Trident cold atmospheric plasma blocks three cancer survival pathways to overcome therapy resistance.

Therapy resistance is responsible for most cancer-related death and is mediated by the unique ability of cancer cells to leverage metabolic conditions, signaling molecules, redox status, and other pathways for their survival. Interestingly, many cancer survival pathways are susceptible to disturbances in cellular reactive oxygen species (ROS) and may therefore be disrupted by exogenous ROS. Here, we explore whether trident cold atmospheric plasma (Tri-CAP), a gas discharge with exceptionally low-level ROS, could inhibit multiple cancer survival pathways together in a murine cell line model of therapy-resistant chronic myeloid leukemia (CML). We show that Tri-CAP simultaneously disrupts three cancer survival pathways of redox deregulation, glycolysis, and proliferative AKT/mTOR/HIF-1α signaling in this cancer model. Significantly, Tri-CAP blockade induces a very high rate of apoptotic death in CML cell lines and in primary CD34+ hematopoietic stem and progenitor cells from CML patients, both harboring the therapy-resistant T315I mutation. In contrast, nonmalignant controls are minimally affected by Tri-CAP, suggesting it selectively targets resistant cancer cells. We further demonstrate that Tri-CAP elicits similar lethality in human melanoma, breast cancer, and CML cells with disparate, resistant mechanisms and that it both reduces tumor formation in two mouse models and improves survival of tumor-bearing mice. For use in patients, administration of Tri-CAP may be extracorporeal for hematopoietic stem cell transplantation therapy, transdermal, or through its activated solution for infusion therapy. Collectively, our results suggest that Tri-CAP represents a potent strategy for disrupting cancer survival pathways and overcoming therapy resistance in a variety of malignancies.

(-)-Epigallocatechin-3-gallate induces apoptosis and differentiation in leukaemia by targeting reactive oxygen species and PIN1.

(-)-Epigallocatechin-3-gallate (EGCG), the major active polyphenol extracted from green tea, has been shown to induce apoptosis and inhibit cell proliferation, cell invasion, angiogenesis and metastasis. Herein, we evaluated the in vivo effects of EGCG in acute myeloid leukaemia (AML) using an acute promyelocytic leukaemia (APL) experimental model (PML/RARα). Haematological analysis revealed that EGCG treatment reversed leucocytosis, anaemia and thrombocytopenia, and prolonged survival of PML/RARα mice. Notably, EGCG reduced leukaemia immature cells and promyelocytes in the bone marrow while increasing mature myeloid cells, possibly due to apoptosis increase and cell differentiation. The reduction of promyelocytes and neutrophils/monocytes increase detected in the peripheral blood, in addition to the increased percentage of bone marrow cells with aggregated promyelocytic leukaemia (PML) bodies staining and decreased expression of PML-RAR oncoprotein corroborates our results. In addition, EGCG increased expression of neutrophil differentiation markers such as CD11b, CD14, CD15 and CD66 in NB4 cells; and the combination of all-trans retinoic acid (ATRA) plus EGCG yield higher increase the expression of CD15 marker. These findings could be explained by a decrease of peptidyl-prolyl isomerase NIMA-interacting 1 (PIN1) expression and reactive oxygen species (ROS) increase. EGCG also decreased expression of substrate oncoproteins for PIN1 (including cyclin D1, NF-κB p65, c-MYC, and AKT) and 67 kDa laminin receptor (67LR) in the bone marrow cells. Moreover, EGCG showed inhibition of ROS production in NB4 cells in the presence of N-acetyl-L-cysteine (NAC), as well as a partial blockage of neutrophil differentiation and apoptosis, indicating that EGCG-activities involve/or are in response of oxidative stress. Furthermore, apoptosis of spleen cells was supported by increasing expression of BAD and BAX, parallel to BCL-2 and c-MYC decrease. The reduction of spleen weights of PML/RARα mice, as well as apoptosis induced by EGCG in NB4 cells in a dose-dependent manner confirms this assumption. Our results support further evaluation of EGCG in clinical trials for AML, since EGCG could represent a promising option for AML patient ineligible for current mainstay treatments.

A high-throughput screen indicates gemcitabine and JAK inhibitors may be useful for treating pediatric AML.

Improvement in survival has been achieved for children and adolescents with AML but is largely attributed to enhanced supportive care as opposed to the development of better treatment regimens. High risk subtypes continue to have poor outcomes with event free survival rates <40% despite the use of high intensity chemotherapy in combination with hematopoietic stem cell transplant. Here we combine high-throughput screening, intracellular accumulation assays, and in vivo efficacy studies to identify therapeutic strategies for pediatric AML. We report therapeutics not currently used to treat AML, gemcitabine and cabazitaxel, have broad anti-leukemic activity across subtypes and are more effective relative to the AML standard of care, cytarabine, both in vitro and in vivo. JAK inhibitors are selective for acute megakaryoblastic leukemia and significantly prolong survival in multiple preclinical models. Our approach provides advances in the development of treatment strategies for pediatric AML.

Deregulated FADD expression and phosphorylation in T-cell lymphoblastic lymphoma.

In the present work, we show that T-cell lymphoblastic lymphoma cells exhibit a reduction of FADD availability in the cytoplasm, which may contribute to impaired apoptosis. In addition, we observe a reduction of FADD phosphorylation that inversely correlates with the proliferation capacity and tumor aggressiveness. The resultant balance between FADD-dependent apoptotic and non-apoptotic abilities may define the outcome of the tumor. Thus, we propose that FADD expression and phosphorylation can be reliable biomarkers with prognostic value for T-LBL stratification.

A novel mouse model identifies cooperating mutations and therapeutic targets critical for chronic myeloid leukemia progression.

The introduction of highly selective ABL-tyrosine kinase inhibitors (TKIs) has revolutionized therapy for chronic myeloid leukemia (CML). However, TKIs are only efficacious in the chronic phase of the disease and effective therapies for TKI-refractory CML, or after progression to blast crisis (BC), are lacking. Whereas the chronic phase of CML is dependent on BCR-ABL, additional mutations are required for progression to BC. However, the identity of these mutations and the pathways they affect are poorly understood, hampering our ability to identify therapeutic targets and improve outcomes. Here, we describe a novel mouse model that allows identification of mechanisms of BC progression in an unbiased and tractable manner, using transposon-based insertional mutagenesis on the background of chronic phase CML. Our BC model is the first to faithfully recapitulate the phenotype, cellular and molecular biology of human CML progression. We report a heterogeneous and unique pattern of insertions identifying known and novel candidate genes and demonstrate that these pathways drive disease progression and provide potential targets for novel therapeutic strategies. Our model greatly informs the biology of CML progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease.

Polycomb Cbx family members mediate the balance between haematopoietic stem cell self-renewal and differentiation.

The balance between self-renewal and differentiation of adult stem cells is essential for tissue homeostasis. Here we show that in the haematopoietic system this process is governed by polycomb chromobox (Cbx) proteins. Cbx7 is specifically expressed in haematopoietic stem cells (HSCs), and its overexpression enhances self-renewal and induces leukaemia. This effect is dependent on integration into polycomb repressive complex-1 (PRC1) and requires H3K27me3 binding. In contrast, overexpression of Cbx2, Cbx4 or Cbx8 results in differentiation and exhaustion of HSCs. ChIP-sequencing analysis shows that Cbx7 and Cbx8 share most of their targets; we identified approximately 200 differential targets. Whereas genes targeted by Cbx8 are highly expressed in HSCs and become repressed in progenitors, Cbx7 targets show the opposite expression pattern. Thus, Cbx7 preserves HSC self-renewal by repressing progenitor-specific genes. Taken together, the presence of distinct Cbx proteins confers target selectivity to PRC1 and provides a molecular balance between self-renewal and differentiation of HSCs.

Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment.

The interactions between the bone marrow (BM) microenvironment and acute myeloid leukemia (AML) is known to promote survival of AML cells. In this study, we used reverse phase-protein array (RPPA) technology to measure changes in multiple proteins induced by stroma in leukemic cells. We then investigated the potential of an mTOR kinase inhibitor, PP242, to disrupt leukemia/stroma interactions, and examined the effects of PP242 in vivo using a mouse model. Using RPPA, we confirmed that multiple survival signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), were up-regulated in primary AML cells cocultured with stroma. PP242 effectively induced apoptosis in primary samples cultured with or without stroma. Mechanistically, PP242 attenuated the activities of mTORC1 and mTORC2, sequentially inhibited phosphorylated AKT, S6K, and 4EBP1, and concurrently suppressed chemokine receptor CXCR4 expression in primary leukemic cells and in stromal cells cultured alone or cocultured with leukemic cells. In the in vivo leukemia mouse model, PP242 inhibited mTOR signaling in leukemic cells and demonstrated a greater antileukemia effect than rapamycin. Our findings indicate that disrupting mTOR/AKT signaling with a selective mTOR kinase inhibitor can effectively target leukemic cells within the BM microenvironment.

Second site mutation in the virus envelope expands the host range of a cytopathic variant of Moloney murine leukemia virus.

Spl574 MLV (murine leukemia virus) is a variant of Moloney ecotropic MLV (MoMLV) that is cytopathic in Mus dunni cells and restricted by other mouse cells. Its host range and cytopathicity are due to a mutation, S82F, at a site critical for binding to the CAT-1 receptor. To identify residues that affect affinity for receptor variants, virus with S82F was passed in restrictive cells. The env genes of the adapted viruses contained 18 novel mutations, including one, E114G, present in 6 of 30 sequenced envs. MoMLV-E114G efficiently infected all mouse cells as well as ecotropic MLV resistant Chinese hamster cells. Virus with E114G and S82F induced large multinucleated syncytia in NIH 3T3 and SC-1 cells as well as M. dunni cells. Inoculation of Mo-S82F,E114G into mice produced lymphomas typical of MoMLV. Residues at env position 114 are thus important determinants of host range, and E114G suppresses host range restriction due to S82F, but does not affect S82F-governed cytopathicity.

Recipient lymphocyte infusion in MHC-matched bone marrow chimeras induces a limited lymphohematopoietic host-versus-graft reactivity but a significant antileukemic effect mediated by CD8+ T cells and natural killer cells.

BACKGROUND: Challenge of MHC-mismatched murine bone marrow chimeras with recipient-type lymphocytes (recipient lymphocyte infusion) produces antileukemic responses in association with rejection of donor chimerism. In contrast, MHC-matched chimeras resist eradication of donor chimerism by recipient lymphocyte infusion. Here, we investigated lymphohematopoietic host-versus-graft reactivity and antileukemic responses in the MHC-matched setting, which is reminiscent of the majority of clinical transplants. DESIGN AND METHODS: We challenged C3H→AKR radiation chimeras with AKR-type splenocytes (i.e. recipient lymphocyte infusion) and BW5147.3 leukemia cells. We studied the kinetics of chimerism using flowcytometry and the mechanisms involved in antileukemic effects using in vivo antibody-mediated depletion of CD8(+) T and NK cells, and intracellular cytokine staining. RESULTS: Whereas control chimeras showed progressive evolution towards high-level donor T-cell chimerism, recipient lymphocyte infusion chimeras showed a limited reduction of donor chimerism with delayed onset and long-term preservation of lower-level mixed chimerism. Recipient lymphocyte infusion chimeras nevertheless showed a significant survival benefit after leukemia challenge. In vivo antibody-mediated depletion experiments showed that both CD8(+) T cells and NK cells contribute to the antileukemic effect. Consistent with a role for NK cells, the proportion of IFN-γ producing NK cells in recipient lymphocyte infusion chimeras was significantly higher than in control chimeras. CONCLUSIONS: In the MHC-matched setting, recipient lymphocyte infusion elicits lymphohematopoietic host-versus-graft reactivity that is limited but sufficient to provide an antileukemic effect, and this is dependent on CD8(+) T cells and NK cells. The data indicate that NK cells are activated as a bystander phenomenon during lymphohematopoietic T-cell alloreactivity and thus support a novel type of NK involvement in anti-tumor responses after post-transplant adoptive cell therapy.

Leukemias induced by altered TRK-signaling are sensitive to mTOR inhibitors in preclinical models.

Rapamycin is a potent allosteric mTORC1 inhibitor with clinical applications as an anticancer agent. However, only a fraction of cancer patients responds to the drug, and no biomarkers are available to predict tumor sensitivity. Recently, we and others have obtained evidence for potential involvement of tropomyosin-related kinase (TRK) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) in leukemia. In the present study, we tested the therapeutic effect of Rapamycin and its analog RAD001 on altered TRK-induced leukemia in a murine model. Daily treatment with Rapamycin (2 mg/kg) or RAD001 (1 mg/kg) significantly prolonged the survival of treated animals (n = 40) compared with the placebo group. Consistently, both mTOR and S6 proteins were strongly dephosphorylated in vitro and in vivo after treatment with Rapamycin or RAD001. However, Rapamycin did not completely inhibit mTORC1-dependent phosphorylation of 4E-BP1. With exception of one mouse showing slight reactivation of Akt after treatment, no reactivation of MAPK or Akt pathways was observed in other resistant tumors. Interestingly, leukemic cells isolated from a Rapamycin-resistant mouse were still highly sensitive to Rapamycin in vitro. Our findings suggest that altered TRK signaling may be a good predictor of tumor sensitivity to mTOR inhibition and that pathways other than MAPK and Akt exist that may trigger resistance of leukemic cells to Rapamycin in vivo.

Dietary effect of Antrodia Camphorate extracts on immune responses in WEHI-3 leukemia BALB/c mice.

Antrodia camphorata has been recognized to be a traditional Chinese medicine for abdominal pain, diarrhea, and to protect against hepatitis virus infection. Several ingredients derived from A. camphorata possess various pharmacological and biological activities such as antioxidant and anticancer. In this study, its ability to promote immune responses and to exhibited antileukemia activity in WEHI-3 leukemia BALB/c mice were investigated. The results indicated A. camphorata significantly prolonged the survival rate and prevented the body weight loss in leukemia mice. Four mg/kg of A. camphorata treatment significantly decreased the weight of the spleen. Both doses (2 and 4 mg/kg) of A. camphorata did not affect Mac-3 marker in leukocytes. However, the 4 mg/kg of A. camphorata decreased the levels of CD11b and both doses of treatment increased CD3 and CD19. With lipopolysaccharide stimulation, the 4 mg/kg of A. camphorata promoted the significant proliferation of leukocytes; but with concanavalin A stimulation, both doses promoted the significant proliferation of leukocytes. YAC-1 target cells were killed by NK cells from the mice after treatment with A. camphorata at 4 mg/kg in target cells at a ratio of 50:1. The percentage of macrophages with phagocyted at A. camphorata treatment increased, and these effects were in dose-dependent manners.

Liposome-incorporated Grb2 antisense oligodeoxynucleotide increases the survival of mice bearing bcr-abl-positive leukemia xenografts.

We previously demonstrated that liposome-incorporated antisense oligodeoxynucleotide specific for the grb2 mRNA (L-Grb2) inhibited Grb2 protein expression and the proliferation of bcr-abl-positive leukemia cell lines. To determine whether L-Grb2 has the potential of being a therapeutic modality against bcr-abl-positive leukemia, we studied the tissue distribution of L-Grb2 in normal mice before studying its effects in mice bearing bcr-abl-positive leukemia xenografts. L-Grb2 was widely distributed in the body. The highest tissue concentrations of L-Grb2 were found in the spleen and liver, which are the organs where the tumor mass of bcr-abl-positive leukemia is mainly found. At 4 h post-injection, the amount of L-Grb2 detected per g of tissue was 64 microg in spleen and 50 microg in liver. Intravenous injection of bcr-abl-positive 32D mouse leukemia cells into radiated NOD/scid mice caused a lethal leukemia syndrome; we determined whether L-Grb2 could prolong the survival of mice bearing such xenografts. One day after leukemia cell inoculation, mice received twice weekly intravenous injections of L-Grb2. At an injection dose of 15 mg of L-Grb2 per kg of mouse body weight, 80% of mice treated with L-Grb2 survived to 48 days (end of study) whereas 0% of mice treated with the same dose of liposomal control oligonucleotide survived; the mean survival duration of these groups was 44 and 20 days, respectively. Our data indicate that L-Grb2 prolonged the survival of mice bearing bcr-abl-positive leukemia xenografts. L-Grb2 may be used as a novel cancer therapeutic modality.

Dual treatment with FLT3 inhibitor SU11657 and doxorubicin increases survival of leukemic mice.

FLT3 is mutated in roughly 30% of human AML. We used our model of APL with activated FLT3 to assess the effectiveness of chemotherapy in combination with SU11657, an inhibitor of FLT3. We found that median survival of untreated and doxorubicin-treated mice was not significantly different. While SU11657 alone increased median of survival to 55 days (P=0.01), dual therapy increased median survival to 62 days (P=0.003) when compared to controls. Neither agent alone or in combination increased survival of control mice. These results suggest that the use of targeted therapeutics can overcome resistance to traditional chemotherapies in AML.

Antileukemic effect of interleukin-7-transduced bone marrow stromal cells in mice following allogeneic T-cell-depleted bone marrow transplantation.

Impaired immune reconstitution following allogeneic bone marrow transplantation (BMT) remains a major obstacle to its clinical application. In this study, interleukin (IL)-7-transduced bone marrow stromal cells (MSC-IL7, 1 x 10(6)/mouse) were transfused into lethally irradiated C57BL/6 recipient mice. By day 40 after transplantation, the recipient mice were challenged with the lymphoma cell line EL4. MSC-IL7 co-transplantation protected recipient mice from leukemic mortality (MST >120 days after BMT vs mean survival time (MST) 70 days in the PBS group) It enhance the PFC count and DTH responses of recipients after transplantation. In conclusion, MSC mediated IL-7 gene therapy and may be a more feasible strategy to restore immune function following allo-TCD-BMT.

Evaluation of nonthreshold leukemogenic response to methyl nitrosourea in p53-deficient C3H/He mice.

The classic controversy of whether genotoxic chemicals induce cancers with or without a certain low-dose limit, i.e., the threshold, is revisited because of a number of current publications available addressing the plausibility of "practical" thresholds even for genotoxic carcinogens, the mechanism of which may be hypothesized to be due, in part, to a repair system composed of ordinarily available various defense mechanisms under the steady-state DNA damage. The question of whether an absolute nonthreshold or a relative nonthreshold, i.e., a "practical" threshold specifically in the low-dose level, is present may not be answered even with the use of a prohibitively large number of wild-type mice. Could the excessive incidence of tumorigenesis in p53-deficient mice contribute to our understanding of the threshold vs nonthreshold issue in genotoxic carcinogenesis? This is considered because an exaggeration of tumorigenesis in p53-deficient mice is hypothesized to reduce or eliminate the range of threshold due to the p53-deficiency-mediated reduction of DNA repair and apoptosis. The present study of chemical leukemogenesis in p53-deficient mice by transplantation assay was designed to answer this question. Briefly, 218 C3H/He mice were lethally irradiated and repopulated with bone marrow cells from wild-type, heterozygous p53-deficient, and homozygous p53-deficient C3H/He mice. This was followed by treatment with a single and graded dose of methyl nitrosourea at 6.6, 14.8, 33.3, 50.0, and 75.0 mg/kg body wt, with the vehicle-treated control groups treated with zero dose for each genotype. Whereas mice repopulated with p53-deficient bone marrow cells showed a marked reduction of the threshold for leukemogenicity, mice repopulated with wild-type bone marrow cells did not exhibit leukemia at a dose of 33.3 mg/kg body wt and showed a curve with a high probability for the linear regression model with a positive dose intercept, predicting a threshold by the likelihood ratio test. Thus, the failure of wild-type mice to show an increase in incidence of leukemogenesis at low doses of genotoxic carcinogens may be due not to a statistical rarity, but to various p53-related pharmacophysiological functions, possibly including DNA repair and apoptosis that may account for a threshold.

[Spontaneous leukosis as a model for an investigation of low and very low physical and physico-chemical effects on oncological process]. / Spontannyi leikoz -- model' dlia izucheniia éffektov malykh i sverkhmalykh doz fizicheskikh i fiziko-khimicheskikh vozdeistvii na opukholevyi protsess.

Kinetic investigation of spontaneous leukosis in AKR mice have been carried in connection with a number of indices: changes in the mass of principal organs of the immune systems (thymus, spleen, lymphatic nodes), liver, alterations of haematological data (the sum of leukocytes, the percentage composition of blood cells, the quantity of undifferentiated cells), changes of physico-chemical conditions in cells (NMR-investigation). The dynamics of some of these indices and also life-spans of animals with leukosis after irradiation with doses 1.2-2.4 cGy (dose-rate 0.6 cGy/day) have been investigated. The enhancement of the frequency of leukosis and shortening the average and maximum life-spans of irradiated mice has been found.

Inhibition of human leukemia in an animal model with human antibodies directed against vascular endothelial growth factor receptor 2. Correlation between antibody affinity and biological activity.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We recently showed that certain 'liquid' tumors such as leukemia not only produce VEGF, but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. A chimeric anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-1C11, was shown to be able to inhibit VEGF-induced proliferation of human leukemia cells in vitro, and to prolong survival of nonobese diabetic-severe combined immune deficient (NOD-SCID) mice inoculated with human leukemia cells. Here we produced two fully human anti-KDR antibodies (IgG1), IMC-2C6 and IMC-1121, from Fab fragments originally isolated from a large antibody phage display library. These antibodies bind specifically to KDR with high affinities: 50 and 200 pM for IMC-1121 and IMC-2C6, respectively, as compared to 270 pM for IMC-1C11. Like IMC-1C11, both human antibodies block VEGF/KDR interaction with an IC(50) of approximately 1 nM, but IMC-1121 is a more potent inhibitor to VEGF-stimulated proliferation of human endothelial cells. These anti-KDR antibodies strongly inhibited VEGF-induced migration of human leukemia cells in vitro, and when administered in vivo, significantly prolonged survival of NOD-SCID mice inoculated with human leukemia cells. It is noteworthy that the mice treated with antibody of the highest affinity, IMC-1121, survived the longest period of time, followed by mice treated with IMC-2C6 and IMC-1C11. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and leukemia. It further underscores the efforts to identify antibodies of high affinity for enhanced antiangiogenic and antitumor activities.

A minimum distance estimation approach to the two-sample location-scale problem.

As reported by Kalbfleisch and Prentice (1980), the generalized Wilcoxon test fails to detect a difference between the lifetime distributions of the male and female mice died from Thymic Leukemia. This failure is a result of the test's inability to detect a distributional difference when a location shift and a scale change exist simultaneously. In this article, we propose an estimator based on the minimization of an average distance between two independent quantile processes under a location-scale model. Large sample inference on the proposed estimator, with possible right-censorship, is discussed. The mouse leukemia data are used as an example for illustration purpose.

Friend leukemia virus infection enhances DNA damage-induced apoptosis of hematopoietic cells, causing lethal anemia in C3H hosts.

Exposure of hematopoietic progenitors to gamma irradiation induces p53-dependent apoptosis. However, host responses to DNA damage are not uniform and can be modified by various factors. Here, we report that a split low-dose total-body irradiation (TBI) (1.5 Gy twice) to the host causes prominent apoptosis in bone marrow cells of Friend leukemia virus (FLV)-infected C3H mice but not in those of FLV-infected DBA mice. In C3H mice, the apoptosis occurs rapidly and progressively in erythroid cells, leading to lethal host anemia, although treatment with FLV alone or TBI alone induced minimal apoptosis in bone marrow cells. A marked accumulation of P53 protein was demonstrated in bone marrow cells from FLV-infected C3H mice 12 h after treatment with TBI. Although a similar accumulation of P53 was also observed in bone marrow cells from FLV-infected DBA mice treated with TBI, the amount appeared to be parallel to that of mice treated with TBI alone and was much lower than that of FLV- plus TBI-treated C3H mice. To determine the association of p53 with the prominent enhancement of apoptosis in FLV- plus TBI-treated C3H mice, p53 knockout mice of the C3H background (C3H p53(-/-)) were infected with FLV and treated with TBI. As expected, p53 knockout mice exhibited a very low frequency of apoptosis in the bone marrow after treatment with FLV plus TBI. Further, C3H p53(-/-) --> C3H p53(+/+) bone marrow chimeric mice treated with FLV plus TBI survived even longer than the chimeras treated with FLV alone. These findings indicate that infection with FLV strongly enhances radiation-induced apoptotic cell death of hematopoietic cells in host animals and that the apoptosis occurs through a p53-associated signaling pathway, although the response was not uniform in different host strains.

Determination of a humane endpoint in the L1210 model of murine leukemia.

The purpose of our study was to derive an alternate end-point to death or moribund appearance for the frequently used L1210 model of murine leukemia. In reviewing the published literature, we were unable to identify a suitable intermediate marker of substantive disease that predicted outcome in the BDF1 recipient of the L1210 leukemia. In an attempt to refine the use of animals in our laboratory, we developed a scoring sheet for behavioral and physical changes that followed intravenous injection of L1210 lymphocytic leukemia cells into BDF1 recipients. At 12-h intervals for the first 2 days after tumor-cell injection and at 6-h intervals thereafter, animals were observed and scored for each parameter. When death was imminent, animals were euthanized by inhalation of methoxyflurane followed by decapitation. Changes in physical and behavioral characteristics then were correlated with the end-point of death. Changes occurred in the mice approximately 7 days after tumor cell inoculation and 24 h before death. The earliest of these signs was hunched posture, followed by one or more other characteristics including decreased activity, increased facial swelling, ears in backward position, abdominal swelling, squinting eyes, and labored breathing. From these data, we were able to develop criteria for early euthanasia. Use of these intermediate end-points likely will substantially reduce the stress on the animals without compromising scientific outcomes in experiments using this or related preclinical models of cancer.