Sublethal Irradiation Research Articles

Abstract 3963: Chemo-resistantregulatory T cells suppress the development of antitumor immunity aftercytotoxic regimens.

Abstract Previous studies demonstrated that naïve T cells transferred into lymphopenic hosts develop into memory like T cells and acquire some effector functions. We and others have shown that sublethal whole body irradiation and transfer of naïve T cells augment antitumor immunity and inhibited tumor progression. Further, we found a significant increase of CD4+CD25+Foxp3+ regulatory T cells (Treg) in irradiated mice. Depletion of those radio-resistant Treg after irradiation and transfer of naïve T cells enhanced generation of antitumor effector T cells and suppressed tumor progression (Baba J, et al. 2012, Blood). The combination of lymphodepletion and transfer of naïve T cells seems to be a promising strategy. However, whole body irradiation has not been routinely used in clinical settings. Previously, we demonstrated that the sublethal doses of cyclophosphamide (CPA) efficiently depleted lymphocytes in mice, and enhanced antitumor effects of transferred naïve T cells. In this study, we examined the effect of lymphodepleting doses of CPA on immune suppressor cells. Similar to the irradiated lymphopenic hosts, a significant increase of Treg was observed in mice treated with CPA. The combination of CPA, transfer of naïve T cells and Treg depletion with anti-CD25 monoclonal antibodies succeeded to cure advanced skin tumors.To evaluate the proliferation and apoptosis of Treg increasing in CPA-treated mice, we performed BrdU incorporation, Ki-67 expression and Annexin V apoptosis assay. The results indicated that the increased percentage of Treg during recovery from lymphopenia is due to the rapid proliferation of Treg that survive CPA treatment. Next, we investigated which subsets of donor T cells are necessary in this combination therapy. CD4+ or CD8+ T cells were depleted from donor T cells before transfer into lymphopenic skin tumor bearing mice. Depletion of CD4+ T cells, but not CD8+ T cells eliminated the antitumor effects. Intracellular cytokine FACS revealed that transfer of CD4+ T cells following CPA treatment induced tumor specific effector CD4+ T cells in lymphopenic hosts. Interestingly, transfer of CD4+ T cells also induced CD8+ effector T cells from CPA treated recipient cells. Depletion of CD8+ recipient cells with anti-CD8 monoclonal antibodies abrogated the antitumor effects of lymphodepletion and CD4+ T cells transfer. Furthermore, Transfer of CD4+ T cells into Rag-2 knockout mice showed no antitumor effects. These results suggested that donor CD4+ T cells induce effector CD8+ T cells originate from recipients after CPA treatment and suppress tumor progression. Our results showed that the combination of CPA treatment, naïve CD4+ T cell transfer, and Treg depletion had potent antitumor efficacy. Both of donor CD4+ T cells and recipient CD8+ T cells were necessary for this augmentation of antitumor immunity. Citation Format: Yu Saida, Satoshi Watanabe, Tomohiro Tanaka, Junko Baba, Koh Satoh, Satoshi Shoji, Natsue Igarashi, Koichirou Nozaki, Masaaki Okajima, Satoru Miura, Junta Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Ichiei Narita. Chemo-resistantregulatory T cells suppress the development of antitumor immunity aftercytotoxic regimens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3963. doi:10.1158/1538-7445.AM2013-3963

Abstract 3964: Appropriate conditioning of CD4+T cells for antitumor immunotherapy.

Abstract It is now evident that lymphodepleting cytotoxic regimens augment antitumor immunity. We and others have demonstrated that transfer of naïve T cells after sublethal whole body irradiation induces antitumor effector T cells and inhibits tumor progression. We recently reported that CD4+CD25+Foxp3+ regulatory T cells (Treg) largely increased after whole body irradiation and suppressed the development of antitumor immune responses during recovery from lymphopenia. Depletion of these radio-resistant Treg following lymphodepletion significantly enhanced antitumor immunity (Baba J, et al. 2012, Blood). The combination of lymphodepletion and transfer of naïve T cells seems to be a promising approach for antitumor immunotherapy. But harvesting sufficient number of T lymphocytes from tumor-bearing patients is difficult. To obtain enough number of T cells that were capable of recognizing tumor-antigens, we expanded naïve T cells in vitro with culturing in CM containing several cytokines. CD4 or CD8 T cells from naïve spleens were activated with immobilized anti-CD3 mAbs for 2 days. Stimulated cells were maintained in CM with the combination of IL-2 (16 U/ml), IL-7 (10 ng/ml), IL-15 (10 ng/ml) and IL-23 (2 ng/ml) for additional 7 days. TCR-Vβ expressions of cultured T cells revealed that they maintained TCR diversity. To evaluate antitumor effects of these cells, mice were irradiated with 500 cGy to deplete lymphocytes and were transferred i.v. with the cultured T cells (4 x 107). On the same day, mice were inoculated s.c. with MCA205 tumor cells (1 x 105). We found that expanded CD4+ T cells mediated regression of s.c. tumors. In contrast, CD8+ T cells alone did not have therapeutic effects even combined with depletion of Treg. Next, we examined T cells proliferation by labeling with the CFSE and evaluated IFN-γ production after stimulation with specific tumor cells. Cultured CD4+ T cells proliferated rapidly and developed into tumor-specific effector T cells after transfer into lymphopenic hosts. Because CD4+ T cells from OT-II mice, that recognize Ovalbumin peptide specifically, did not show any antitumor effects after transfer into lymphopenic mice, this antitumor activity of CD4+ T cells is more likely to be tumor-antigen specific. Interestingly, using irradiated Rag-2 knockout mice as recipient abrogated antitumor effects of transferred cultured CD4+ T cells. Our results indicated that the combination of lymphodepletion and transfer of in vitro expanded CD4+ T cells has a potent antitumor efficacy, and recipient cells from irradiated hosts play a role in this treatment model. Citation Format: Tomohiro Tanaka. Appropriate conditioning of CD4+ T cells for antitumor immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3964. doi:10.1158/1538-7445.AM2013-3964

Murine Cytomegalovirus Immediate-Early 1 Gene Expression Correlates with Increased GVHD after Allogeneic Hematopoietic Cell Transplantation in Recipients Reactivating from Latent Infection

The success of allogeneic (allo) hematopoietic cell transplantation (HCT) is limited by its treatment related complications, mostly graft versus host disease (GVHD) and fungal and viral infections. CMV reactivation after HCT has been associated with increased morbidity and mortality, and a causal relation between GVHD, immunosuppressive therapy and vice versa has been postulated. Using a low GVHD severity murine HCT model, we assessed the role of MCMV reactivation and GVHD development. BALB/c mice were infected with either murine CMV (MCMV) or mock and monitored for 25 weeks to establish latency, followed by sublethal irradiation conditioning and infusion of bone marrow plus splenocytes from either syngeneic (syn) BALB/c or allo B10.D2 donors. Engraftment of allo donor cells was confirmed by PCR for D2Mit265 gene product size. Day+100 mortality and overall GVHD severity in allo MCMV pre-infected recipients was higher than in allo mock controls. Pathologic changes of lung and liver GVHD in immediate-early gene 1 (IE1) positive recipients were significantly increased compared to mock controls, and were only slightly increased in IE1 negative. No significant gut injury was seen in any group. Aggravated lung injury in IE1 positive recipients correlated with higher BAL cell counts both for total cells and for CD4+ T cells when compared with mock controls, and also with protein expression of lung IFN-gamma and liver TNF. No evidence for CMV specific morphologic changes was seen on histopathology in any organ of IE1 positive recipients, suggesting that CMV reactivation is related to increased GVHD severity but does not require active CMV disease, strengthening the concept of a reciprocal relationship between CMV and GVHD.

Molecular mechanisms of platelet and stem cell rebound after 5-fluorouracil treatment

Sublethal irradiation and 5-fluorouracil (5-FU) treatment are two commonly used myelosuppressive methods used in the study of hematopoiesis. These methods have been considered interchangeable by some researchers because the morphological changes in the bone marrow to these treatments are similar. Here, we sought to compare the responses of hematopoietic cells, stem and progenitor cells and the bone marrow microenvironment to these treatments. Although bone marrow cellularity decreased after both treatments, the underlying mechanism of the bone marrow cell regression and recovery were very different between the two models. We found: 1. Myeloid cells and lymphoid cells had different sensitivity to the different treatments. 2. Following an initial decrease in stem cell number, 5-FU treated mice had profound thrombopoietin (Tpo) dependent stem cell rebound above baseline levels. 3. Platelet rebound in 5-FU treated animals was not the result of stem cell rebound. 4. Stem cell and platelet rebound did not occur in sub-lethally irradiated mice. 5. Platelet rebound resulted from an indirect effect of 5-FU on the microenvironment cells, but not a direct effect on the stem cells. 6. Microarray studies demonstrated that up-regulation of the angiopoietin-1/Tie2 signaling pathway coincided with platelet rebound. 7. Suppression of genes involved in chromosomal organization coincided with stem cell and platelet rebound.

Changes in Gene Expression ofStreptococcus mutansin Planktonic Environment Following CO2Laser Irradiation

Abstract Objective: The aim of this work was to study the effect of CO2 laser light on gene expression of Streptococcus mutans in a planktonic environment irradiated by sublethal energies. The use of the CO2 laser for oral clinical applications has great benefits over the traditional procedures, including a reduced bacterial environment. In order to determine the sublethal irradiation dose, S. mutans in suspension was irradiated with a continuous wave CO2 laser (wavelength 10.6 μm) for duration times of 0.5-1.5 min at fluences ranging from 65 to 536 J/cm(2), following bacterial growth and viability measurements. The effect of CO2 laser irradiation at sublethal dose on gene expression was evaluated by DNA microarray. Temperature rise in the suspension caused by laser irradiation was measured using thermocouple electrodes. Bacterial growth and viability began to decrease after exposure to laser irradiation using 284 J/cm(2). Following irradiation with sublethal fluences of 134 and 268 J/cm(2), changes in gene expression were found with seven downregulated genes being common to both fluences. The temperature rise in the bacterial sample after irradiation with 268 J/cm(2) was from room temperature to only 35°C. CO2 laser irradiation inhibited viability and growth of S. mutans in suspension in a dose-dependent manner. Irradiation of planktonic bacterial suspensions with sublethal fluences resulted in changes mostly of downregulation in gene expression in a fashion similar to S. mutans in biofilm, except that different genes were involved.

Radiation‐induced loss of cell surface CD47 enhances immune‐mediated clearance of human papillomavirus‐positive cancer

The increasing incidence of human papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (OSSC) demands development of novel therapies. Despite presenting at a more advanced stage, HPV(+) oropharyngeal squamous cell carcinoma (OSCC) have a better prognosis than their HPV(-) counterparts. We have previously demonstrated that clearance of HPV(+) OSCC during treatment with radiation and chemotherapy requires an immune response which is likely responsible for the improved clinical outcomes. To further elucidate the mechanism of immune-mediated clearance, we asked whether radiation therapy induces tumor cell changes that allow the body to recognize and aid in tumor clearance. Here, we describe a radiation-induced change in tumor surface protein expression that is critical for immune-mediated clearance. Radiation therapy decreases surface expression of CD47, a self-marker. CD47 is frequently overexpressed in head and neck squamous cell carcinoma and radiation induces a decrease of CD47 in a dose-dependent manner. We show that both in vitro and in vivo tumor cell CD47 protein levels are restored over time after sublethal radiation exposure and that protein levels on adjacent, normal tissues remain unaffected. Furthermore, reduction of tumor cell CD47 increases phagocytosis of these cells by dendritic cells and leads to increased interferon gamma and granzyme production from mixed lymphocytes. Finally, decreasing tumor cell CD47 in combination with standard radiation and chemotherapy results in improved immune-mediated tumor clearance in vivo. These findings help define an important mechanism of radiation-related immune clearance and suggest that decreasing CD47 specifically on tumor cells may be a good therapeutic target for HPV related disease.

Radiation tolerance of Bacillus cereus pre-treated with carvacrol alone or in combination with nisin after exposure to single and multiple sub-lethal radiation treatment

Currently little information exists on the response of Bacillus cereus after repetitive exposure to combined mild treatments involving antimicrobials and gamma irradiation. Therefore, the aim of this present study was to evaluate the radiation stress on growth and physiology of B. cereus LSPQ 2872 vegetative cells at stationary phase, following exposure to single and repetitive sub-lethal γ-radiation treatment at 1 kGy simultaneously with carvacrol alone or combined with nisin at sub-inhibitory concentrations. Results indicated that the combination of carvacrol and nisin significantly enhance the radiation sensitivity of B. cereus since lower D10 values were recorded after both single and repetitive irradiation treatments. Flow cytometric analysis of radiation-stressed B. cereus cells following repetitive treatment revealed the heterogenic behaviour of the bacterium leading to the induction of a radiation tolerance response. When compared to carvacrol alone, the combination of carvacrol and nisin developed also increased radioresistance if repetitively processed with γ-radiation at 1 kGy, since the decrease percentage of dead cells was accompanied by an increase in the number of injured cells. However, good agreement was not found between classical plate counting (log cfu reductions) and flow cytometry method. For both antimicrobials, the increase of radioresistance after repetitive mild treatment was not accompanied by augmentations of lag phase or growth rate. The structural changes of the outer membranes were assessed by TEM analysis and results revealed that radioresistance might be related to changes in the cell wall.

Prostaglandin E2 Increases Hematopoietic Stem Cell Survival and Accelerates Hematopoietic Recovery After Radiation Injury

Hematopoietic stem and progenitor cells (HSPCs), which continuously maintain all mature blood cells, are regulated within the marrow microenvironment. We previously reported that pharmacologic treatment of naïve mice with prostaglandin E2 (PGE2) expands HSPCs. However, the cellular mechanisms mediating this expansion remain unknown. Here, we demonstrate that PGE2 treatment in naïve mice inhibits apoptosis of HSPCs without changing their proliferation rate. In a murine model of sublethal total body irradiation (TBI), in which HSPCs are rapidly lost, treatment with a long-acting PGE2 analog (dmPGE2) reversed the apoptotic program initiated by TBI. dmPGE2 treatment in vivo decreased the loss of functional HSPCs following radiation injury, as demonstrated both phenotypically and by their increased reconstitution capacity. The antiapoptotic effect of dmPGE2 on HSPCs did not impair their ability to differentiate in vivo, resulting instead in improved hematopoietic recovery after TBI. dmPGE2 also increased microenvironmental cyclooxygenase-2 expression and expanded the α-smooth muscle actin-expressing subset of marrow macrophages, thus enhancing the bone marrow microenvironmental response to TBI. Therefore, in vivo treatment with PGE2 analogs may be particularly beneficial to HSPCs in the setting of injury by targeting them both directly and also through their niche. The current data provide rationale for in vivo manipulation of the HSPC pool as a strategy to improve recovery after myelosuppression.

Hematopoietic Stem Cell Regeneration Enhanced by Ectopic Expression of ROS-detoxifying Enzymes in Transplant Mice

High levels of reactive oxygen species (ROS) can exhaust hematopoietic stem cells (HSCs). Thus, maintaining a low state of redox in HSCs by modulating ROS-detoxifying enzymes may augment the regeneration potential of HSCs. Our results show that basal expression of manganese superoxide dismutase (MnSOD) and catalase were at low levels in long-term and short-term repopulating HSCs, and administration of a MnSOD plasmid and lipofectin complex (MnSOD-PL) conferred radiation protection on irradiated recipient mice. To assess the intrinsic role of elevated MnSOD or catalase in HSCs and hematopoietic progenitor cells, the MnSOD or catalase gene was overexpressed in mouse hematopoietic cells via retroviral transduction. The impact of MnSOD and catalase on hematopoietic progenitor cells was mild, as measured by colony-forming units (CFUs). However, overexpressed catalase had a significant beneficial effect on long-term engraftment of transplanted HSCs, and this effect was further enhanced after an insult of low-dose γ-irradiation in the transplant mice. In contrast, overexpressed MnSOD exhibited an insignificant effect on long-term engraftment of transplanted HSCs, but had a significant beneficial effect after an insult of sublethal irradiation. Taken together, these results demonstrate that HSC function can be enhanced by ectopic expression of ROS-detoxifying enzymes, especially after radiation exposure in vivo.

Mitigating effects of hUCB-MSCs on the hematopoietic syndrome resulting from total body irradiation

This study evaluated the clinical and pathologic effects of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in the recovery from total body irradiation by comparing it with the effects of granulocyte-colony stimulating factor (G-CSF), an efficacious drug in the treatment of acute bone marrow radiation syndrome. BALB/c mice were treated with G-CSF or hUCB-MSCs after they were irradiated with 7 Gy cobalt-60 γ-rays. Circulating blood counts, histopathologic changes in the bone marrow, and plasma level of Flt-3L and transforming growth factor (TGF-β1) were monitored in the postirradiation period. Hematologic analysis revealed that the peripheral leukocyte counts were markedly increased in the hUCB-MSCs-treated group, whereas G-CSF-treated mice did not recover significantly. Moreover, differential counts showed that hUCB-MSC treatment has regenerative effects on white blood cells, lymphocytes, and monocytes compared with the irradiated group. Treatment with hUCB-MSCs or G-CSF significantly increased immunoreactivity of Ki-67 until 3 weeks after total body irradiation. However, at 3 weeks, the number of Ki-67 immunoreactive cells significantly increased in the hUCB-MSCs-treated group compared with the G-CSF-treated group. Furthermore, hUCB-MSC treatment significantly modulated plasma levels of the hematopoietic cytokines Flt-3L and TGF-β1, whereas G-CSF treatment failed to decrease the plasma Flt-3L levels at 2 weeks after irradiation. Based on the differences in circulating blood cell reconstitution and cell density of bone marrow, the authors suggest that MSC treatment is superior to G-CSF treatment for hematopoietic reconstitution following sublethal dose radiation exposure.

Turning T cells on: epigenetically enhanced expression of effector T-cell costimulatory molecules on irradiated human tumor cells.

BackgroundSub-lethal doses of radiation can alter the phenotype of target tissue by modulating gene expression and making tumor cells more susceptible to T-cell-mediated immune attack. We have previously shown that sub-lethal tumor cell irradiation enhances killing of colorectal carcinoma cells by tumor-specific cytotoxic T cells by unknown mechanisms. Recent data from our lab indicates that irradiation of tumor cells results in the upregulation of OX40L and 41BBL, and that T cells incubated with irradiated tumor cells displayed improved CTL survival, activation and effector activity. The objective of this current study was to determine the mechanism of enhanced OX40L and 41BBL expression in human colorectal tumor cells.MethodsTwo colorectal carcinoma cell lines, HCT116 and SW620, were examined for changes in the expression of 41BBL and OX40L in response to inhibition of histone deacetylases (using TSA) and DNA methyltransferases (using 5-Aza-2′-deoxycytidine) to evaluate if epigenetic mechanisms of gene expression can modulate these genes. Tumor cells were treated with radiation, TSA, or 5-Aza-dC, and subsequently evaluated for changes in gene expression using RT-qPCR and flow cytometry. Moreover, we assessed levels of histone acetylation at the 41BBL promoter using chromatin immunoprecipitation assays in irradiated HCT116 cells.ResultsOur data indicate that expression of 41BBL and OX40L can indeed be epigenetically regulated, as inhibition of histone deacetylases and of DNA methyltransferases results in increased OX40L and 41BBL mRNA and protein expression. Treatment of tumor cells with TSA enhanced the expression of these genes more than treatment with 5-Aza-dC, and co-incubation of T cells with TSA-treated tumor cells enhanced T-cell survival and activation, similar to radiation. Furthermore, chromatin immunoprecipitation experiments revealed significantly increased histone H3 acetylation of 41BBL promoters specifically following irradiation.ConclusionsFull understanding of specific mechanisms of immunogenic modulation (altered expression of immune relevant genes) of irradiated tumor cells will be required to determine how to best utilize radiation as a tool to enhance cancer immunotherapy approaches. Overall, our results suggest that radiation can be used to make human tumors more immunogenic through epigenetic modulation of genes stimulatory to effector T-cells.

Cyclophilin A stimulates recovery of the hematopoietic and the immune system of the organism after radiotherapy

Event Abstract Back to Event Cyclophilin A stimulates recovery of the hematopoietic and the immune system of the organism after radiotherapy Anastasia A. Kalinina1*, Yulia Y. Silaeva1, Dmitry B. Kazansky1 and Ludmila M. Khromykh1 1 Federal State Budgetary Institution”N.N.Blokhin Cancer Research Center”, Russian Academy of Medical Sciences, Russia Radiotherapy is accompanied with severe side effects: damage and death of hematopoietic cells and decrease of leukocytes in blood. Preparations, commonly used for restoration of the hemogram after such treatment, are colony stimulating factors, but their application is limited. Search of new factors, promoting neutralization of side effects after radiotherapy, is of great practical interest. Previously in our laboratory it was shown, that Cyclophilin A (Cyp A) (a 18 kD protein) is a chemoattractant for stem cells and progenitors of different lineages, localized in bone marrow (Khromykh L., 2007). It was also shown that Cyp A induces differentiation of stem cells in granulocytes and thrombocytes (unpublished data). These findings suggest that the studied protein can promote recovery of the hematopoietic and the immune systems of the organism after radiotherapy. We have shown that Cyp A stimulates restoration of bone marrow in mice post sublethal irradiation, promoting reconstitution of stem cells and granulocytopoiesis. Post irradiation Cyp A stimulates migration of bone marrow stem cells, committed toward granulocytes, in blood. On the third week post sublethaly irradiation Cyp A promotes full restoration of T lymphocytes in mice spleens that exceed 2-fold the percent of these cells in control irradiated animals. The level of humoral response in mice on the third week post irradiation restores to the level of non-irradiated mice under Cyp A treatment. Thus, our data suggest that Cyp A is a factor, taking part in recovery of the hematopoietic and the immune systems of the organism after radiotherapy. References Ludmila M. Khromykh et al. Cyclophilin A produced by thymocytes regulates the migration of murine bone marrow cells. Cellular Immunology 249 (2007) 46–53. Keywords: Cyclophilin A, Radiotherapy, Bone Marrow, Recovery, Hematopoietic System Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Kalinina AA, Silaeva YY, Kazansky DB and Khromykh LM (2013). Cyclophilin A stimulates recovery of the hematopoietic and the immune system of the organism after radiotherapy. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00363 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 15 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Miss. Anastasia A Kalinina, Federal State Budgetary Institution”N.N.Blokhin Cancer Research Center”, Russian Academy of Medical Sciences, Moscow, Russia, aakalinina89@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Anastasia A Kalinina Yulia Y Silaeva Dmitry B Kazansky Ludmila M Khromykh Google Anastasia A Kalinina Yulia Y Silaeva Dmitry B Kazansky Ludmila M Khromykh Google Scholar Anastasia A Kalinina Yulia Y Silaeva Dmitry B Kazansky Ludmila M Khromykh PubMed Anastasia A Kalinina Yulia Y Silaeva Dmitry B Kazansky Ludmila M Khromykh Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Enhancement of radiation effect using beta-lapachone and underlying mechanism

Beta-lapachone (β-Lap; 3,4-dihydro-2, 2-dimethyl-2H-naphthol[1, 2-b]pyran-5,6-dione) is a novel anti-cancer drug under phase I/II clinical trials. β-Lap has been demonstrated to cause apoptotic and necrotic death in a variety of human cancer cells in vitro and in vivo. The mechanisms underlying the β-Lap toxicity against cancer cells has been controversial. The most recent view is that β-Lap, which is a quinone compound, undergoes two-electron reduction to hydroquinone form utilizing NAD(P)H or NADH as electron source. This two-electron reduction of β-Lap is mediated by NAD(P)H:quinone oxidoreductase (NQO1), which is known to mediate the reduction of many quinone compounds. The hydroquinone forms of β-Lap then spontaneously oxidizes back to the original oxidized β-Lap, creating futile cycling between the oxidized and reduced forms of β-Lap. It is proposed that the futile recycling between oxidized and reduced forms of β-Lap leads to two distinct cell death pathways. First one is that the two-electron reduced β-Lap is converted first to one-electron reduced β-Lap, i.e., semiquinone β-Lap (SQ)·- causing production of reactive oxygen species (ROS), which then causes apoptotic cell death. The second mechanism is that severe depletion of NAD(P)H and NADH as a result of futile cycling between the quinone and hydroquinone forms of β-Lap causes severe disturbance in cellular metabolism leading to apoptosis and necrosis. The relative importance of the aforementioned two mechanisms, i.e., generation of ROS or depletion of NAD(P)H/NADH, may vary depending on cell type and environment. Importantly, the NQO1 level in cancer cells has been found to be higher than that in normal cells indicating that β-Lap may be preferentially toxic to cancer cells relative to non-cancer cells. The cellular level of NQO1 has been found to be significantly increased by divergent physical and chemical stresses including ionizing radiation. Recent reports clearly demonstrated that β-Lap and ionizing radiation kill cancer cells in a synergistic manner. Indications are that irradiation of cancer cells causes long-lasting elevation of NQO1, thereby sensitizing the cells to β-Lap. In addition, β-Lap has been shown to inhibit the repair of sublethal radiation damage. Treating experimental tumors growing in the legs of mice with irradiation and intraperitoneal injection of β-Lap suppressed the growth of the tumors in a manner more than additive. Collectively, β-Lap is a potentially useful anti-cancer drug, particularly in combination with radiotherapy.

IB-MECA, an Adenosine A3 Receptor Agonist, Does Not Influence Survival of Lethally γ-Irradiated Mice

In our previous studies, IB-MECA, an adenosine A(3) receptor agonist, was found to stimulate proliferation of hematopoietic progenitor and precursor cells in mice. This property of IB-MECA was considered to be responsible for its ability to support regeneration of suppressed hematopoiesis after irradiation with sublethal doses of γ-rays when the drug was given in a post-irradiation treatment regimen. This study was aimed at assessing the ability of IB-MECA to influence a 30-day survival of lethally irradiated mice. In a series of experiments, IB-MECA was administered following various lethal radiation doses in various numbers of drug doses and various administration routes. Though in some of these experiments a moderate increase in 30-day survival was observed in IB-MECA-treated mice, the differences in comparison with the controls were not significantly different. It can be inferred from these results and those of previous studies assessing the effects of IB-MECA after sublethal radiation doses that IB-MECA can probably influence only a substantially preserved hematopoiesis like that remaining after sublethal irradiation. Future studies should be aimed at evaluation of the abilities of IB-MECA to influence post-irradiation survival when administered as a part of combined treatment regimens.

Effect of Human Parathyroid Hormone on Hematopoietic Progenitor Cells in NOD/SCID Mice Co-Transplanted with Human Cord Blood Mononuclear Cells and Mesenchymal Stem Cells

PurposeWe evaluated the effect of human parathyroid hormone (hPTH) on the engraftment and/or in vivo expansion of hematopoietic stem cells in an umbilical cord blood (UCB)-xenotransplantation model. In addition, we assessed its effect on the expression of cell adhesion molecules.Materials and MethodsFemale NOD/SCID mice received sublethal total body irradiation with a single dose of 250 cGy. Eighteen to 24 hours after irradiation, 1×107 human UCB-derived mononuclear cells (MNCs) and 5×106 human UCB-derived mesenchymal stem cells (MSCs) were infused via the tail vein. Mice were randomly divided into three groups: Group 1 mice received MNCs only, Group 2 received MNCs only and were then treated with hPTH, Group 3 mice received MNCs and MSCs, and were treated with hPTH.ResultsEngraftment was achieved in all the mice. Bone marrow cellularity was approximately 20% in Group 1, but 70-80% in the hPTH treated groups. Transplantation of MNCs together with MSCs had no additional effect on bone marrow cellularity. However, the proportion of human CD13 and CD33 myeloid progenitor cells was higher in Group 3, while the proportion of human CD34 did not differ significantly between the three groups. The proportion of CXCR4 cells in Group 3 was larger than in Groups 1 and 2 but without statistical significance.ConclusionWe have demonstrated a positive effect of hPTH on stem cell proliferation and a possible synergistic effect of MSCs and hPTH on the proportion of human hematopoietic progenitor cells, in a xenotransplantation model. Clinical trials of the use of hPTH after stem cell transplantation should be considered.

Mer Receptor Tyrosine Kinase Is A Potential Therapeutic Target in Acute Myeloid Leukemia

AbstractAbstract 1317Acute myelogenous leukemia (AML) is difficult to treat successfully in both adult and pediatric patients using conventional chemotherapy. Mutated or aberrantly expressed proteins on the cell surface of myeloblasts provide a focus for targeted therapy which could potentially augment therapeutic outcome, decrease toxicity to normal tissues, and/or provide a therapy option for those who are not able to tolerate conventional therapy. We report here that the Mer receptor tyrosine kinase is upregulated in approximately 80% of AML cell lines and patient samples, and explore the therapeutic potential of Mer inhibition.We assessed the prevalence of Mer expression in AML. Western blot and flow cytometric analysis demonstrated expression of Mer in greater than 85% (12/14) of AML cell lines. Mer expression was also assessed at the time of diagnosis and relapse in both pediatric and adult patient samples using flow cytometry. We found that Mer was expressed on leukemic blasts in 80% of 36 pediatric and 100% of 10 adult patients at the time of diagnosis with AML. Additionally, 100% of 11 patients expressed Mer at the time of relapse. Furthermore, when analyzing patient samples at relapse compared to the same patient's diagnostic sample, there was a trend toward increased Mer expression. This is in contrast to normal bone marrow myeloid progenitors from healthy donors, which express little or no Mer.Using two independent shRNA constructs directed against Mer, we analyzed the effects of Mer inhibition in two Mer expressing AML cell lines. Mer knock-down and control cell lines were assessed for apoptosis by flow cytometry after serum starvation and staining with Yo-Pro-1 iodide and propidium iodide. Compared to AML cell lines transduced with a non-silencing control shRNA (shControl), cell lines expressing reduced levels of Mer protein demonstrated significantly more apoptosis (p<0.05). Additionally, when these cell lines were plated in equal number in methylcellulose, cell lines with reduced Mer expression demonstrated decreased colony forming potential compared to shControl cells (p<0.01). Mer knock-down and control cell lines were injected into NOD-SCID-gamma mice after sublethal irradiation and the mice were monitored for development of leukemia. Mice injected with myeloblasts expressing decreased levels of Mer demonstrated significantly prolonged symptom-free survival compared to mice transplanted with shControl AML cells (p<0.001).To further explore the effects of Mer inhibition in AML, we used a novel small molecule tyrosine kinase inhibitor (UNC1666), which has high specificity to Mer. Three Mer expressing AML cell lines were treated with UNC1666 in vitro; treatment reduced phosphorylation of Mer and the downstream signaling molecules ERK1/2 and STAT6. Additionally, treatment with UNC1666 resulted in significant induction of apoptosis (p<0.05) by flow cytometric analysis after staining with Yo-Pro-1 iodide and propidium iodide, and dose-dependent inhibition of colony formation in soft agar, when compared to vehicle treated cellsIn summary, the upregulation of Mer expression in patient samples and the functional effects on survival with Mer shRNA knockdown help validate Mer as a new and attractive AML therapeutic target. Furthermore, a novel Mer tyrosine kinase inhibitor decreased myeloblast cell survival, providing evidence that Mer is a druggable target in AML. Disclosures:Kireev:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Liu:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Wang:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Frye:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Graham:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties.

Immune Injury by Allogeneic CD4+ T Cells Leads to Host Hematopoietic Stem Cell Dormancy and Prevents Engraftment of Donor Cells.

Abstract 2366The bone marrow (BM) is a complex microsystem that supports lifelong blood production and dynamically responds to injury, blood cell loss and senescence. At steady-state most hematopoietic stem cells (HSC) are quiescent. However, in situations of increased demand, their activation is triggered by an array of signals, such as cytokines released during infections. The susceptibility of BM to injury by T cells (TC) is long recognized, e.g. in aplastic anemia, and BM failure syndromes, and the inhibitory effect of IFNg on the proliferative activity of (HSC) is established. How donor TC given as part of an allogeneic hematopoietic cell transplantation (HCT) affect the recipient BM microenvironment and influence HSC activity has not been elucidated. Yet, graft TC are believed to be essential to overcome host barriers, especially following lower intensity (non-myeloablative) conditioning that spares host HSC and residual host immunity. We examined in an MHC-matched, minor-antigen-mismatched mouse model of non-myeloablative HCT the impact of graft TC subsets on HSC and hematopoietic reconstitution. BALB.K (H2k) and BALB.B (H2b) mice were prepared with sublethal radiation at a dose that allows donor cells (AKR/J [H2k] and C57BL/6 [H2b]) to engraft but also permits survival of host HSC and immune populations. Grafts consisted of purified HSC (KTLS; cKit+Thy1.1loLin−Sca-1+) given alone or supplemented with donor TC or TC subsets. Rather than improve engraftment, donor TC, specifically CD4+ cells, resulted in detainment of host HSC in their niches and consequent failure of donor HSC to engraft. These events appeared to be initiated specifically by conventional alloreactive donor CD4+CD25− cells. These CD4+CD25− cells interacted with immune-competent host cells, stimulating CD11c+ dendritic cells to upregulate the activation markers CD40 and MHC II. Following their activation dendritic cells produced excessive amounts of IL-12. IL-12, in turn, activated donor CD4+ cells to produce IFNg. This Th1-response was prominent in BM but absent in the spleen. In this Th1-cytokine environment, high levels of quiescent host HSC were observed, arrested at the short-term HSC stage, while more mature, multipotent progenitors were absent. Moreover, HSC arrest correlated with marrow aplasia, analogous to that seen in BM failure syndromes. This hypocellularity was only observed in recipients of CD4+ containing grafts, while recipients given HSC alone or HSC in combination with CD8+ TC had prompt blood regeneration. When resting HSC were FACS-purified from the BM of recipients of HSC+CD4+ and infused into secondary recipients they promptly expanded and gave rise to all blood lineages. Although recipients of HSC+CD4+ cells ultimately recovered blood formation, B lymphopoiesis was markedly delayed. No clinical signs of graft-versus-host disease were noted in these mice. Lymphocytes that recovered in these recipients of HSC+CD4+ cells were of host type only. In contrast, recipients of pure HSC demonstrated prompt donor cell engraftment, and mixed chimerism in all lymphoid lineages, including T cells. Our model corroborates the importance of the cytokine microenvironment in hematopoietic reconstitution and marrow failure post-transplant. Moreover, our findings underscore the importance of IFNg in BM aplasia and support the idea that IFNg can, under physiologic conditions, exert regulatory effects on hematopoiesis by controlling the exit of ST-HSC out of the marrow niche. Our studies shed new light on understanding the physiology of engraftment and marrow failure after HCT using nonmyeloablative conditioning, and indicate that the composition of the graft is of critical importance due to its effects on the marrow microenvironment We postulate that in some naturally occurring settings of BM inflammation (e.g., infections) detainment of resting HSC in their niche may provide protection of hematopoiesis and that comparable events are relevant for engraftment post-HCT. Disclosures:No relevant conflicts of interest to declare.

Activation of K-Ras in Arf-Null Mice Is Not Sufficient for Malignant Transformation of Post-Germinal Center B-Cells

Abstract 5016Activating mutations in Ras (N- and K-) are the most common mutations in multiple myeloma (MM), and are associated with advanced clinical stage and poor outcomes. As previously reported, we re-sequenced coding regions of highly expressed cytokine signaling candidate genes from MM patient samples and found the most common mutations to affect Ras oncogenes (N and K-). To test the role of Ras activation in MM pathogenesis directly, we generated mice harboring a mutant K-ras allele, Lox-stop-lox(LSL)- K-ras G12D (K-ras+/G12D) with Cγ1-Cre knock-in mice (Cγ1+/Cre), activated specifically in germinal center (GC) cells. K-ras+/+/Cγ1+/Cre mice served as negative controls. We found 58% of unstimulated K-ras+/G12D/Cγ1+/Cre mice developed fatal double-positive CD4/8 T-cell lymphomas (n=12) and 42% developed lung adenocarcinomas (n=12), phenotypes that have previously been reported for K-ras+/G12D mice. Tumor cells demonstrated successful recombination of the K-Ras locus. We showed recombination in splenic germinal center cells (B220+, GL7+ and IgG1+), as well as in post-germinal center cells (B220-, CD138+). No evidence of plasma cell proliferation was seen by serum ELISA, SPEP, flow cytometry or histological examination. We confirmed these results by crossing the K-ras+/G12D mice to a second strain, AID-Cre, which expresses the recombinase earlier and more specifically in the GC. We found 100% (n=20) of K-ras+/G12D/AID-Cre-YFP mice possessed visible, small benign papillomas on the ventral neck, as early as 3 weeks of age. To stimulate malignant transformation, vitamin D deficient chow and/or sub-lethal radiation was given to K-ras+/G12D/AID-Cre-YFP and negative control, K-ras+/G12D mice. Dual treatment increased the size and number of papillomas, giving a median survival of 26 weeks, compared to 52 weeks with control. However, no evidence of MM was detected by serum ELISA, SPEP, flow cytometry or histological examination. Finally, we generated AID-Cre-K-ras+/G12D triple transgenic mice in a tumor prone Arf-null background. After 13 wks, we found sarcomas in 66% (n=3) of K-ras+/G12D/AID-Cre-YFP/Arf−/− mice. Additionally, 100% (n=3) of these mice had rapid growing benign papillomas and only a minimal increase in the plasma cell compartment, compared to K-ras+/G12D/AID-Cre/Arf +/− control. Together, these data demonstrate that Ras activation is not sufficient to transform primary germinal center or plasma cells, even in an Arf-null context, and suggests that specific myeloma events, yet to be identified, are required for malignant transformation. Disclosures:No relevant conflicts of interest to declare.

SDF-1 Acutely Promotes the Physical Association of Megakaryocytes with Vascular Endothelium in the Bone Marrow and Increases the Number of Circulating Platelets.

Abstract 2306Thrombocytopenia complicates many diseases and can be a life-threatening consequence of genotoxic treatments including chemotherapy and radiation therapy. It is well established that thrombopoiesis occurs in the bone marrow where mature megakaryocyte (MK) precursor cells associate with sinusoidal endothelial cells and extrude pro-platelets into the vasculature. There has been much interest in elucidating mechanisms that control megakaryopoiesis and in utilizing these pathways to increase platelet output. The leading paradigm of megakaryopoiesis centers on the ability of cytokines, chiefly thrombopoietin (TPO), to promote MK progenitor proliferation and MK precursor maturation. More recently, attention has been focused on the ability of the bone marrow microenvironment to promote MK maturation and platelet formation. The chemokine stromal-derived factor-1 (SDF-1, also known as CXCL12), signaling through its receptor CXCR4, is implicated in the chemotaxis of MKs toward sinusoidal vessels, and in vivo evidence demonstrates that sustained plasma elevation of SDF-1 can increase platelet counts (Avecilla et al. Nature Medicine, 2004). To more specifically determine the short-term effects of SDF-1, we injected mice with a single 400ng intravenous dose of SDF-1 and enumerated the progenitor, precursor, and platelet compartments of the MK lineage. At 24 hours, SDF-1 induced a 30% increase in platelets compared to vehicle control (p<0.05). However, MK progenitors, defined functionally by the formation of acetylcholinesterase-positive colonies in vitro, and MK precursors, enumerated by imaging flow cytometry, were both unchanged (p>0.7 and p>0.5). To quantitatively determine if SDF-1 regulates the physical interactions of MK precursors with sinusoidal endothelium, we developed a double immunohistochemistry assay using Gp1Bβ to distinguish MK precursors and MECA32 to identify vascular endothelial cells. In vehicle-treated mice, 39% of MKs in the marrow localized to the sinusoidal endothelium, and this increased to 53% 24 hours following SDF-1 treatment (p<0.01). Thus, a single dose of SDF-1 acutely increases the number of MKs in the vascular niche as well as peripheral platelet counts. Given these results, we tested whether a single dose of SDF-1 could improve thrombocytopenia in the setting of radiation-induced marrow injury. Mice were treated with SDF-1 4 days after sublethal 4Gy total body irradiation (TBI), when radiosensitive MK progenitors are drastically reduced and radioresistant MK precursors and platelets are just beginning to decline. At 5 days post-TBI (24 hours post-SDF-1), SDF-1 treatment increased the number of circulating platelets by 15% (p<0.01) as well as the percentage of MK precursors in the vascular niche by over 15% (p<0.02) without changing the total number of MK progenitors or precursors in the marrow compared to irradiated vehicle controls (p>0.4 and p>0.7). As the platelet response following SDF-1 was less robust in the setting of TBI injury, we hypothesized that SDF-1-induced thrombopoiesis may improve if the number of MK precursors available to move to the vascular niche is increased. To test this, we administered TPO and SDF-1 at 2 hours and at 4 days, respectively, following TBI. TPO treatment alone resulted in 15% more MK precursors than irradiated vehicle controls at 5 days post-TBI (p<0.02), but did not significantly change the platelet count (p>0.2). In contrast, mice receiving both TPO and SDF-1 had over 20% more platelets than irradiated vehicle controls (p<0.01) and over 12% more platelets than mice receiving TPO alone (p<0.05) with the same increase in MK precursors (p>0.9). Correcting for differences in MK precursor numbers, mice receiving TPO and SDF-1 post-TBI had 1.8-fold more MKs in the vascular niche than irradiated vehicle controls (p<0.03) and 1.4-fold more than mice treated with TPO only (p<0.02). Taken together, we provide quantitative data in support of the concept that SDF-1 acutely promotes increases in the number of MK precursors in the vascular niche as well as peripheral platelet counts, and this effect correlates with the number of MK precursors in the marrow. Therapeutic approaches combining agents that first increase MK number and secondly increase MKs poised for thrombopoiesis by localization within the vascular niche may be a novel strategy to maximally increase peripheral platelet counts. Disclosures:No relevant conflicts of interest to declare.

BCR-ABL-Induced Transcriptional Repression of the Interferon Regulatory Factor 8 (IRF-8/ICSBP) Leads to Depletion of Plasmocytoid Dendritic Cells (PDC), Which May Contribute to Leukemogenesis in a Murine Model of Chronic Myeloid Leukemia

AbstractAbstract 36 Introduction:Chronic myeloid leukemia (CML) is caused by the BCR-ABL oncogene. CML patients lack expression of IRF-8 - an interferon-regulated transcription factor that has been shown to exert tumor suppressor functions. IRF-8 is also critical for the development of a rare dendritic cell population, so called plasmocytoid dendritic cells (PDC). PDC are quantitatively significantly reduced or absent in the peripheral blood of first diagnosis CML patients. PDC are also the major producers of IFN-alpha (IFNa) in man. IFNa is a cytokine that has significant therapeutic efficiency in the treatment of CML patients.We here wished to experimentally test, whether BCR-ABL expression and loss of IRF-8 may be causally linked to a reduction of PDC in murine CML and whether there could be any functional relevance for PDC loss in CML development or treatment. Methods:PDC counts were studied from peripheral blood samples of primary CML patients at diagnosis, at the time of remission or from healthy donors. PDC function was assessed in vitro by treatment of magnetic bead-enriched PDC with Toll-like receptor 9-specific oligos (ODN 2216) and subsequent assessment of the intracellular IFNa expression in stimulated PDC. A supposed link between BCR-ABL expression, IRF-8 repression and loss of PDC counts was studied in vivo using a murine CML transduction-transplantation model (C57/Bl6 mice, 7Gy sub-lethal irradiation for conditioning). Multiparameter flow cytometry and cell sorting were performed to analyze and enrich, BCR-ABL-positive (GFP+) hematopoietic subpopulations and PDC in order to then quantitate their IRF-8 and BCR-ABL transcript level by RT-PCR. In order to also test the functional relevance of PDC during CML leukemogenesis, CML mice were injected intravenously, weekly from day +5 after transplantation with in vitro generated PDC. Mice were simultaneously also s.c.-injected weekly with ODN 2216 to stimulate IFNα secretion in adoptively transferred PDC in vivo. Results:As previously reported, newly diagnosed CML patients displayed a significantly reduced PDC count when compared to healthy donors (p<0.001). Upon remission induction with imatinib, PDC counts restored partially, but to a much lesser extend in patients successfully treated with IFNa therapy. Importantly, albeit significantly reduced in number, BCR-ABL-positive first diagnosis CML PDC seem to be functionally intact: CML and healthy donor PDC produced comparable amounts of IFNa in response to Toll-like receptor 9 -specific CpG ODN 2216 stimulation. This suggested that BCR-ABL may compromise PDC function by quantitative rather than qualitative dysregulation.CML mice developed a fatal, BCR-ABL-positive myeloproliferation within 13 to 29 days with 88% penetrance. Compared to control mice (n=8), CML mice (n=14) showed a 7-fold and 3-fold reduction of the frequency of B220+mPDCA-1+ PDC in bone marrow and spleen, respectively. This was associated with a statistically significant (4-fold) suppression of IRF8 mRNA expression in sorted BCR-ABL(GFP)-positive PDC relative to BCR-ABL-negative PDC from the same mice (n=3) or from control transplantations (n=5). By RT-PCR, there was a trend also for lower IRF8 expression in CML progenitor cells (Lin− c-Kit+ Sca-1- GFP+), but not in the stem cell enriching population (Lin− c-Kit+ Sca-1+ GFP+). This implied that IRF8 expression is lost during BCR-ABL-induced leukemogenesis in more mature compartments, which supposedly include PDC precursors.Intriguingly, a once weekly adoptive transfer of in vitro generated (to > 30% enriched) PDC for three successive weeks combined with a once weekly subcutaneous injection of CpG ODN 2216 for three weeks was sufficient to almost double survival of CML mice. Conclusions:Using a murine model of CML, we provide first experimental evidence that BCR-ABL induced myeloproliferation is causally linked to a quantitative suppression of PDC, and that this is associated with a BCR-ABL-mediated suppression of IRF8 transcription. Since adoptively transferred PDC were capable of counteracting murine CML development, BCR-ABL may facilitate leukemogenesis in part by obstructing PDC maturation. PDC could thus be a novel immunological effector cell population that exerts and/or integrates anti-leukemic immune responses in CML. Disclosures:No relevant conflicts of interest to declare.