Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells: Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression

Summary We have examined the role of cyclin D1 and cyclin-dependent kinase-4 (CDK4) in the cell cycle progression and proliferation of MCF-7 breast cancer cells. Forced expression of cyclin D1 using a tetracycline-regulated expression system, and suppression of endogenous cyclin D1 and CDK4 using small interfering RNA (siRNA) were used to validate this protein complex as a drug target in cancer drug discovery. Overexpression of cyclin D1 increased both phosphorylation of the retinoblastoma gene product (RB) and passage through the G1–S phase transition, resulting in increased proliferation of cells. When cyclin D1 expression was shut off, growth rates fell below those seen in control cell lines transfected with the vector, indicating an increased dependence on this protein for proliferation. Inhibition of endogenous cyclin D1 or CDK4 expression by RNA interference resulted in hypophosphorylation of RB and accumulation of cells in G1. These results support the prevailing view that pharmacological inhibition of cyclin D1/CDK4 complexes is a useful strategy to inhibit the growth of tumors. Furthermore, since MCF-7 cells appear to be dependent on this pathway for their continued proliferation, it is a suitable cell line to test novel cyclin D1/CDK4 inhibitors.     

Wavefront analysis and contrast sensitivity of aspheric and spherical intraocular lenses: a randomized prospective study

PURPOSE: To compare visual performance, total and high order wavefront aberrations (coma, spherical aberration, and other terms), and contrast sensitivity in 120 eyes implanted with one monofocal aspheric intraocular lens (IOL) and two spherical IOLs. DESIGN: Randomized prospective study. METHODS: Sixty patients were randomized to receive three IOL types: Alcon AcrySofIQ (40 eyes), AcrySofNatural (40 eyes), and advanced medical optic (AMO)Sensar (40 eyes). Complete ophthalmologic examination including uncorrected visual acuity (UCVA), best-spectacle corrected visual acuity (BSCVA), corneal topography, and wavefront analysis were performed preoperatively, 30 days, and 90 days postoperatively. Pelli-Robson chart test and functional acuity contrast testing (FACT-Optec6500) were performed approximately 50 days after surgery. Statistical analyses were performed using analysis chi(2), analysis of variance (ANOVA), and multiple comparisons Tukey test. RESULTS: After 90 days, all eyes had postoperative BSCVA > or =20/32. The AcrySofIQ IOL showed statistically significant less induction of spherical aberration (P < .001) when compared with the AMOSensar and the AcrySofNatural IOLs. The AMOSensar presented significantly less spherical aberration then the AcrySofNatural (P < .05). The AcrySofIQ also had lower values of total and high-order aberration (HOA) (P < .05) when compared with the AMOSensar and the AcrySofNatural. The mean values of trefoil 9, coma, and HOA root mean square (RMS) decreased between one and three months (P < .001, P < .001, P = .023, P < .001, respectively) in all groups. Mean Pelli-Robson contrast sensitivity values in photopic condition were similar between the groups. The AcrySofIQ showed better results in 3cpd spatial frequency in mesopic condition using FACT-Optec 6500 (P = .008), although there were no statistical differences in photopic and mesopic with glare conditions.     

Is maternal obesity a predictor of shoulder dystocia?

OBJECTIVE: To explore the relationship between maternal obesity and shoulder dystocia while controlling for the potential confounding effects of other variables associated with obesity. METHODS: We performed a case-control study of provincial delivery records audited by the Northern and Central Alberta Perinatal Outreach Program. Risk factors evaluated were selected based on previously published studies. Cases and controls were drawn from 45,877 live singleton cephalic vaginal deliveries weighing more than 2500 g between January 1995 and December 1997. There were 413 cases of shoulder dystocia (0.9% incidence). Controls (n = 845) were randomly chosen from the remainder of the target population to create a 1:2 case/control ratio. Univariate analysis with calculation of odds ratios (ORs) was used to determine which of the chosen risk factors were significantly related to the incidence of shoulder dystocia. Multivariable regression analyses were then used to determine the independently associated variables, and the adjusted ORs were obtained for each relevant risk factor. RESULTS: Maternal obesity was not significant as an independent risk factor for shoulder dystocia after adjusting for confounding variables (adjusted OR 0.9; 95% confidence interval [CI] 0.5, 1.6). Fetal macrosomia was the single most powerful predictor. The adjusted ORs were 39.5 (95% CI 19.1, 81.4) for birth weight greater than 4500 g and 9.0 (95% CI 6.5, 12.6) for birth weight between 4000 and 4499 g. CONCLUSION: The strongest predictors of shoulder dystocia are related to fetal macrosomia. For obese nondiabetic women carrying fetuses whose weights are estimated to be within normal limits, there is no increased risk of shoulder dystocia.     

Avoidance and management of positive surgical margins before,during and after radical prostatectomy

Positive surgical margins after radical prostatectomy lead to an increased risk of progression and reduced disease free survival. Earlier detection of prostate cancer, appropriate patient selection and improved operative techniques can reduce the incidence of positive margins, though the risk can not be eliminated as pre-operative staging techniques are not sufficiently sensitive. Nerve sparing and bladder neck sparing do not adversely affect margin status in appropriately selected men. Once positive margins have been diagnosed the optimal management and the timing of treatment remains controversial. Adjuvant radiotherapy or salvage radiotherapy in men with a low PSA may improve local control and PSA free survival in some individuals, a survival benefit has not yet been established.     

PATTERNS OF INTELLECTUAL FUNCTIONING IN NON-RETARDED AUTISTIC AND SCHIZOPHRENIC CHILDREN

The WISC-R factor scores of non-retarded children meeting DSM III criteria for autism and schizophrenia were compared. The scores of the schizophrenic children on factor 3 were significantly lower than those of the autistic children, below the range of normal children, and significantly lower than the scores they obtained on the verbal comprehension and perceptual organization factors. The autistic children scored in the superior range on the block design subtest and did not show gross impairments in language function as indexed by scores on the verbal comprehension factor. The only subtest autistic children were impaired on was the comprehension subtest.     

Functional reconstitution of ICln in lipid bilayers

Reconstitution of purified ICln in lipid bilayer leads to functional ion channels showing varying rectification. The reconstituted single channels have a conductance of approximately equal to 3 pS and their open probability is sensitive to nucleoside analogues. Mutation of a putative nucleotide binding site identified at the predicted extracellular mouth of the ICln channel protein leads to the reduction of the nucleoside-analogue sensitivity. Reconstituted ICln channels can be permeated both by cations and anions. The relative permeability of cations over anions depends on the presence of calcium. In the presence of calcium reconstituted ICln channels are more permeable to bromide than chloride, and more permeable to potassium than sodium. Similarly in NIH3T3 fibroblasts, the relative permeability of cations over anions of swelling-dependent chloride channels depends on extracellular calcium. Site-directed mutagenesis revealed the calcium-binding site responsible for the shift of the selectivity from cations towards anions of reconstituted ICln channels. Additional indirect structural information has been obtained by mutating a histidine in the predicted pore region of ICln. This histidine seems to have access to the ion-conducting tunnel of the pore. Our experiments show that ICln can act as an ionic channel, which does not exclude additional functions of the protein in regulatory mechanisms of the cell. Since knocking down the ICln protein in fibroblasts and epithelial cells leads to an impaired regulatory volume decrease (RVD) after cytoplasmic swelling and reconstituted ICln channels show several biophysical features of ion channels activated after swelling, ICln is a molecular candidate for these channels.     

Uncoupling of Na+H+ from Cl−HCO 3 − exchange under some steady state conditions in rabbit gallbladder

The transapical Cl^– influx and transepithelial Na⁺ transport were measured in rabbit gallbladder. Only 11.7% of the transported Na⁺ was found to be accompanied by HCO ₃ ^– . 10^–4 M SITS eliminated the HCO ₃ ^– dependent fraction of Cl^– influx (50%) but did not significantly alter intracellular Na⁺ activity and Na⁺ transport. Exposure to HCO₃-free salines or to 10^–4 M acetazolamide about halved Cl^– influx and Na⁺ transport. 25 mM SCN^– reduced Cl^– influx to zero, decreased intracellular Na⁺ activity, but only halved Na⁺ transport which under these conditions was abolished only in the absence of HCO ₃ ^– . Exposure to a Cl^–-free saline produced effects similar to those caused by SCN^–. These resuits suggest that when Cl^–/HCO ₃ ^– exchange is inhibited at the apical membrane, Na⁺/H⁺ exchange and transepithelial Na⁺ transfer are unmodified if HCO ₃ ^– is available for transport. The permanent uncoupling of the exchangers and the elevated transepithelial transport of Na⁺ are not due to an increased activity of the parallel Na⁺–Cl^– cotransport but to a redirection of HCO ₃ ^– flux toward the basolateral side.     

Psychiatric disorders among at-risk consumers of alcohol in the general population

OBJECTIVE: At-risk consumption of alcohol has increasingly become the focus of primary and secondary prevention efforts. Little is known about the co-occurrence of psychiatric disorders with at-risk drinking. We examined patterns of Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM-IV) lifetime co-occurrence of psychiatric disorders in individuals in the general population with at-risk consumption of alcohol, alcohol abuse, alcohol dependence and moderate drinking/abstention, considering potential gender differences. METHOD: Cross-sectional data of a representative general population-based study were analyzed. Based on DSM-IV criteria, participants aged 18-64 (N = 4,074; 2,045 men) were diagnosed using a standardized computer-assisted version of the Munich Composite International Diagnostic Interview (M-CIDI). Nonpsychotic Axis-I lifetime diagnoses were examined. At-risk consumption of alcohol was defined as an average of more than 20 g (0.71 oz) pure alcohol consumption per day for women and 30 g (1.06 oz) for men, with alcohol abuse or alcohol dependence excluded. RESULTS: Almost 9% of participants were identified as at-risk drinkers. Prevalence rates for at-risk drinkers were 16.9% for affective, 18.1% for anxiety and 17.8% for somatoform disorders. Compared with moderate drinkers/abstainers, at-risk drinkers showed a twofold increased risk of having a psychiatric disorder. Subjects with alcohol abuse showed a comparable level of risk and individuals with alcohol dependence showed an even greater risk. Female at-risk drinkers were twice as likely to have a psychiatric disorder as their male counterparts. The odds ratios for psychiatric disorders in at-risk drinkers compared with moderate drinkers/abstainers, however, did not differ in men and women. CONCLUSIONS: Rates of psychiatric co-occurrence among at-risk drinkers were considerably elevated when compared with moderate drinkers/abstainers. These findings underline the relevance of at-risk consumption of alcohol and represent an important challenge to public health efforts regarding screening of psychiatric disorders and referral to appropriate treatment services.