Suberoylanilide Hydroxamic Acid as a Radiosensitizer through Modulation of RAD51 Protein and Inhibition of Homology-Directed Repair in Multiple Myeloma

Histone deacetylase inhibitors (HDI) have shown promise as candidate radiosensitizers for many types of cancers. However, the mechanisms of action are not well understood, and whether they could sensitize multiple myeloma (MM) to radiation therapy is unclear. In this study, we show that suberoylanilide hydroxamic acid (SAHA) at low concentrations has minimal cytotoxic effects, yet can significantly increase radiosensitivity of MM cells. SAHA seems to block RAD51 protein response to ionizing radiation, consistent with an inhibitory effect on the formation of RAD51 focus in irradiated MM cells. These effects of SAHA on RAD51 focus are independent of cell-cycle distribution changes. Furthermore, we show that SAHA selectively inhibits the homology-directed repair (HDR) pathway. The results of this study suggest that SAHA, a recently approved HDI in clinical trials for malignancies, at lower concentrations may act as a radiosensitizer via disruption of the RAD51-dependent HDR pathway. Mol Cancer Res; 10(8); 1052-64. ?2012 AACR.     

Histone H2AX phosphorylation as a predictor of radiosensitivity and target for radiotherapy

Based on the role of phosphorylation of the histone H2A variant H2AX in recruitment of DNA repair and checkpoint proteins to the sites of DNA damage, we have investigated gammaH2AX as a reporter of tumor radiosensitivity and a potential target to enhance the effectiveness of radiation therapy. Clinically relevant ionizing radiation (IR) doses induced similar patterns of gammaH2AX focus formation or immunoreactivity in radiosensitive and radioresistant human tumor cell lines and xenografted tumors. However, radiosensitive tumor cells and xenografts retained gammaH2AX for a greater duration than radioresistant cells and tumors. These results suggest that persistence of gammaH2AX after IR may predict tumor response to radiotherapy. We synthesized peptide mimics of the H2AX carboxyl-terminal tail to test whether antagonizing H2AX function affects tumor cell survival following IR. The peptides did not alter the viability of unirradiated tumor cells, but both blocked induction of gammaH2AX foci by IR and enhanced cell death in irradiated radioresistant tumor cells. These results suggest that H2AX is a potential molecular target to enhance the effects of radiotherapy.     

Stochastic model for tumor control probability: effects of cell cycle and (a)symmetric proliferation

Background

Estimating the required dose in radiotherapy is of crucial importance since the administrated dose should be sufficient to eradicate the tumor and at the same time should inflict minimal damage on normal cells. The probability that a given dose and schedule of ionizing radiation eradicates all the tumor cells in a given tissue is called the tumor control probability (TCP), and is often used to compare various treatment strategies used in radiation therapy.

Method

In this paper, we aim to investigate the effects of including cell-cycle phase on the TCP by analyzing a stochastic model of a tumor comprised of actively dividing cells and quiescent cells with different radiation sensitivities. Moreover, we use a novel numerical approach based on the method of characteristics for partial differential equations, validated by the Gillespie algorithm, to compute the TCP as a function of time.

Results

We derive an exact phase-diagram for the steady-state TCP of the model and show that at high, clinically-relevant doses of radiation, the distinction between active and quiescent tumor cells (i.e. accounting for cell-cycle effects) becomes of negligible importance in terms of its effect on the TCP curve. However, for very low doses of radiation, these proportions become significant determinants of the TCP. We also present the results of TCP as a function of time for different values of asymmetric division factor.

Conclusion

We observe that our results differ from the results in the literature using similar existing models, even though similar parameters values are used, and the reasons for this are discussed.

     

Computational Models of HIV-1 Resistance to Gene Therapy Elucidate Therapy Design Principles

Gene therapy is an emerging alternative to conventional anti-HIV-1 drugs, and can potentially control the virus while alleviating major limitations of current approaches. Yet, HIV-1''s ability to rapidly acquire mutations and escape therapy presents a critical challenge to any novel treatment paradigm. Viral escape is thus a key consideration in the design of any gene-based technique. We develop a computational model of HIV''s evolutionary dynamics in vivo in the presence of a genetic therapy to explore the impact of therapy parameters and strategies on the development of resistance. Our model is generic and captures the properties of a broad class of gene-based agents that inhibit early stages of the viral life cycle. We highlight the differences in viral resistance dynamics between gene and standard antiretroviral therapies, and identify key factors that impact long-term viral suppression. In particular, we underscore the importance of mutationally-induced viral fitness losses in cells that are not genetically modified, as these can severely constrain the replication of resistant virus. We also propose and investigate a novel treatment strategy that leverages upon gene therapy''s unique capacity to deliver different genes to distinct cell populations, and we find that such a strategy can dramatically improve efficacy when used judiciously within a certain parametric regime. Finally, we revisit a previously-suggested idea of improving clinical outcomes by boosting the proliferation of the genetically-modified cells, but we find that such an approach has mixed effects on resistance dynamics. Our results provide insights into the short- and long-term effects of gene therapy and the role of its key properties in the evolution of resistance, which can serve as guidelines for the choice and optimization of effective therapeutic agents.     

Sublethal irradiation promotes invasiveness of neuroblastoma cells

Neuroblastoma is the most frequent extracranial solid tumour of childhood. Despite multiple clinical efforts, clinical outcome has remained poor. Neuroblastoma is considered to be radiosensitive, but some clinical studies including the German trial NB90 failed to show a clinical benefit of radiation therapy. The mechanisms underlying this apparent discrepancy are still unclear. We have therefore investigated the effects of radiation on neuroblastoma cell behaviour in vitro. We show that sublethal doses of irradiation up-regulated the expression of the hepatocyte growth factor (HGF) and its receptor c-Met in some neuroblastoma cell lines. The increase in HGF/c-Met expression was correlated with enhanced invasiveness and activation of proteases degrading the extracellular matrix. Thus, irradiation at sublethal doses may promote the metastatic dissemination of neuroblastoma cells through activating the HGF/c-Met pathway and triggering matrix degradation.     

Repair of gamma-ray-induced base damage in L5178Y sublines is damage type-dependent and unrelated to radiation sensitivity.

The L5178Y (LY) murine lymphoma sublines LY-R and LY-S are differentially sensitive to ionizing radiation. The high radiation sensitivity of LY-S cells is related to impaired rejoining of DNA double strand breaks. We found previously that the gamma-ray-induced base damage is higher in the more radiosensitive LY-S subline. Here, we examine the role of the repair of ionizing radiation induced base damage in relation to the radiosensitivity difference of these sublines. We used the GS/MS technique to estimate the repair rates of six types of base damage in gamma-irradiated LY cells. All modified DNA bases identified in the course of this study were typical for irradiated chromatin. The total amount of initial base damage was higher in the radiation sensitive LY-S subline than in the radiation resistant LY-R subline. The repair rates of 5-OHMeUra, 5-OHCyt, 8-OHAde were similar in both cell lines, the repair rates of FapyAde and 8-OHGua were higher in the radiosensitive LY-S cell line, whereas the repair of 5-OHUra was faster in its radioresistant counter, the LY-R. Altogether, the repair rates of the y-ray-induced DNA base damage in LY sublines are related neither to the initial amounts of the damaged bases nor to the differential lethal or mutagenic effects of ionizing radiation in these sublines.     

Integrating cell-cycle progression, drug penetration and energy metabolism to identify improved cancer therapeutic strategies

The effectiveness of chemotherapeutic drugs in tumors is reduced by multiple effects including drug diffusion and variable susceptibility of local cell populations. We hypothesized that quantifying the interactions between drugs and tumor microenvironments could be used to identify more effective anti-cancer strategies. To test this hypothesis we created a mathematical model that integrated intracellular metabolism, nutrient and drug diffusion, cell-cycle progression, cellular drug effects, and drug pharmacokinetics. To our knowledge, this is the first model that combines these elements and has coupled them to experimentally derived parameters. Drug cytotoxicity was assumed to be cell-cycle phase specific, and progression through the cell cycle was assumed to be dependent on ATP generation. The model consisted of a coupled set of nonlinear partial differential, ordinary differential and algebraic equations with an outer free boundary, which was solved using orthogonal collocation on a moving grid of finite elements. Model simulations showed the existence of an optimum drug diffusion coefficient: a low diffusivity prevents effective penetration before the drug is cleared from the blood and a high diffusivity limits drug retention. This result suggests that increasing the molecular weight of the anti-cancer drug paclitaxel from 854 to approximately 20,000 by nano-particle conjugation would improve its efficacy. The simulations also showed that fast growing tumors are less responsive to therapy than are slower tumors with more quiescent cells, demonstrating the competing effects of regrowth and cytotoxicity. The therapeutic implications of the simulation results are that (1) monolayer cultures are inadequate for accurately determining therapeutic effects in vitro, (2) decreasing the diffusivity of paclitaxel could increase its efficacy, and (3) measuring the proliferation fraction in tumors could enhance the prediction of therapeutic efficacy.     

Individual variability in tree allometry determines light resource allocation in forest ecosystems: a hierarchical Bayesian approach

Tree species differences in crown size and shape are often highlighted as key characteristics determining light interception strategies and successional dynamics. The phenotypic plasticity of species in response to light and space availability suggests that intraspecific variability can have potential consequences on light interception and community dynamics. Species crown size varies depending on site characteristics and other factors at the individual level which differ from competition for light and space. These factors, such as individual genetic characteristics, past disturbances or environmental micro-site effects, combine with competition-related phenotypic plasticity to determine the individual variability in crown size. Site and individual variability are typically ignored when considering crown size and light interception by trees, and residual variability is relegated to a residual error term, which is then ignored when studying ecological processes. In the present study, we structured and quantified variability at the species, site, and individual levels for three frequently used tree allometric relations using fixed and random effects in a hierarchical Bayesian framework. We focused on two species: Abies alba (silver fir) and Picea abies (Norway spruce) in nine forest stands of the western Alps. We demonstrated that species had different allometric relations from site to site and that individual variability accounted for a large part of the variation in allometric relations. Using a spatially explicit radiation transmission model on real stands, we showed that individual variability in tree allometry had a substantial impact on light resource allocation in the forest. Individual variability in tree allometry modulates species’ light-intercepting ability. It generates heterogeneous light conditions under the canopy, with high light micro-habitats that may promote the regeneration of light-demanding species and slow down successional dynamics.     

Radiation survival of vascular smooth muscle cells as a function of age

Late damage to normal tissues is an important consideration in determining the dose of radiation which can be delivered to a given target volume in clinical radiation therapy. The response of large blood vessels to radiation injury is undoubtedly complex and is influenced by (1) the cellular composition of the vessel wall, (2) the slow turnover of vascular cells, and (3) vascular repair mechanisms. As a first order model for radiation effects in large vessels, we have studied the radiobiologic properties of cultured vascular smooth muscle cells. We have measured survival curves and repair of sublethal radiation damage in exponentially growing cultures of rat aortic smooth muscle cells as a function of animal age and site of origin (thoracic versus abdominal aorta). Radiation survival parameters (utilizing two different mathematical models for the survival curve) and repair of sublethal damage did not appear to vary significantly as a function of animal age (3-23 months) or site or origin.     

Radioresistance of human carcinoma cells is correlated to a defect in raft membrane clustering

In addition to DNA damage, exposure to irradiation involves the plasma membrane in the early phases of gamma-ray-induced cell death. The involvement of raft microdomains following gamma-radiation derives essentially from the role of ceramide as a critical component leading to apoptosis. It is demonstrated here that gamma-irradiation of a radiosensitive human head and neck squamous carcinoma cell line (SCC61) results in the triggering of raft coalescence to larger membrane platforms associated with the externalization of an acid sphingomyelinase (A-SMase), leading to ceramide release in raft, 30 min postirradiation. For the first time, we show that this structural rearrangement is defective in the radioresistant SQ20B cells and associated with the lack of A-SMase activation and translocation, a result which could explain in part their resistance to apoptosis following ionizing radiation. Moreover, we show that SQ20B are protected against radiation injury through a fivefold upper level of endogenous glutathione compared to SCC61. Overcoming the endogenous antioxidant defenses of SQ20B through either H(2)O(2) treatment or GSH depletion triggers A-SMase activation and translocation, raft coalescence, and apoptosis. On the contrary, ROS scavengers abolished these events in radiosensitive SCC61 cells. Translation of this concept to tumor biology suggests that manipulation of rafts through redox equilibrium may provide opportunities for radiosensitization of tumor cells.     

X-irradiation--induced changes in the progression of type B spermatogonia and preleptotene spermatocytes

In response to induced DNA damage, proliferating cells arrest in their cell cycle or go into apoptosis. Ionizing radiation is known to induce degeneration of mammalian male germ cells. The effects on cell-cycle progression, however, have not been thoroughly studied due to lack of methods for identifying effects on a particular cell-cycle phase of a specific germ cell type. In this study, we have utilized the technique for isolation of defined segments of seminiferous tubules to examine the cell-cycle progression of irradiated rat mitotic (type B spermatogonia) and meiotic (preleptotene spermatocytes) G1/S cells. Cells irradiated as type B spermatogonia in mitotic S phase showed a small delay in progression through meiosis. Thus, it seems that transient arrest in the progression can occur in the otherwise strictly regulated progression of germ cells in the seminiferous epithelium. Contrary to the arrest observed in type B spermatogonia and in previous studies on somatic cells, X-irradiation did not result in a G1 delay in meiotic cells. This lack of arrest occurred despite the presence of unrepaired DNA damage that was measured when the cells had progressed through the two meiotic divisions.     

Time ordering of gene coexpression

Temporal microarray gene expression profiles allow characterization of gene function through time dynamics of gene coexpression within the same genetic pathway. In this paper, we define and estimate a global time shift characteristic for each gene via least squares, inferred from pairwise curve alignments. These time shift characteristics of individual genes reflect a time ordering that is derived from ob- served temporal gene expression profiles. Once these time shift characteristics are obtained for each gene, they can be entered into further analyses, such as clustering. We illustrate the proposed methodology using Drosophila embryonic development and yeast cell-cycle gene expression profiles, as well as simulations. Feasibility is demonstrated through the successful recovery of time ordering. Estimated time shifts for Drosophila maternal and zygotic genes provide excellent discrimination between these two categories and confirm known genetic pathways through the time order of gene expression. The application to yeast cell-cycle data establishes a natural time order of genes that is in line with cell-cycle phases. The method does not require periodicity of gene expression profiles. Asymptotic justifications are also provided.     

Epistatic interactions alter dynamics of multilocus gene-for-gene coevolution

Fitness costs associated with resistance or virulence genes are thought to play a key role in determining the dynamics of gene-for-gene (GFG) host-parasite coevolution. However, the nature of interactions between fitness effects of multiple resistance or virulence genes (epistasis) has received less attention. To examine effects of the functional form of epistasis on the dynamics of GFG host-parasite coevolution we modified a classic multilocus GFG model framework. We show that the type of epistasis between virulence genes largely determines coevolutionary dynamics, and that coevolutionary fluctuations are more likely with acceleratingly costly (negative) than with linear or deceleratingly costly (positive) epistasis. Our results demonstrate that the specific forms of interaction between multiple resistance or virulence genes are a crucial determinant of host-parasite coevolutionary dynamics.     

Radiosensitivity of normal and polyunsaturated fatty acid supplemented fibroblasts after depletion of glutathione

Mouse fibroblast LM cells have been modified with respect to their phospholipid composition in all subcellular fractions, including the nuclear membrane. The content of polyunsaturated fatty acids (PUFA) was significantly increased but no difference in cell survival after X-irradiation could be observed between the normal and PUFA enriched cells. It is concluded that the radiosensitive PUFAs in the membranes are well protected against radiation damage. This protection of the PUFA cells could not be caused by vitamin E, because this membrane protector was not present in these fibroblasts. The content of glutathione (GSH) was about the same in the normal and the modified cells. Reduction of the cellular GSH content to less than 5 per cent of that for non-treated cells did not alter cellular survival after radiation of either normal or PUFA enriched cells under oxic or anoxic conditions. The radiosensitive lipids present in the membranes of the PUFA enriched cells proved to be vulnerable to radiation-induced lipid peroxidation when extracted from the cells and reconstituted into liposomes, indicating that the fatty acids per se are peroxidizable. It is concluded that the lipids in the membranes of mammalian cells are not the principal target in radiation-induced reproductive death, and that no generalization is permitted with respect to glutathione, as being the major hydrogen donating species in mammalian cells responsible for the repair of those target molecules responsible for cell survival after radiation.     

Ant 4,4, a polyamine-anthracene conjugate, induces cell death and recovery in human promyelogenous leukemia cells (HL-60)

One of the major problems in cancer therapy is the lack of specificity of chemotherapeutic agents towards cancer cells, resulting in adverse side effects. One means to counter this is to selectively deliver the drug to the cancer cell. Cancer cells accumulate increased concentrations of polyamines compared to normal cells, mainly through an increased uptake of preformed polyamines via the polyamine transport system (PTS). Furthermore, the non-stringent structural requirements of the PTS enable the transport of a range of polyamine-based molecules. Thus, the PTS can be used to transport compounds linked to polyamines selectively to cancer cells. In our laboratory, polyamine–anthracene conjugates have shown potent anti-tumour activity towards HL-60 cells. The aim of this study was to determine the cytotoxicity of Ant-4,4, a homospermidine–anthracene conjugate, and assess the long-term effects by determining whether cancer cells were able to recover from treatment. During exposure, Ant-4,4 was an effective growth-inhibitory agent in HL-60 cells decreasing viable cell number, protein and polyamine content. Evidence indicates concomitant cell-cycle arrest and increased apoptosis. Once the drug was removed, HL-60 cells recovered gradually over time. Increasing cell number, protein content and polyamine content, as well as diminished effects on cell-cycle and apoptotic stimuli were observed over time. These data suggest that, despite being an effective way of delivering anthracene, these polyamine conjugates do not exert long-lasting effects on HL-60 cells.     

Dimensional reduction of a V1 ring model with simple and complex cells

In this paper, we extend a framework for constructing low-dimensional dynamical systems models of mammalian primary visual cortex to a cortical network model that incorporates the full nonlinear effects of complex cells. The procedure consists of capturing the essential dynamics in a low-dimensional subspace using empirical methods, then recasting the equations in the reduced vector space. Previously, we considered visual cortical network models consisting of only simple cells with nearly linear responses to external stimuli. Here we show that fully nonlinear effects can be incorporated by examining the dimensional reduction of an idealized ring model of V1 with both simple and complex cells. We found it expedient to divide the subspace into four separate neuronal populations: excitatory simple, excitatory complex, inhibitory simple and inhibitory complex. In order to reproduce the fluctuation-driven dynamics in this reduced space, we incorporated (1) white noises with different intensities into individual neuronal populations, and (2) firing rate estimates to capture the probability of firing due to subthreshold fluctuations. With a more accurate, fitted connectivity, our modified dimensional reduced models can reproduce the firing rates, circular variances and modulation ratios observed in the original ring model.     

Discriminating Survival Outcomes in Patients with Glioblastoma Using a Simulation-Based,Patient-Specific Response Metric

Accurate clinical assessment of a patient''s response to treatment for glioblastoma multiforme (GBM), the most malignant type of primary brain tumor, is undermined by the wide patient-to-patient variability in GBM dynamics and responsiveness to therapy. Using computational models that account for the unique geometry and kinetics of individual patients'' tumors, we developed a method for assessing treatment response that discriminates progression-free and overall survival following therapy for GBM. Applying these models as untreated virtual controls, we generate a patient-specific “Days Gained” response metric that estimates the number of days a therapy delayed imageable tumor progression. We assessed treatment response in terms of Days Gained scores for 33 patients at the time of their first MRI scan following first-line radiation therapy. Based on Kaplan-Meier analyses, patients with Days Gained scores of 100 or more had improved progression-free survival, and patients with scores of 117 or more had improved overall survival. Our results demonstrate that the Days Gained response metric calculated at the routinely acquired first post-radiation treatment time point provides prognostic information regarding progression and survival outcomes. Applied prospectively, our model-based approach has the potential to improve GBM treatment by accounting for patient-to-patient heterogeneity in GBM dynamics and responses to therapy.