Programmed and altruistic ageing

Ageing is widely believed to be a non-adaptive process that results from a decline in the force of natural selection. However, recent studies in Saccharomyces cerevisiae are consistent with the existence of a programme of altruistic ageing and death. We suggest that the similarities between the molecular pathways that regulate ageing in yeast, worms, flies and mice, together with evidence that is consistent with programmed death in salmon and other organisms, raise the possibility that programmed ageing or death can also occur in higher eukaryotes.     

Caspase-independent cytochrome c release is a sensitive measure of low-level apoptosis in cell culture models

Age-associated loss of tissue function and several chronic diseases may derive in part from the cumulative effects of subtle changes in the level of apoptotic cell death. Because apoptosis is rapid and undetectable once complete, small changes in its incidence are difficult to detect, even in well-controlled cell cultures. We describe a new apoptosis assay that provides greater sensitivity than conventional assays because it measures the accumulation of apoptotic cells. Human and mouse fibroblasts and human mammary epithelial cells that initiated apoptosis were preserved for 3 days by inhibiting caspase activity using the chemical inhibitor Q-VD-OPH (QVD). Cells suspended in the process of apoptosis were scored by immunostaining for cytochrome c, which redistributed from mitochondria in healthy cells to the cytoplasm in dying cells. This caspase-independent cytochrome c release (CICR) assay was more sensitive than several conventional assays when apoptosis was induced by actinomycin D, and detected cumulative background levels of apoptosis over a 3-day interval. Using this assay, we show that normal fibroblasts undergo very little apoptosis upon X-irradiation, indicating dominance of the senescence response in this cell type. Further, apoptosis increased subtly but measurably when human mammary epithelial and skin fibroblast cells entered crisis, indicating that cell death during crisis is largely non-apoptotic.     

Angiogenesis in male breast cancer

BACKGROUND: Male breast cancer is a rare but aggressive and devastating disease. This disease presents at a later stage and in a more advanced fashion than its female counterpart. The immunophenotype also appears to be distinct when compared to female breast cancer. Angiogenesis plays a permissive role in the development of a solid tumor and provides an avenue for nutrient exchange and waste removal. Recent scrutiny of angiogenesis in female breast cancer has shown it to be of significant prognostic value. It was hypothesized that this holds true in invasive ductal carcinoma of the male breast. In the context of male breast cancer, we investigated the relationship of survival and other clinico-pathological variables to the microvascular density of the tumor tissue. METHODS: Seventy-five cases of primary male breast cancer were identified using the records of the Saskatchewan Cancer Agency over a period of 26 years. Forty-seven cases of invasive ductal carcinoma of the male breast had formalin-fixed paraffin-embedded tissue blocks that were suitable for this study. All cases were reviewed. Immunohistochemical staining was performed for the angiogenic markers (cluster designations 31 (CD31), 34 (CD34) and 105 (CD105), von Willebrand factor (VWF), and vascular endothelial growth factor (VEGF)). Microvascular density (MVD) was determined using average, centre, and highest microvessel counts (AMC, CMC, and HMC, respectively). Statistical analyses compared differences in the distribution of survival times and times to relapse between levels of MVD, tumor size, node status and age at diagnosis. In addition, MVD values were compared within each marker, between each marker, and were also compared to clinico-pathological data. RESULTS: Advanced age and tumor size were related to shorter survival times. There were no statistically significant differences in distributions of survival times and times to relapse between levels of MVD variables. There was no significant difference in MVD between levels of the different clinico-pathological variables. MVD was strongly and significantly correlated between AMC, CMC and HMC for CD31, CD34, and CD105 (p < 0.01) and remained moderate to weak for VWF and VEGF. CONCLUSION: Microvascular density does not appear to be an independent prognostic factor in male breast cancer. However, the likelihood of death for men with breast cancer is increased in the presence of increased age at diagnosis and advanced tumor size. This is perhaps linked to inherent tumor vasculature, which is strongly related throughout a tumor section.     

Functional conservation and specialization among eukaryotic anti-silencing function 1 histone chaperones

Chromatin disassembly and reassembly, mediated by histone chaperones such as anti-silencing function 1 (Asf1), are likely to accompany all nuclear processes that occur on the DNA template. In order to gain insight into the functional conservation of Asf1 across eukaryotes, we have replaced the budding yeast Asf1 protein with Drosophila Asf1 (dAsf1) or either of the two human Asf1 (hAsf1a and hAsf1b) counterparts. We found that hAsf1b is best able to rescue the growth defect of Saccharomyces cerevisiae lacking Asf1. Moreover, dAsf1 and hAsf1b but not hAsf1a can replace the role of yeast Asf1 in protecting against replicational stress and activating the PHO5 gene, while only hAsf1a can replace the role of Asf1 in protecting against double-stranded-DNA-damaging agents. Furthermore, it appears that the interaction between Asf1 and the DNA damage checkpoint protein Rad53 is not required for Asf1's role in maintaining genomic integrity. In addition to indicating the functional conservation of the Asf1 proteins across species, these studies suggest distinct roles for the two human Asf1 proteins.     

An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways

We made substantial advances in the implementation of a rapamycin-triggered heterodimerization strategy. Using molecular engineering of different targeting and enzymatic fusion constructs and a new rapamycin analog, Rho GTPases were directly activated or inactivated on a timescale of seconds, which was followed by pronounced cell morphological changes. As signaling processes often occur within minutes, such rapid perturbations provide a powerful tool to investigate the role, selectivity and timing of Rho GTPase-mediated signaling processes.     

Interaction of the mammalian endosomal sorting complex required for transport (ESCRT) III protein hSnf7-1 with itself, membranes, and the AAA+ ATPase SKD1

SKD1/VPS4B is an AAA+ (ATPase associated with a variety of cellular activities) protein involved in multivesicular body (MVB) biogenesis. In this study, we show that the impairment in MVB biogenesis caused by the ATP hydrolysis-deficient mutant SKD1(E235Q) is accompanied by assembly of a large detergent-insoluble protein complex that includes normally soluble endogenous components of mammalian endosomal sorting complex required for transport (ESCRT) I and ESCRT-III complexes. Membrane-bound ESCRT-III complex has been proposed to be the substrate that recruits SKD1 to nascent MVBs. To explore this relationship, we studied interactions among the human ESCRT-III components hSnf7-1 and hVps24, membranes, and SKD1. We found that a significant portion of overexpressed hSnf7-1 associated with membranes where it formed a large protein complex that recruited SKD1 and perturbed normal MVB biogenesis. Overexpressed hVps24 also associated with membranes and perturbed endosome structure but only when fused to green fluorescent protein. Domain analysis revealed that the basic N-terminal half of hSnf7-1 localized to membranes and formed detergent-resistant polymers, some of which looked like filopodia extending into the lumen of swollen endosomes or out from the plasma membrane. The C-terminal acidic half of hSnf7-1 did not associate with membranes and was required for interaction of hSnf7-1 with SKD1. Together with earlier studies, our work suggests that a variety of ESCRT-III-containing polymers can assemble on membranes and recruit SKD1 during formation of the MVB.