pyrophosphate and Liver-Neoplasms

We previously showed that human hepatic intrasinusoidal (HI) natural killer (NK) T cells selectively eliminate hepatocellular carcinoma (HCC) cell lines. In this study, we investigated the underlying mechanisms on how HI γδ T cells, expanded with zoledronate, exhibit a superior cytotoxic effect on HI NK-resistant Huh7 HCC cells.. γδ T cells were obtained from living liver transplant donors or from peripheral blood mononuclear cells (PBMC) of healthy volunteers and were expanded in the presence of IL-2, IL-15, and zoledronate for 2 weeks. Cytotoxicity was measured using the lactate dehydrogenase (LDH) assay in vitro and by flow cytometry using carboxyfluorescein succinimidyl ester (CFSE) in vivo.. The cytotoxicity of expanded HI γδ T cells against Huh7 cells was associated with a higher pyrophosphate expression in Huh7 cells compared to SNU398 cells. In contrast, the cytotoxicity of HI γδ T cells against SNU398 cells depended on NKG2D. HI γδ T cells expressed less PD-1 than PB γδ T cells. The cytotoxicity of HI γδ T cells against Du145 and PC3 prostate cancer cells was also associated with pyrophosphate expression in these cells, as well as NKG2D and DNAM-1.. The expression levels of phospho-antigen in tumor cells determined the cytotoxicity of HI γδ T cells, although the NK activating receptors, death ligands, and immune checkpoint molecules also contribute to their cytotoxicity. γδ T cells are attractive candidates for cancer immune cell therapy.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cytotoxicity, Immunologic; Diphosphates; Humans; Leukocytes, Mononuclear; Liver Neoplasms; Male; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Antigen, T-Cell, gamma-delta; Zoledronic Acid

Cyclin-dependent kinase 9 (CDK9) inhibitors are a novel category of anticancer treatment for cancers. However, their effects on hepatocellular carcinoma (HCC) are rarely investigated. Human ribonucleotide reductase (RR, which consists of RRM1 and RRM2 subunits) catalyzes the conversion of ribonucleoside diphosphate into 2'-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools, which play essential roles in DNA synthesis and DNA repair. In this study, we identified that CDK9 protein expression in adjacent non-tumor tissues predicted HCC patients' overall and progression-free survivals. The anticancer activity of a CDK9-selective inhibitor, LDC000067, on HCC cells was positively associated with its ability to inhibit the expression of RRM1 and RRM2. LDC000067 downregulated RRM1 and RRM2 expression through post-transcriptional pathway. Specifically, LDC000067 triggered RRM2 protein degradation via multiple pathways, including proteasome-, lysosome-, and calcium-dependent pathways. Furthermore, CDK9 positively correlates with RRM1 or RRM2 expression in HCC patients, and the expressions of these three genes were associated with the higher infiltration of immune cells in HCC. Taken together, this study identified the prognostic relevance of CDK9 in HCC and the molecular mechanism for the anticancer effect of CDK9 inhibitors on HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin-Dependent Kinase 9; Diphosphates; Humans; Liver Neoplasms; Ribonucleotide Reductases

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Diphosphates; Humans; Liver Neoplasms; Mutation Rate; Recurrence; Retrospective Studies; Tumor Suppressor Protein p53

Preclinical research indicate that vascular disrupting agent (VDA) treatment induces extensive tumor death but also a systemic mobilization of bone marrow derived cells including endothelial progenitor cells (EPC) leading to revascularization and renewed growth within the residual tumor. This study investigates if combination of VDA with the anti-angiogenic agent Sunitinib increases the treatment efficacy in a colorectal liver metastases mouse model.. CBA mice with established liver metastases were given a single dose of OXi4503 at day 16 post tumor induction, a daily dose of Sunitinib starting at day 14 or day 16 post tumor induction or a combination of Sunitinib given daily from day 14 or day 16 post tumor induction in combination with a single dose of OXi4503 at day 16. Treatment was terminated at day 21 post tumor induction and its effects were assessed using stereological and immunohistochemical techniques. Long term effects were assessed in a survival study.. Combination with long (7 day) Sunitinib treatment lead to liver toxicity but this was ameliorated in the shorter (5 day) treatment without significantly altering the effects on tumor reduction. Combination treatment resulted in significant reduction of viable tumor, reduction in tumor vasculature, reduction in tumor proliferation, increase in tumor apoptosis and prolonged mouse survival compared to control and single arm treatments. Complete tumor eradication was not achieved. Redistribution of E-cadherin and strong up regulation of ZEB1 and Vimentin were observed in the surviving tumor; indicative of epithelial to mesenchymal transition (EMT), a mechanism that could contribute to tumor resistance.. Combination treatment significantly reduces viable tumor and prolongs animal survival. EMT in the surviving tumor may prevent total tumor eradication and could provide novel targets for a more lasting treatment.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cadherins; Chemical and Drug Induced Liver Injury; Colorectal Neoplasms; Diphosphates; Epithelial-Mesenchymal Transition; Humans; Indoles; Liver Neoplasms; Male; Mice; Mice, Inbred CBA; Protein Kinase Inhibitors; Pyrroles; Stilbenes; Sunitinib; Vimentin; Xenograft Model Antitumor Assays; Zinc Finger E-box-Binding Homeobox 1

We aimed to test the ability of texture analysis to differentiate the spatial heterogeneity of (125)I-A5B7 anti-carcinoembryonic antigen antibody distribution by nano-single photon emission computed tomography (SPECT) in well-differentiated (SW1222) and poorly differentiated (LS174T) hepatic metastatic colorectal cancer models before and after combretastatin A1 di-phosphate anti-vascular therapy.. Nano-SPECT imaging was performed following tail vein injection of 20 MBq (125)I-A5B7 in control CD1 nude mice (LS174T, n=3 and SW1222, n=4), and CA1P-treated mice (LS174T, n=3; SW1222, n=4) with liver metastases. Grey-level co-occurrence matrix textural features (uniformity, homogeneity, entropy and contrast) were calculated in up to three liver metastases in 14 mice from control and treatment groups.. Before treatment, the LS174T metastases (n=7) were more heterogeneous than SW1222 metastases (n=12) (uniformity, P=0.028; homogeneity, P=0.01; contrast, P=0.045). Following CA1P, LS174T metastases (n=8) showed less heterogeneity than untreated LS174T controls (uniformity, P=0.021; entropy, P=0.006). Combretastatin A1 di-phosphate-treated SW1222 metastases (n=11) showed no difference in texture features compared with controls (all P>0.05).. Supporting the potential for novel imaging biomarkers, texture analysis of (125)I-A5B7 SPECT shows differences in spatial heterogeneity of antibody distribution between well-differentiated (SW1222) and poorly differentiated (LS174T) liver metastases before treatment. Following anti-vascular treatment, LS174T metastases, but not SW1222 metastases, were less heterogeneous.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Carcinoembryonic Antigen; Cell Line, Tumor; Colorectal Neoplasms; Diphosphates; Female; Heterografts; Humans; Iodine Radioisotopes; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Phenotype; Radiopharmaceuticals; Stilbenes; Tomography, Emission-Computed, Single-Photon

Epithelial to mesenchymal transition (EMT) is considered an important mechanism in tumor resistance to drug treatments; however, in vivo observation of this process has been limited. In this study we demonstrated an immediate and widespread EMT involving all surviving tumor cells following treatment of a mouse model of colorectal liver metastases with the vascular disruptive agent OXi4503. EMT was characterized by significant downregulation of E-cadherin, relocation and nuclear accumulation of β-catenin as well as significant upregulation of ZEB1 and vimentin. Concomitantly, significant temporal upregulation in hypoxia and the pro-angiogenic growth factors hypoxia-inducible factor 1-alpha, hepatocyte growth factor, vascular endothelial growth factor and transforming growth factor-beta were seen within the surviving tumor. The process of EMT was transient and by 5 days after treatment tumor cell reversion to epithelial morphology was evident. This reversal, termed mesenchymal to epithelial transition (MET) is a process implicated in the development of new metastases but has not been observed in vivo histologically. Similar EMT changes were observed in response to other antitumor treatments including chemotherapy, thermal ablation, and antiangiogenic treatments in our mouse colorectal metastasis model and in a murine orthotopic breast cancer model after OXi4503 treatment. These results suggest that EMT may be an early mechanism adopted by tumors in response to injury and hypoxic stress, such that inhibition of EMT in combination with other therapies could play a significant role in future cancer therapy.

Topics: Angiogenic Proteins; Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Cadherins; Cell Hypoxia; Colorectal Neoplasms; Diphosphates; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Neoplasm Proteins; Neoplasm Transplantation; Neoplasm, Residual; Stilbenes; Up-Regulation

Treatment of solid tumors with vascular disrupting agent OXi4503 results in over 90% tumor destruction. However, a thin rim of viable cells persists in the tumor periphery following treatment, contributing to subsequent recurrence. This study investigates inherent differences in the microenvironment of the tumor periphery that contribute to treatment resistance.. Using a murine colorectal liver metastases model, spatial morphological and molecular differences within the periphery and the center of the tumor that may account for differences in resistance to OXi4503 treatment were investigated. H&E staining and immunostaining were used to examine vessel maturity and stability, hypoxia and HIF1α levels, accumulation of immune cells, expression of proangiogenic factors/receptors (VEGF, TGF-β, b-FGF, and AT1R) and expression of EMT markers (ZEB1, vimentin, E-cadherin and β-catenin) in the periphery and center of established tumors. The effects of OXi4503 on tumor vessels and cell kinetics were also investigated.. Significant differences were found between tumor periphery and central regions, including association of the periphery with mature vessels, higher accumulation of immune cells, increased growth factor expression, minimal levels of hypoxia and increased evidence of EMT. OXi4503 treatment resulted in collapse of vessels in the tumor center; however vasculature in the periphery remained patent. Similarly, tumor apoptosis and proliferation were differentially modulated between centre and periphery after treatment.. The molecular and morphological differences between tumor periphery and center may account for the observed differential resistance to OXi4503 treatment and could provide targets for drug development to totally eliminate metastases.

Topics: Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Cadherins; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Diphosphates; Drug Resistance, Neoplasm; Fibroblast Growth Factors; Homeodomain Proteins; Hypoxia-Inducible Factor 1, alpha Subunit; Kruppel-Like Transcription Factors; Liver Neoplasms; Male; Mice; Mice, Inbred CBA; Stilbenes; Transforming Growth Factor beta; Tumor Microenvironment; Vascular Endothelial Growth Factor A; Vimentin; Zinc Finger E-box-Binding Homeobox 1

Hepatocellular carcinoma (HCC) and colorectal carcinoma with hepatic metastases (mCRC) are cancers with poor prognosis and limited therapeutic options. New approaches are needed and adoptive immunotherapy with Vgamma9Vdelta2 T lymphocytes represents an attractive strategy. Indeed, Vgamma9Vdelta2 T cells were shown to exhibit efficient lytic activity against various human tumor cell lines, and in vitro Vgamma9Vdelta2 T expansion protocol based on single phosphoantigen stimulation could be easily performed for healthy donors. However, a low proliferative response of Vgamma9Vdelta2 T cells was observed in about half of the cancer patients, leading to an important limitation in the development of Vgamma9Vdelta2 T cell-based immunotherapy. Here, for the first time in the context of cancer patients, Vgamma9Vdelta2 T cell expansions were performed by co-culturing peripheral blood mononuclear cell (PBMCs) with autologous dendritic cells (DCs) pretreated with aminobisphosphonate zoledronate. For patients not responding to the conventional culture protocol, co-culture of PBMC with zoledronate-pretreated DCs induced strong cell expansion and allowed reaching a minimal rate of purity of 70% of Vgamma9Vdelta2 T cells. The potent immunostimulatory activity of zoledronate-treated DCs was associated with higher amount of isopentenyl pyrophosphate (IPP) in the culture and was correlated with better ability to activate Vgamma9Vdelta2 T cells as measured by IFN-gamma production. Moreover, we demonstrated that the cytotoxic level of Vgamma9Vdelta2 T cells against freshly autologous tumor cells isolated from patients could be significantly increased by pretreating the tumor cells with zoledronate. Thus, this method of generating Vgamma9Vdelta2 T cells leads eligible for Vgamma9Vdelta2 T cell adoptive immunotherapy the HCC and mCRC patients.

Topics: Adenocarcinoma; Aged; Blotting, Western; Bone Density Conservation Agents; Carcinoma, Hepatocellular; Cell Differentiation; Cell Proliferation; Coculture Techniques; Colorectal Neoplasms; Cytotoxicity, Immunologic; Dendritic Cells; Diphosphates; Diphosphonates; Female; Flow Cytometry; Hemiterpenes; Humans; Imidazoles; Immunotherapy, Adoptive; Liver Neoplasms; Male; Middle Aged; Organophosphorus Compounds; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocyte Subsets; Zoledronic Acid

OXi4503 retards tumor growth in a dose-dependent manner and improves survival in a murine model of colorectal liver metastases. This agent causes extensive vascular shutdown by selectively altering the tubulin cytoskeleton within the endothelial cells of tumor vessels. The destruction of tumor vessels is incomplete, however, and tumor revascularization occurs after the treatment. This study evaluates the pattern of microcirculatory changes and alterations to the ultrastructural properties of the tumor vasculature that result from OXi4503 treatment. Male CBA mice were induced with liver metastases via an intrasplenic injection of a murine-derived colorectal cell line. After administering a single intraperitoneal dose of OXi4503, changes in tumor perfusion, microvascular architecture and permeability were assessed at various time points. One hour after a 100-mg/kg dose of OXi4503, a significant decrease in the percentage of tumor perfusion (63.96+/-1.98 in controls versus 43.77+/-2.71 in treated mice, P<0.001) was observed, which was still evident 5 days after the treatment. Substantial tumor microvascular damage and minimal normal liver injury were observed. Tumor vascular permeability was significantly elevated 45 min after the OXi4503 treatment (67.5+/-3.60 in controls versus 80.5+/-2.24 microg/g, P<0.05). The findings suggest that OXi4503 selectively targets tumor vessels and causes immediate microvascular destruction. Even at the maximum tolerated dose, however, residual patent tumor vessels were still present after treatment, implying incomplete tumor destruction. A combination of OXi4503 with other chemotherapeutic modalities might achieve complete tumor eradication and improve long-term survival.

Topics: Angiogenesis Inhibitors; Animals; Blood Vessels; Capillaries; Capillary Permeability; Carcinogens; Colorectal Neoplasms; Dimethylhydrazines; Diphosphates; Evans Blue; Laser-Doppler Flowmetry; Liver Neoplasms; Male; Mice; Mice, Inbred CBA; Microscopy, Confocal; Microscopy, Electron, Scanning; Regional Blood Flow; Stilbenes; Tissue Fixation

The objective of this study is to correlate the presence of bone and liver metastases in patients with breast cancer with respect to the results of bone and liver scans, axillary nodal status, and serum alkaline phosphatase levels. One hundred ninety-seven patients with breast cancer treated by modified radical mastectomy between the years 1978 and 1981 were studied. Fifty-nine (30%) of the total group had distant metastases during the course of observation of 60 to 96 months; of 35 patients in whom bone metastases developed, 30 had normal preoperative bone scan results. Of 21 patients who had liver metastases, 19 had normal preoperative liver scans. Nineteen (70%) of the 27 patients with abnormal bone scans had normal alkaline phosphatase levels. Seven (63%) of the 11 patients who had abnormal liver scans had a normal alkaline phosphatase. The study supports the concept that preoperative bone and liver scans are ineffective indicators of metastatic involvement. Selection of patients for screening by bone and liver scans according to alkaline phosphatase determinations was not supported by this study. The appropriate use of bone scans for screening in patients with breast carcinoma is suggested as a follow-up device in patients with positive lymph nodes.

Topics: Alkaline Phosphatase; Bone Neoplasms; Breast Neoplasms; Diphosphates; Female; Humans; Liver Neoplasms; Mastectomy; Radionuclide Imaging; Technetium; Technetium Tc 99m Pyrophosphate; Technetium Tc 99m Sulfur Colloid

Sequential liver scintiphotography with Tc-99m pyrophosphate (PYP) was used to prospectively evaluate its uptake patterns in hepatoma. The scintiphotos and time-activity curves of 40 cases were analyzed. Two distinct patterns of tumor activity were noted: gradual but complete extraction and trapping of Tc-99m PYP in hepatoma in 38% of the patients (group 1), and absence of subsequent Tc-99m PYP uptake in hepatoma after initial blood pool activity in 62% of the patients (group 2). Since extraction and trapping of Tc-99m PYP occur approximately in two fifths of the patients with hepatoma, we conclude that Tc-99m PYP liver scintigraphy is not worthwhile supplementing the conventional radionuclide studies for diagnosing hepatoma, even in the selected patients in the countries where the prevalence of hepatoma is high.

Topics: Carcinoma, Hepatocellular; Diphosphates; Humans; Liver; Liver Neoplasms; Prospective Studies; Radionuclide Imaging; Technetium; Technetium Tc 99m Pyrophosphate; Technetium Tc 99m Sulfur Colloid; Time Factors

Topics: Carcinoma; Diphosphates; Female; Humans; Liver Neoplasms; Radionuclide Imaging; Technetium; Technetium Tc 99m Pyrophosphate; Thyroid Neoplasms

Staging bone scans or skeletal surveys were obtained of 97 patients with endometrial carcinoma. Of the 77 patients with Stage I or II disease, no metastases were identified at staging. Three patients in the entire series demonstrated bony metastases; all of these metastases were detectable by radionuclide bone scan and radiographic bone survey. Eighty-nine patients were examined with radionuclide liver/spleen scanning at the time of staging. Four of the 89 initial scans were interpreted as demonstrating hepatocellular disease, and all four patients had abnormal liver function studies. Only one patient demonstrated a possible hepatic metastasis at initial diagnosis. This patient also had abnormal liver function studies. Based on these results, bone surveys and radionuclide bone scans are not indicated as screening procedures in endometrial carcinoma. It is suggested that screening for liver metastases in patients with endometrial carcinoma is not warranted in patients with normal liver function studies.

Topics: Bone Neoplasms; Carcinoma; Diphosphates; Diphosphonates; Female; Humans; Liver Neoplasms; Neoplasm Staging; Radionuclide Imaging; Sulfur; Technetium; Technetium Tc 99m Medronate; Technetium Tc 99m Sulfur Colloid; Uterine Neoplasms

A case of small cell anaplastic carcinoma of the lung with hepatic secondaries has been found to accumulate Tc-99m-pyrophosphate in both lung primary and hepatic metastases and to our knowledge is the first such case reported in the literature. There was no radiographic evidence of calcification in the tumor or hepatic metastases.

Topics: Aged; Bone and Bones; Diphosphates; Humans; Liver Neoplasms; Lung Neoplasms; Male; Radionuclide Imaging; Technetium; Technetium Tc 99m Pyrophosphate

Previously, we reported that the rate of metabolism of methyl sterol intermediates of cholesterol biosynthesis by broken-cell preparations of Morriss hepatoma 7777 is very slow, whereas the intact tumors are known to synthesize cholesterol quite efficiently. Active preparations have now been obtained by substitution of pyrophosphate for phosphate buffer. Although substitution of pyrophosphate buffer markedly enhances microsomal methyl sterol demethylation rates 3- to 4-fold in hepatoma 7777, other microsomal enzymes and electron carriers in either liver or a more slowly growing hepatoma appear to be unaffected by pyrophosphate. Several properties of the active microsomal methyl sterol demethylase have now been compared for control rat liver, host liver, tumor 7777, and tumor 5123C. Conditions necessary for the assay of initial velocities of enzymic reactions in the tumor microsomes have been established with respect to the amount of protein, time-course, concentrations of cofactors and substrate, pH, and other variables. The K'm and the responses to the variables studied above are very similar for methyl sterol demethylase of microsomes isolated from control liver, host liver, tumor 5123C, and tumor 7777. The multienzymic demethylase in the various preparations has been found to be inhibited similarly by in vitro additions of cyanide, cytochrome c, and bile salts. Thus, the enzymes of the microsomal-bound 4-methyl sterol demethylase of cholesterol biosynthesis appear to be very similar in liver and these 2 Morris hepatomas. When xenobiotic inducers of microsomal oxidases, such as phenobarbital and methylcholanthrene, are administered to normal and tumor-bearing rats, elevated rates of methyl sterol demethylation are observed with isolated liver microsomes obtained from both normal and tumor-bearing rats. Similar increases are not observed in the tumors. Furthermore, daily administration of an intestinal bile acid sequestrant elevates hepatic methyl sterol demethylase, but statistically significant changes were not observed in tumors 7777 and 5123C. Since the enzymes of methyl sterol demethylase appear to be grossly similar in liver and these hepatomas, regulation of the activity of the multienzymic system contained in the tumors may be altered. On the other hand, these agents in vivo simply may not affect liver and the hepatomas similarly, due to a lack of uptake of the foreign substances by the tumor that has been transplanted to the thighs.

Topics: Animals; Buffers; Carcinoma, Hepatocellular; Cholesterol; Cholestyramine Resin; Cyanides; Cytochrome c Group; Diphosphates; Edetic Acid; Liver; Liver Neoplasms; Male; Methylcholanthrene; Microsomes, Liver; NADH, NADPH Oxidoreductases; Neoplasms, Experimental; Phenobarbital; Phosphates; Rats; Steroid Hydroxylases; Sterols

99Tcm-Sn pyrophosphate bone scans of 250 patients referred for skeletal metastatic survey were analysed to determine the frequency of abnormal extra-osseous localization and the various pathological causes. Twenty-six patients demonstrated abnormal extra-osseous concentration. There were three false positives. Sixty-five per cent of the extra-osseous lesions concentrating pyrophosphate were malignant (carcinoma of lung and breast, metastatic hepatic carcinoma, chondrosarcoma) and the remainder were benign lesions, e.g. sarcoidosis, soft-tissue calcification, post-surgical and irradiation sites. An incidental finding was the unusual frequency of accumulation of pyrophosphate in various joints, especially the knees and shoulders in asymptomatic patients above 70 years of age.

Topics: Age Factors; Aged; Bone Neoplasms; Breast Neoplasms; Diphosphates; False Positive Reactions; Humans; Joints; Liver Neoplasms; Lung Neoplasms; Middle Aged; Neoplasm Metastasis; Neoplasms; Radionuclide Imaging; Sarcoidosis; Technetium; Tin

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; Cells, Cultured; Cycloheximide; Cytidine; Deamination; Deoxyadenosines; Deoxyribonucleosides; Deoxyuridine; Diphosphates; Dipyridamole; DNA, Neoplasm; Guanine Nucleotides; Hydroxyurea; Hypoxanthines; Inosine; Inosine Nucleotides; Liver Neoplasms; Neoplasms, Experimental; Phosphates; Rats; RNA, Neoplasm; Thymidine; Time Factors; Tritium

Topics: Animals; Carbon Isotopes; Carcinoma, Hepatocellular; Centrifugation, Density Gradient; Cholesterol; Citrates; Diphosphates; Electrophoresis, Polyacrylamide Gel; Half-Life; Leucine; Liver; Liver Neoplasms; Male; Methods; Microsomes, Liver; Molecular Weight; Neoplasm Proteins; Neoplasms, Experimental; Phospholipids; Polytetrafluoroethylene; Proteins; Rats; Sodium Dodecyl Sulfate; Urea

Topics: Adenosine Triphosphate; Amino Acids; Animals; Ascitic Fluid; Carbon Radioisotopes; Carcinoma, Hepatocellular; Cell Nucleolus; Centrifugation, Density Gradient; Cytoplasm; Diphosphates; Hydroxamic Acids; Hydroxylamines; Liver Neoplasms; Neoplasm Proteins; Neoplasms, Experimental; Phosphates; Phosphorus Radioisotopes; Rats; RNA, Transfer; Transfer RNA Aminoacylation

Topics: Aging; Animals; Animals, Newborn; Carbon Isotopes; Carcinoma, Hepatocellular; Cytoplasm; Diphosphates; Liver; Liver Neoplasms; Male; Neoplasms, Experimental; Nucleic Acids; Orotic Acid; Pentosyltransferases; Rats; Ribose; Thymidine; Tritium; Uridine

A system for study and measurement of the attachment in vitro of exogenous polyribosomes to membranes has been presented. Its main features are use of low temperature, post-microsomal supernatant, pyrophosphate and citric acid to remove ribosomes from the surface of rough endoplasmic reticulum, and a method for quantitative separation of unattached from membrane-associated polyribosomes. The following were found. (1) Rough endoplasmic reticulum, from which ribosomes had been removed by treatment with pyrophosphate and citrate, bound over 50% of added polyribosomes, whereas the untreated (or control) rough and smooth endoplasmic reticulum and the smooth endoplasmic reticulum treated with pyrophosphate-citrate did not bind polyribosomes. (2) The polyribosome-binding capacity of rough endoplasmic reticulum stripped of its ribosomes decayed upon storage of the membranes at 0-4 degrees C. The half-life of this decay was about 6 days whereas that of the polyribosome-binding capacity of hepatoma stripped rough endoplasmic reticulum was about 1.5 days. (3) Preparations of stripped rough endoplasmic reticulum after reassociation with polyribosomes in vitro were quite similar to preparations of native rough endoplasmic reticulum as viewed with the electron microscope. Evidence is presented to support the contention that association of polyribosomes with membranes was the result of polyribosomal reattachment to the membranes rather than trapping of the polyribosomes between vesicles of the membranes.

Topics: Animals; Carcinoma, Hepatocellular; Citrates; Diphosphates; Endoplasmic Reticulum; In Vitro Techniques; Liver; Liver Neoplasms; Male; Methods; Microscopy, Electron; Microsomes, Liver; Neoplasms, Experimental; Phosphorus Isotopes; Rats; Ribosomes; Temperature

Topics: Adenine Nucleotides; Adenosine Triphosphate; Ammonium Chloride; Animals; Asparagine; Aspartic Acid; Carbon Isotopes; Carcinoma, Hepatocellular; Diphosphates; Female; Glutamine; Hydrogen-Ion Concentration; Hydroxamic Acids; Hydroxylamines; Liver Neoplasms; Magnesium; Manganese; Neoplasms, Experimental; Potassium; Rats

Topics: Animals; Bile Acids and Salts; Carcinoma, Hepatocellular; Cell Fractionation; Cell Line; Centrifugation, Density Gradient; Centrifugation, Zonal; Culture Techniques; Diphosphates; Encephalomyocarditis virus; Liver Neoplasms; Microsomes; Mitochondria; Rats; Ribonucleases; RNA Nucleotidyltransferases; RNA, Viral; Temperature; Time Factors

Topics: Carbon Isotopes; Carcinoma, Hepatocellular; Chemistry Techniques, Analytical; Choline; Cytosine Nucleotides; Deoxycytosine Nucleotides; Diphosphates; Lecithins; Lipid Metabolism; Liver; Liver Neoplasms; Neoplasms, Experimental; Phosphatidylcholines; Rats; Research; Ribonucleases