Breast cancer, side population cells and ABCG2 expression.

Recurrent metastatic breast cancer may arise in part due to the presence of drug resistant adult stem cells such as Side Population (SP) cells, whose phenotype has been demonstrated to be due to the expression of ABCG2. We hypothesised that SP may be identified in Fine Needle Aspirates (FNAs) and their presence may be determined by expression of ABCG2 in breast tumours. SP and non-side population cells (NSP) were isolated using dual wavelength flow cytometry combined with Hoechst 33342 dye efflux and analysed for expression of ABCG2 and chemoresistance. FNA samples used in SP analysis were matched with paraffin-embedded tissue which was used in immunohistochemical analysis to assess ABCG2 expression. Results were correlated to the pathobiology of the tumour. MCF7 and MDA-MB-231 cell lines contain SP cells. MCF7 SP have increased expression of ABCG2 and increased resistance to mitoxantrone compared to NSP cells. The presence of SP in FNAs were significantly associated with ER-negative (p=0.008) and with triple negative breast cancers (p=0.011) which were also found to have a significant increase in ABCG2 protein expression. ABCG2 transcript was detected in some but not all SP cell populations isolated from FNA samples.

Agonist- and nerve-induced phasic activity in the isolated whole bladder of the guinea pig: evidence for two types of bladder activity.

Spontaneous localised propagating waves of contraction and localised stretches have been reported to occur in the isolated whole bladder of the guinea pig. The physiological role and the cellular processes underlying these events are unknown. In order to gain insight into the mechanisms generating this complex activity, experiments were performed to examine and compare the responses of the whole bladder preparation to (i) the muscarinic agonists carbachol and arecaidine, (ii) the nicotinic ligand lobeline and (iii) nerve stimulation. High concentrations of the muscarinic agonists (>3 micro M) induced a slow rise in intra-vesical pressure upon which were superimposed pressure transients, while low concentrations (< 300 nM) induced only phasic rises in pressure. One interpretation of these data is that there are two separate mechanisms activated by muscarinic agonists: one generating contracture and the other phasic activity. Immunocytochemical staining revealed M(3) muscarinic receptors on smooth muscle cells within trabeculae and a second population of positive cells in the sub-urothelial layer. This observation raises the possibility that the actions of muscarinic agonists are a consequence of activating different cell types. Lobeline (1-60 micro M) activated phasic contractions but did not cause a rise in basal pressure. Atropine did not inhibit the lobeline-induced responses but abolished the muscarinic responses. Also, hexamethonium or tetrodotoxin did not affect the lobeline-induced responses. These observations suggest that the mechanism generating phasic activity is activated by a nicotinic stimulus that does not involve ganglia, nerves or the neuromuscular junction. Stimulation of the bladder nerve at frequencies between 20 and 30 Hz for 5 s resulted in a rapid rise in intra-vesical pressure. Prolonged nerve stimulation (10-200 s) at frequencies between 1 and 10 Hz activated phasic rises in pressure. Low frequency nerve stimulation increased the frequency of agonist-induced phasic activity. Thus, nerve stimulation can also produce two forms of activity and low frequency stimulation can augment the processes generating phasic activity. These observations suggest that there are two distinct types of bladder activity: global contractions involving most of the bladder wall and phasic contractions comprising propagating waves of contraction. The mechanisms generating these contractile events appear to be different and they may involve cells located in different regions of the bladder. The nature of these mechanisms and their possible physiological significance is discussed.

Autonomous activity in the isolated guinea pig bladder.

Phasic changes in pressure have been reported to occur in the bladder which are not associated with micturition. Spontaneous intravesical pressure changes can be recorded from bladders in vitro or bladders in vivo isolated from the central nervous system suggesting that the bladder itself is capable of autonomous activity. Experiments using isolated cells and muscle strips indicate that the smooth muscle can generate spontaneous activity. Whether this is the origin of phasic changes in the intact organ remains unknown. The present study set out to establish the presence and characteristics of autonomous activity in the isolated guinea pig bladder. Multiple-point motion analysis and concurrent intravesical pressure recording were used to identify and quantify spontaneous and evoked activity. Highly complex autonomous activity was observed in unstimulated bladders. This activity comprised localised micro-contractions in single or multiple discrete regions, waves of activity and micro-stretches. Low-amplitude phasic 'micro-transients' were seen in the intravesical pressure trace in association with micro-contractions. Incremental increases in the intravesical volume recruited additional areas of activity. Atropine and tetrodotoxin had no effect on the micro-transients or micro-contractions. Exposure to the muscarinic agonist arecaidine (10-300 nM) initially increased the incidence of micro-contractions which subsequently became co-ordinated into phasic pressure rises and contraction waves, interspersed with periods of total quiescence. The findings describe the generation and co-ordination of autonomous activity in the bladder wall and also demonstrate complex phasic activity. This approach has shown the importance of assessing the integrative properties of the entire organ in studies of the physiology and patho-physiology of the bladder.

A putative alternatively spliced variant of the P2X(1) purinoreceptor in human bladder.

Activation of purinergic P2X receptors, putatively P2X1, may be important in the initiation of contraction in human detrusor. Purinergic transmission may be more important in muscle taken from patients with bladder instability. In this study the presence of the P2X1 receptor subtype was confirmed using RT-PCR. In addition, the results indicate, at the mRNA level, the presence of a splice variant of P2X1 that is lacking part of the second transmembrane domain. It is therefore possible that human bladder expresses multiple isoforms of the P2X1 receptor which may be potential sites for modifying or regulating putative purinergic activation of the human bladder.

Spatial and biological characterisation of the complete quinic acid utilisation gene cluster in Aspergillus nidulans.

Heterologous probing of restriction digests of chromosomal DNA from Aspergillus nidulans with radioactively labelled probes encoding dehydroshikimate dehydratase (QA-4) and a repressor gene (QA1-S) from Neurospora crassa revealed a pattern of hybridisation inconsistent with an equivalent single copy of each gene in A. nidulans. Screening of size-selected and total genome A. nidulans DNA libraries allowed the isolation of four unique classes of sequence, two of which hybridised to the QA-4 probe, and two of which hybridised to the QA1-S probe. In each case, one of each pair of unique sequences was able to complement the equivalent mutations qutC (= QA-4) and qutR (= QA1-S) in A. nidulans, whereas the second of each pair was unable to complement the same mutation. The complementing sequences were physically mapped relative to the previously cloned A. nidulans QUT gene cluster, demonstrating that QUTR is distal and divergently transcribed from QUTA with approximately 3.6 kb between the ATG translational start codons, and that QUTC is transcribed in the same direction as QUTD on the other side of the cluster, approximately 1.65 kb downstream of the QUTD TAA translational stop signal. The physical and genetic maps of the QUT gene cluster correlate precisely. The non-complementing A. nidulans DNA sequences that hybridise to the N. crassa QA-4 (= QUTC) and QA1-S (= QUTR) fulfill many of the criteria characteristic of pseudogenes.(ABSTRACT TRUNCATED AT 250 WORDS)