Synthesis and half-life of a serum factor in normal and tumor-bearing animals.

The synthesis and disappearance of a serum factor from the circulating blood of rats bearing Morris hepatomas 7777 or 7800 were described. We provided evidence that the factor was synthesized by the livers of normal rats and continued to be synthesized in tumor-bearing animals. Despite very low levels in the sera of tumor-bearing animals, the factor was synthesized at twice the rate in these animals compared to that in normal rats. The half-life of the total serum protein was 2 days in normal rats and 2.9 days in hepatoma 7777-bearing animals. The half-life of the serum factor, on the other hand, was 23 hours in normal rats but only 9 hours in hepatoma-bearing rats. This rapid disappearance of the factor from the circulating blood, despite an increased synthesis, accounts for the complete absence of the factor in the sera of hepatoma-bearing animals with tumor weights larger than the liver weights.

Effect of a normal serum protein absent from hepatoma-bearing animals on cell cultures.

The effect of a serum factor purified from normal rat serum on growth and incorporation of 3-H-thymidine and 3-H-leucine was investigated with the use of 3T3 and L cells. The factor was not present in serum of hepatoma-bearing animals at a time when the weight of the hepatomas was equal to or greater than liver weight. The factor disappeared gradually from the serum of hepatoma-bearing animals, and the disappearance was proportional to the increase in the size of the hepatomas. Diminished amounts of the factor could be detected as early as 10 days after hepatoma transplant, and the factor was absent at 30 days after hepatoma transplant from the serum of rats bearing hepatoma 7777. Medium supplemented with 5% normal rat serum supported incorporation of lavel into 3T3 and L cells and growth of these cells, as did medium supplemented with 10% calf serum. Serum from hepatoma-bearing animals was only approximately equal to 50% as efficient as normal rat serum in supporting growth and label incorporation. The addition of purified factor to the serum from hepatoma-bearing animals restored the ability of the serum to support growth and label incorporation to between 70 and 90% of that found with normal rat serum as a medium supplement. The factor also enhanced incorporation of 3-H-thymidine following release of 3T3 cells from contact inhibition.

Control of hepatocyte replication by two serum factors.

Serum proteins from hepatectomized or control rats were separated by gel permeation chromatography and assayed for stimulation of hepatocyte proliferation in primary cultures of hepatocytes. Two peaks of activity were seen in the areas of large (greater than 120,000) and small (less than 3,000) molecular weight. These activities are different from insulin, epidermal growth factor, or vasopressin and are empirically termed hepatopoietin A and B, respectively. The two activities interact in a synergistic manner to stimulate hepatocyte proliferation at rates comparable to that of the whole serum.

Evaluation of a serum protein in pregnant rats.

The increase of a serum protein in the circulation of rats during pregnancy has been described. The protein is different from the human pregnancy zone protein previously described. This new serum protein factor is present in normal rat serum and accounts for approximately 1% of the total serum protein. The protein factor disappears from the serum of animals bearing Morris hepatomas 7777 and 7800. The amount of factor diminishes as the tumors increase in size. The levels of this protein in the serum increased several fold during pregnancy with highest levels found shortly before parturition. Levels remained elevated for at least 12 days post partum. No corresponding increase in the levels of this serum factor in the fetal circulation was observed. Increased levels in the maternal circulation were due to an increased synthesis of this serum protein during pregnancy. A corresponding band was observed on polyacrylamide gels for normal human female and pregnant human sera or plasma. However, there was no increase during pregnancy as judged by polyacrylamide gel electrophoresis. Quantitative determination of the protein in human sera was not possible since the band corresponding to the serum protein in rats did not cross react with a rabbit anti-rat serum factor.

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.