The canonical E2Fs together with RETINOBLASTOMA-RELATED are required to establish quiescence during plant development.

Maintaining stable and transient quiescence in differentiated and stem cells, respectively, requires repression of the cell cycle. The plant RETINOBLASTOMA-RELATED (RBR) has been implicated in stem cell maintenance, presumably by forming repressor complexes with E2F transcription factors. Surprisingly we find that mutations in all three canonical E2Fs do not hinder the cell cycle, but similarly to RBR silencing, result in hyperplasia. Contrary to the growth arrest that occurs when exit from proliferation to differentiation is inhibited upon RBR silencing, the e2fabc mutant develops enlarged organs with supernumerary stem and differentiated cells as quiescence is compromised. While E2F, RBR and the M-phase regulatory MYB3Rs are part of the DREAM repressor complexes, and recruited to overlapping groups of targets, they regulate distinct sets of genes. Only the loss of E2Fs but not the MYB3Rs interferes with quiescence, which might be due to the ability of E2Fs to control both G1-S and some key G2-M targets. We conclude that collectively the three canonical E2Fs in complex with RBR have central roles in establishing cellular quiescence during organ development, leading to enhanced plant growth.

MYB3R-mediated and cell cycle-dependent transcriptional regulation of a tobacco ortholog of SCARECROW-LIKE28 in synchronized cultures of BY-2 cells.

Although it is well known that hierarchical transcriptional networks are essential for various aspects of plant development and environmental response, little has been investigated about whether and how they also regulate the plant cell cycle. Recent studies on cell cycle regulation in Arabidopsis thaliana identified SCARECROW-LIKE28 (SCL28), a GRAS-type transcription factor, that constitutes a hierarchical transcriptional pathway comprised of MYB3R, SCL28 and SIAMESE-RELATED (SMR). In this pathway, MYB3R family proteins regulate the G2/M-specific transcription of the SCL28 gene, of which products, in turn, positively regulate the transcription of SMR genes encoding a group of plant-specific inhibitor proteins of cyclin-dependent kinases. However, this pathway with a role in cell cycle inhibition is solely demonstrated in A. thaliana, thus leaving open the question of whether and to what extent this pathway is evolutionarily conserved in plants. In this study, we conducted differential display RT-PCR on synchronized Nicotiana tabacum (tobacco) BY-2 cells and identified several M-phase-specific cDNA clones, one of which turned out to be a tobacco ortholog of SCL28 and was designated NtSCL28. We showed that NtSCL28 is expressed specifically during G2/M and early G1 in the synchronized cultures of BY-2 cells. NtSCL28 contains MYB3R-binding promoter elements, so-called mitosis-specific activator elements, and is upregulated by a hyperactive form of NtmybA2, one of the MYB3R proteins from tobacco. Our study indicated that a part of the hierarchical pathway identified in A. thaliana is equally operating in tobacco cells, suggesting the conservation of this pathway across different families in evolution of angiosperm.

The DREAM complex represses growth in response to DNA damage in Arabidopsis.

The DNA of all organisms is constantly damaged by physiological processes and environmental conditions. Upon persistent damage, plant growth and cell proliferation are reduced. Based on previous findings that RBR1, the only Arabidopsis homolog of the mammalian tumor suppressor gene retinoblastoma, plays a key role in the DNA damage response in plants, we unravel here the network of RBR1 interactors under DNA stress conditions. This led to the identification of homologs of every DREAM component in Arabidopsis, including previously not recognized homologs of LIN52. Interestingly, we also discovered NAC044, a mediator of DNA damage response in plants and close homolog of the major DNA damage regulator SOG1, to directly interact with RBR1 and the DREAM component LIN37B. Consistently, not only mutants in NAC044 but also the double mutant of the two LIN37 homologs and mutants for the DREAM component E2FB showed reduced sensitivities to DNA-damaging conditions. Our work indicates the existence of multiple DREAM complexes that work in conjunction with NAC044 to mediate growth arrest after DNA damage.

Identification of two tobacco genes encoding MYB3R proteins with repressor function and showing cell cycle-regulated transcript accumulation.

MYB3R family transcription factors play a central role in the regulation of G2/M-specific gene transcription in Arabidopsis thaliana. Among the members of this family, MYB3R3 and MYB3R5 are structurally closely related and are involved in the transcriptional repression of target genes in both proliferating and quiescent cells. This type of MYB3R repressor is widespread in plants; however, apart from the studies on MYB3Rs in Arabidopsis thaliana, little information about them is available. Here we isolated tobacco cDNA clones encoding two closely related MYB3R proteins designated as NtmybC1 and NtmybC2 and determined the nucleotide sequences of the entire coding regions. Phylogenetic analysis suggested that NtmybC1 and NtmybC2 can be grouped into a conserved subfamily of plant MYB3Rs that also contains MYB3R3 and MYB3R5. When transiently expressed in protoplasts prepared from tobacco BY-2 cells, NtmybC1 and NtmybC2 repressed the activity of target promoters and blocked promoter activation mediated by NtmybA2, a MYB3R activator from tobacco. Unlike MYB3R3 and MYB3R5, NtmybC1 and NtmybC2 showed cell cycle-regulated transcript accumulation. In synchronized cultures of BY-2 cells, mRNAs for both NtmybC1 and NtmybC2 were preferentially expressed during the G2 and M phases, coinciding with the expression of NtmybA2 and G2/M-specific target genes. These results not only broadly confirm our fundamental view that this type of MYB3R protein acts as transcriptional repressor of G2/M-specific genes but also suggest a possible divergence of MYB3R repressors in terms of the mechanisms of their action and regulation.

Glucose concentration-dependent potentiation of insulin secretion by a new chemical entity, KCP256.

The insulinotropic activity of KCP256 [(R)-8-benzyl-2-cyclopentyl-7, 8-dihydro-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one hydrochloride] was examined using MIN6 cells (a pancreatic beta-cell line) and pancreatic islets isolated from rats. Unlike sulfonylurea anti-diabetic drugs, KCP256 dose-dependently (0.1-10 microM) enhanced insulin secretion from MIN6 cells and its insulinotropic effect was exerted only at high concentrations of glucose (8.3-22 mM) but not at low concentrations of glucose (3.3-5.5 mM). Furthermore, the action mechanism of KCP256 was different because, unlike sulfonylurea drugs, KCP256 did not displace the binding of [3H]glibenclamide, and did not inhibit the 86Rb+ efflux nor K(ATP) channel activity. In isolated islets, KCP256 also enhanced insulin secretion in a dose- and a glucose-concentration-dependent manner. Plasma levels of insulin after glucose challenge in KCP256-administrated rats were higher than those in vehicle-administrated animals, indicating that KCP256 can enhance insulin secretion in vivo. Since the insulinotropic activity of KCP256 only occurs at high concentrations of glucose, this novel drug may exhibit a decreased risk of drug-induced hypoglycemia compared with sulfonylurea drugs when treating patients with diabetes.

Effects of combination treatment with dipeptidyl peptidase IV inhibitor and sulfonylurea on glucose levels in rats.

The effects of orally administered dipeptidyl peptidase IV (DPP-IV) inhibitor on the glucose-lowering effect of glibenclamide are still unknown. We evaluated the effects of combination treatment with a long-lasting DPP-IV inhibitor, K579 ((S)-1-[4-methyl-1-(2-pyrimidinyl)-4-piperidylamino]acetyl-2-pyrrolidinecarbonitrile), and glibenclamide on the glycemic responses to glucose loading in rats. Treatment with K579 inhibited the plasma DPP-IV activity even 8 h after the administration. K579 significantly suppressed the blood glucose elevation in glibenclamide-pretreated rats without excessive hypoglycemia. These profiles of K579 indicate that it could be useful agent to correct the postprandial glucose excursion in type 2 diabetes patients by combination treatment with glibenclamide.

K579, a slow-binding inhibitor of dipeptidyl peptidase IV, is a long-acting hypoglycemic agent.

Dipeptidyl peptidase IV inhibitors are expected to be categorized in a new type of antidiabetic drugs. We had developed a long-acting dipeptidyl peptidase IV inhibitor, K579 [(S)-1-[4-methyl-1-(2-pyrimidinyl)-4-piperidylamino]acetyl-2-pyrrolidinecarbonitrile]. The aim of present study was to characterize the pharmacological profiles of K579. In normal rats, K579 suppressed the blood glucose elevation after an oral glucose tolerance test with the increment of plasma insulin and active forms of glucagon-like peptide-1 (GLP-1). During repetitive glucose loading using Zucker fatty rats, pretreatment with K579 attenuated the glucose excursion after the second glucose loading as well as the first glucose loading without inducing hypoglycemia. The kinetic study using cell extract revealed that K579 was a more potent and slower binding inhibitor than the existing dipeptidyl peptidase IV inhibitor (NVP-DPP728, 1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrrolidine). These profiles of K579 might be advantageous over the existing dipeptidyl peptidase IV inhibitor with respect to less dosing frequency.

Potent and selective inhibitors of platelet-derived growth factor receptor phosphorylation. 1. Synthesis, structure-activity relationship, and biological effects of a new class of quinazoline derivatives.

A new series of 4-[4-(N-substituted carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives were found to show potent and selective inhibition of platelet-dervied growth factor (PDGF) receptor phosphorylation. In this exploration of the structure-activity relationships (SARs) of the prototype inhibitor KN1022, the 4-nitrophenylurea moiety was probed. We found that 4-substitution on the phenyl ring was optimal and the introduction of more than two substituents on the phenyl ring decreased activities. Bulky substituents on the phenyl ring enhanced activities. Thiourea analogues were also prepared, and the SARs were found to be slightly different from those of the urea derivatives. Through this research, we obtained some potent KN1022 derivatives such as 4-(4-methylphenoxy)phenyl (36, IC(50) 0.02 micromol/L), 4-tert-butylphenyl (16, IC(50) 0.03 micromol/L), and 4-phenoxyphenyl (21, IC(50) 0.08 micromol/L) analogues, which had almost a 10-fold increase in activity against KN1022. These potent compounds retained their high selectivity against the PDGF receptor family similar to KN1022. We also observed that these compounds could inhibit the PDGF-BB-induced proliferation of porcine vascular smooth muscle cells without cell toxicity almost at the same IC(50) values observed for PDGF receptor phosphorylation. To evaluate the biological effects in vivo, we selected some analogues on the basis of the measurement of the plasma drug concentration after oral administration to rats. Oral administration of the 4-chlorophenyl (6), 4-bromophenyl (9), or 4-isopropoxyphenyl (20) analogue to Sprague-Dawley rats (30 mg/kg, twice daily) resulted in significant inhibition (24-38%) of neointima formation in the carotid artery of the balloon catheter deendothelialized vessel in the rats. Therefore, 4-[4-(N-substituted carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives, which are potent inhibitors of PDGFR phosphorylation, may be expected to represent a new therapeutic approach for the treatment of various aspects of atherosclerosis and other cellular proliferative disorders.