Effects of interaction between estradiol-17 beta and progesterone on the proliferation of cloned breast tumor cells (MCF-7 and T47D)

We have previously reported that the proliferation of cloned MCF-7 and T47D human mammary tumor cells can be inhibited by increasing concentrations of charcoal-dextran stripped female human serum (CDFHS). The maximal proliferation rate was restored by the addition of 3 X 10(-11) M estradiol-17 beta to the culture media. These observations suggest that the proliferation of T47D and MCF-7 cells is regulated by a blood-borne inhibitor whose effects are neutralized by estrogens. In the present report we explore the possibility that progesterone alters the estrogenic response. MCF-7 cells were grown in DME containing 2-40% CDFHS. Progesterone, at 3 X 10(-7) M to 3 X 10(-12) M, had no effect on the yield of MCF-7 or T47D cells that were cultured in the presence or absence of estradiol-17 beta.     

Expression of novel genes linked to the androgen-induced, proliferative shutoff in prostate cancer cells

Androgens control cell numbers in the prostate through three separate pathways: (a) inhibition of cell death, (b) induction of cell proliferation (Step-1) and (c) inhibition of cell proliferation (Step-2, proliferative shutoff). The mechanisms underlying these phenomena are incompletely understood. The human prostate carcinoma LNCaP variants express these pathways as follows: LNCaP-FGC express both steps, LNCaP-LNO expresses Step-2, LNCaP-TAC expresses Step-1, and LNCaP-TJA cells express neither step. These cells facilitated the search for mediators of the androgen-induced proliferative shutoff pathway. Androgen exposure for 24 h or longer induced an irreversible proliferative shutoff in LNCaP-FGC cells. The Wang and Brown approach for identifying differentially expressed mRNAs was used to search for mediators of Step-2. Ten unique inserts were identified and from those ten, three genes were further studied. The basal expression of these genes in shutoff-negative variants was not affected by androgen exposure. They were induced by androgens in shutoff-positive LNCaP variants and the androgen receptor-transfected, shutoff-positive, MCF7-AR1 cells. These genes were induced only in the range of androgen concentrations that elicited the shutoff response. Time course analysis showed that their induction precedes the commitment point by 12–18 h. In addition, they were expressed in the normal prostate during proliferative shutoff. These features suggest that the candidate genes have a role in the regulation cascade for proliferative shutoff.     

Estrogen target cells. Establishment of a cell line derived from the rat pituitary tumor MtT/F4.

Pretreatment of rabbit kidney cells with cytochalasins B and D (CB, CD) enhanced herpes simplex virus type 2 (HSV-2) DNA infectivity 3- to 6-fold over values obtained using the standard CaCl₂ technique. Cells were pretreated with CB for 4–6 h to achieve infectivity enhancement. A lower concentration of CD, and shorter pretreatment periods, resulted in comparable DNA infectivity. Separate exposure of cells to colchicine, colcemid, or vinblastine increased DNA infectivity 7-, 6-, and 5-fold, respectively, over control values. Additional enhancement was obtained when CD was used together with any one of the aforementioned drugs. Maximal enhancement of HSV-2 DNA infectivity was obtained by pretreating recipient cells with a drug mixture containing colchicine, colcemid, and CD. This treatment maximized infectivity levels 20- to 30-fold over CaCl₂ control values.     

Inhibition of cholesteryl ester transfer protein by anacetrapib does not impair the anti-inflammatory properties of high density lipoprotein

Cholesteryl ester transfer protein (CETP) is a target of therapeutic intervention for coronary heart disease. Anacetrapib, a potent inhibitor of CETP, has been shown to reduce LDL-cholesterol by 40% and increase HDL-cholesterol by 140% in patients, and is currently being evaluated in a phase III cardiovascular outcomes trial. HDL is known to possess anti-inflammatory properties, however with such large increases in HDL-cholesterol, it is unclear whether CETP inhibition perturbs HDL functionality such as anti-inflammatory effects on endothelial cells. The purpose of the present study was to determine whether CETP inhibition by anacetrapib affects the anti-inflammatory properties of HDL. HDL was isolated from either hamsters treated with vehicle or anacetrapib for 2 weeks, or from normal human subjects treated either placebo, 20 mg, or 150 mg anacetrapib daily for 2 weeks. Anacetrapib treatment increased plasma HDL cholesterol levels by 65% and between 48 and 82% in hamsters and humans, respectively. Pre-incubation of human aortic endothelial cells with HDL isolated from both control and anacetrapib treated hamsters suppressed TNFα induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin. Similar results were obtained with human HDL samples pre and post treatment with placebo or anacetrapib. Further, HDL inhibited TNFα-induced MCP-1 secretion, monocyte adhesion and NF-κB activation in endothelial cells, and the inhibition was similar between control and anacetrapib treated groups. These studies demonstrate that anacetrapib treatment does not impair the ability of HDL to suppress an inflammatory response in endothelial cells.     

The aging of the 2000 and 2011 Hallmarks of Cancer reviews: A critique

Two review articles published in 2000 and 2011 by Hanahan and Weinberg have dominated the discourse about carcinogenesis among researchers in the recent past. The basic tenets of their arguments favour considering cancer as a cell-based, genetic disease whereby DNA mutations cause uncontrolled cell proliferation. Their explanation of cancer phenotypes is based on the premises adopted by the somatic mutation theory (SMT) and its cell-centered variants. From their perspective, eight broad features have been identified as so-called ‘Hallmarks of Cancer’. Here, we criticize the value of these features based on the numerous intrinsic inconsistencies in the data and in the rationale behind SMT. An alternative interpretation of the same data plus data mostly ignored by Hanahan and Weinberg is proposed, based instead on evolutionarily relevant premises. From such a perspective, cancer is viewed as a tissue-based disease. This alternative, called the tissue organization field theory, incorporates the premise that proliferation and motility are the default state of all cells, and that carcinogenesis is due to alterations on the reciprocal interactions among cells and between cells and their extracellular matrix. In this view, cancer is development gone awry.     

Predicting and validating a model of suppressor of IKKepsilon through biophysical characterization

Suppressor of IKKepsilon (SIKE) is a 207 residue protein that is implicated in the TLR3‐TANK‐binding kinase‐1‐mediated response to viral infection. SIKE's function in this pathway is unknown, but SIKE forms interactions with two distinct cytoskeletal proteins, α‐actinin and tubulin, and SIKE knockout reduces cell migration. As structure informs function and in the absence of solved structural homologs, our studies were directed toward creating a structural model of SIKE through biochemical and biophysical characterization to probe and interrogate SIKE function. Circular dichroism revealed a primarily (73%) helical structure of minimal stability (<T_m > =32°C) but reversibly denatured. Limited proteolysis (LP) and chemical modification identified the N‐terminal 2/3 of the protein as dynamic and accessible, whereas size exclusion chromatography (SEC) confirmed three homo‐oligomeric species. SEC coupled to chemical crosslinking characterized the primary species as dimeric, a secondary hexameric species, and a higher order aggregate/polymer. Fluorescence polarization using intrinsic tryptophan fluorescence contextualized the anisotropy value for the SIKE dimer (molecular weight 51.8 kDa) among proteins of known structure, bovine serum albumin (BSA; 66 kDa), and glutamate dehydrogenase (GDH; 332 kDa). Radii of gyration for BSA and GDH provided exclusionary values for SIKE tertiary and dimeric quaternary models that otherwise conformed to secondary structure, LP, and modification data. Dimeric quaternary models were further culled using acrylamide quenching data of SIKE's single tryptophan that showed a single, protected environment. The low cooperativity of folding and regions of dynamic and potentially disordered structure advance the hypothesis that SIKE forms a conformational ensemble of native states that accommodate SIKE's interactions with multiple, distinct protein‐binding partners.     

Spindle pole fragmentation due to proteasome inhibition

During interphase, the centrosome concentrates cell stress response molecules, including chaperones and proteasomes, into a proteolytic center. However, whether the centrosome functions as proteolytic center during mitosis is not known. In this study, cultured mammalian cells were treated with the proteasome inhibitor MG 132 and spindle morphology in mitotic cells was characterized in order to address this issue. Proteasome inhibition during mitosis leads to the formation of additional asters that cause the assembly of multipolar spindles. The cause of this phenomenon was investigated by inhibiting microtubule-based transport and protein synthesis. These experimental conditions prevented the formation of supernumerary asters during mitosis. In addition, the expression of dsRed without proteasome inhibition led to the fragmentation of spindle poles. These experiments showed that the formation of extra asters depends on intact microtubule-based transport and protein synthesis. These results suggest that formation of supernumerary asters is due to excessive accumulation of proteins at the spindle poles and consequently fragmentation of the centrosome. Together, this leads to the conclusion that the centrosome functions as proteolytic center during mitosis and proteolytic activity at the spindle poles is necessary for maintaining spindle pole integrity.