Tomoki Okazaki

Prediction of prognosis of estrogen receptor‐positive breast cancer with combination of selected estrogen‐regulated genes

Estrogen receptor (ER)‐positive breast cancer is a distinct subpopulation of breast cancer exhibiting a high response rate to endocrine therapy. However, not all ER‐positive patients respond to the therapy, and a subgrouping of ER‐positive patients based on the physiology of estrogen signaling is expected to be useful for predicting the prognosis. This study has revealed that selected estrogen‐regulated genes (ERGs) are useful in identification of a poor‐prognosis population among ER‐positive breast cancer patients. First, the expression levels of 11 ERGs, selected based on our earlier microarray study in cultured cells, were analyzed by means of real‐time reverse transcription‐PCR in 14 ER‐positive human breast cancer tissues. The patients were clearly divided into two groups in cluster analysis. Then, we examined the expression levels of two representative ERGs, histone deacetylase 6 (HDAC6) and insulin‐like growth factor binding protein 4 (IGFBP‐4), In 62 ER‐positive patients with immunohistochemistry to assess the impact of ERG expression on prognosis (median follow‐up 4409 days). Positive HDAC6 staining was significantly correlated with a lower disease‐free survival rate. Moreover, when the expression level of HDAC6 was assessed in combination with IGFBP‐4 expression in the nucleus, the poor‐prognosis patients were more accurately identified. This study has identified new candidate ERGs for prediction of prognosis, and we suggest that combined assessment of the expression levels of these ERGs will contribute to the clinically useful stratification of ER‐positive breast cancer patients.

The Interaction between Ku Antigen and REF1 Protein Mediates Negative Gene Regulation by Extracellular Calcium

Through the specific binding of a negative calcium-responsive element to its binding protein in response to extracellular Ca (Ca), negative calcium-responsive element-bearing genes, such as the human parathyroid hormone gene, are negatively regulated by Ca. The Ku antigen mediated negative gene regulation by Ca by interacting with a redox factor protein, REF1. Although sequence-nonspecific DNA binding activity of the Ku antigen has been well characterized, the mechanism of its sequence-specific DNA binding remained obscure. Here, we report that the specific binding of the Ku antigen to another protein, REF1, leads to DNA-protein complex formation with a novel sequence specificity and thereby regulates gene expression.

A negative vitamin D response DNA element in the human parathyroid hormone-related peptide gene binds to vitamin D receptor along with Ku antigen to mediate negative gene regulation by vitamin D.

We found that the human parathyroid hormone-related peptide (hPTHrP) gene contained a DNA element (nVDREhPTHrP) homologous to a negative vitamin D response element in the human parathyroid hormone gene. It bound to vitamin D receptor (VDR) but not retinoic acid Xα receptor (RXRα) in the human T cell line MT2 cells. VDR binding to this element was confirmed by the Southwestern assay combined with immunodepletion using anti-VDR monoclonal antibody, and this binding activity was repressed by 1,25-dihydroxyvitamin D3. Such a repression was reversed by acid phosphatase treatment, suggesting that 1,25-dihydroxyvitamin D3 phosphorylates VDR to weaken its binding activity to nVDREhPTHrP. In electrophoretic mobility shift assay, we found anti-Ku antigen antibody specifically supershifted the MT2 nuclear proteinnVDREhPTHrP complex. The nVDREhPTHrP-bearing reporter plasmid produced vitamin D-dependent inhibition of the reporter activity in MT2 cells, which was markedly masked by the introduction of the Ku antigen expression vector in the antisense orientation. On the other hand, such a procedure did not perturb the vitamin D response element-mediated gene stimulation by vitamin D. These results indicate that nVDREhPTHrP interacts with Ku antigen in addition to VDR to mediate gene suppression by vitamin D.

Ossification of the paravertebral ligaments: A frequent complication of hypoparathyroidism

Various forms of paravertebral ligamentous ossification (PVLO) were detected radiologically in 9 (53%) of 17 consecutive patients with hypoparathyroidism. A significant correlation was found between the period during which the patient was untreated and the incidence of ossification. Serum levels of calcium, phosphate, and their ionic product appeared not to influence the incidence. All the patients with PVLO exhibited evidence of ectopic calcification. The fact that ectopic ossification, which is histologically distinct from ectopic calcification, is frequently associated with hypoparathyroidism suggests that similar pathogenetic mechanisms are responsible for the development of these two conditions. The high incidence of PVLO in hypoparathyroid patients in this study may explain some of the neurologic findings in hypoparathyroidism that have been hard to explain simply on the basis of altered neuromuscular excitability.

Synergistic activation of NF-?b and inducible isoform of nitric oxide synthase induction by interferon-? and tumor necrosis factor-? in INS-1 cells

Interferon‐γ (IFN‐γ) is known to exert deleterious effects on pancreatic β‐cells and is implicated in the development of type 1 (autoimmune) diabetes mellitus. In this study, we investigated signaling mechanisms mediating the effects of IFN‐γ in pancreatic β‐cells using a differentiated rat insulin‐secreting cell line, INS‐1, with special reference to the activation of transcription factors STAT (signal transducers and activators of transcription)1 and NF‐κB. Exposure of INS‐1 cells to 100 IU/ml IFN‐γ for 24 h resulted in significant inhibition of nutrient‐induced insulin secretion associated with impaired metabolism. In combination with tumor necrosis factor‐α (TNF‐α) (50 ng/ml), IFN‐γ elicited severe cytotoxicity and induced the expression of the inducible isoform of nitric oxide synthase (iNOS) mRNA. IFN‐γ promoted tyrosine phosphorylation and DNA‐binding of STAT1 through Janus kinase (JAK)1 activation without apparent phosphorylation of JAK2. TNF‐α did not affect STAT1 activation, but stimulated DNA‐binding and transcriptional activity of NF‐κB, both of which were further increased by IFN‐γ. These effects of IFN‐γ and TNF‐α seem physiologically relevant, because either inhibition of STAT1 by the tyrosine kinase inhibitor herbimycin A or that of NF‐κB by sulfasalazine resulted in the reduction of iNOS mRNA expression. In conclusion, IFN‐γ activates STAT1 and potentiates TNF‐α–induced NF‐κB activation in INS‐1 cells, thereby inducing iNOS and cell destruction. J. Cell. Physiol. 184:46–57, 2000.

Vitamin D-dependent recruitment of DNA-PK to the chromatinized negative vitamin D response element in the PTHrP gene is required for gene repression by vitamin D.

The mechanism of transcriptional repression by nuclear hormone receptors, especially in the presence of the ligands, is largely unknown. We previously reported that 1,25-dihydroxyvitamin D(3) (1,25 vitamin D3) inhibited expression of the parathyroid hormone-related polypeptide (PTHrP) gene through the interaction between the liganded monomeric vitamin D receptor (VDR) and the negative DNA element in the PTHrP gene (nVDRE(RP)). In this study, we employed chromatin immunoprecipitation (ChIP) assay and confirmed that 1,25 vitamin D3 recruited DNA-dependent protein kinase (DNA-PKcs) to the chromatinized nVDRE(RP). Conversely, the regulatory subunits of DNA-PK were associated with the nVDRE(RP) sequences only when 1,25 vitamin D3 was absent. VDR was constitutively associated with these chromatinized nVDRE(RP) sequences. Furthermore, DNA-PKcs could phosphorylate VDR in vitro. We raise a possibility that a conformational change of VDR through its phosphorylation mediated by DNA-PKcs underlies the mechanism of gene repression by 1,25 vitamin D3-bound VDR.

Gonadotropins Stimulate Growth of MCF-7 Human Breast Cancer Cells by Promoting Intracellular Conversion of Adrenal Androgens to Estrogens

Estrogen receptor (ER)-positive breast cancers initially respond well to estrogen ablation treatment but finally acquire refractoriness, the phenomenon that is a major clinical problem. Because some breast cancers synthesize estradiol (E2) and E2 synthesis is regulated by gonadotropins in normal ovaries, and because circulating gonadotropins are elevated in postmenopausal women and during estrogen ablation treatment, we hypothesized that gonadotropins might modulate estrogen synthesis/metabolism in breast cancer tissue as well. To test this possibility, MCF-7 cells were treated with dehydroepiandrosterone (DHEA) or human chorionic gonadotropin (hCG; ∼LH), each alone or in combination. Cell growth (3-day treatment) was assayed by the MTT method and estrogen synthesis (24-hour treatment) was measured using the ERE-luciferase reporter system. First, MCF-7 cell growth was stimulated by DHEA in a concentration-dependent manner with a maximal effect at 10–4M. Although hCG alone did not have a significant proliferative effect, hCG significantly and dose dependently stimulated MCF-7 cell growth in the presence of a submaximal concentration of DHEA (10–7 M). This stimulatory effect of DHEA and hCG was blocked by a pure antiestrogen ICI182,780 and an aromatase inhibitor, arimidex. Using MCF-7 cells transfected with the ERE-luciferase reporter system, hCG treatment was shown to increase ERE-mediated transcription. These results indicate that MCF-7 cells intrinsically converted DHEA into E2 upon hCG stimulation, then grew their own cells DHEA- and hCG-dependently. We conclude that gonadotropins can act on breast cancer cells and accelerate conversion of DHEA into estrogens, thereby stimulating growth of estrogen-dependent tumor cells. This phenomenon, at least in part, could explain: (1) an increased tissue concentration of E2 in postmenopausal breast cancer; (2) acquisition of hormone refractoriness during estrogen ablation treatment, and (3) the effectiveness of GnRH antagonist/superagonist in some postmenopausal breast cancer patients.

Hyperosmolarity-induced Gene Stimulation Is Mediated by the Negative Calcium Responsive Element

The negative calcium responsive elements of the parathyroid hormone gene bind to a specific set of nuclear proteins in an extracellular calcium (Ca2+ e )-dependent manner. We have found that one of the negative calcium responsive elements, named oligo B, is found in the 5′-flanking region of such vasoactive genes as the vasopressin and atrial natriuretic polypeptide genes. Furthermore, the oligo B-like sequence in the former gene is conserved throughout evolution. Because expression of some of these vasoactive genes is altered by external stimuli which change cell volume, we examined whether oligo B is involved in gene regulation by hyperosmolarity. Here, we demonstrate that the binding between oligo B and its binding nuclear proteins including a redox factor 1 was reduced by hyperosmolarity generated by sodium chloride but not by urea. Such attenuated binding was reversed by dephosphorylating nuclear proteins by a potato acid phosphatase, suggesting that NaCl treatment elicited phosphorylation of these nuclear proteins to weaken their binding activity to oligo B. Furthermore, these nuclear events led to hyperosmolarity-mediated transcriptional stimulation of the genes bearing this DNA element in the cultured cells.

GH Inhibits Interferon-γ-Induced Signal Transducer and Activator of Transcription-1 Activation and Expression of the Inducible Isoform of Nitric Oxide Synthase in INS-1 Cells

Interferon-γ and TNFα synergistically induce the inducible isoform of nitric oxide synthase and elicit severe cytotoxicity in pancreatic β-cells. We demonstrate here that GH, the well knownβ -cell mitogen, inhibits nitric oxide production by reducing inducible nitric oxide synthase gene induction by the two cytokines and counteracts their cytotoxic effect in insulin-secreting INS-1 cells. To elucidate the underlying mechanism, we examined activation of the transcription factors implicated in the induction of inducible nitric oxide synthase, signal transducer and activator of transcription-1, and nuclear factor-κB. GH inhibited tyrosine phosphorylation and DNA binding of signal transducer and activator of transcription-1 promoted by interferon-γ, whereas nuclear factor-κB activation by TNFα was not affected by GH. GH was found to induce suppressor of cytokine signaling-1 and -3, both of which are able to inhibit interferon-γ activation of signal transducer and activator of transcription-1, suggesting that ...

Degradation of p21Cip1 through Anaphase-promoting Complex/Cyclosome and Its Activator Cdc20 (APC/CCdc20) Ubiquitin Ligase Complex-mediated Ubiquitylation Is Inhibited by Cyclin-dependent Kinase 2 in Cardiomyocytes

Background: p21Cip1 is controlled by both a transcriptional and post-translational mechanism during cell cycle progression and differentiation. Results: CDK2 blocks APC/CCdc20-mediated ubiquitylation of p21 at the N terminus, causing G2 arrest in mitogen-stimulated cardiomyocytes. Conclusion: The regulation of p21 ubiquitylation is required to maintain a terminal differentiation state of cardiomyocytes. Significance: This provides a novel insight on the control of cell cycle arrest in terminal differentiation. Cyclin-dependent kinase inhibitor p21Cip1 plays a crucial role in regulating cell cycle arrest and differentiation. It is known that p21Cip1 increases during terminal differentiation of cardiomyocytes, but its expression control and biological roles are not fully understood. Here, we show that the p21Cip1 protein is stabilized in cardiomyocytes after mitogenic stimulation, due to its increased CDK2 binding and inhibition of ubiquitylation. The APC/CCdc20 complex is shown to be an E3 ligase mediating ubiquitylation of p21Cip1 at the N terminus. CDK2, but not CDC2, suppressed the interaction of p21Cip1 with Cdc20, thereby leading to inhibition of anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20)-mediated p21Cip1 ubiquitylation. It was further demonstrated that p21Cip1 accumulation caused G2 arrest of cardiomyocytes that were forced to re-enter the cell cycle. Taken together, these data show that the stability of the p21Cip1 protein is actively regulated in terminally differentiated cardiomyocytes and plays a role in inhibiting their uncontrolled cell cycle progression. Our study provides a novel insight on the control of p21Cip1 by ubiquitin-mediated degradation and its implication in cell cycle arrest in terminal differentiation.