Faculty Bibliography

Previous studies showed that the WTH3 gene functioned as a negative regulator during multidrug resistance (MDR) development in vitro. To understand whether this gene is also involved in clinical drug resistance, hypermethylation at its promoter region observed in cultured MDR MCF7/AdrR cells was examined in primary drug-resistant breast cancer epithelial cells isolated from effusions of breast cancer patients. The results showed that this event also occurred in drug-resistant breast cancer epithelial cells and a newly induced drug-resistant cell line, MCF7/inR. Interestingly, we found that a CpG (CpG 23) that was close to the TATA-like box was constantly methylated in the WTH3 promoter of drug-resistant breast cancer epithelial and cultured MDR cells. Mutagenic study suggested that this CpG site had a functional effect on promoter activity. We also discovered that MCF7/AdrR cells treated with trichostatin A, a histone deacetylase inhibitor, exhibited higher WTH3, but lower MDR1, expression. A reverse correlation between WTH3 and MDR1 gene expression was also observed in MCF7/AdrR, and its non-MDR parental cell line, MCF7/WT. This result indicated that both DNA methylation and histone deacetylase could act in concert to inhibit WTH3 and consequently stimulate MDR1 expression. This hypothesis was supported by data obtained from introducing the WTH3 transgene into MDR cell lines, which reduced endogenous MDR1 expression. Therefore, our studies suggested that the behavior of WTH3 in primary drug-resistant breast cancer epithelial cells was similar to that in a model system where epigenetic regulation of the WTH3 gene was linked to the MDR phenotype.

Apaf-1 and cytochrome c coassemble in the presence of dATP to form the apoptosome. We have determined a structure of the apoptosome at 12.8 A resolution by using electron cryomicroscopy and single-particle methods. We then docked appropriate crystal structures into the map to create an accurate domain model. Thus, we found that seven caspase recruitment domains (CARDs) form a central ring within the apoptosome. At a larger radius, seven copies of the nucleotide binding and oligomerization domain (NOD) associate laterally to form the hub, which encircles the CARD ring. Finally, an arm-like helical domain (HD2) links each NOD to a pair of beta propellers, which bind a single cytochrome c. This model provides insights into the roles of dATP and cytochrome c in assembly. Our structure also reveals how a CARD ring and the central hub combine to create a platform for procaspase-9 activation

Radiation rapidly and persistently alters the soluble and insoluble components of the tissue microenvironment. This affects the cell phenotype, tissue composition and the physical interactions and signalling between cells. These alterations in the microenvironment can contribute to carcinogenesis and alter the tissue response to anticancer therapy. Examples of these responses and their implications are discussed with a view to therapeutic intervention

Chronic limb-threatening ischemia is a devastating disease with limited surgical options. However, inducing controlled angiogenesis and enhancing reperfusion holds therapeutic promise. To gain a better understanding of the mechanisms that contribute to limb reperfusion, we examined the temporal biochemical and structural changes occurring within the extracellular matrix of ischemic skeletal muscle. Both the latent and active forms of MMP-2 and -9 significantly increased during the active phase of limb reperfusion. Moreover, small but significant alterations in tissue inhibitors of metalloproteinase levels also occurred during a similar time course, consistent with a net increase in extracellular matrix remodeling. This temporal increase in MMP activity coincided with enhanced exposure of the unique HU177 cryptic collagen epitope. Although the HUIV26 cryptic collagen epitope has been implicated in angiogenesis, little is known concerning such epitopes within ischemic muscle tissue. Here, we provide the first evidence that a functionally distinct cryptic collagen epitope (HU177) is temporally exposed in ischemic muscle tissue during the active phase of reperfusion. Interestingly, the exposure of the HU177 epitope was greatly diminished in MMP-9 null mice, corresponding with significantly reduced limb reperfusion. Therefore, the regulated exposure of a unique cryptic collagen epitope within ischemic muscle suggests an important role for collagen remodeling during the active phase of ischemic limb reperfusion

The TRIM/RBCC proteins are defined by the presence of the tripartite motif composed of a RING domain, one or two B-box motifs and a coiled-coil region. These proteins are involved in a plethora of cellular processes such as apoptosis, cell cycle regulation and viral response. Consistently, their alteration results in many diverse pathological conditions. The highly conserved modular structure of these proteins suggests that a common biochemical function may underlie their assorted cellular roles. Here, we review recent data indicating that some TRIM/RBCC proteins are implicated in ubiquitination and propose that this large protein family represents a novel class of 'single protein RING finger' ubiquitin E3 ligases.

Telomeres protect chromosomes from degradation and loss of vital sequence, block end-end fusion, and allow the cell to distinguish between broken ends and chromosome ends. Mammalian telomeres end in single-stranded (TTAGGG)-rich 3'-overhangs that are tucked back into the preceding double stranded region to form a T-loop. The end structure of mammalian telomeres has just started to be elucidated and through this extra views we highlight one aspect of that structure. We have recently identified the terminal nucleotides of both the C-rich and G-rich telomere strands in human cells and showed that approximately 80% of the C-rich strands terminate precisely in ATC-5', while the last base of the G-strand is less precise. This finding has important implications for the processing events that act on the telomere ends post-replication. While the mechanism behind this phenotype is yet to be unraveled, we discuss potential models that could explain the last base specificity

Plant secondary metabolites with estrogenic activity (phyto-estrogens) have been studied in the past as a potential alternative to classical hormone-replacement therapy (HRT) in menopausal women. No final verdict on the efficacy of soy or red clover based pharmaceutical preparations has been reached despite numerous clinical studies. We have studied the novel and most potent phyto-estrogen 8-prenylnaringenin (8-PN) in adult ovariectomized rats, an established animal model to mimic hormone dependent osteoporosis in menopausal women. Our results demonstrate that 8-PN can completely protect from ovariectomy induced bone-loss while exhibiting minimal, (dose independent) trophic effects on uterus and endometrium. It is estimated that at equivalent bone protective doses of 17beta-estradiol and 8-PN, the phyto-estrogen has a 10-fold lower stimulatory effect on uterus and endometrium. The bone tissue specific effect of 8-PN was confirmed in a transgenic reporter mouse model (ERE-Luc mice). Here we also found pronounced estrogenic activity in prostate. Present results add important aspects to the pharmacological profile of 8-PN and position this compound as an interesting alternative new candidate for treatment of peri- and postmenopausal symptoms.

Chronic wounds present a significant challenge, because there are few available treatment options for timely healing. Topical negative pressure devices have been used in a number of different types of wounds, including chronic wounds. They are believed to hasten wound healing by (1) maintaining a moist environment, (2) removing wound exudates, (3) increasing local blood flow, (4) increasing granulation tissue formation, (5) applying mechanical pressure to promote wound closure, and (6) reducing bacterial loads in the wound. Multiple nonrandomized, noncontrolled studies have reported that the use of these devices results in faster healing times and more successful closures. Five small randomized, controlled trials have also shown favorable outcomes with the use of topical negative pressure devices compared with conventional treatment. Adverse effects include discomfort, pain, and excessive tissue growth into the dressing. Complications are limited if the device is used properly. In light of the current treatment options, topical negative pressure devices may be considered useful as adjuvant therapy for chronic wounds; however, there is inadequate definitive evidence that wound healing is substantially better with these devices than with traditional therapy

PURPOSE: This study determines the analytic accuracy of a Luminex bead-based commercial analyte-specific reagent for the simultaneous analysis of 30 mutations prevalent in Ashkenazi Jews at eight genetic disease loci. METHODS: DNA from 20 samples with known abnormal genotypes were run a total of 109 times. DNA from 820 patients with unknown genotypes submitted for Ashkenazi Jewish testing panels were analyzed using our current laboratory techniques. The 820 samples were then stripped of identifiers, coded, and reanalyzed using the Tm Biosciences (Toronto, Canada) Ashkenazi Jewish panel analyte-specific reagent in a blinded fashion. For the controls, comparisons were made with their known genotypes. For the patient samples, the results of the Tm assay were compared with the results of our current assay. For 24 of the 30 mutations, we had genomic DNA controls or detected patients' samples heterozygous for these mutations. RESULTS: There were no discrepant results in the control or patient samples. In the patient samples, 19,680 genotyping reactions were performed without error in both our laboratory-developed single-disease assays and the Tm multiplex assay. Including the controls, 22,296 genotypes were determined without error. CONCLUSION: The Tm Biosciences Ashkenazi Jewish analyte-specific reagent is capable of performing accurate analyses of 24 different mutations in eight different genes in a single multiplex reaction and can be used with confidence in the clinical molecular genetics laboratory.