Kaoru Segawa

Cell density-dependent increase in chromatin-associated ADP-ribosyltransferase activity in simian virus 40-transformed cells

Abstract ADP-ribosyltransferase activity associated with chromatin is two- to tenfold higher in simian virus 40 (SV40)-transformed cells than in untransformed cells. When confluent transformed cells were subcultured, their specific enzyme activity first decreased two- to fourfold and the rapidly increased during the logarithmic phase of growth. This increase ceased or slowed down when the cells entered the stationary phase. In contrast, the activity in the untransformed cells remained low throughout the growth cycle. In SV40tsA-transformed cells (ts = temperature sensitive), this density-dependent increase in the enzyme activity was observed when the cells were cultivated at the permissive temperature, whereas the activity remained low at the restrictive temperature. The enzyme activity did not increase during induction of cellular DNA synthesis in quiescent cells either by addition of fresh medium or by infection with SV40. The chromatin-associated enzyme activity extracted with 1 m NaCl was eluted together with almost all the DNA-binding proteins from a phosphocellulose column with 0.6 m NaCl. The enzyme activity in this fraction from transformed cells, measured with or without added DNA and histones, was higher than that in a similar fraction from untransformed cells, reflecting the difference in the original activities present in the nuclei of these cells. The chain lengths of poly(ADP-ribose) formed by chromatin from SV40-transformed and untransformed cells were not significantly different. These results suggest that the number of initiation sites for ADP-ribosylation is increased in the chromatin of SV40-transformed cells compared to that of untransformed cells.

In vitro translation of adenovirus type 12-specific mRNA complementary to the transforming gene

Abstract Specific mRNA complementary to the adenovirus type 12 (AM) transforming gene was selected from Ad12-infected cells by hybridization with Eco RI-C (0–16.5 map units) and AccI -H (0–4.7 map units) DNA restriction fragments, and translated in vitro in [ 35 S]methio-nine-containing reticulocyte lysate. The products were analyzed by two-dimensional gel electrophoresis. The result showed that the Eco RI-C region encoded at least three polypeptides with molecular weights of 40,000 (40K), 38K, and 10K, while the AccI -H region encoded only 40K and 38K polypeptides. According to molecular weights and p I values, it is found that 40K or 38K and 10K polypeptides correspond to T antigen g and f, respectively. The result indicates that T antigen g is coded for by the left end of the transforming gene (0–4.7 map units, region la) and suggests that T antigen f is coded by the rest region (4.7–6.8 map units, region Ib). The size of mRNA for T antigen g was examined by translation with mRNA fractionated by sedimentation through a sucrose gradient and electrophoresis of 32 P-labeled mRNA selected by the Accl-H fragment. The results showed that the size of mRNA for T antigen g was about 13 S. The structure of the 13 S mRNA was examined by nuclease S1 mapping described by Berk and Sharp, and two :kinds of mRNA were detected. These two mRNAs are considered to be translated into two species of T antigen g polypeptides (40K and 38K).

Protein kinases and their protein substrates associated with chromatin and ribosomes in SV40-transformed rat cells

The level of endogenous protein phosphorylation in non-histone chromosomal and ribosomal wash proteins is 7--10 times greater in SV40-transformed rat cells than in untransformed parental cells. Protein kinase activity in these proteins was fractionated by either phosphocellulose or DEAE-cellulose chromatography. One major and one minor component were detected in non-histone proteins and only one component in ribosomal wash proteins when the activity in each fraction was measured with an exogenous substrate, casein. These enzymes prefer casein to whole histone as substrate and are cyclic AMP-independent. The enzyme activity in a major peak of non-histone proteins and in ribosomal wash proteins measured with casein as substrate is 3 times greater in transformed cells than in untransformed cells, whereas pH optimum, cation requirements and apparent Km values for casein and ATP are identical or very similar in the two cell types. No significant phosphatase was detected in non-histone and ribosomal wash proteins from the two types of cell. The patterns of endogenous protein phosphorylation in these protein fractions analysed by gel electrophoresis are significantly different between these cells. These results suggest that the high level of endogenous protein phosphorylation in non-histone and ribosomal wash proteins from SV40-transformed cells is caused mainly by the increased activity of protein kinase and the nature of protein substrates.

Cooperation of mitogenic growth factors with polyoma virus middle T antigen in transformation of secondary cultured rat cells.

Platelet derived growth factor cooperated with middle T antigen in inducing growth in agarose medium of secondary cultured rat embryo cells transfected with a polyoma virus middle T antigen cDNA clone. In contrast, epidermal growth factor and a conditioned medium containing transforming growth factor did not stimulate the colony-forming efficiency of such cells in the agarose medium.

The effect of viral transformation on prostaglandin production depends on cell type.

The role of prostaglandins in cellular differentiation and transformation has been widely studied. We have found previously that prostaglandin E2 production was greatly diminished in dog kidney cells (MDCK) after transformation by Harvey murine sarcoma virus. In the present study, we have shown that viral transformation can have differing effects in the ability to modify the production of prostaglandin in cultured cells. For example, the prostaglandin E2 production in rat kidney cells (NRK) is decreased after transformation by Rous sarcoma virus, while production in 3T3 cells is increased markedly after transformation by the same virus. Similarly, SV40 transformation increases prostaglandin E2 production of 3T3 cells and decreases the production in rat thyroid cells (FRTL). These results indicate that the biosynthetic pathway for prostaglandin production has varying susceptibility following viral transformation and the effect of transformation depends more on the type of cell than virus. Taking advantage of the well-defined transforming proteins encoded by polyomavirus, we have further studied the relationship between prostaglandin production in cells and the expression of T antigens in transformed cells. We showed that the expression of middle T antigen, which is associated with a protein kinase and is responsible for phenotype of transformed cells, is required for the change in prostaglandin production in cells. How these changes of prostaglandin production relate to the progression of viral transformation remains to be explored.