Nonapical and Cytoplasmic Expression of Interleukin-8, CXCR1, and CXCR2 Correlates with Cell Proliferation and Microvessel Density in Prostate Cancer

Purpose: We characterized interleukin-8 (IL-8) and IL-8 receptor expression (CXCR1 and CXCR2) in prostate cancer to address their significance to this disease. Experimental Design: Immunohistochemistry was conducted on 40 cases of human prostate biopsy containing histologically normal and neoplastic tissue, excised from patients with locally confined or invasive androgen-dependent prostate cancer, and 10 cases of transurethral resection of the prostate material from patients with androgen-independent disease. Results: Weak to moderate IL-8 expression was strictly localized to the apical membrane of normal prostate epithelium. In contrast, membranous expression of IL-8, CXCR1, and CXCR2 was nonapical in cancer cells of Gleason pattern 3 and 4, whereas circumferential expression was present in Gleason pattern 5 and androgen-independent prostate cancer. Each of IL-8, CXCR1, and CXCR2 were also increasingly localized to the cytoplasm of cancer cells in correlation with advancing stage of disease. Cytoplasmic expression (but not apical membrane expression) of IL-8 in Gleason pattern 3 and 4 cancer correlated with Ki-67 expression (R = 0.79; P <0.001), cyclin D1 expression (R = 0.79; P <0.001), and microvessel density (R = 0.81; P <0.001). In vitro studies on androgen-independent PC3 cells confirmed the mitogenic activity of IL-8, increasing the rate of cell proliferation through activation of both CXCR1 and CXCR2 receptors. Conclusions: We propose that the concurrent increase in IL-8 and IL-8 receptor expression in human prostate cancer induces autocrine signaling that may be functionally significant in initiating and promoting the progression of prostate cancer by underpinning cell proliferation and angiogenesis.

Targeted cytoplasmic irradiation induces bystander responses.

The observation of radiation-induced bystander responses, in which cells respond to their neighbors being irradiated, has important implications for understanding mechanisms of radiation action particularly after low-dose exposure. Much of this questions the current dogma of direct DNA damage driving response in irradiated systems. In this study, we have used a charged-particle microbeam to target individual helium ions ((3)He(2+)) to individual cells within a population of radioresistant glioma cells cultured alone or in coculture with primary human fibroblasts. We found that even when a single cell within the glioma population was precisely traversed through its cytoplasm with one (3)He(2+) ion, bystander responses were induced in the neighboring nonirradiated glioma or fibroblasts so that the yield of micronuclei was increased by 36% for the glioma population and 78% for the bystander fibroblast population. Importantly, the yield of bystander-induced micronuclei was independent of whether the cytoplasm or nucleus of a cell was targeted. The bystander responses were fully eliminated when the populations were treated with 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide or filipin, which scavenge nitric oxide (NO) and disrupt membrane rafts, respectively. By using the probe 4-amino-5-methylamino-2',7'-difluorofluorescein, it was found that the NO level in the glioma population was increased by 15% after 1 or 10 cytoplasmic traversals, and this NO production was inhibited by filipin. This finding shows that direct DNA damage is not required for switching on of important cell-signaling mechanisms after low-dose irradiation and that, under these conditions, the whole cell should be considered a sensor of radiation exposure.

Cytoplasmic Irradiation Induces Mitochondrial-Dependent 53BP1 Protein Relocalization in Irradiated and Bystander Cells

The accepted paradigm for radiation effects is that direct DNA damage via energy deposition is required to trigger the downstream biological consequences. The radiation-induced bystander effect is the ability of directly irradiated cells to interact with their nonirradiated neighbors, which can then show responses similar to those of the targeted cells. p53 binding protein 1 (53BP1) forms foci at DNA double-strand break sites and is an important sensor of DNA damage. This study used an ionizing radiation microbeam approach that allowed us to irradiate specifically the nucleus or cytoplasm of a cell and quantify response in irradiated and bystander cells by studying ionizing radiation-induced foci (IRIF) formation of 53BP1 protein. Our results show that targeting only the cytoplasm of a cell is capable of eliciting 53BP1 foci in both hit and bystander cells, independently of the dose or the number of cells targeted. Therefore, direct DNA damage is not required to trigger 53BP1 IRIF. The use of common reactive oxygen species and reactive nitrogen species (RNS) inhibitors prevent the formation of 53BP1 foci in hit and bystander cells. Treatment with filipin to disrupt membrane-dependent signaling does not prevent the cytoplasmic irradiation-induced 53BP1 foci in the irradiated cells, but it does prevent signaling to bystander cells. Active mitochondrial function is required for these responses because pseudo-rho(0) cells, which lack mitochondrial DNA, could not produce a bystander signal, although they could respond to a signal from normal rho(+) cells.

Patterns of cytoplasmic variation in Arabidopsis thaliana (Brassicaceae) revealed by polymorphic chloroplast microsatellites

Despite being the model organism for plant molecular genetic studies, little is known about the origins and evolutionary history of extant natural populations of Arabidopsis thaliana. We have analysed phylogenetic relationships between worldwide populations of Arabidopsis using polymorphic chloroplast microsatellites. These highly variable markers have revealed previously undetected levels of cytoplasmic variation and confirm previous hypotheses of a recent and rapid expansion of the species from its centre of origin. Furthermore, the results seem to verify previous nuclear analyses that call into question the true origin of several individual Arabidopsis ecotypes.

Micron-scale fast electron filaments and recirculation determined from rear-side optical emission in high-intensity laser-solid interactions

The transport of relativistic electrons generated in the interaction of petawatt class lasers with solid targets has been studied through measurements of the second harmonic optical emission from their rear surface. The high degree of polarization of the emission indicates that it is predominantly optical transition radiation (TR). A halo that surrounds the main region of emission is also polarized and is attributed to the effect of electron recirculation. The variation of the polarization state and intensity of radiation with the angle of observation indicates that the emission of TR is highly directional and provides evidence for the presence of mu m-size filaments. A brief discussion on the possible causes of such a fine electron beam structure is given.

SPECTRALLY MODULATED 2ND-HARMONIC EMISSION FROM LASER-PLASMA FILAMENTS

Spectra of forward emitted second harmonic light from laser interaction with filamentary plasmas have been experimentally studied. Rather regular modulations in the frequency domain have been observed into overall red-shifted spectra. The observed spectral features are consistent with self-phase-modulation of the intense laser light in growing filaments. A model accounts for this effect.

Physico-chemical characterization of a recombinant cytoplasmic form of lysine:N6-hydroxylase

A recombinant cytoplasmic preparation of lysine: N6-hydroxylase, IucD398, with a deletion of 47 amino acids at the N-terminus, was purified to homogeneity. IucD398 is capable of N-hydroxylation of L-lysine upon supplementation with FAD and NADPH. The enzyme is stringently specific with L-lysine and (S)-2-aminoethyl-L-cysteine serving as substrates. Protonophores, FCCP and CCCP, as well as cinnamylidene, have been found to serve as potent inhibitors of lysine: N6-hydroxylation by virtue of their ability to interfere in the reduction of the flavin cofactor.

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)