Kohtaro Kawashima

Comparison of ADP-ribosylation of chromosomal proteins between intact and broken cells.

ADP-ribosylation of nonhistone high mobility group (HMG) proteins and histone H1 in intact cells was markedly different from that in the broken cell systems using isolated nuclei and chromatin. (i) The amounts of (ADP-ribose)n on these proteins in intact cells were less 10(-2) - 10(-3) of those in the broken cell systems. (ii) The modified protein molecules in intact cells were about 2 orders of magnitude less extent than those in the broken cells. (iii) The (ADP-ribose)n chains synthesized in intact cells, nuclei and chromatin were mainly mono, oligo and poly, respectively. (iv) The principal acceptor molecules in intact cells were concentrated in HMG 1, 2, 14 and 17, and histone H1, whereas the four HMG proteins were minor acceptors in nuclei, and HMG 1 and 2 and histone H1 were major acceptors in chromatin.

Effect of KCA-098 on the function of osteoblast-like cells and the formation of TRAP-positive multinucleated cells in a mouse bone marrow cell population.

KCA-098 (3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinol ine-6-one), an analogue of coumestrol (a naturally occurring weak phytoestrogen), dose-dependently increased alkaline phosphatase activity of osteoblastic ROS 17/2.8 cells and freshly-isolated osteoblasts from neonatal mouse calvaria, and reduced cell proliferation. In addition, KCA-098 increased the synthesis of collagenese-digestible protein (CDP) of ROS 17/2.8 cells. On the other hand, KCA-098 had no effect on the basal synthesis of osteocalcin but reduced the 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)(2)D3)-induced increase in osteocalcin synthesized by ROS 17/2.8 cells. Therefore, KCA-098 had a bidirectional effect on the differentiation of osteoblasts (i.e., stimulating both alkaline phosphatase activity and synthesis of CDP and inhibiting osteocalcin synthesis). However, as KCA-098 stimulated the mineralization of chick embryonic bone in organ culture and recovered the bone density reduced by ovariectomy of rats, it would serve overall to stimulate the differentiation of osteoblasts. On the other hand, KCA-098 inhibited the formation of tartrate-resistant, acid phosphate-positive multinucleated cells (TRAP(+)MNC) induced by 1 alpha,25(OH)(2)D(3), parathyroid hormone (PTH), and prostaglandin E2 (PGE2) in cultures of mouse bone marrow cells, showing that it inhibits the formation of osteoclast-like cells. Coumestrol and 17beta-estradiol had no effect on the proliferation and alkaline phosphatase activity of ROS 17/2.8 cells. They did, however, dose-dependently inhibit osteoclast-like cell formation as well as KCA-098 did, indicating that the main action of coumestrol and 17beta-estradiol on bone tissue is the inhibition of bone resorption.

Identification of two activities of (ADP-ribose)N glycohydrolase in hela S3 cells

HeLa S3 cells contained two activities (form I and II) that degrade (ADP-ribose)n exo-glycosidically. Form I was extracted from nuclei only by sonication in high ionic strength, while form II was soluble in cytosol. The two active forms differed in chromatographic behaviors, in their Km values for (ADP-ribose)n, and in their pH and salt requirements for optimal activity, although both forms exhibited properties characteristic of (ADP-ribose)n glycohydrolase such as requirement of sulfhydryl compounds and sensitivity of ADP-ribose and cAMP. Form I and II had apparent molecular weights of 72,000 and 53,000, respectively, as determined by gel filtration on Sepharose CL-6B.

In vitro effect of KCA-098, a derivative of coumestrol, on bone resorption of fetal rat femurs

The effects of 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoli ne-6-one (KCA-098), a derivative of coumestrol, on bone resorption was studied in organ cultures of 20-day fetal rat femora. KCA-098 increased the length, dry weight, and calcium and phosphorus contents of parathyroid hormone (PTH)-treated fetal rat femur. As PTH significantly reduced the calcium and phosphorus contents of the femora, probably by stimulating bone resorption, KCA-098 seems to inhibit bone resorption. In fact, KCA-098 inhibited the PTH-induced release of 45Ca from pre-labeled fetal rat femora into the medium in organ culture. Coumestrol also inhibited the release of 45Ca from bone into the medium. However, KCA-098 did not increase the uterine weight of ovariectomized rats, whereas coumestrol did so. Thus KCA-098 is a unique, new inhibitor of bone resorption that has no estrogenic activity.

Evidence of a regulatory role of the level of poly (ADP-ribose) in chromosomal proteins in metallothionein gene expression by glucocorticoids but not by heavy metals

The induction capacity of dexamethasone, a synthetic glucocorticoid, for the synthesis of metallothionein was about the same as that of 3-aminobenzamide, which is an inhibitor of ADP-ribosylation of chromosomal proteins, in cultured mouse mammary tumor cells. Both inductions of metallothionein were temporally correlated with a decrease in the amount of endogenous poly (ADP-ribose) on nonhistone high-mobility-group 14 and 17 proteins. In contrast, the extent of cadmium-induced metallothionein synthesis was 2-3-times that of dexamethasone or 3-aminobenzamide. However, cadmium had essentially no effect on de-ADP-ribosylation of these proteins.

Poly(ADP-ribose) glycohydrolase is present in metaphase chromosomes of HeLa S3 cells

Abstract Poly(ADP-ribose) glycohydrolase was found in metaphase chromosomes of HeLa S3 cells. Adenosine diphosphate ribose and 3′, 5′-cyclic AMP inhibited the glycohydrolase activity, whereas ADP, ATP, NAD and 3′,5′-cyclic GMP did not. The hydrolytic product of poly(ADP-ribose) bound to metaphase chromosomes with this enzyme was identified as adenosine diphosphate ribose.

Establishment of permanent cell lines exhibiting vitamin D-dependent expression of β-galactosidase activity

The active hormonal form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), has been described as a principal mediator of skeletal homeostasis. Treatment of rat osteosarcoma (ROS)17/2.8, an osteoblast-like cell line, with 1alpha,25(OH)2D3 results in a ligand-dependent increase in transcription of the bone-specific osteocalcin gene. We isolated permanent cell lines that were established by transfecting ROS 17/2.8 cells with plasmids consisting of the human osteocalcin gene promoter containing the vitamin D responsive element linked to a bacterial beta-galactosidase gene. In one of many cell lines, especially in clone NK-31, 1alpha,25(OH)2D3 strongly stimulated beta-galactosidase activity. Reverse transcription-polymerase chain reaction analysis also showed endogenous osteocalcin gene expression and beta-galactosidase gene expression in clone NK-31 cells, which paralleled the increase in beta-galactosidase activity. Using a synthetic analogue of 1alpha,25(OH)2D3, 24,24-difluoro-1alpha,25-dihydroxyvitamin D3, we found that the levels of this activity and these gene expressions were nearly parallel to those of 1alpha,25(OH)2D3. 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 at high doses (concentration: 10(-7) M) also induced beta-galactosidase activity in clone NK-31. These cell lines, harboring the plasmid-carrying beta-galactosidase gene under the control of the osteocalcin gene promoter, may contribute to studies on the regulation by 1alpha,25(OH)2D3 or to the development of synthetic analogues of 1alpha,25(OH)2D3.