Isolation and characterization of a cytosol protein (64/7.2) present in large amounts in rapidly growing hepatomas (original) (raw)
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Purification and Characterization of Cytosoi Protein 45/7.8 Present in Rapidly Growing Hepatomas1
Protein 45/7.8 (molecular weight x 10~3/isoelectric point) was found in the cytosol of several rapidly growing hepatomas including Morris hepatomas 3924A and 9618A2 and Novikoff hepatoma. It was not found in Morris hepatomas 7794A and 8999, which have intermediate growth rates, or in the slow- growing Morris hepatoma 9618A, normal rat liver, 18-hr regen erating rat liver, or livers of fetal rats. Protein 45/7.8 was isolated under nondenaturing conditions from Morris hepato mas 3924A and 9618A2 and Novikoff hepatoma in high purity by purification in three steps: ammonium sulfate fractionation; diethylaminoethyl cellulose chromatography; and hydroxylap- atite chromatography. The protein was highly purified as shown by two-dimensional, isoelectric-focusing sodium dodecyl sul- fate:polyacrylamide gels. The amino acid composition of pro tein 45/7.8 from the three tumors studied was very similar; the acidic:basic amino acid ratio was 1.4. The NH2terminus amino acid of protein 45/7.8 was p...
Cancer research, 1978
Following the recent demonstration of differences in mRNA species in the Novikoff hepatoma and normal rat liver (2), a search was made for differences in the abun dant cytosol proteins of these tissues as well as of several Morris hepatomas and other rat tissues. With the aid of 2dimensional electrophoresis, it was found that each of the nontumorous tissues had a distinctive pattern of abundant proteins. Protein 56/8.3 (molecular weight/isoelectric point) was present in all of the tissues studied; its spot size and density were proportional to protein synthesis and/or growth rate. The 2-dimensional electrophoretic migration of Protein 56/8.3 is very similar to Elongation Factor 1«of protein synthesis. The fastest-growing tu rners, Morris hepatoma 3924A and the Novikoff hepatoma, contain a large number of proteins that have not been found in normal liver; their 13 common proteins included Proteins 72/6.8, 64/7.3, 45/7.8, and 35/7.7, which were not found in other tissues. Interestingly, these fast-growing tumors also differed markedly in the other proteins of their patterns. Only Protein 79/6.7 was common to all of the tumors studied and was not present in nontumorous tissues. In the slow-growing Morris hepatomas 9618A and 8999, the protein patterns were very similar to that of normal liver; only 4 abundant proteins differed from those of the normal liver. A group of 11 proteins in the 61-69/6.5-7.5 region was prominent in the liver and slowly growing hepatomas; this group of proteins was absent from the rapidly growing tumors. These results are in agreement with previous reports that have demonstrated many biochemical similarities of slow-growing Morris hepatomas and normal liver. Also, it is apparent that increasing growth rate is associated with concomitant alteration in the abundance of special pro teins. These special proteins differ from those of normal liver and slow-growing hepatomas.
Cancer research, 1979
The nuclear proteins of regenerating and fetal rat liver, slow growing Morris hepatoma 961 8A, and fast-growing Morris hepatoma 3924A, sequentially extracted from nuclei with (a) 0.075 M NaCl/0.O25 M EDTA, (b) 10 mMTnis,(c) 0.35 M NaCl, (d) 0.6 M NaCI, and (e) 3 M NaCI/7 M urea, were analyzed by two-dimensional, isoelectric-focusing sodium dodecyl sulfate gel electrophoresis. Many of the protein spots were common to these tissues as well as to the Novikoff hepatoma and normal liver. The protein patterns of the regenerating liver and slow growing 961 8A hepatoma were more similar to that of normal liver than to those of other tissues. Many similarities were found between the fetal liver and the fast-growing 3924A and Novikoff hepatomas. Four protein spots, 64/5.9, 60/6.3, 51 /5.3, and 38/7.3 (M.W. x 1O'3/pI), were only found in the fast-growing 3924A and Novikoff hepatomas. Proteins 79/6.4 and 61/7.2 were only found in the three hepatomas. Protein 37/6.3 was dense in the three hepatornas and much less dense in the regenerating and fetal livers. Two proteins, 28/5.0 and 27/ 4.9, were detected only in the fast-growing 3924A and Novikoft hepatomas and fetal liven. Proteins 125/8.2 and 98/8.4 were found in the fetal liver and the three hepatomas; these proteins may be â€ẫ€˜oncofetal' â€p roteins. Three proteins, 61 /5.5, 56/5.8, and 53/7.5, that were absent from other tissues were present in the regenerating liver, fast-growing 3924A and Novikoff hepatomas, fetal liver, and slow-growing 961 8A hepatoma. These proteins may be related to growth processes of normal and tumor tissues studied.
International Journal of Cancer, 1995
Both freshly-isolated rat hepatocytes and Morris hepatoma 7777 cells synthesized cathepsin D as a precursor that was either processed intracellularly to smaller mature forms or secreted into the medium. The pattern of mature enzyme forms was different in the 2 cell types. In addition, the relative amount of precursor secreted was much higher for hepatoma cells. Monensin strongly enhanced the secretion and also impaired the intracellular transport-linked maturation of procathepsin D in hepatocytes, while it markedly inhibited intracellular maturation and only slightly increased secretion of the pro-enzyme in hepatoma cells. Ammonium chloride influenced the intralyso-soma1 segregation and maturation of procathepsin D in hepatocytes but not in hepatoma cells. Our observations indicate that (i) the lysosornal segregation of cathepsin D was less efficient and its fractional secretion higher in hepatoma cells than in hepatocytes; (ii) in the 2 cell types, delivery to lysosomes and processing of procathepsin D were differently sensitive to increases in the vacuolar pH.
Biochemical Society Transactions, 1974
Upon exposure of hepatoma D23 or D30 embryonic antigens to 1 % (w/v) sodium dodecyl sulphate and 1 % (w/v) 2-mercaptoethanol and after electrophoresis on 5 or 10 % (w/v) polyacrylamide gels in the presence of detergent, identical mobilities for the two antigens were calculated. Again only one band of protein stained with Coomassie Blue was observed on each gel. By calibration of the gels with standard proteins, it was determined that the molecular weight of hepatoma D23 or D30 antigens was within the range 65000 to 70000. Preliminary measurement of the isoelectric point of the hepatoma D23 antigen preparation indicated that the material focussed as a single band with a pH range of approx. 4.8-5.0. These studies demonstrate that it is possible to isolate tumour-associated embryonic antigens as relatively homogeneous products. Although these are suitable for further chemical characterization, it is considered that their availability will allow a more precise definition of their involvement in neoplasia and their relationship to other tumourassociated antigens expressed upon 4-dimethylaminoazobenzene-induced rat hepatomas.
Expression of 65-kDa oncofetal protein in experimental hepatoma after anticancer therapy
Neoplasma
We have tested the expression of a 65-kDa oncofetal protein (p65) after combined treatment with menadione and methotrexate in hamsters transplanted with Kirkman-Robbins hepatoma. The treatment of tumor-bearing animals with these compounds significantly inhibited both the tumor development and the expression of p65. This inhibition in tumor tissue was calculated from densitograms of Western blots. The inhibition of p65 expression was also confirmed in the serum of hepatoma bearing animals by using solid-phase radioimmunoassay (RIA) to quantify the specificity of polyclonal antibodies to fetal p65 molecules. Additionally, p65 was shown to localize both in cytoplasm and in the nuclear extracts prepared from hepatoma tissue.
Immunolocalization of alpha-fetoprotein and albumin in morris 7777 hepatoma cells grown in vivo
Cancer Letters, 1979
Cytoimmunoperoxidase analyses of Morris 7777 hepatoma cells grown in viva revealed no cells specifically stained for albumin: the 7777 is thus a non or very low albumin producer, as opposed to other protein producer/non-secretor hepatomas. A majority of cells were alpha-fetoprote~n (AFP) positive, pointing, with other data in the literature, to a high degree of clonal homogeneity of the 7777 in regard of its .AFP production. In electron microscopy, AFP was found on the membranes and in the lumina of the rough endoplasmic reticulum, in the smooth endoplasmic reticulum, in the Golgi apparatus, and in vesicles in proximity of, or fusing with, the plasma membrane. This subcellular distribution indicates that the AFP production and secretion path in the Morris 7777 hepatoma cells follows the normal scheme for export proteins.
Cytotechnology, 2003
Rat ascites hepatoma cell line of AH109A proved to be divided into two subpopulations with different invasive and metastatic potentials, when cultured in the medium containing allogeneic rat sera. One population adheres to the culture dish, actively extending pseudopodia, and the other remains in a floating state. Utilizing this character, we have separated these two populations. After three successive separation steps, adhesive AH109A cells and floating AH109A cells were obtained. Adhesive AH109A cells proliferated more rapidly and invaded more actively than did floating AH109A cells. Adhesive AH109A cells metastasized mainly to lung, while floating AH109A cells to mesentery,when intravenously injected into tail veins. Histological studies revealed that adhesive AH109A cells showed lymphatic metastases to lung. These results suggest that the two populations separated from parental AH109A cells provide good models for the study of tumor invasion and tissue-specific metastasis and that adhesive AH109A cells can be used for the creation of lymphatic metastasis model of rats.