Regulatory role of endogenous regucalcin in the enhancement of nuclear deoxyribonuleic acid synthesis with proliferation of cloned rat hepatoma cells (H4‐II‐E)

Abstract

The role of endogenous regucalcin in the regulation of deoxyribonuleic acid (DNA) synthesis in the nuclei of the cloned rat hepatoma cells (H4-II-E) with proliferative cells was investigated. Cells were cultured for 6-96 h in a alpha-minimum essential medium (alpha-MEM) containing fetal bovine serum (FBS; 1 or 10%). Cell number was significantly increased between 24 and 96 h after culture with 10% FBS; cell proliferation was markedly stimulated by culture with 10% FBS as compared with that of 1% FBS. In vitro DNA synthesis activity in the nuclei of cells was significantly elevated 6 h after culture with 10% FBS and its elevation was remarkable at 12 and 24 h after the culture. Nuclear DNA synthesis activity was significantly reduced in the presence of various protein kinase inhibitors (PD98059, staurosprine, or trifluoperazine) in the reaction mixture containing the nuclei of cells cultured for 12 and 24 h with FBS (1 and 10%). The addition of regucalcin (10(-7) and 10(-6)M) in the reaction mixture caused a significant inhibition of nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (25-100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS resulted in a significant increase in nuclear DNA synthesis activity. This increase was completely blocked by the addition of regucalcin (10(-6) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing nuclear DNA synthesis activity was significantly inhibited in the presence of various protein kinase inhibitors. DNA synthesis activity was significantly enhanced in the presence of anti-regucalcin antibody (100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS in the presence of Bay K 8644 (2.5 x 10(-6) M). Culture with Bay K 8644 did not cause a significant increase in DNA synthesis activity in the absence of anti-regucalcin antibody. The present study demonstrates that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis with proliferative cells.

Regucalcin is broadly expressed in male reproductive tissues and is a new androgen-target gene in mammalian testis

“…We think that the use of different fixation protocols may be causing this histological artifact. RGN immunostaining is visible in cytoplasm as well as in nucleus, which is in accordance with reports showing that RGN is able to translocate to the nucleus regulating DNA synthesis and gene expression (Inagaki & Yamaguchi 2001, Tsurusaki & Yamaguchi 2004, Maia et al 2009). This is the first report describing RGN expression and localization in testis of any vertebrate.…”

Suppression of cell proliferation and deoxyribonucleic acid synthesis in the cloned rat hepatoma H4‐II‐E cells overexpressing regucalcin

“…Endogenous regucalcin has been shown to have a suppressive effect on protein phosphatase, protein kinase, and DNA synthesis activities in proliferative H4-II-E cells following stimulation with FBS , 2001a, 2001b. Regucalcin may play a suppressive role for overexpression of cell proliferation.…”

“…This cell may be a tool to study a role of regucalcin in the regulation of cell proliferation. More recently, it has been shown that regucalcin can inhibit protein kinase and protein phosphatase activities which are raised with a proliferation of hepatoma H4-II-E cells Yamaguchi, 2000, 2001a], and that regucalcin has an inhibitory effect on nuclear DNA synthesis in the proliferation of H4-II-E [Inagaki and Yamaguchi, 2001b]. Endogenous regucalcin may have a suppressive effect on proliferation of the cloned rat hepatoma cells.…”

Suppressive role of endogenous regucalcin in the enhancement of deoxyribonucleic acid synthesis activity in the nucleus of regenerating rat liver

“…Regucalcin has also been shown to inhibit DNA synthesis in the nuclei of cloned rat hepatoma cells with proliferation after serum stimulation [Inagaki and Yamaguchi, 2001b]. Regucalcin may play a suppressive role in the proliferation of liver cells, because of regulating overexpression of cell proliferation.…”

“…Regucalcin can inhibit protein kinase [Inagaki and Yamaguchi, 2001a] and protein tyrosine phosphatase [Inagaki and Yamaguchi, 2000] activities which are increased in the proliferation of the cloned rat hepatoma cells cultured with fetal bovine serum. More recently, it has been demonstrated that endogenous regucalcin has an inhibitory effect on DNA synthesis in the nuclei of cloned rat hepatoma cells cultured with serum stimulation [Inagaki and Yamaguchi, 2001b]. Regucalcin may play an inhibitory role in the signaling pathway, which is related to protein kinase and protein phosphatases in proliferative cells, suggesting a suppressive role of regucalcin in cell proliferation.…”