A new model for imaging and radiation therapy quality assurance in the National Clinical Trials Network of the National Cancer Institute
In March 2014, the National Cancer Institute (NCI) will consolidate the current cooperative group clinical trials program and establish a National Clinical Trials Network (NCTN).
The molecular circuitries controlling osseous prostate metastasis are known to depend on the activity of multiple pathways, including integrin signaling. Here, we demonstrate that the alphavbeta6 integrin is upregulated in human prostate cancer bone metastasis. In prostate cancer cells, this integrin is a functionally active receptor for fibronectin and latency-associated peptide-TGF-beta1; it mediates attachment and migration upon ligand binding and is localized in focal contacts. Given the propensity of prostate cancer cells to form bone metastatic lesions, we investigated whether the alphavbeta6 integrin promotes this type of metastasis. We show for the first time that alphavbeta6 selectively induces matrix metalloproteinase 2 (MMP2) in vitro in multiple prostate cancer cells and promotes osteolysis in vivo in an immunodeficient mouse model of bone metastasis through upregulation of MMP2, but not MMP9. The effect of alphavbeta6 on MMP2 expression and activity is independent of androgen receptor in the analyzed prostate cancer cells. Increased levels of parathyroid hormone-related protein (PTHrP), known to induce osteoclastogenesis, were also observed in alphavbeta6-expressing cells. However, by using MMP2 short hairpin RNA, we demonstrate that the alphavbeta6 effect on bone loss is due to upregulation of soluble MMP2 by the cancer cells, not due to changes in tumor growth rate. Another related alphav-containing integrin, alphavbeta5, fails to show similar responses, underscoring the significance of alphavbeta6 activity. Overall, these mechanistic studies establish that expression of a single integrin, alphavbeta6, contributes to the cancer cell-mediated program of osteolysis by inducing matrix degradation through MMP2. Our results open new prospects for molecular therapy for metastatic bone disease.
Bending the cost curve: a unique collaboration between radiation oncologists and Blue Cross Blue Shield of Massachusetts to optimize the use of advanced technology
PURPOSE: Intensity-modulated radiation therapy (IMRT) limits the dose of radiation to critical normal tissue structures and can be applied to the management of most cancers treated with radiation therapy. Because of increased treatment planning time and quality assurance, IMRT is costly. Blue Cross Blue Shield of Massachusetts (BCBSMA) and the Massachusetts Radiation Oncology Physicians Advisory Council (PAC) developed a strategy to develop standards for the appropriate use of IMRT.
METHODS: Normal tissue volume guidelines were established in multiple oncology disease areas and body site regions. Guidelines were activated in September 2011, and the use of IMRT per case was tracked quarterly by BCBSMA staff.
RESULTS: During the first year of activation of the volume-based guidelines, use of IMRT decreased by 17% in Massachusetts, in contrast to a 20% increase during the previous year.
CONCLUSIONS: The normal tissue-based guidelines have decreased the use of IMRT in Massachusetts; increased the use of 3D treatment; continued communication between treating radiation oncologists and an insurance organization responsible for cost and quality in medicine; increased cost savings; enabled an efficient appeal process; and provided optimal, cost-effective patient care. This may prove to be an effective model for other disciplines and other developing and maturing radiation technologies.
Dosimetric impact of the AeroForm tissue expander in postmastectomy radiation therapy: an ex vivo analysis
PURPOSE: To evaluate the effect of the AeroForm (AirXpanders Inc, Palo Alto, CA) tissue expander on the dose distribution in a phantom from a simulated postmastectomy radiation treatment for breast cancer.
METHODS AND MATERIALS: Experiments were conducted to determine the effect on the dose distribution with the metallic reservoir irradiated independently and with the entire AeroForm tissue expander placed on a RANDO phantom (The Phantom Laboratory, Salem, NY). The metallic reservoir was irradiated on a block of solid water with film at various depths ranging from 0 to 8.2 cm from the surface. The intact 400 cc AeroForm was inflated to full capacity and irradiated while positioned on a RANDO phantom, with 12 optically stimulated luminescent dosimeters (OSLDs) placed at clinically relevant expander-tissue interface points.
RESULTS: Film dosimetry with the reservoir perpendicular to film reveals 40% transmission at a depth of 0.7 cm, which increases to 60% at a depth of 8.2 cm. In the parallel position, the results vary depending on which area under the reservoir is examined, indicating that the reservoir is not a uniformly dense object. Testing of the intact expander on the phantom revealed that the average percent difference (measured vs expected dose) was 2.7%, sigma = 6.2% with heterogeneity correction and 3.7%, sigma = 2.4% without heterogeneity correction. The only position where the OSLD readings were consistently higher than the calculated dose by > 5% was at position 1, just deep to the canister at the expander-phantom interface. At this position, the readings varied from 5.2% to 14.5%, regardless of heterogeneity correction.
CONCLUSIONS: Film dosimetry demonstrated beam attenuation in the shadow of the metallic reservoir in the expander. This decrease in dose was not reproduced on the intact expander on the phantom designed to replicate a clinical setup. Inc.
A wealth of physical interaction data between transcription factors (TFs) and DNA has been generated, but these interactions often do not have apparent regulatory consequences. Thus, equating physical interaction data with gene regulatory networks (GRNs) is problematic. Here, we comprehensively assay TF activity, rather than binding, to construct a network of gene regulatory interactions in the C. elegans intestine. By manually observing the in vivo tissue-specific knockdown of 921 TFs on a panel of 19 fluorescent transcriptional reporters, we identified a GRN of 411 interactions between 19 promoters and 177 TFs. This GRN shows only modest overlap with physical interactions, indicating that many regulatory interactions are indirect. We applied nested effects modeling to uncover information flow between TFs in the intestine that converges on a small set of physical TF-promoter interactions. We found numerous cell nonautonomous regulatory interactions, illustrating tissue-to-tissue communication. Altogether, our study illuminates the complexity of gene regulation in the context of a living animal.
Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization.
Chromatin interaction analysis reveals changes in small chromosome and telomere clustering between epithelial and breast cancer cells
BACKGROUND: Higher-order chromatin structure is often perturbed in cancer and other pathological states. Although several genetic and epigenetic differences have been charted between normal and breast cancer tissues, changes in higher-order chromatin organization during tumorigenesis have not been fully explored. To probe the differences in higher-order chromatin structure between mammary epithelial and breast cancer cells, we performed Hi-C analysis on MCF-10A mammary epithelial and MCF-7 breast cancer cell lines.
RESULTS: Our studies reveal that the small, gene-rich chromosomes chr16 through chr22 in the MCF-7 breast cancer genome display decreased interaction frequency with each other compared to the inter-chromosomal interaction frequency in the MCF-10A epithelial cells. Interestingly, this finding is associated with a higher occurrence of open compartments on chr16-22 in MCF-7 cells. Pathway analysis of the MCF-7 up-regulated genes located in altered compartment regions on chr16-22 reveals pathways related to repression of WNT signaling. There are also differences in intra-chromosomal interactions between the cell lines; telomeric and sub-telomeric regions in the MCF-10A cells display more frequent interactions than are observed in the MCF-7 cells.
CONCLUSIONS: We show evidence of an intricate relationship between chromosomal organization and gene expression between epithelial and breast cancer cells. Importantly, this work provides a genome-wide view of higher-order chromatin dynamics and a resource for studying higher-order chromatin interactions in two cell lines commonly used to study the progression of breast cancer.
High-Affinity Sites Form an Interaction Network to Facilitate Spreading of the MSL Complex across the X Chromosome in Drosophila
Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, we show that high-affinity sites (HAS), landing platforms of the male-specific lethal (MSL) complex, are enriched around topologically associating domain (TAD) boundaries on the X chromosome and harbor more long-range contacts in a sex-independent manner. Ectopically expressed roX1 and roX2 RNAs target HAS on the X chromosome in trans and, via spatial proximity, induce spreading of the MSL complex in cis, leading to increased expression of neighboring autosomal genes. We show that the MSL complex regulates nucleosome positioning at HAS, therefore acting locally rather than influencing the overall chromosomal architecture. We propose that the sex-independent, three-dimensional conformation of the X chromosome poises it for exploitation by the MSL complex, thereby facilitating spreading in males.
To accommodate genomes in the limited space of the cell nucleus and ensure the correct execution of gene expression programs, genomes are packaged in complex fashion in the three-dimensional cell nucleus. As a consequence of the extensive higher-order organization of chromosomes, distantly located genomic regions on the same or distinct chromosomes undergo long-range interactions. This article discusses the nature of long interactions, mechanisms of their formation, and their emerging functional roles in gene regulation and genome maintenance.
Recent studies have shown that chromosomes in a range of organisms are compartmentalized in different types of chromatin domains. In mammals, chromosomes form compartments that are composed of smaller Topologically Associating Domains (TADs). TADs are thought to represent functional domains of gene regulation but much is still unknown about the mechanisms of their formation and how they exert their regulatory effect on embedded genes. Further, similar domains have been detected in other organisms, including flies, worms, fungi and bacteria. Although in all these cases these domains appear similar as detected by 3C-based methods, their biology appears to be quite distinct with differences in the protein complexes involved in their formation and differences in their internal organization. Here we outline our current understanding of such domains in different organisms and their roles in gene regulation.
Myosin-binding protein C corrects an intrinsic inhomogeneity in cardiac excitation-contraction coupling
The beating heart exhibits remarkable contractile fidelity over a lifetime, which reflects the tight coupling of electrical, chemical, and mechanical elements within the sarcomere, the elementary contractile unit. On a beat-to-beat basis, calcium is released from the ends of the sarcomere and must diffuse toward the sarcomere center to fully activate the myosin- and actin-based contractile proteins. The resultant spatial and temporal gradient in free calcium across the sarcomere should lead to nonuniform and inefficient activation of contraction. We show that myosin-binding protein C (MyBP-C), through its positioning on the myosin thick filaments, corrects this nonuniformity in calcium activation by exquisitely sensitizing the contractile apparatus to calcium in a manner that precisely counterbalances the calcium gradient. Thus, the presence and correct localization of MyBP-C within the sarcomere is critically important for normal cardiac function, and any disturbance of MyBP-C localization or function will contribute to the consequent cardiac pathologies.
How cells maintain nuclear shape and position against various intracellular and extracellular forces is not well understood, although defects in nuclear mechanical homeostasis are associated with a variety of human diseases. We estimated the force required to displace and deform the nucleus in adherent living cells with a technique to locally pull the nuclear surface. A minimum pulling force of a few nanonewtons--far greater than typical intracellular motor forces--was required to significantly displace and deform the nucleus. Upon force removal, the original shape and position were restored quickly within a few seconds. This stiff, elastic response required the presence of vimentin, lamin A/C, and SUN (Sad1p, UNC-84)-domain protein linkages, but not F-actin or microtubules. Although F-actin and microtubules are known to exert mechanical forces on the nuclear surface through molecular motor activity, we conclude that the intermediate filament networks maintain nuclear mechanical homeostasis against localized forces.
Prepubertal Serum Concentrations of Organochlorine Pesticides and Age at Sexual Maturity in Russian Boys
BACKGROUND: Few human studies have evaluated the impact of childhood exposure to organochlorine pesticides (OCP) on pubertal development.
OBJECTIVE: To evaluate associations of serum OCP concentrations [hexachlorobenzene (HCB), beta-hexachlorocyclohexane (beta-HCH), and p,p-dichlorodiphenyldichloroethylene (p,p'-DDE)] with age at attainment of sexual maturity among boys.
METHODS: From 2003-2005, 350 8-9 year-old boys from Chapaevsk, Russia with measured OCPs were enrolled and followed annually for eight years. We used multivariable interval-censored models to evaluate associations of OCPs (quartiles) with three physician-assessed measures of sexual maturity: Tanner stage 5 for genitalia growth, Tanner stage 5 for pubic hair growth, or testicular volume (TV) > /=20 mL in either testis.
RESULTS: In adjusted models, boys with higher HCB concentrations achieved sexual maturity reflected by TV > /=20 mL a mean of 3.1 months (95% CI: -1.7, 7.8), 5.3 months (95% CI: 0.6, 10.1), and 5.0 months (95% CI: 0.2, 9.8) later for quartiles Q2, Q3, and Q4, respectively compared to Q1 (trend p=0.04). Tanner stage 5 for genitalia growth was attained a mean of 2.2 months (95% CI: -3.1, 7.5), 5.7 months (95% CI: 0.4, 11.0), and 3.7 months (-1.7, 9.1) later for quartiles Q2, Q3, and Q4 respectively of beta-HCH as compared to Q1 (trend p=0.09). Tanner stage 5 for pubic hair growth occurred 6-9 months later on average for boys in the highest vs. lowest quartile for HCB (trend p < 0.001), beta-HCH (trend p=0.01), and p,p'-DDE (trend p=0.04). No associations were observed between p,p'-DDE and Tanner stage 5 for genitalia growth or TV > /=20 mL.
CONCLUSIONS AND RELEVANCE: Higher prepubertal serum HCB and beta-HCH concentrations were associated with a later age at attainment of sexual maturity. Only the highest quartile of serum p,p'-DDE was associated with later pubic hair maturation.
TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport
The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions.
C-ing the Genome: A Compendium of Chromosome Conformation Capture Methods to Study Higher-Order Chromatin Organization
Three-dimensional organization of the chromatin has important roles in transcription, replication, DNA repair, and pathologic events such as translocations. There are two fundamental ways to study higher-order chromatin organization: microscopic and molecular approaches. In this review, we briefly introduce the molecular approaches, focusing on chromosome conformation capture or "3C" technology and its derivatives, which can be used to probe chromatin folding at resolutions beyond that provided by microscopy techniques. We further discuss the different types of data generated by the 3C-based methods and how they can be used to answer distinct biological questions.
Intraflagellar transport (IFT) moves IFT trains carrying cargoes from the cell body into the flagellum and from the flagellum back to the cell body. IFT trains are composed of complexes IFT-A and IFT-B and cargo adaptors such as the BBSome. The IFT-B core proteins IFT74 and IFT81 interact directly through central and C-terminal coiled-coil domains, and recently it was shown that the N termini of these proteins form a tubulin-binding module important for ciliogenesis. To investigate the function of IFT74 and its domains in vivo, we have utilized Chlamydomonas reinhardtii ift74 mutants. In a null mutant, lack of IFT74 destabilized IFT-B, leading to flagella assembly failure. In this null background, expression of IFT74 lacking 130 amino acids (aa) of the charged N terminus stabilized IFT-B and promoted slow assembly of nearly full-length flagella. A further truncation (lacking aa 1-196, including part of coiled-coil 1) also stabilized IFT-B, but failure in IFT-A/IFT-B interaction within the pool at the base of the flagellum prevented entry of IFT-A into the flagellum and led to severely decreased IFT injection frequency and flagellar-assembly defects. Decreased IFT-A in these short flagella resulted in aggregates of stalled IFT-B in the flagella. We conclude that IFT74 is required to stabilize IFT-B; aa 197-641 are sufficient for this function in vivo. The N terminus of IFT74 may be involved in, but is not required for, tubulin entry into flagella. It is required for association of IFT-A and IFT-B at the base of the flagellum and flagellar import of IFT-A.
Opposing calcium-dependent signalling pathways control skeletal muscle differentiation by regulating a chromatin remodelling enzyme
Calcium signalling is important for differentiation-dependent gene expression, but is also involved in other cellular functions. Therefore, mechanisms must exist to distinguish calcium signalling relevant to differentiation. Calcineurin is a calcium-regulated phosphatase that is required for myogenic gene expression and skeletal muscle differentiation. Here, we demonstrate that inhibition of calcineurin blocks chromatin remodelling and that the Brg1 ATPase of the SWI/SNF chromatin remodelling enzyme, which is required for the activation of myogenic gene expression, is a calcineurin substrate. Furthermore, we identify the calcium-regulated classical protein kinase C beta (PKCbeta) as a repressor of myogenesis and as the enzyme that opposes calcineurin function. Replacement of endogenous Brg1 with a phosphomimetic mutant in primary myoblasts inhibits myogenesis, whereas replacement with a non-phosphorylatable mutant allows myogenesis despite inhibition of calcineurin signalling, demonstrating the functionality of calcineurin/PKC-modified residues. Thus, the Brg1 chromatin remodelling enzyme integrates two antagonistic calcium-dependent signalling pathways that control myogenic differentiation.
The establishment of the head to tail axis at early stages of development is a fundamental aspect of vertebrate embryogenesis. In mice, experimental embryology, genetics and expression studies have suggested that the visceral endoderm, an extra-embryonic tissue, plays an important role in anteroposterior axial development. Here we show that absence of Wnt3 in the posterior visceral endoderm leads to delayed formation of the primitive streak and that interplay between anterior and posterior visceral endoderm restricts the position of the primitive streak. Embryos lacking Wnt3 in the visceral endoderm, however, appear normal by E9.5. Our results suggest a model for axial development in which multiple signals are required for anteroposterior axial development in mammals.
Cell senescence, the permanent withdrawal of a cell from the cell cycle, is characterized by dramatic, cytological scale changes to DNA condensation throughout the genome. While prior emphasis has been placed on increases in heterochromatin, such as the formation of compact Senescent Associated Heterochromatin Foci (SAHF) structures, our recent findings showed that SAHF formation is preceded by the unravelling of constitutive heterochromatin into visibly extended structures, which we have termed Senescent Associated Distension of Satellites or SADS. Interestingly, neither of these marked changes in DNA condensation appear to be mediated by changes in canonical, heterochromatin-associated histone modifications. Rather, several observations suggest that these events may be facilitated by changes in LaminB1 levels and/or other factors that control higher-order chromatin architecture. Here, we review what is known about senescence-associated chromatin reorganization and present preliminary results using high-resolution microscopy techniques to show that each peri/centromeric satellite in senescent cells is comprised of several condensed domains connected by thin fibrils of satellite DNA. We then discuss the potential importance of these striking changes in chromatin condensation for cell senescence, and also as a model to provide a needed window into the higher-order packaging of the genome.
Centriole function has been difficult to study because of a lack of specific tools that allow persistent and reversible centriole depletion. Here we combined gene targeting with an auxin-inducible degradation system to achieve rapid, titratable, and reversible control of Polo-like kinase 4 (Plk4), a master regulator of centriole biogenesis. Depletion of Plk4 led to a failure of centriole duplication that produced an irreversible cell cycle arrest within a few divisions. This arrest was not a result of a prolonged mitosis, chromosome segregation errors, or cytokinesis failure. Depleting p53 allowed cells that fail centriole duplication to proliferate indefinitely. Washout of auxin and restoration of endogenous Plk4 levels in cells that lack centrioles led to the penetrant formation of de novo centrioles that gained the ability to organize microtubules and duplicate. In summary, we uncover a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth after centriole duplication failure.