K. Tamiva-Koizumi's scientific contributions

Two forms of phospholipase D (PLD) have been found to be present in nuclei isolated from rat hepatocytes by measuring phosphatidylbutanol (PBul) produced from exogenous radiolabeled phosphatidylcholinc in the presence of butanol. In nuclear lysatcs from either rat liver or asciles hepaioma AH 7974 cells, the PLD activity was markedly stimulated by a recombinant ADP-ribosylation factor (rARF) in the presence of the guanosine 5′-0-(3-thiotriphosphate) (GTPγS) and phosphatidylinositol 4,5bisphosphate. ATP and phorbol-12-myristatc 13-acelatc (PMA) had no syncrgistic effect on tins PLD activity On the other hand, the nuclear PLD was stimulated by unsaturated fatty acids, especially by oleic acid The ARF-dependcnt nuclear PLD activity was increased in the S-phase of the regenerating rat liver after partial hcpateciomy and also was much higher in AH 7974 cells than in the resting rat liver In contrast, the levels of the oleale-dependent PI,D activity remained constant throughout the cell cycle in liver regeneration The intranuclear levels of the stimulating proteins of the nuclear PLD activity c g ARF, RhoA, and PKCδ . but not Cdc42 and PKOβII. increased in the S-phasc of the regenerating liver. These results suggested that the nuclear ARF-dcpendcnt PLD activity ma> be associated with cell proliferation.

A human leukemia celt line, TF1 is induced to apoptosis by the depletion of GM-CSF, but its bcl2 transfectant, TF1 -bc!2, is resistant to apoptosis. Here we analyzed the process of apoptosis induced by various DNA damages to determine the critical pathway for the sensitivity to apoptosis. Treatments including GM-CSF depletion, methyl methanesulfonate:MMS (lOOfig/ml), UV dSJ/m2), X-ray (20Gy). and sphingostne (5a M) produced the typical feature of apoptosis (DNA ladder formation) in TF1 with aJI treatments as described above, while TF1 -bc!2 was resistant to these treatments except for sphingosine. Furthermore, analysis of cellular sphingolipids showed that all of sphingomyelm, ceramide, and sphingosine in TF1 cells significantly increased after DNA damage but not in TF1-bcl2. This results confirmed the involvement of sphingosine in these process of apoptosis as well as ceramide. Then we focused-on the gene expression related with apoptosis such as p21/WAF1, bax, gadd45/gadd153 and early expression gene such as c-myc, c-myb, c-fos, c-jun. Gadd45 strongly expressed in both celts treated by GM-CSF depletion, MMS, and UV but very weak in both cells treated by X-ray and sphingosine, suggesting that there were some heterogeneity on the DNA damaging effect among these treatments and that gadd gene expression is not always the sensitive indicator of apoptosis.