Mona L. Coetzee

Different response to epidermal growth factor of hepatocytes in cultures isolated from male or female rat liver

Deoxyribonucleic acid (DNA) synthesis in hepatocytes isolated from the livers of male and female rats has been compared in monolayer culture. Plating efficiency, DNA and protein content, viability, and morphologic appearance were the same in cultures prepared with hepatocytes isolated from male or female rats. Epidermal growth factor (EGF)-induced DNA synthesis was significantly higher in hepatocytes from male rats than in hepatocytes from female rats. This was the case whether hepatocytes were isolated from normal or partially hepatectomized male or female rats. Hepatocytes isolated from regenerating liver synthesize more DNA than those isolated from normal liver in response to EGF. This increased response to EGF in hepatocytes derived from regenerating liver was relatively the same for male- and female-derived hepatocytes, but the magnitude of the response was considerably higher in male-derived hepatocytes. In contrast, in vivo DNA synthesis in the liver remnant after partial hepatectomy was similar in male and female rats if measured 24 h after the operation. A comparison of EGF binding to male- and female-derived hepatocytes maintained in primary culture indicated a lower number of high-affinity receptors for EGF in the female hepatocytes. The addition of estrogen to primary cultures of hepatocytes isolated from male rats inhibited EGF binding as well as EGF-induced DNA synthesis. Our studies show significant differences in DNA synthesis in response to EGF when male and female hepatocytes are compared in primary culture. The regenerative response after partial hepatectomy, on the other hand, was the same in male and female rats. Thus, our studies indicate that the sex of the donor, rat is important when hepatocytes in culture are used for a variety of studies, such as hepatocyte metabolism, induction and control of DNA synthesis, and hepatocarcinogenesis. In addition, our results indicate that caution is advised when inferences are made from in vitro findings for in vivo conditions.

A comparison of DNA repair synthesis in primary hepatocytes from young and old rats

DNA repair synthesis has been compared in primary hepatocyte cultures obtained from 3-month-old and 16-20-month-old rats. Several morphological and metabolic characteristics were determined to assure cultures of comparable quality. DNA damage was induced by the addition of bleomycin or the exposure of the culture to UV irradiation. DNA repair (unscheduled DNA synthesis) was determined by measuring [3H]thymidine incorporation. After UV irradiation, there was almost twice as much [3H]thymidine incorporation in cells obtained from young rats as in those obtained from old rats. Equal amounts of bleomycin resulted in substantially greater damage to DNA in cells from old rats than from young rats. For equal amounts of DNA damage there was again diminished [3H]thymidine incorporation in cells obtained from old rats. Finally equal amounts of bleomycin resulted in equal damage to DNA when the bleomycin was added to isolated rat liver nuclei from young or old rats. Bleomycin treated nuclei from young rats incorporated substantially more [3H]thymidine triphosphate (TTP) than bleomycin treated nuclei from old rats. The results indicate that hepatocytes from old rats are much more susceptible to bleomycin than hepatocytes from young rats and that the capacity for DNA repair synthesis is impaired in hepatocytes from old rats.

A difference in bleomycin-induced DNA synthesis between liver nuclei from mature and old rats

Abstract Nuclei from the livers of mature and old rats have been compared in a nuclear incorporating system. Regular incorporation of 3 H-TTP was found to be the same with the two nuclear preparations. The addition of bleomycin resulted in increased 3 H-TTP incorporation with both nuclear preparations. Nuclei from mature rats, however, responded twice as well to bleomycin than did nuclei from old rats. The addition of 50 μg bleomycin to 100 mg nuclear DNA resulted in a 13.8 fold stimulation in nuclei from mature rats and a 6.2 fold stimulation in nuclei from old rats. Bleomycin has previously been shown to stimulate repair synthesis and our finding suggests that there might be diminished repair capacity in old rat liver cells. Repair of DNA from nuclei exposed to bleomycin has been demonstrated, but the difference between mature and old nuclei was not apparent in that determination. DNA polymerase β seems to be the enzyme responsible for increased incorporation due to bleomycin. The amounts of DNA polymerase β that could be extracted from nuclei of mature and old rats was found to be the same. The difference in bleomycin- induced incorporation found with the nuclear incorporation system might depend on the accessibility of the damaged DNA in the chromosomes to DNA polymerase β.

Loss of a Serum Protein from Hepatoma-Bearing Animals

A serum protein that is present in normal rat serum, and can be detected if serum is fractionated on polyacrylamide gel electrophoresis, disappears from the serum of hepatoma-bearing animals as the he

Synthesis of the 60-S ribosomal subunit in yeast

Abstract We have previously shown that the process of ribosomal RNA synthesis in yeast is similar in many respects to that observed in other nucleated cells. This investigation concerns the formation of the large (60 S) subunit of the yeast ribosome. We demonstrate that a precursor to this subunit exists. The characteristics of this particle are: (1) it sediments at about 55 S in high-ionic-strength sucrose gradients; it sediments coincidentally with the 60-S subunit in low-ionic strength sucrose gradients; (3) the kinetics of its formation are those of a precursor particle; (4) it contains mature (25 S) ribosomal RNA; (5) the addition of protein to the mature ribosomal RNA takes longer than the processing of the ribosomal RNA precursor to 25-S RNA; and (6) the protein associated with the precursor particle is apparently obtained from a pool of ribosomal protein in the cell. We also show that this pool does not contain all the ribosomal proteins necessary for completion of the 60-S subunit.

Purification of thymidine kinase by affinity chromatography with an enzyme inhibitor as the ligand.

Abstract An affinity column for the purification of thymidine kinase is described. The ligand in this column is a glycoprotein isolated from rat kidney. This glycoprotein inhibits phosphorylation of thymidine in cultured cells and in a cell-free assay system. With an affinity column containing the glycoprotein as a ligand, a 24-fold purification of thymidine kinase from an ammonium sulfate fraction of a crude tissue extract can be obtained. Thymidine kinase eluted from the affinity column migrates as one major band on polyacrylamide and as one diffuse major band on sodium dodecyl sulfate-polyacrylamide. The affinity column, with thymidine kinase bound to the inhibitor, can also be used as an assay system. When the glycoprotein is covalently attached to Sepharose, it retains its binding capacity for thymidine kinase but has apparently lost its ability to inhibit the enzyme. Thymidine kinase eluted from the affinity column is again sensitive to the glycoprotein. It seems to be a carbohydrate moiety of the glycoprotein that is responsible for the inhibition.

The Effect of Several Antitumor Agents on 3H-TTP Incorporation in Host Liver and Hepatoma Nuclei

Six different tumor antibiotics have been investigated in a nuclear incorporating system for their ability to inhibit 3H-TTP incorporation. Both host liver nuclei and nuclei prepared from two different Morris hepatomas have been used in the investigation. Three of these anti-tumor agents inhibit 3H-TTP incorporation equally in host liver and hepatoma nuclei, two preferentially inhibit incorporation in hepatoma nuclei and one stimulates incorporation preferentially in host liver nuclei. The effects of these compounds on nuclear DNA has been analyzed on neutral and alkaline sucrose density gradients. The nuclear incorporation system appears to be useful as a screening test system for potential anti-tumor agents.

Inhibition of growth of Morris hepatomas 7777 and 7800 by corn oil.

Intraperitoneal injection of trace amounts of corn oil prior to and following the injection of 40-50 mg of tissue from hepatoma 7777 or 7800 into the thigh of adult male Buffalo rats resulted in a marked decrease in the growth rate of both tumors. Exhaustive extraction of the corn oil with water indicated that the active component was not water soluble. Similar injections of safflower oil or isotonic saline had no effect on tumor growth rate. Analysis of the tissue phospholipid fatty acids revealed that the injected corn oil caused no change in the esterified fatty acids in this lipid fraction.

Stimulatory effect of a serum factor on DNA synthesis in isolated hepatoma nuclei.

A serum protein present in normal rat serum and absent from the serum of hepatoma-bearing animals at advanced stages has a stimulatory effect on 3H-thymidine incorporation into hepatoma cells in suspension. Liver cells maintained in a similar suspension are not affected by the factor. The stimulation appears to be at the level of chromatin or DNA. Isolated membrane-denuded nuclei from Morris hepatoma 7777 incorporate more 3H-TTP when the factor is present in the incubation mixture. Nuclei from host liver are not stimulated. The factor also stimulates incorporation of 3H-TTP in a system using calf thymus DNA as primer and an extracted DNA polymerase. In this system incorporation is stimulated with DNA polymerase from both tissues, host liver and hepatoma 7777. It is concluded that the factor does not act on the DNA polymerase but on chromatin or DNA.