Role of beta-catenin in epidermal stem cell expansion, lineage selection, and cancer.

The mammalian epidermis is an excellent model with which to analyze the factors that regulate adult stem cell renewal, lineage selection, and tumor formation. One of the key regulators of all three processes is beta-catenin, the main cytoplasmic effector of the canonical Wnt signaling pathway. In this chapter, we review some of the ways in which beta-catenin exerts its effects on cultured human epidermal cells and in genetically modified mice. We highlight the importance of the timing and level of activation and discuss some of the pathways activated downstream from beta-catenin. Finally, we demonstrate the importance of Lef/Tcf-independent beta-catenin signaling through interaction with the vitamin D receptor.

Processing, characterisation and biocompatibility of iron-phosphate glass fibres for tissue engineering.

Iron-phosphate glass fibres based on the CaO-Na2O-Fe2O3-P2O5 system have been processed and characterised via thermal, XRPD, dissolution rates, diameter and biocompatibility studies. The compositions investigated were fixed at 50mol% P2O5, and the CaO content was varied between 30, 35 and 40mol%. The Fe2O3 was added in low amounts from 1-5mol%, substituting it for the Na2O mol%. The number of Tc (crystallisation temperature) peaks detected from the thermal analysis traces only showed correlation with XRPD analysis, for five out of the 15 compositions investigated. It has been suggested that either the crystalline phases had very similar Tc temperatures or that the other phase(s) were present in very small quantities. There was a good match seen with number of Tm (melting temperature) peaks picked up from the DTA traces, with the number of phases identified from XRPD analysis. The main phases identified from XRPD were NaCa(PO3)3, CaP2O6 and NaFeP2O7. Using network connectivity (NC), predictions on Qn species present within the compositions investigated were made. The predicted species (metaphosphates) matched with phases identified from XRPD analysis. A decrease in dissolution rates for the bulk glass and glass fibres was seen with an increase in CaO mol%, along with an increase in Fe2O3 mol%. An increase in fibre dissolution rates was seen with a decrease in diameter size. The biocompatibility studies were conducted using a conditionally immortal muscle precursor cell line derived from the H-2Kb-tsA58 immortomouse. It was found that iron-phosphate glass fibres containing 4-5mol% Fe2O3 was sufficient for cell attachment and differentiation. It was seen that myotubes formed along the axis of the fibres (which was indicative of differentiation). The biocompatibility of these compositions was attributed to the enhanced chemical durability of the glass fibres.

Duchenne's muscular dystrophy: animal models used to investigate pathogenesis and develop therapeutic strategies.

Duchenne's muscular dystrophy (DMD) is a lethal childhood disease caused by mutations of the dystrophin gene, the protein product of which, dystrophin, has a vital role in maintaining muscle structure and function. Homologues of DMD have been identified in several animals including dogs, cats, mice, fish and invertebrates. The most notable of these are the extensively studied mdx mouse, a genetic and biochemical model of the human disease, and the muscular dystrophic Golden Retriever dog, which is the nearest pathological counterpart of DMD. These models have been used to explore potential therapeutic approaches along a number of avenues including gene replacement and cell transplantation strategies. High-throughput screening of pharmacological and genetic therapies could potentially be carried out in recently available smaller models such as zebrafish and Caenorhabditis elegans. It is possible that a successful treatment will eventually be identified through the integration of studies in multiple species differentially suited to addressing particular questions.

Form or function? Part 1. Subjective cosmetic and functional correlates of quality of life in women treated with breast-conserving surgical procedures and radiotherapy.

BACKGROUND: Quality of life (QOL) after a diagnosis of cancer varies considerably across individuals. Treatment-related factors that predict QOL for women who are diagnosed with breast carcinoma require further specification. This study was designed to develop a measure of perceived aesthetic (e.g., breast shape) and functional status (e.g., pain, mobility) after breast-conserving surgical treatment (BCT) and radiotherapy, to examine the relations of these indicators with patient QOL, and to determine whether these relations varied as a function of diagnosis duration. METHODS: Women (n = 185 patients) who underwent BCT and radiotherapy for Stage 0-II disease for whom the diagnosis duration ranged from 3 months to 18 years completed assessments of background information, perceived cosmetic and functional treatment outcomes, and QOL. Medical data also were obtained from medical charts. RESULTS: The Breast Cancer Treatment Outcome Scale (BCTOS) produced a coherent factor structure and three internally consistent subscales (i.e., cosmetic status, functional status, and breast specific pain) that demonstrated predictive validity. With patient age, diagnosis duration, and other BCTOS subscales controlled, greater breast specific pain predicted greater depressive symptoms (P < 0.01) and lower QOL related to mental health (P < 0.05) and physical health (P < 0.05). Cosmetic status predicted QOL related to physical health (P < 0.05). The relations of breast specific pain with QOL indicators varied somewhat as a function of diagnosis duration. CONCLUSIONS: Although considerable research on treatment-relevant outcomes has addressed appearance-related concerns, functional parameters have not been explored fully. Findings suggest that functional consequences of treatment, and particularly breast specific pain, also are significant influences on patient QOL.

Form or function? Part 2. Objective cosmetic and functional correlates of quality of life in women treated with breast-conserving surgical procedures and radiotherapy.

BACKGROUND: Treatment-related factors that influence quality of life (QOL) for women who are diagnosed with breast carcinoma require study. This study was designed to examine the convergent validity of objective and subjective indices of cosmetic and functional status after patients undergo breast-conserving treatment (BCT) and to test the relations of the objective indicators with QOL. METHODS: Women (n = 54 patients) who had received BCT and radiotherapy for Stage 0-II disease for whom the diagnosis duration ranged from 9 months to 18 years completed assessments of background information, perceived cosmetic and functional outcomes, and QOL. They also were administered a clinical breast examination, including objective ratings of arm edema and breast cosmesis. The patients were a subsample from the study reported by the authors in an accompanying article that is presented in this issue. RESULTS: The findings revealed positive cosmetic and functional treatment outcomes, such that 82% of patients had no or minimal arm edema, and 70% of patients evidenced good or excellent cosmetic results. Convergent validity was demonstrated for the objective and subjective assessments of cosmetic and functional treatment outcomes. In addition, women who had more arm edema reported poorer QOL with regard to depressive symptoms (P < 0.05) and fear of disease recurrence (P < 0.05). CONCLUSIONS: The findings from this study and those reported in the accompanying article reveal that functional treatment outcomes, such as arm edema and breast specific pain, are important correlates of QOL even many years after patients undergo BCT and radiotherapy. Both subjective and objective indicators of treatment outcomes are useful predictors of QOL.

Modulation of cytoplasmic dynein ATPase activity by the accessory subunits.

The microtubule-based motor molecule cytoplasmic dynein has been proposed to be regulated by a variety of mechanisms, including phosphorylation and specific interaction with the organelle-associated complex, dynactin. In this study, we examined whether the intermediate chain subunits of cytoplasmic dynein are involved in modulation of ATP hydrolysis, and thereby affect motility. Treatment of testis cytoplasmic dynein under hypertonic salt conditions resulted in separation of the intermediate chains from the remainder of the dynein molecule, and led to a 4-fold enhancement of ATP hydrolysis. This result suggests that the accessory subunits act as negative regulators of dynein heavy chain activity. Comparison of ATPase activities of dyneins with differing intermediate chain isoforms showed significant differences in basal ATP hydrolysis rates, with testis dynein 7-fold more active than dynein from brain. Removal of the intermediate chain subunits led to an equalization of ATPase activity between brain and testis dyneins, suggesting that the accessory subunits are responsible for the observed differences in tissue activity. Finally, our preparative procedures have allowed for the identification and purification of a 1:1 complex of dynein with dynactin. As this interaction is presumed to be mediated by the dynein intermediate chain subunits, we now have defined experimental conditions for further exploration of dynein enzymatic and motility regulation.

Genetic interactions between the 5' and 3' splice site consensus sequences and U6 snRNA during the second catalytic step of pre-mRNA splicing.

The YAG/ consensus sequence at the 3' end of introns (the slash indicates the location of the 3' splice site) is essential for catalysis of the second step of pre-mRNA splicing. Little is known about the interactions formed by these three nucleotides in the spliceosome. Although previous observations have suggested that the G of the YAG/ interacts with the first nucleotide of the /GUA consensus sequence at the 5' end of the intron, additional interactions have not been identified. Here we report several striking genetic interactions between A+3 of the 5' /GUA with Y-3 of the 3' YAG/ and G50 of the highly conserved ACAGAG motif in U6 snRNA. Two mutations in U6 G50 of the ACAGAG can weakly suppress two mutations in A+3 of the 5' /GUA. This suppression is significantly enhanced upon the inclusion of a specific mutation Y-3 in the 3' YAG/. RNA analysis confirmed that the severe splicing defect observed in A+3 and Y-3 double mutants can be rescued to near wild-type levels by the mutations in U6 G50. The contributions of each mutation to the genetic interaction and the strong position specificity of suppression, combined with previous findings, support a model in which the 5' /GUA and the GAG of U6 function in binding the 3' YAG/ during the second catalytic step.

Emotionally expressive coping predicts psychological and physical adjustment to breast cancer.

This study tested the hypothesis that coping through emotional approach, which involves actively processing and expressing emotions, enhances adjustment and health status for breast cancer patients. Patients (n = 92) completed measures within 20 weeks following medical treatment and 3 months later. Women who, at study entry, coped through expressing emotions surrounding cancer had fewer medical appointments for cancer-related morbidities, enhanced physical health and vigor, and decreased distress during the next 3 months compared with those low in emotional expression, with age, other coping strategy scores, and initial levels on dependent variables (except medical visits) controlled statistically. Expressive coping also was related to improved quality of life for those who perceived their social contexts as highly receptive. Coping through emotional processing was related to one index of greater distress over time. Analyses including dispositional hope suggested that expressive coping may serve as a successful vehicle for goal pursuit.

The question remains: is the spliceosome a ribozyme?

The two phosphoryl transfer steps of pre-mRNA splicing are catalyzed within the large ribonuclear protein machine called the spliceosome. The highly dynamic nature of the spliceosome has presented many challenges to a structural and mechanistic understanding of its catalytic core. While much evidence supports the popular hypothesis that the catalytic steps of pre-mRNA splicing are mediated by spliceosomal RNA, a role for protein in catalysis cannot yet be ruled out. A highly conserved protein, Prp8, is a component of the catalytic core. We review data consistent with the hypothesis that Prp8 functions as a cofactor to an RNA enzyme.

Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome.

The highly conserved spliceosomal protein Prp8 is known to cross-link the critical sequences at both the 5' (GU) and 3' (YAG) ends of the intron. We have identified prp8 mutants with the remarkable property of suppressing exon ligation defects due to mutations in position 2 of the 5' GU, and all positions of the 3' YAG. The prp8 mutants also suppress mutations in position A51 of the critical ACAGAG motif in U6 snRNA, which has been observed previously to cross-link position 2 of the 5' GU. Other mutations in the 5' splice site, branchpoint, and neighboring residues of the U6 ACAGAG motif are not suppressed. Notably, the suppressed residues are specifically conserved from yeast to man, and from U2- to U12-dependent spliceosomes. We propose that Prp8 participates in a previously unrecognized tertiary interaction between U6 snRNA and both the 5' and 3' ends of the intron. This model suggests a mechanism for positioning the 3' splice site for catalysis, and assigns a fundamental role for Prp8 in pre-mRNA splicing.

Suspected spinal cord compression in breast cancer patients: a multidisciplinary risk assessment.

Breast cancer is the most common cause of metastatic epidural spinal cord compression (SCC) in women, and this condition results in significant neurologic dysfunction and morbidity. Prior studies of patients with suspected SCC did not employ multivariate analysis techniques, often included persons with a wide variety of malignancies, and generally focused on identifying associated neurologic and radiologic features. We therefore conducted a study examining a more comprehensive set of potential clinical risk factors in breast cancer patients with suspected SCC. We retrospectively analysed 123 episodes of suspected SCC among 93 breast cancer patients evaluated by spine computed tomography (CT) scanning. Multiple logistic regression analysis was employed to identify independent predictors of SCC. Clinically significant metastatic epidural cancer was defined as thecal sac compression (TSC), which occurred in 33 episodes (27%). Four independent predictors of TSC were identified and included oncologic features (known bone metastases > or = 2 years, metastatic disease at initial diagnosis) in addition to neurologic and radiologic features (objective weakness, vertebral compression fracture on spine radiograph). These four predictors stratified episodes into subgroups with widely varying risks of TSC, ranging from 12% (0 risk factors) to 85% (> or = 3 risk factors). These results suggest that the evaluation of breast cancer patients with suspected SCC should include clinical information about their disease course in addition to neurologic examination and prior imaging studies. If confirmed, these predictors may help clinicians assess risk in this patient population.

Comparison of the intracellular distribution of cytoplasmic dynein and kinesin in cultured cells: motor protein location does not reliably predict function.

While immunolocalization methods have been used as a reasonable means to judge where a given molecule may be active in the cellular milieu, the correlation between distribution and function for proteins involved in intracellular transport may not be clear cut. To address the question of specificity and reproducibility of immunolocalization of microtubule-based motor proteins, we have co-localized cytoplasmic dynein and kinesin by immunofluorescence microscopy using two specific antibodies for each motor molecule. The results indicate that cytoplasmic dynein and kinesin appear to co-localize on a small subset of vesicles, but largely reside or accumulate on morphologically distinct organelles. In addition, anti-kinesin antibodies differing in their epitope specificity label different cellular compartments. To address the question of whether the distribution of motor molecules is representative of organelles that are undergoing active transport, we have altered the activity of vesicle trafficking pathways in fibroblasts using several different methods, including cytoplasmic acidification and disruption of cellular compartments with brefeldin A, nocodazole and okadaic acid. Analysis of the distribution of cytoplasmic dynein and kinesin under these conditions indicates that immunolocalization data alone are not reliable indicators of sites of likely function for these microtubule-based motors.

Dynamin forms polymeric complexes in the presence of lipid vesicles. Characterization of chemically cross-linked dynamin molecules.

Dynamin is a GTP-binding protein that is involved in the release of coated endocytic vesicles from the plasma membrane. We have been characterizing the enzymatic properties of purified rat brain dynamin to better understand how GTP binding and hydrolysis relate to its proposed function. Previously, we have demonstrated that activation of dynamin GTPase results from positive cooperative associations between dynamin molecules as they are bound to a polymeric surface. Our present report has extended these studies and has examined the structural features of dynamin self-association. After treatment with the zero-length protein cross-linking reagent, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, dynamin in solution was found cross-linked into dimers. This homodimer likely reflects the native soluble state of the molecule. After binding to brain vesicles, dynamin was cross-linked into higher order oligomers of greater than 800 kDa. Dynamin, copurified on brain membranous organelles, also formed multimeric complexes when cross-linked suggesting dynamin exists in polymeric form in vivo. No cross-linked species other than homo-oligomers were observed, providing no evidence for close interactions between dynamin and membrane proteins. From experiments examining the effects of GTP, GDP, guanosine 5'-3-O-(thio)triphosphate, and 5'-guanylyl-beta,gamma-imidodiphosphate on cross-linking, we have determined that both dynamin membrane binding and self-association occur independently from the nucleotide-bound state of the enzyme. An 80-kDa dynamin fragment that is lacking its carboxyl-terminal domain is not cross-linked into higher order oligomers, suggesting that this domain is required for binding of dynamin to membranes and the subsequent enhancement of oligomerization. However, the dynamin fragment was found to form dimers indicating that this domain is not required for dynamin dimerization. Cross-linked dynamin was able to cooperatively bind microtubules, but did not exhibit GTPase activation. We propose that intramolecular cross-links in the dynamin monomer impart structural constraints that prevent the enhancement of GTP hydrolysis. We describe a model of the dynamin activation process to be considered in further investigations of the role for dynamin in endocytic vesicle formation.

Microtubule-independent phospholipid stimulation of cytoplasmic dynein ATPase activity.

In this study we report that phospholipid vesicles activate ATP hydrolysis by cytoplasmic dynein but not kinesin, consistent with reported differences in the organelle/vesicle binding of these motor proteins. Dynein activation by phospholipids was comparable with that seen in the presence of microtubules but was not sensitive to moderate salt concentrations and was independent of the net charge of the phospholipid, suggesting that the means of interaction between dynein and the lipid vesicle was not strictly ionic in nature. Based on this result, previous data that show that the interaction between dynein and vesicles is not ATP sensitive, and the concentration dependence observed for lipid activation of cytoplasmic dynein, it is likely that the binding interaction between dynein and liposomes is a stable one. In contrast to a previous report, microtubules increased the hydrolysis rate of all naturally occurring nucleotides tested, whereas only ATPase activity was stimulated by phospholipids. As ATP is the physiologically relevant substrate and is the only nucleotide to promote motility, the activation of only the ATPase by phospholipids may represent a means of discriminating between coupled and uncoupled nucleotide hydrolysis in vitro.

Activation of dynamin GTPase is a result of positive cooperativity.

Dynamin is a GTP-binding protein thought to be involved in the early stages of endocytosis. Presently, it is not known how dynamin GTP binding and hydrolysis are related to its role in this process. We previously characterized the ability of acidic phospholipid vesicles and microtubules to strongly stimulate the GTPase activity of purified brain dynamin. In a further analysis of dynamin enzymatic properties, we have found that the increase of dynamin GTP hydrolysis in the presence of activating agent depends on enzyme concentration. At low enzyme concentration, little or no activation is observed. Plots of dynamin GTPase activity with increasing enzyme concentration in the presence of either activating agent are strongly sigmoidal, indicating that positive cooperativity is responsible for the increased activity observed. A Hill coefficient of 2.3 was determined, implying that at least two dynamin molecules associate for maximal GTPase activity. No cooperative effects in GTP binding were observed. Linear transformation of reaction velocity versus enzyme concentration data indicate an apparent Km for dynamin-dynamin interactions of 37 nM, which is significantly lower than the physiological concentration of dynamin in brain. These results suggest that cooperative interactions between dynamin molecules are responsible for the apparent activation of GTPase observed and are likely involved in dynamin function in vivo.

Cytoplasmic dynein undergoes intracellular redistribution concomitant with phosphorylation of the heavy chain in response to serum starvation and okadaic acid.

Cytoplasmic dynein is a microtubule-binding protein which is considered to serve as a motor for retrograde organelle movement. In cultured fibroblasts, cytoplasmic dynein localizes primarily to lysosomes, membranous organelles whose movement and distribution in the cytoplasm have been shown to be dependent on the integrity of the microtubule cytoskeleton. We have recently identified conditions which lead to an apparent dissociation of dynein from lysosomes in vivo, indicating that alterations in membrane binding may be involved in the regulation of retrograde organelle movement (Lin, S. X. H., and C. A. Collins. 1993. J. Cell Sci. 105:579-588). Both brief serum withdrawal and low extracellular calcium levels induced this alteration, and the effect was reversed upon addition of serum or additional calcium. Here we demonstrate that the phosphorylation state of the dynein molecule is correlated with changes in its intracellular distribution in normal rat kidney fibroblasts. Dynein heavy chain phosphorylation level increased during serum starvation, and decreased back to control levels upon subsequent addition of serum. We found that okadaic acid, a phosphoprotein phosphatase inhibitor, mimicked the effects of serum starvation on both phosphorylation and the intracellular redistribution of dynein from a membrane-associated pool to one that was more soluble, with similar dose dependence for both phenomena. Cell fractionation by differential detergent extraction revealed that a higher proportion of dynein was present in a soluble pool after serum starvation than was found in comparable fractions from control cells. Our data indicate that cytoplasmic dynein is phosphorylated in vivo, and changes in phosphorylation state may be involved in a regulatory mechanism affecting the distribution of this protein among intracellular compartments.

Activation of dynamin GTPase by acidic phospholipids and endogenous rat brain vesicles.

Dynamin is a GTPase thought to play a role in endocytosis based on genetic analysis of its homolog in Drosophila melanogaster shibire. Previous studies have stressed an in vitro association with microtubules, though additional evidence suggests that dynamin associates with membranous organelles. In an analysis of the enzymatic and membrane binding properties of dynamin, we have found that the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, are able to stimulate GTP hydrolysis in a manner similar to activation previously shown with microtubules. A neutral phospholipid, phosphatidylcholine, had no effect on dynamin GTPase. Activation of dynamin was biphasic, with a decrease in activity back to basal levels with increased concentrations of either microtubules or liposomes. A comparison between GTPase stimulation induced by microtubules and that by phospholipids suggests that ionic interactions between the basic C-terminal domain of dynamin and the negatively charged microtubule or phospholipid head group are important. In support of this, GTPase stimulation by these agents in combination was not additive. A salt-extracted membrane fraction from brain tissue also activated dynamin GTPase, though to a lower extent than pure phospholipids. These results suggest that membrane components could be responsible for some aspects of the regulation of dynamin function in vivo.

Regulation of the intracellular distribution of cytoplasmic dynein by serum factors and calcium.

Previous work has indicated that cytoplasmic dynein localizes primarily to lysosomes in cultured fibroblasts, consistent with a function for dynein in retrograde movement. We now show that dynein can be redistributed from a lysosome-associated pool to a more diffuse cytoplasmic pool upon shifting fibroblasts to culture medium lacking serum for several hours. This effect on dynein localization is readily reversed upon addition of serum, with a substantial return to a control appearance of punctate staining within 10 minutes. The serum effect appears to be selective for dynein, in that the localization of kinesin and the overall morphology of intracellular organelles does not change. However, the distribution of kinesin-positive vesicles and lysosomes does appear to be altered during serum starvation, in that these organelles are located to greater extents in the peripheral regions of the cell. Dynein is also associated with the mitotic apparatus, but this localization does not change in response to serum starvation. Removal of calcium from the extracellular medium also results in the loss of punctate dynein staining, which can be recovered upon addition of calcium to calcium-free medium. The redistribution of dynein observed under these experimental conditions may reflect the activity of a regulatory process controlling the association of dynein with organelles, thereby providing one means of modulating intracellular transport.