Citations

Abstract Purpose: PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer. Experimental Design: We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and array CGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort of ovarian cancer patients. Results: Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, RB, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration-and time-dependent manner, and enhanced the effects of chemotherapy. Rb-proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and was independently associated with poor progression-free survival (adjusted relative risk 1.49, 95% CI 1.00–2.24, P = 0.052). Conclusions: PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Clin Cancer Res; 17(6); 1591–602. ©2011 AACR.

Coronary artery disease is a leading cause of death. Hypertension (HT) increases the incidence of cardiac events, but its effect on cardiac adaptation to coexisting coronary artery stenosis (CAS) is unclear. We hypothesized that concurrent HT modulates microvascular function in chronic CAS and aggravates microvascular remodeling and myocardial injury. Four groups of pigs ( n = 6 each) were studied: normal, CAS, HT, and CAS+HT. CAS and HT were induced by placing local irritant coils in the left circumflex coronary artery and renal artery, respectively. Six weeks later multidetector computerized tomography (CT) was used to assess systolic and diastolic function, microvascular permeability, myocardial perfusion, and responses to adenosine in the “area at risk.” Microvascular architecture, inflammation, and fibrosis were then explored in cardiac tissue. Basal myocardial perfusion was similarly decreased in CAS and CAS+HT, but its response to adenosine was significantly more attenuated in CAS. Microvascular permeability in CAS+HT was greater than in CAS and was accompanied by amplified myocardial inflammation, fibrosis, and microvascular remodeling, as well as cardiac systolic and diastolic dysfunction. On the other hand, compared with normal, micro-CT-derived microvascular (20–200 μm) transmural density decreased in CAS but not in HT or CAS+HT. We conclude that the coexistence of early renovascular HT exacerbated myocardial fibrosis and vascular remodeling distal to CAS. These changes were not mediated by loss of myocardial microvessels, which were relatively preserved, but possibly by exacerbated myocardial inflammation and fibrosis. HT modulates cardiac adaptive responses to CAS and bears cardiac functional consequences.

Abstract The low efficiency of reprogramming and genomic integration of virus vectors obscure the potential application of induced pluripotent stem (iPS) cells; therefore, identification of chemicals and cooperative factors that may improve the generation of iPS cells will be of great value. Moreover, the cellular mechanisms that limit the reprogramming efficiency need to be investigated. Through screening a chemical library, we found that two chemicals reported to upregulate E-cadherin considerably increase the reprogramming efficiency. Further study of the process indicated that E-cadherin is upregulated during reprogramming and the established iPS cells possess E-cadherin-mediated cell–cell contact, morphologically indistinguishable from embryonic stem (ES) cells. Our experiments also demonstrate that overexpression of E-cadherin significantly enhances reprogramming efficiency, whereas knockdown of endogenous E-cadherin reduces the efficiency. Consistently, abrogation of cell–cell contact by the inhibitory peptide or the neutralizing antibody against the extracellular domain of E-cadherin compromises iPS cell generation. Further mechanistic study reveals that adhesive binding activity of E-cadherin is required. Our results highlight the critical role of E-cadherin-mediated cell–cell contact in reprogramming and suggest new routes for more efficient iPS cell generation.

Identified in an RNA interference screen, proteins that prevent spindle multipolarity in human cancer cells may prove to be promising drug targets.

Objective— Hypertension and hypercholesterolemia might interfere with renal repair mechanisms. We hypothesized that simvastatin improves the survival of endothelial progenitor cells (EPC) in the renal microenvironment imposed by concurrent renovascular hypertension and dietary hypercholesterolemia (HTC). Methods and Results— Pigs were studied after 12 weeks of no intervention (n=6), HTC (n=6), or HTC+ oral simvastatin supplementation (80 mg/day, n=5). EPC were also isolated and studied in vitro after exposure to the proapoptotic oxidized low-density lipoprotein with or without coincubation with simvastatin. Renal hemodynamics, function, and endothelial function were evaluated in vivo, and the number of CD34+/KDR+ EPC, apoptosis, oxidative stress, inflammation, and fibrosis in renal tissue studied ex vivo. Compared with normal kidney, the HTC kidney showed endothelial dysfunction and increased oxidative stress, interstitial macrophage filtration, and fibrosis. The number of EPC in the kidney increased, as did their apoptosis (0.85±0.24% versus 0.22±0.07%, P <0.05 versus normal). Simvastatin did not affect blood pressure, cholesterol levels, basal renal function, or number of renal EPC in HTC, but it improved endothelial function; blunted renal oxidative stress, inflammation, and fibrosis; and attenuated EPC apoptosis (to 0.37±0.09%, P <0.05 versus HTC). Simvastatin also significantly decreased oxidized low-density lipoprotein–induced EPC apoptosis in vitro. Conclusion— EPC are recruited but undergo apoptosis in the HTC kidney, likely because of a hostile microenvironment. Simvastatin rescues renal repair mechanisms in HTC and counteracts renal damage, which may account for its protective effects on the kidney during exposure to cardiovascular risk factors.

Background— Monocyte chemoattractant proteins (MCPs) play an important role in mediating inflammatory processes. Hypertension (HTN) is associated with inflammation as well as impaired cardiac microcirculatory function and structure, but the contribution of MCPs to these alterations remained unclear. This study tested the hypothesis that MCPs regulate cardiac microvascular function and structure in experimental HTN. Methods and Results— Pigs (n=6 per group) were studied after 10 weeks of normal, renovascular HTN, or renovascular HTN+ bindarit (MCPs inhibitor, 50 mg/kg/d PO). Left ventricular (LV) function, myocardial microvascular permeability, and fractional vascular volume were assessed by fast computed tomography before and after adenosine infusion (400 μg/kg/min). Myocardial fibrosis, inflammation, and microvascular remodeling were determined ex vivo. Hypertension was not altered by bindarit, but LV hypertrophy and diastolic function were improved. In response to adenosine, myocardial microvascular permeability increased in HTN (from 0.0083±0.0009 to 0.0103±0.0011 AU, P =0.038 versus baseline) and fractional vascular volume decreased, whereas both remained unchanged in normal and HTN+bindarit pigs. HTN upregulated endothelin-1 expression, myocardial inflammation, and microvascular wall thickening, which were inhibited by bindarit. Conclusions— MCPs partly mediate myocardial inflammation, fibrosis, vascular remodeling, and impaired vascular integrity induced by hypertension. Inhibition of MCPs could potentially be a therapeutic target in hypertensive cardiomyopathy.

Mutations in the electrogenic Na+/ nHCO3− cotransporter (NBCe1, SLC4A4) cause severe proximal renal tubular acidosis, glaucoma, and cataracts in humans, indicating NBCe1 has a critical role in acid-base homeostasis and ocular fluid transport. To better understand the homeostatic roles and protein ontogeny of NBCe1, we have cloned, localized, and downregulated NBCe1 expression in zebrafish, and examined its transport characteristics when expressed in Xenopus oocytes. Zebrafish NBCe1 (zNBCe1) is 80% identical to published mammalian NBCe1 cDNAs. Like other fish NBCe1 clones, zebrafish NBCe1 is most similar to the pancreatic form of mammalian NBC (Slc4a4-B) but appears to be the dominant isoform found in zebrafish. In situ hybridization of embryos demonstrated mRNA expression in kidney pronephros and eye by 24 h postfertilization (hpf) and gill and brain by 120 hpf. Immunohistochemical labeling demonstrated expression in adult zebrafish eye and gill. Morpholino knockdown studies demonstrated roles in eye and brain development and caused edema, indicating altered fluid and electrolyte balance. With the use of microelectrodes to measure membrane potential ( Vm), voltage clamp (VC), intracellular pH (pHi), or intracellular Na+ activity ( aNai), we examined the function of zNBCe1 expressed in Xenopus oocytes. Zebrafish NBCe1 shared transport properties with mammalian NBCe1s, demonstrating electrogenic Na+ and HCO3− transport as well as similar drug sensitivity, including inhibition by 4,4′-diiso-thiocyano-2,2′-disulfonic acid stilbene and tenidap. These data indicate that NBCe1 in zebrafish shares many characteristics with mammalian NBCe1, including tissue distribution, importance in systemic water and electrolyte balance, and electrogenic transport of Na+ and HCO3−. Thus zebrafish promise to be useful model system for studies of NBCe1 physiology.