strategy for high-resolution separation of membrane proteins, the chromatographic and electrophoretic separation of proteins from the plasma membranes of the liver and Morris hepatomas is shown. Three groups of proteins are chosen, with different degrees of hydrophobicity. Membrane-associated annexins [calcium-binding protein (CBP) 65/67, CBP 35, and CBP 33] are less hydrophobic and can be solubilized, without using detergents. These proteins can be phosphorylated, but they are not glycosylated. Dipeptidyl-peptidase IV (DPP IV; EC 3.4.14.5) and the cell-cell adhesion protein cell-CAM (cell adhesion molecule or gp110) are intrinsic membrane proteins, and they are also glycosylated. Other membrane proteins, e.g., from eukaryotic cells or of bacterial and viral origin, can be assigned to one of the above-mentioned groups, in accordance with their behavior in chromatographic as well as electrophoretic separations.

Electrophoretic pattern of cytosolic pyruvate kinase fractions A and B (type L and M 2) from normal rat liver and Morris hepatoma 7777

Cytosolic pyruvate kinase fractions A and B obtained by salting out procedure from normal rat liver and Morris hepatoma 7777, purified by affinity chromatography on Blue Sepharose CL-6B, have shown similar electrophoretic patterns in polyacrylamide gel at pH 8.3, to previously studied pyruvate kinase extracts from chromatin of cell nuclei. Three variants ( α 1, β 1, γ 1) from normal liver pyruvate kinase fraction A (type L) had the greatest electrophoretic mobility, showed sigmoidal kinetics in relation to 2-phospho enolpyruvate (PEP), and sensitivity to ATP and fructose 1,6-diphosphate (FDP). The fraction A dominated over normal liver fraction B (type M 2), which in electrophoresis showed a slower y2 variant, similar to the fraction A of hepatoma. All variants from fractions B of normal liver and A of hepatoma had linear kinetics and were sensitive to ATP but not to FDP. The greatest differences showed pyruvate kinase fraction B from Morris hepatoma. Its all variants α 2, β 2, γ 3 were more cathodic and had linear kinetics in relation to PEP. They all were insensitive to normal signal molecules (ATP and FDP). The γ 3 alkaline variant acquired sensitivity to inhibition by l-cysteine. Showing several-fold higher activity, much greater affinity to the main substrate, and a lack of sensitivity to feed-back inhibition by ATP, it was responsible for a high rate of aerobic glycolysis and diminution of the Pasteur effect in metabolic studies. It was probably encoded during oncogene activation and plays a special role in different metabolic strategies of tumour cells.

Protein Metabolism in Cachectic Tumor-Bearing Rats: Effect of Tumor Excision

The effect of tumor resection on protein metabolism in cancer-bearing subjects is poorly documented. We explored changes in nitrogen (N) and protein metabolism after excision of tumors both at the whole body level (N balance) and at the tissue level, including skeletal muscle, small intestine, and liver. Sixteen male Sprague–Dawley rats (∼375 g) bearing subcutaneous Morris hepatoma 7777 for 6 weeks were either operated for tumor excision and studied for 10 days postoperatively (n= 10) or sacrificed on the day of surgery as tumor-bearing controls; operated and unoperated tumor-bearing rats were compared with healthy rats (n= 16). Tumors, which grew to a mass of 74 ± 7 g (mean ± SEM), induced significant loss of body mass (−27 ± 13 g) and protein depletion in epitrochlearis muscle (EPI) (−38%) and small intestine (−42%) vs healthy rats. Tumor significantly decreased muscle protein synthesis vs healthy rats (7.14 ± 0.5 vs 10.7 ± 0.5 nmol phenylalanine (Phe)/EPI/3 hr), net degradation (21.7 ± 2.9 vs 30.6 ± 2.5 nmol Phe/EPI/3 hr) and degradation (28.8 ± 2.7 vs 41.4 ± 2.5 nmol Phe/EPI/3 hr). In 50% of operated rats, tumor removal was followed immediately by increased food intake, body weight, and N balance; in other rats, this was delayed by 2–4 days. By 6 days postoperative, all rats were gaining weight and had normal food intake; wasting was abolished in small intestine, but not in skeletal muscle (protein mass −43% vs healthy rats,P< 0.05). Postoperative rats maintained lower muscle protein degradation (28.2 ± 2.0 vs 41.4 ± 2.5 nmol Phe/EPI/3 hr,P< 0.05) than healthy rats; protein synthesis was no longer reduced. In skeletal muscle, protein synthesis and protein deposition were related to levels of postoperative food intake (r= 0.91 and 0.98, respectively;P< 0.05). Following tumor excision, reversal of cancer cachexia appeared to be highly dependent on the level of postoperative food intake.

Influence of extracellular matrices on ganglioside pattern of two hepatoma cell lines with different adhesive properties

A cell culture model was developed to investigate the involvement of gangliosides in cell-matrix adhesion. Two cell lines with different adhesive properties derived from solid Morris hepatoma 7777 were established. Cultured in horse serum-containing medium, the adhesive cell line (MH 7777 A) adheres and spreads on uncoated culture dishes, whereas the revertant cell line (MH 7777 A>N) does not adhere and grows in suspension. The adhesiveness of both cell lines is dependent on the coating protein used (none, bovine serum albumin, fibronectin or collagen I) and the horse serum concentration in the culture medium. Both cell lines, although of the same origin, differed in their ganglioside composition. The most abundant ganglioside of both MH 777 A and MH 7777 A>N cell lines was fucosyl-G M1, 0.78 and 0.72 μg per mg cellular protein, respectively. The G M3 and G D1a content of MH 7777 A>N cells was significantly higher than that of MH 7777 A cells. Furthermore, a matrix-dependency of the ganglioside pattern of both cell lines was demonstrated.

CT imaging of morris hepatoma with liver-specific polyiodinated triglycerides

CT imaging of morris hepatoma with liver-specific polyiodinated triglycerides

Supplemental nutrition with ornithine alpha-ketoglutarate in rats with cancer-associated cachexia: surgical treatment of the tumor improves efficacy of nutritional support.

We investigated the use of ornithine alpha-ketoglutarate in treatment of rats bearing Morris hepatoma 7777. Rats received diets containing either ornithine alpha-ketoglutarate, which has been used in other catabolic states (i.e. injury, sepsis), or an isonitrogenous, isocaloric diet containing glycine. Untreated tumors grew to a mass of 11 g/100 g body weight over the 3-wk period after implantation and induced progressive anorexia, negative nitrogen balance, and body and tissue wasting. Compared with glycine, ornithine alpha-ketoglutarate had no effect on tumor growth, but also did not alter the catabolic effects of the tumor on its host. We hypothesized that capture of amino acids by the tumor limited the efficacy of supplemental nutrition here and in published reports in which tumor burden comprised 4-30% of body weight. This is supported by our observation that a 3-wk of implantation the rate of protein deposition plus amino acid oxidation by the tumor was equivalent to approximately 70% of the host's daily protein intake. To parallel the clinical situation in which tumor burden is small at diagnosis and initiation of treatment, the same diets were tested in rats treated by excision of the tumor at a limited stage of the disease. Rats received 3 d preoperative nutrition with ornithine alpha-ketoglutarate or glycine, and continued on the same diets for 3 or 6 d postoperatively. Compared with glycine-fed rats, ornithine alpha-ketoglutarate-fed rats showed a more positive nitrogen balance, higher concentrations of glutamine and branched-chain amino acids in muscle, and accelerated protein deposition in small intestine (P < 0.05). Our results explain the lack of success of nutritional support in untreated cancer and underline the need for clinically relevant animal models for further studies.

Inhibition of Glycosylation Induces Formation of Open Connexin-43 Cell-to-Cell Channels and Phosphorylation and Triton X-100 Insolubility of Connexin-43

We transfected the cDNA for the cell-to-cell channel protein connexin-43 (Cx43) into Morris hepatoma H5123 cells, which express little Cx43 and lack gap junctional communication (open cell-to-cell channels). We found that cells overexpressing Cx43 nonetheless lacked open cell-to-cell channels, but that inhibition of glycosylation by tunicamycin induced open channels in these cells. Tunicamycin also induced biochemical changes in Cx43 protein; the level increased, and a considerable fraction became phosphorylated and Triton X-100 insoluble, in contrast to untreated cells where Cx43 was non-phosphorylated and Triton X-100 soluble. Although tunicamycin caused the formation of open channels, channels were not found aggregated into gap junctional plaques, as they are when they have been induced by elevation of intracellular cAMP. The results suggest that although Cx43 itself is not glycosylated, other glycosylated proteins influence Cx43 posttranslational modification and the formation of Cx43 cell-to-cell channels.

Synergistic induction of acyl-CoA oxidase activity, an indicator of peroxisome proliferation, by arachidonic acid and retinoic acid in Morris hepatoma 7800C1 cells

Morris hepatoma 7800C1 cells (a Wistar rat cell line) were exposed to 100 μM arachidonic acid in the medium for seven days. This treatment resulted in 150% and 60% increases (above control activities) in acyl-CoA oxidase (which catalyzes the first step in peroxisomal β-oxidation) and catalase activities, respectively. Arachidonic acid (C20:4) can be metabolized to 20- and 19-hydroxyarachidonic acid by cytochrome P-450IVA and it was shown that our cells are capable of forming 20-hydroxyarachidonic acid. However, 20-hydroxyarachidonic acid (0.l–0.8 μM, 4 days) had no effects on lauroyl-CoA oxidase and catalase activities in Morris hepatoma cells. Treatment of 7800C1 cells with 100 μM all- trans-retinoic acid resulted in inductions of catalase (160% above the control activity) and carnitine acetyltransferase (140% above the control activity) activities. The activity of lauroyl-CoA oxidase was often, but not always, slightly induced by treatment with all- trans-retinoic acid. When all- trans-retinoic acid was administered together with arachidonic acid, these two compounds had a synergistic effect on the induction of acyl-CoA oxidase activity (almost 700% above the control activity). However, treatment of Morris hepatoma cells with the man-made peroxisome proliferator, perfluorooctanoic acid, together with all- trans-retinoic acid did not result in any synergistic effect on this same enzyme activity. In summary, this study (1) corroborates findings from transfection experiments indicating that the heterodimer PPAR-RXR α activates transcription of the acyl-CoA oxidase gene using the Morris hepatoma cell line; (2) shows that arachidonic acid induces the activity of lauroyl-CoA oxidase; (3) suggests that transcription of the catalase gene is not regulated by a PPAR-RXR α heterodimer in this system; and (4) demonstrates that peroxisome proliferation in Morris hepatoma cells by perfluorooctanoic acid is not as dependent on the level of retinoic caused by arachidonic acid.

Manganese Content and High-Affinity Transport in Liver and Hepatoma

Morris hepatomas 3924A and 9618A have much lower endogenous contents of Mn than normal rat liver. This work studied the uptake of Mn by slices of these three tissues over a range of concentrations from 0.05 to 100 μM. The influx was assessed with 54Mn while atomic absorption measurements determined the total content. At medium Mn from 0.05 to 5 μM, entry of 54Mn in 2 min was taken as the initial rate and within this period the apparent concentration of Mn in the cell water exceeded that in the medium. Liver showed three apparently saturable uptake systems, the medium concentrations of Mn for half-maximal uptake rate being 0.075, approximately 2, and 100 μM. Hepatoma 3924A appeared to have only two systems, the half-maximal concentration for the higher affinity mechanism being, at 0.34 μM, substantially greater than that for liver. At no concentration was the uptake rate of Mn by hepatoma 3924A less than that of liver although there was some indication that Mn uptake by 9618A was somewhat less than that by the other two tissues. It is concluded that liver and hepatoma 3924A have systems for Mn uptake with affinities that enable them to be active at the plasma concentration (∼0.1 μM) as well as uptake systems of less affinity. However, differences in these systems between liver and hepatomas do not account for the differences in endogenous Mn content.

Negative impact of cancer chemotherapy on protein metabolism in healthy and tumor-bearing rats

Although chemotherapeutic agents are widely used in the treatment of cancer, few experimental data are available on their effects on host N metabolism. We studied the effects of a single intraperitoneal (IP) injection of cyclophosphamide ([CYP] 120 mg/kg), 5-fluorouracil ([5-FU], 50 mg/kg), cisplatinum ([CDDP], 5 mg/kg), or methotrexate ([MTX], 30 mg/kg). N balance was studied for 6 days following chemotherapy in healthy rats (n = 40) and in rats bearing Morris Hepatoma 7777 ([MH7777] n = 40) in a situation comparable to that of human cancer (tumor burden <0.2% of body weight, moderate anorexia, and weight loss). In healthy rats, all drugs induced transient body weight loss, anorexia, and poor N balance. At day 6 posttreatment, all animals had resumed normal feed intake and positive N balance except CDDP-treated rats, which showed continued weight loss and poor N balance. CDDP and MTX exhibited antitumor activity; however, CDDP induced diarrhea in six of eight tumor-bearing rats. Drug-induced anorexia was more severe in tumor-bearing than in healthy treated rats. N balance was more severely decreased in MH7777-bearing rats than in healthy treated animals in response to 5-FU (159 ± 36 v 273 ± 27 mg N/2 d) and MTX (−66 ± 36 v 153 ± 37 mg N/2 d) at days 3 to 4 postinjection. These results establish the presence of drug-specific effects on host N balance and the existence of a drug-tumor interaction for N metabolism in the tumor-bearing host.

Inhibiting morris hepatoma growth in rats by blocking late cholesterol synthesis: [formula omitted] GI Lab VA Medical Center, East Orange, NJ 0701B

Inhibiting morris hepatoma growth in rats by blocking late cholesterol synthesis: [formula omitted] GI Lab VA Medical Center, East Orange, NJ 0701B

Mapping the biodistribution and catabolism of 5-fluorouracil in tumor-bearing rats by chemical-shift selective 19F MR imaging.

A chemical-shift selective (CHESS) 19F MR imaging technique was used to map selectively the antineoplastic drug 5-fluorouracil (5-FU) and its major catabolite alpha-fluoro-beta-alanine (FBAL) in tumor-bearing rats. The pulse sequence employed a CHESS RF saturation pulse to suppress either the 5-FU or the FBAL resonance before the other component in the two-line 19F MR spectra was measured. Selective 5-FU and FBAL images with a spatial resolution of 10 x 10 x 15 mm3 (1.5 ml) were obtained in 40 min from six ACl rats with implanted Morris hepatoma. Because the transmitter frequency could always be set to the Larmor frequency of the 19F resonance employed for imaging, the images were free of chemical-shift artifacts in readout and slice-selection direction. Whereas FBAL appeared only in the liver, the kidneys, and the bladder, 5-FU could also be detected in all major organs and in the muscular system. In the Morris hepatomas, a small 5-FU uptake and no FBAL accumulation were measured. The CHESS 19F MRI technique provides useful physiological and biochemical data on the biodistribution of the antineoplastic drug 5-FU and on the different catabolic activities of the tissues.

Utilization and preferred metabolic pathway of ketone bodies for lipid synthesis by isolated rat hepatoma cells

Morris hepatoma 7777 cells freshly isolated from highly malignant tumors grown in the hindlimb of buffalo rats actively convert ketone bodies to cholesterol and fatty acids. On the basis of results obtained with (-)-hydroxycitrate, an inhibitor of the ATP citrate lyase enzyme, the metabolic pathway for acetoacetate conversion to lipids is exclusively cytoplasmic, whereas that for 3-hydroxybutyrate involves both extra- and intramitochondrial compartments. Subcellular distribution studies indicated accumulation and compartmentation of 3-hydroxybutyryl CoA primarily in the cytoplasm of hepatoma cells incubated with either ketone body. In contrast, the compartmentation of acetoacetyl CoA is dependent on whether the substrate is acetoacetate or 3-hydroxybutyrate. With acetoacetate, the acetoacetyl CoA is entirely cytoplasmic, whereas with 3-hydroxybutyrate, it is equally divided between the intra- and extramitochondrial compartments. The results are discussed in terms of the known and proposed metabolic pathways for lipid synthesis from ketone bodies, particularly that from 3-hydroxybutyrate.

Inhibitory effect of the human recombinant tumor necrosis factor on the growth of the Morris hepatoma in rats

The effect of the human recombinant tumor necrosis factor alpha (h rec TNF-alpha) on the transplantable Morris hepatoma 5123 was studied in Buffalo rats. The cytokine was repeatedly administered intratumorly (i.t.) in a dose of 1.5 x 10(4) U once a day in a cycle of four and eight days. The control groups consisted of animals given saline i.t. The experiments revealed an inhibitory effect of the h rec TNF-alpha upon the growth of neoplastic tumors. The biometric parameters of the tumors indicated that the inhibition of the Morris hepatoma was most effective after eight repeated doses of TNF. After injections of TNF-alpha, the tumors presented extensive hemorrhagic necrosis, the regressive alterations being found mainly in the central and intermediate tumor zones. In the early phase of the tumor growth, neoplastic tissue necrosis prevailed, as well as hemorrhages within the necrotic masses, necrosis of the blood vessel walls and thrombi in their lumina. In the later period, numerous fibres of the fibrous tissue, richly vascularized, occurred in the peripheral and intermediate zones. Clusters of eosinophilic granulocytes and macrophages with apoptotic bodies in the cytoplasm were seen on the border of the necrotic foci.

Polyamine acetylations in normal and neoplastic growth processes

The expression patterns of cytosolic and nuclear polyamine acetyltransferases were studied in normal and neoplastic growth processesin vivo andin vitro to evidentiate the roles played by these enzymes in cell proliferation. In regenerating liver, cytosolic spermidine/spermine N(1)-acetyltransferase showed similar augments of mRNA level and enzymatic activity during the prereplicative period (4-8 h), whereas spermidine N(8)-acetyltransferase activity increased later (24 h) when DNA synthesis was maximally enhanced. In fibroblasts continuously dividing, the messenger for spermidine/spermine N(1)-acetyltransferase rapidly accumulated after serum-stimulation. In cultured Morris hepatoma cells stimulated to logarithmic growth, spermidine N(8)-acetyltransferase activity remained at plateau for 1 day declining thereafter, while spermidine/spermine N(1)-acetyltransferase activity immediately decreased. In Yoshida AH-130 hepatoma cells transplanted in rat peritoneum, spermidine N(8)-acetyltransferase and spermidine/spermine N(1)-acetyltransferase activities rose, respectively, in concomitance with elevated proliferation-rate and quasi-stationary phase of growth. Since the expression of cytosolic and nuclear acetyltransferases underwent different temporal activation, an involvement of these enzymes in separate metabolic processes controlling normal and neoplastic growth may be suggested.

Application of high-performance membrane chromatography for separation of annexins from the plasma membranes of liver and isolation of monospecific polyclonal antibodies.

The separation of annexins, calcium-binding plasma membrane-associated proteins from rat liver and Morris hepatoma 7777 by high-performance membrane chromatography (HPMC) is described. The annexins with low molecular masses, CBP 33 and CBP 35, and the annexin with a high molecular mass, CBP 65/67, can be separated within 10 min from one another by anion-exchange HPMC under non-denaturing conditions. The separation devices used consist of compact, porous disks (QuickDisk) on the one hand and of bundled membranes made of cellulose fibers (MemSep) on the other. Both have been found to be equally well suited for this separation. The annexins obtained in this way are subsequently bound to epoxy-activated porons disks and used for the separation of monospecific polyclonal antibodies against the annexin CBP 65/67.

Microheterogeneity of cytosolic and membrane-bound hexokinase II in Morris hepatoma 3924A

Phosphorylation of glucose by hexokinase is the key step in glucose and energy metabolism of the cell. In the Morris hepatoma 3924A, hexokinase II is the predominant hexokinase isoenzyme and occurs in the cytosol as well as bound to membranes. Hexokinase II was isolated by DEAE-cellulose chromatography from both the cytosolic and the mitochondria-enriched fractions and further resolved by hydrophobic-interaction chromatography on phenyl-Sepharose into two components designated hexokinase IIa and IIb. In both the soluble and the mitochondria-enriched fractions, type IIb was the predominant form, but the IIb/IIa ratio was higher in the particulate (6-8) as compared with the cytosolic fraction (1.5-2.0). Binding of the isolated forms of the enzyme to rat liver mitochondria resulted in a 2-10-fold activation of both subtypes. Biochemical characterization showed that both subtypes are closely related to the isoenzyme commonly referred to as hexokinase II, and that the microheterogeneity was not a consequence of contamination with hexokinase I or III. Both subtypes had a molecular mass of 110 kDa, they were inhibited by Pi at concentrations higher than 5 mM, and activated by the detergent CHAPS. The two subtypes differed in electrophoretic mobility (IIa > IIb), in Km values for glucose (IIa, 0.109 mM; IIb, 0.216 mM), in Ki values for glucose 6-phosphate (IIa, 25 microM; IIb, 0.106 mM), and in Ki values for glucose 1,6-biphosphate (IIa, 12.2 microM; IIb, 5.5 microM). An artificial proteolytic cleavage as cause of the hexokinase II microheterogeneity can be excluded, since both subtypes show the same molecular mass and the ability to bind to mitochondria and phenyl-Sepharose. In addition, the relative proportions of the two subtypes did not vary markedly between several enzyme preparations. Northern-blot analysis with a hexokinase II-specific cDNA probe revealed two distinct mRNA transcripts of 5.2 and 6.3 kb in length, which offers the possibility that hexokinase II microheterogeneity is due to differential RNA transcription and/or processing.

Effects of perfluorooctanoic acid — a potent peroxisome proliferator in rat — on Morris hepatoma 7800C1 cells, a rat cell line

In this study, Morris hepatoma 7800C1 cells (from rat) were exposed to 500 μM perfluorooctanoic acid (PFOA) in the culture medium for 7 days. This treatment resulted in inductions of catalase, lauroyl-CoA oxidase (which catalyzes the first step in peroxisomal β-oxidation) and of cytochrome P-450IVA (specialized for omega-and omega-1 hydroxylation of fatty acids). Northern blot analysis revealed that the level of mRNA for peroxisomal fatty acyl-CoA oxidase was enhanced in cells treated with PFOA. Inductions of the enzymes mentioned above are generally connected with peroxisome proliferation in vivo. This work also includes a comparison between the activities of catalase, lauroyl-CoA oxidase, DT-diaphorase and glutathione transferase in rat liver homogenate and 7800C1 cells in order to investigate to what extent this cell line differs from the situation in vivo. The findings suggest that the cells selectively lost most of their peroxisomes during transformation into a cell line and subsequent propagation. The control activities of catalase and lauroyl-CoA oxidase (marker enzymes for peroxisomes) were only about 2% of the corresponding enzyme activities in rat liver. In addition, a morphological study revealed that the frequency of peroxisomes in 7800C1 cells is very low. The control activity of glutathione transferase in 7800C1 cells was 11% of the corresponding activity in rat liver homogenate, whereas the level of DT-diaphorase was virtually the same in 7800C1 cells as in rat liver. Electron microscopic investigation of the control cultures revealed all signs of viable cells, with well-developed cell organelles. Treatment of 7800C1 cells with 500 μM PFOA has little effect on cellular morphology.

Cell-Collagen Adhesion Is Inhibited by Monoclonal Antibody 33.4 against the Rat α 1-Integrin Subunit

A monoclonal antibody (mAb 33.4) is described which inhibits the adhesion and spreading of an adherent cell line (A-cells), established from Morris hepatoma 7777 and isolated hepatocytes on collagen IV but not on laminin, fibronectin, and vitronectin. mAb 33.4 retains its immunological activity after immobilization and covalent cross-linking to Protein G-Sepharose and is therefore a suitable tool for the preparative equimolar purification of two proteins with M r of 130 and 190 kDa from detergent-solubilized membrane fractions by immunoaffinity chromatography. The 190-kDa protein was identified as rat α 1-integrin subunit by N-terminal amino acid sequencing, while the 130-kDa protein was specifically stained by a β 1-integrin subunit-specific antiserum. In immunoblot analysis mAb 33.4 recognized the 190-kDa band, suggesting that it is specific for the rat α 1-integrin subunit. The epitope recognized by mAb 33.4 is conformation-dependent because the staining in immunoblots was very strong if the SDS-PAGE was performed in the absence of reducing agents. The expression of α 1β 1-integrin in sinusoidal hepatocyte membrane domains of liver sections is shown by mAb 33.4 and antisera raised against the rat α 1- and β 1-integrin subunits.