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.

p21 is essential for normal myogenic progenitor cell function in regenerating skeletal muscle.

Despite the ability of myogenic progenitor cells (MPCs) to completely regenerate skeletal muscle following injury, little is known regarding the molecular program that regulates their proliferation and differentiation. Although mice lacking the cyclin-dependent kinase inhibitor p21 (p21-/-), develop normally, we report here that p21-/- MPCs display increased cell number and enhanced cell cycle progression compared with wild-type MPCs. Therefore, we hypothesized that p21-/- mice would demonstrate temporally enhanced regeneration following myotrauma. In response to cardiotoxin-induced injury, p21-/- skeletal muscle regeneration was significantly attenuated vs. regenerating wild-type muscle, contrary to the hypothesis. Regenerating p21-/- skeletal muscle displayed increased proliferative (PCNA positive) nuclei coincident with increased apoptotic nuclei (TUNEL positive) compared with wild-type muscle up to 3 wk after injury. Differentiation of p21-/- MPCs was markedly impaired and associated with increased apoptosis compared with wild-type MPCs, confirming that the impaired differentiation of the p21-/- MPCs was a cell autonomous event. No dysregulation of p27, p53, or p57 protein expression in differentiating p21-/- MPCs compared with wild-type MPCs was observed, suggesting that other compensatory mechanisms are responsible for the regeneration that ultimately occurs. On the basis of these findings, we propose that p21 is essential for the coordination of cell cycle exit and differentiation in the adult MPC population and that in the absence of p21, skeletal muscle regeneration is markedly impaired.

Adaptive mechanisms that preserve cardiac function in mice without myoglobin.

Mice lacking myoglobin survive to adulthood and meet the circulatory demands of exercise and pregnancy without cardiac decompensation. In the present study, we show that many myoglobin-deficient embryos die in utero at midgestation with signs of cardiac failure. Fetal mice that survive to gestational day 12.5, however, suffer no subsequent excess mortality. Survival in the absence of myoglobin is associated with increased vascularity and the induction of genes encoding the hypoxia-inducible transcription factors 1alpha and 2, stress proteins such as heat shock protein 27, and vascular endothelial growth factor. These adaptations are evident in late fetal life, persist into adulthood, and are sufficient to maintain normal myocardial oxygen consumption during stressed conditions. These data reveal that myoglobin is necessary to support cardiac function during development, but adaptive responses evoked in some animals can fully compensate for the defect in cellular oxygen transport resulting from the loss of myoglobin.

VEGF deprivation-induced apoptosis is a component of programmed capillary regression.

The pupillary membrane (PM) is a transient ocular capillary network, which can serve as a model system in which to study the mechanism of capillary regression. Previous work has shown that there is a tight correlation between the cessation of blood flow in a capillary segment and the appearance of apoptotic capillary cells throughout the segment. This pattern of cell death is referred to as synchronous apoptosis (Lang, R. A., Lustig, M., Francois, F., Sellinger, M. and Plesken, H. (1994) Development 120, 3395-3404; Meeson, A., Palmer, M., Calfon, M. and Lang, R. A. (1996) Development 122, 3929-3938). In the present study, we have investigated whether the cause of synchronous apoptosis might be a segmental deficiency of either oxygen or a survival factor. Labeling with the compound EF5 in a normal PM indicated no segmental hypoxia; this argued that oxygen deprivation was unlikely to be the cause of synchronous apoptosis. When rat plasma was used as a source of survival factors in an in vitro PM explant assay, inhibition of vascular endothelial growth factor (VEGF) all but eliminated the activity of plasma in suppressing apoptosis. This argued that VEGF was an important plasma survival factor. Furthermore, inhibition of VEGF in vivo using fusion proteins of the human Flk-1/KDR receptor resulted in a significantly increased number of capillaries showing synchronous apoptosis. This provides evidence that VEGF is necessary for endothelial cell survival in this system and in addition, that VEGF deprivation mediated by flow cessation is a component of synchronous apoptosis.

The distribution of inflammatory demyelinated lesions in the central nervous system of rats with antibody-augmented demyelinating experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) has long been studied as an animal model of the human demyelinating disease Multiple Sclerosis. However, EAE induced in the Lewis rat by injection of myelin basic protein (MBP), or MBP-specific T-lymphocytes, is primarily an inflammatory condition of the central nervous system (CNS) with little or no demyelination. In EAE models in which demyelination does result, it is either not very widespread or is unpredictable in its degree and location. In this study we have produced antibody-augmented demyelinating EAE (ADEAE) in the Lewis rat by injection of activated MBP-specific T-lymphoblasts, followed by injection 4 days later of a monoclonal antibody against myelin/oligodendrocyte glycoprotein, an extrinsic protein of myelin. We have documented the extent and location of inflammatory cell infiltrates and demyelination throughout the CNS using histochemistry, immunofluorescence, and image analysis. Perivascular inflammatory infiltrates were seen in the deep cerebellar white matter and in the folia. Perivascular, periventricular, and subpial inflammation was widespread throughout the pons/medulla and at all levels of the spinal cord. Very little inflammation was apparent in the forebrain. MBP immunofluorescence demonstrated extensive areas of periventricular demyelination in the forebrain around the third ventricle. Both periventricular and perivascular lesions were commonly observed in the cerebellum and pons/medulla. The extent of demyelination in the spinal cord increased caudally with large confluent areas of subpial demyelination seen throughout the lumbar cord. The extensive and reproducible distribution of inflammatory demyelinating lesions in ADEAE provide the possibility to select areas of the CNS for more detailed analysis of the cellular changes that accompany demyelination and remyelination.