Effect of narrow fractions of chromatin non-histone proteins on the expression of membrane tumor-associated antigen MA-50 and phosphorylation of proteins of cultured rat hepatocytes

As earlier reported, the main component of narrow fractions of chromosomal non-histone proteins (NHP) of kidney and of Zaidel hepatoma cells has its own protein kinase activity, and is identified as a heteroorgan NHP-antigen, which is intrinsic to the definite renal tissue and absent in the liver. Effects of narrow fractions of kidney and Zaidel hepatoma NHP on biosynthetic processes and sizes of hepatocytes were studied in vitro. It has been shown that as a result of a 5 h incubation of rat hepatocytes with a narrow fraction of renal NHP the proportion of small hepatocytes increases approximately by 12% as compared with that of cells cultivated without NHP. Besides, binding of organ-specific anti-kidney immune serum with a small hepatocyte population rises by more than 20%, which results from the expression of tumor-associated heteroorgan kidney-specific antigen on the hepatocyte surface. According to immunoprecipitation and subsequent electrophoresis, the molecular mass of a membrane heteroorgan antigen on the surface of hepatocytes amounts approximately to 65 kDa, and an active phosphorylation of cellular proteins takes place. The same effect on hepatocytes is produced by a narrow NHP fraction of chromatin of Zaidel hepatoma cells, whereas no phosphorylation is observed in the presence of liver NHP as well as in the absence of NHP. It is suggested that the heteroorgan NHP-antigen induces biosynthetic processes including synthesis of membrane tumorassociated antigen on the surface of hepatocytes cultivated in vitro by activation of cellular protein phosphorylation, which can lead to changes in size of cultivated cells.     

Studies in rapidly labelled ribonucleic acid in cell fractions and in tumour tissues

1. Twenty minutes after injection of [(3)H]orotic acid into rats the rapidly labelled RNA from the liver is mainly associated with the nuclear fraction and little with the ribosomal cytoplasmic fraction. 2. The thermal denaturation of RNA from the fractions was not as reversible as that of the RNA extracted from whole liver. 3. Rapidly labelled RNA is synthesized by cells from a transplantable hepatoma when incubated in the presence of [(3)H]uridine and, after extraction and centrifugation, the label is present in three main fractions: one which sediments to the bottom of a gradient and is associated with DNA, a second which sediments to the heavy side of the 28s RNA, and a third which has a peak of activity between 28s RNA and 18s RNA and is associated with DNA. 4. After labelling and extraction of the RNA from Ehrlich ascites cells the distribution of radioactive components is similar to that of the material from the hepatoma cells. 5. The difference between the tumour cells and liver is due to some extent to the method of homogenizing the tissues and the nature of the components is discussed.     

Molecular characterization of non-histone chromatin proteins from experimental tumours

Using two-dimensional (2-D) electrophoresis, two non-histone chromatin protein fractions (NHCP1 and NHCP2) from three animal tumours (Kirkman-Robbins hepatoma, Morris hepatoma 7777 and Ehrlich ascites cells) and normal hamster liver were analyzed. Apart from many common components several tissue specific polypeptides of the NHCP1 and NHCP2 fractions were detected. It was found that some spots present in electropherograms of non-histone proteins of tumour cells (M X 10(-3)/pI): 17-24/4.9-6.5 (NHCP1 and NHCP2); 34-41/4.9-6.0 (HCP1 and NHCP2); 44-46/5.3-7.5 (HCP2); 46-49/5.0-7.5 (NHCP1); 49/5.9-7.5 (NHCP2) and 102-134/5.6-7.0 (NHCP1) were absent from normal liver.     

Identification of a nuclear antigen with molecular weight of 48,000 differentially expressed in tumour and normal cells

A non-histone protein with mol. wt of 48,000 differentially expressed in normal and tumour cells was identified using immunological criteria. Antibodies were raised against a component specific for Kirkman-Robbins hepatoma of mol. wt about 48,000 separated from hepatoma non-histone proteins by preparative electrophoresis in polyacrylamide gel. It was demonstrated by immunoblotting that Morris hepatoma 7777 and Ehrlich ascites cells share an antigenic non-histone protein with Kirman-Robbins hepatoma. Tumour cells when compared with normal cells, i.e. hamster and rat liver, are characterized by significant enrichment of this component. Intracellular distribution of the polypeptide with mol. wt 48,000 suggests that this component may be a structural protein the biosynthesis of which increases or the antigenic determinants of which change in tumour cells.     

Action of embryonic thymus and liver cells on the development of the ascitic form of Ehrlich's carcinoma

The influence of the cells of embryonic thymus and liver on the development of Ehrlich carcinoma was studied. The intraperitoneal injection of the embryonic cells in the adult mice infested by the Ehrlich carcinoma resulted in a marked lengthening of the life time of animals and an increase of the survival percentage. The embryonic cells of thymus and liver inhibited sharply the growth of carcinoma cells in the diffusion chambers as well. In contrast to this, the thymus and bone marrow cells of adult animals, taken in the same concentrations as the embryonic cells, exhibited only a slight inhibiting effect on the growth of tumour cells. On the basis of these data a suggestion is put forward to the effect that the embryonic immunocompetent cells determine the stronger inhibition of tumour growth in the embryos as compared with the adult animals.     

The role of mitochondria in modifying the cellular ionic environment. Calcium-induced respiratory activities in mitochondria isolated from various tumour cells

Cyclic stimulation by Ca(2+) of respiration in mitochondria isolated from Ehrlich ascites-tumour cells occurs only when low phosphate concentrations (approx. 0.5mm) are also included in the incubation system. Under these circumstances the extra oxygen consumed is related stoicheiometrically to the amount of Ca(2+) taken up by the mitochondria; the values are similar to those obtained with mitochondria from rat liver in the absence of added phosphate. In contrast with liver mitochondria, up to 280nmol of Ca(2+)/mg of protein can be added to ascites mitochondria without causing any deleterious effect. Respiration in mitochondria isolated from the Yoshida ascites hepatoma (HA 130) and from the Morris hepatomas 5123C and 9618A is also stimulated by Ca(2+) in a cyclic manner. However, that in mitochondria from regenerating rat liver responds to Ca(2+) in the same way as those from normal rat liver. ADP-stimulated respiration in mitochondria from Ehrlich ascites tumour cells, but not from rat liver, is inhibited by low amounts of Ca(2+).     

Transport of proteins into hepatic and nonhepatic mitochondria: specificity of uptake and processing of precursor forms of carbamoyl-phosphate synthetase I

An in vitro system reconstituted with mouse liver polysome translation products was used to study the nature of polypeptide species imported into mitochondria from different mouse tissues such as liver, kidney, brain, and heart, as well as from Ehrlich ascites, Novikoff hepatoma, and Morris hepatoma 3924A tumor lines. Mouse hepatic mitochondria import a number of proteins including 160-kilodalton (kDa) carbamoyl-phosphate synthetase I (CPS-I). Two other proteins of 63 and 57 kDa of unknown function are also imported as major components by mouse liver mitochondria. Under these in vitro conditions, however, mitochondria from non-CPS-I expressing tissues such as brain, kidney, and heart failed to import and process the precursor forms of CPS-I (pCPS-I). Furthermore, mitochondria from three different tumor lines (Novikoff hepatoma, Morris hepatoma, and Ehrlich ascites) containing negligible CPS-I activity were also unable to import and process pCPS-I to any significant level. Similarly, the 63-kDa protein was selectively transported into liver and kidney mitochondria and also into Ehrlich ascites mitochondria at reduced levels, but not into mitochondria from heart and brain. Nevertheless, the 57-kDa protein and a number of proteins of less than 45 kDa are transported efficiently by all of the mitochondrial types studied. These results provide evidence for tissue- or cell-specific selectivity at the mitochondrial membrane level for the transport of some proteins. The transports of 63- and 57-kDa proteins are differentially inhibited by mouse liver mitochondrial matrix and membrane fractions, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunogenic proteins of nuclease-sensitive regions of chromatin of the normal rat liver and N-nitrosodiethylamine-induced hepatoma]

The localization of immunogenic proteins of chromatin in the nuclease-hypersensitive regions was investigated by means of polyclonal antibodies against the total chromatin from cells N-nitrosodiethylamine-induced hepatoma of rats and from hepatocytes. The fractions released after digestion by Ca-dependent endonuclease and by exogenous DNasa-1 were considerably enriched with immunogenic determinants. The enrichment of nuclease-hypersensitive sites of chromatin with tumour-associated antigens was observed in experiments with antihepatoma antibodies preincubated with chromatin from the normal liver cells. Higher immunoreactivity was typical of fractions obtained during Ca-dependent endonuclease segregation of the hepatoma chromatin, while products of digestion by exogenous DNasa-1 possessed higher tumour specificity.     

Correlation of membrane-bound and free ribosomes in normal rat liver, Zajdela hepatoma rat liver and ascite cells proper]

The content of membrane-bound ribosomes in normal rat liver cells is 3 times as high as compared to that of free ribosomes. (K=membrane-bound ribosome RNAs divided by free ribosome RNAs=3, the opposite effect being observed in case of ascites hepatoma cells. A considerable increase in the free ribosome fraction in the liver of hepatoma-bearing rats occurs by the sixth day due to a decrease in the content of hepatoma-bearing rats occurs by the sixth day due to a decrease in the content of membrane-bound ribosomes (K=0.6). Similar, but less-pronounced changes were observed in liver cells of control animals after 48-hour starvation (K=0.9), simulating the condition occurring during the last days of tumour animals' life. Thus, changes in the rativ of membrane-bound to free ribosomes in liver during the ascites tumour growth are probably specifics and are not only due to anorexia in Zajdela hepatoma animals.     

A simple procedure for the purification of calmodulin bound to membranes; calmodulin bound to the particulate fraction of AH-66 hepatoma ascites cells

We have demonstrated the identity of calmodulin tightly bound to the particulate fractions of AH-66 hepatoma cells and normal liver with authentic soluble calmodulin and have compared the particulate calmodulin content of AH-66 cells with that of normal liver. Calmodulin bound to the particulate fractions of the hepatoma and normal liver cells was purified to electrophoretic homogeneity by a simple procedure which involves solubilization of the particulate fractions with LIS, extraction of the solubilized solution with 37.5% phenol, gel filtration, and affinity chromatography on Fluphenazine-Sepharose. There were no detectable differences between the particulate calmodulin thus purified and authentic soluble calmodulin of rat brain. The particulate calmodulin in the hepatoma and normal liver cells was assayed based on its ability to activate calmodulin-deficient phosphodiesterase after partial purification of calmodulin from the particulate fractions by solubilization with LIS and extraction with phenol as described above. In addition, the particulate calmodulin content in the hepatoma and normal liver cells was also measured after solubilization of the particulate fractions with Lubrol PX. The results obtained by these two different procedures indicate that calmodulin content in the particulate fraction as well as in the soluble fraction of the hepatoma is significantly higher than that in the corresponding fractions of normal liver.     

Elevated cholesterol and decreased sterol carrier protein-2 in peroxisomes from AS-30D hepatoma compared to normal rat liver

Peroxisomes were isolated from AS-30D hepatoma and compared to normal rat liver cells for the purpose of investigating the cholesterol accumulation in the hepatoma cells. Cholesterol was found to be approximately 10-fold higher relative to protein in AS-30D peroxisomes as compared to peroxisomes from normal liver. The peroxisomes from the hepatoma cells were found to be more stable; catalase was not released from these peroxisomes during isolation or osmotic shock of the peroxisomal fraction. The elevated cholesterol level may stabilize the peroxisomal membrane. Sterol carrier protein-2 (SCP-2) levels were measured using a radioimmunoassay (RIA), which indicated the highest concentration of SCP-2 to be in peroxisomes. Hepatoma peroxisomes had a lower concentration of SCP-2 (2.5 micrograms/mg) than normal liver peroxisomes (8 micrograms/mg). Approximately half of all SCP-2 detected was found to be soluble in both hepatoma and normal rat liver cells. Immunoblots from both rat liver and AS-30D fractions demonstrated the presence of the 14-kDa form of SCP-2. The liver fractions also had a 57-kDa immunoreactive protein, which was barely detectable in the AS-30D fractions. The low abundance of the high molecular weight form of SCP-2 from hepatoma peroxisomes and the lower amounts of SCP-2 detected in the AS-30D peroxisomes may be related to the accumulation of cholesterol in the cells.     

Proteinase activity of nuclei from various tissues towards endogenous histones]

The proteinase activities of nuclei isolated from tissues differing in their mitotic activities (brain, thymus, liver, ascite lymphoma) towards the histones and non-histone acid -- extractable proteins were studied. The sensitivity of different histone fractions to nuclear proteinase depends on temperature and time of nuclei incubation under conditions providing for complete dissociation of chromatin proteins from DNA (2 M NaCl--5 M urea). The proteinase activity in the brain and thymus nuclei is revealed only under prolonged (43 hrs) incubation of the nuclei at 25 degrees C, which is accompanied by partial proteolysis of histone H1. Histone H4 from brain nuclei and histone H2a from thymus nuclei are preferably degraded. In the nuclei isolated from the mice ascite cell lymphoma NK/ly and from rat liver the enzyme activity is revealed mainly towards the arginine-enriched histones H3 and H4. The proteolysis of the arginine-enriched histones in tumour cell nuclei is more complete. A high sensitivity to proteolysis was observed for non-histone acid-extractable proteins with low electrophoretic mobility, which were found in brain and tumour cell nuclei.     

Lactate dehydrogenase-elevating agent is responsible for interferon induction and enhancement of natural killer cell activity by inoculation of Ehrlich ascites carcinoma cells into mice

Inoculation of Ehrlich ascites carcinoma cells (EAC) into the peritoneal cavities of outbred ddY mice induced interferon (IFN) in the circulation. The maximum titer (1,280 U) was obtained at 24 hr after inoculation. This induced IFN had the characteristics of type I IFN, i.e., stability at pH2 and lability at 56 C. An increase in natural killer cell (NK) activity was also observed for the first 3 days after inoculation. In addition, plasma lactate dehydrogenase (LDH) activity was elevated in these mice. Inoculation of ascitic fluid or serum of EAC-bearing mice into normal mice increased plasma LDH activity six- to sevenfold over normal levels and elevated activities persisted throughout the life of the mice. These results suggest that the LDH-elevating agent was responsible for IFN induction and for enhancing NK activity. Because lactate dehydrogenase-elevating virus (LDV) can be eliminated from tumor cells by passage in vitro, we attempted to grow EAC in tissue culture for several months and re-examined whether the inoculation of such cells could elevate plasma LDH activity induce IFN and enhance NK activity. The results showed that inoculation of the passaged cells had no effect on these activities in normal mice. Therefore, we concluded that the IFN inducer was LDV which contaminated the EAC and then enhanced the NK activity. N-tropic murine leukemia virus also contaminated EAC, but this virus was not responsible because cultured cells of EAC still shed this virus.     

Uridine kinase activities in developing, adult and neoplastic rat tissues.

Uridine kinase activities were found chiefly in the soluble fractions of rat tissues. In normal adults the activities ranged from 13 munits/g in skeletal muscle to 178 munits/g in colon. Enzyme activities in several rat neoplasms were significantly higher (e.g. in a fibrosarcoma, mammary carcinoma, renal carcinoma, pancreatic carcinoma and lymphocytic lymphoma, but not in a fast-growing Morris hepatoma). The activities were not related to tumour growth rates or sizes. In normal foetal liver, lung, brain, heart and kidney, uridine kinase concentrations equalled or exceeded those in the adult homologous tissue, but maximal activities in liver were reached 3--5 days post partum. In suckling rats the intestinal activity decreased substantially immediately after birth and normally did not rise again until late in the third postnatal week. Premature upsurges could be evoked by an injection of cortisol or by starvation of the pups overnight. Pancreatic activity was absent from 1-day-old rats, and only about 5% of the adult activity was reached by day 20; adult activities were attained rapidly after weaning. In pancreas, precocious formation or uridine kinase was elicited by overnight starvation of 2-week-old rats.     

The inhibitory effect of l-cysteine and its derivatives on glycolysis in Ehrlich ascites tumour cells

(1) l-Cysteine inhibits aerobic glycolysis and restores the Pasteur effect in Ehrlich ascites tumour cells or in their supernatants, while d-cysteine has no effect on this process. (2) Other compounds which have configuration l at the α-carbon and a thiol group in the β-position (penicillamine) or restore them in vivo (3-mercaptopyruvate, cystine or l-serine together with l-homocysteine) also show inhibitory properties. (3) dl-Homocysteine with a free thiol group in the γ-position, reduced glutathione, methionine and products of cysteine oxidation (cysteic acid, taurine) do not inhibit tumour aerobic glycolysis. (4) Glycolysis of normal tissue supernatants (mouse liver and muscle) is not sensitive to the inhibitory effect of cysteine. (5) Metabolic studies showing a cysteine-induced decrease in ATP content, coupled with cross-over of the pyruvate and 2-phosphoenolpyruvate concentrations in Ehrlich ascites tumour cells, indicate that tumour pyruvate kinase is an enzyme sensitive to cysteine inhibition. (6) Enzymatic studies carried out both after preincubation of Ehrlich ascites tumour cells with cysteine or during direct action of this substance on tumour and normal tissue supernatants indicate the presence of a cysteine-sensitive isoenzyme besides the normal cysteine-insensitive pyruvate kinase in tumour material.     

A study of the cell surface of tumour, foetal and lymph-node cells by cell electrophoresis after antibody and enzymic treatment

1. Rabbit anti-(rat foetal liver) serum, absorbed with adult rat liver cells, decreased the electrophoretic mobility of foetal liver cells by 51% and rat hepatoma cells by 45%, indicating the presence of a foetal-type antigen on the hepatoma cell membrane. 2. The chemical nature of the surface antigen was investigated. Incubation with neuraminidase had no effect on adult liver cells but decreased the electrophoretic mobility of foetal liver cells by 51% and of hepatoma cells by 34%; the effect of antiserum was decreased to one-fifth. 3. Sialic acid, or the supernatant from neuraminidase-treated cells, partially blocked the decrease in electrophoretic mobility induced by antiserum. 4. The pH-electrophoretic mobility curves of hepatoma cells treated with antisera were consistent with a sialic acidcontaining antigen on the surface of the tumour cells. 5. Treatment with ribonuclease did not decrease the electrophoretic mobility of adult-liver cells, but decreased that of the foetal liver cells by 17% and hepatoma cells by 29%. 6. In parallel studies made with mouse BP8 ascites-tumour cells ribonuclease decreased the electrophoretic mobility by 39%, that of normal mouse lymph-node cells by 4.8% and allergized mouse lymph-node cells by 13.3%. 7. Trypsin treatment also decreased the electrophoretic mobility of hepatoma cells by 22%.     

AFP基因细胞专一性的表达依赖于某些核蛋白

Southern blotting分析没有发现成年大鼠肝、胚肝及肝癌细胞AFP基因5′端及上游有任何不同。以AFP基因转录起始点到5′端上游255 bp DNA片段为探针进行Southwestern blotting分析,发现表达AFP基因的细胞核蛋白中存在与其结合的核蛋白,这些在成年大鼠肝、肺、脾、心和肾细胞核蛋白中不存在。含有结合蛋白的肝癌核蛋白部分能使作为RNA聚合酶Ⅱ来源的成年大鼠肝细胞核蛋白部分具备较高的体外转录活性,表明基因细胞专一的表达确与某些结合蛋白有关。     

Effects of polyamines on DNA synthesis using various subcellular DNA polymerases extracted from normal rat liver, tumour-bearing rat liver, and tumour cells

The effects of polyamines on DNA synthesis in vitro using various subcellular DNA polymerase fractions from normal and tumour-bearing rat livers, and tumour cells were investigated. When nuclear and mitochondrial DNA polymerase fractions were used, DNA synthesis on activated DNA was increased 3.5-8-fold by the addition of 20 mM putrescine or cadaverine. However, DNA synthesis was not stimulated by the addition of spermidine or spermine at any concentration tested. In contrast, DNA synthesis using the cytoplasmic DNA polymerase fraction was not stimulated at various concentrations of any of the four polyamines tested. The stimulatory effects of putrescine and cadaverine were absent when nuclear fractions from tumour-bearing rat liver or from tumour cells were used. In addition, in vitro DNA synthesis was not stimulated by 20 mM putrescine or cadaverine when nuclear extracts from the livers of rats administered putrescine subcutaneously were used. The specific activities of DNA polymerases extracted from tumour cells and tumour-bearing rat liver were already fully stimulated. These results suggest that DNA polymerases in tumour cells and tumour-bearing liver cells are stimulated by trapped putrescine produced in tumour cells and are thus no longer activated by exogenous putrescine.     

The effect of insulin on the growth of transplanted tumors in mice

The growth of four murine transplantable tumours, Ehrlich ascites or solid tumour, an aplastic carcinoma, B-16 melanoma, and a thymoma, were suppressed in mice treated daily with insulin (2 IU). Since insulin increased the number of plaque-forming cells and the phagocytic activity of the liver and spleen cells, the retardation of the tumour growth was ascribed to immunological mechanisms.