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.

Nonmyeloablative allogeneic bone marrow transplantation as immunotherapy for hematologic malignancies and metastatic solid tumors in preclinical models.

OBJECTIVE: We previously demonstrated that a combination of mild total lymphoid irradiation (TLI) with selective depletion of the host's donor-reactive cells allows for stable and graft-vs-host disease (GVHD)-free engraftment of allogeneic bone marrow (BM). In this study, we investigated the efficacy of this nonmyeloablative strategy for BM transplantation (BMT) as immunotherapy for minimal residual disease. MATERIALS AND METHODS: BALB/c mice inoculated with leukemia (BCL1) or breast carcinoma (4T1) cells were conditioned for BMT with TLI (200 cGy) followed by priming with donor (C57BL/6) BM cells on day 1, and by injection with 200 mg/kg cyclophosphamide on day 2. After conditioning (day 3), recipients were transplanted with BM cells from the same donor. Treated animals were monitored for 230 days for survival, development of leukemia/solid tumor, and GVHD. RESULTS: BMT converted the mice to complete chimeras and prevented development of leukemia in 90% of recipients and locally growing breast carcinoma in 40% of the mice. Immunization of donors of the second BM with 4T1 cells prevented development of breast carcinoma in 80% of 4T1 inoculated mice. Fewer animals treated for malignancy by nonmyeloablative BMT died of GVHD than those treated by myeloablative BMT. However, late GVHD-related mortality in mice treated for leukemia was higher than after nonmyeloablative BMT to naive recipients (p < 0.00001). Infusion of host-type anti-donor immune lymphocytes 8 days after BMT improved the survival of recipients treated for leukemia without affecting engraftment and the graft-vs-leukemia potential of donor BM. CONCLUSIONS: Effective eradication of malignant cells can be achieved following allogeneic BMT after nonmyeloablative conditioning.

The influence of radiation and chemotherapy-related DNA strand breaks on carcinogenesis: an evaluation.

PURPOSE: DNA strand breaks are believed to induce carcinogenesis. This study was conducted to analyze induction and repair of irradiation- and chemotherapy-related strand breaks in vitro. METHODS: Friend Leukemia cells were exposed to irradiation and various chemotherapeutic agents at different doses and concentrations. Occurrence of strand breaks was determined fluorometrically, measuring the rate of DNA unwinding immediately after exposure and 24 hours later. RESULTS: The amount of double-stranded DNA decreased significantly for irradiation, doxorubicin, dactinomycin and etoposide (p < or = 0.05, t-test). After 24 hours free of exposure, the persistent damage was detectable for all of these agents but not for irradiated cells, with DNA strand breaks being decreased for etoposide, unchanged for doxorubicin and increased for methotrexate as well as for dactinomycin. CONCLUSIONS: Severe DNA damage is induced by various chemotherapeutic agents and by irradiation. While repair of chemotherapy-related strand breaks may remain incomplete or prolonged for some chemotherapeutic agents, repair of radiation induced strand breaks is faster and more complete. Therefore chemotherapy-related carcinogenesis may partially be explained by prolonged persistence of DNA strand breaks.

Interactions of steroid, methotrexate, and radiation determine neurotoxicity in an animal model to study therapy for childhood leukemia.

Children with leukemia receive CNS therapy to improve long-term survival. Neurotoxic effects, such as cognitive impairment, have been associated with this therapy. A rat model was developed to determine which agent, or combination of agents, in CNS therapy causes neurotoxicity. The agents examined were cranial irradiation (1000 cGy), methotrexate (2 or 4 mg/kg, intraperitoneally), and prednisolone (18 or 36 mg/kg, intraperitoneally). Young Sprague-Dawley rats were exposed to each agent alone or to two- or three-agent combinations. Each therapy had matched controls that received sham radiation and/or intraperitoneal saline. Subsequent to exposure, spontaneous behavior was tested using a computer pattern recognition system, which recorded and classified behavior in a novel environment. Behavioral initiations, total times, and time structures were compared in therapy and control groups. Combined rather than single-agent therapies had more behavioral effects, and these were dose- and sex-dependent. Synergistic interactions between agents caused behavioral deficits, and components of the combination determined the abnormality. Some combinations interacted antagonistically, and thus mitigated behavioral deficits. Prednisolone was clearly pivotal to behavioral outcome. A low prednisolone dose antagonized methotrexate preventing deficits, whereas a higher prednisolone dose altered behavior by enhancing effects of methotrexate and radiation. These findings emphasize that steroids are important in agent interactions. Their role in morbidity associated with leukemia treatment protocols may be equally important as that of methotrexate and cranial irradiation.

Hemopoietic neoplasms in lethally irradiated mice repopulated with bone marrow cells carrying the human c-myc oncogene: a repopulation assay.

Bone marrow (BM) cells from two transgenic mice carrying the human c-myc oncogene were separately harvested, and each sample was injected into 25 lethally irradiated mice. We observed the contribution of the myc gene to the occurrence of hemopoietic neoplasms in the BM-repopulated mice, establishing a new experimental system for analyzing oncogene expression in the hemopoietic system in vivo. The hybrid gene that was transferred into the original transgenic mice was a combination of the human c-myc gene with a regulatory unit consisting of a murine immunoglobulin-heavy chain with an SV40 early-T promoter gene (Ig/Tp-myc). Among the transgenic lines, the tested BM cells were chosen from two lines that had been low-prone in leukemia; in these lines hemopoietic neoplasms did not appear for greater than or equal 200 days after birth. Lethally irradiated controls received BM cells from litters of transgenic mice that did not carry c-myc. The lifetime incidence of hemopoietic neoplasms was 94% and 91% in the two groups of mice repopulated with myc+ BM. By contrast, only 15% of control mice with myc- BM developed hemopoietic lesions. The incidence of hemopoietic malignancies combined with nonthymic lymphomas and myeloma cases (88% and 65%) was higher in the repopulated mice than the incidence of pre-B cell lymphomas in the original transgenic lines (56%). Thirty-two of the 40 myc+ mice that were examined showed the presence of the transferred gene in either the normal hemopoietic tissue or in the hemopoietic neoplasm. Furthermore, 18 of 22 hemopoietic neoplasms studied by Northern hybridization expressed mRNA from the transgenic gene; in other four neoplasms, expression was weak or absent.

Effect of cyclophosphamide, total body irradiation, and zidovudine on retrovirus proliferation and disease progression in murine AIDS.

Murine acquired immunodeficiency syndrome (MAIDS) develops when C57B1/6 mice are inoculated with LP-BM5 murine leukemia viruses. Disease progression in these animals is characterized by lymphadenopathy, polyclonal B-cell activation, severe immunodeficiency, and death. Mice with MAIDS have been used to examine the efficacy of antiretroviral therapies for possible use in AIDS patients. In the present work, MAIDS mice were employed to test the hypothesis that established retroviral infection might be cured by the combined use of a cytotoxic agent (cyclophosphamide) and total body irradiation--a regimen reported to have successfully cured HIV-1 infection in one AIDS patient. Results indicate that the ablation of retrovirus-infected lymphoid cells reduced but did not eliminate LP-BM5 infection. Moreover, this regimen was no more effective at controlling virus proliferation or preventing the polyclonal IgG activation characteristic of murine AIDS than was AZT alone.

Time-gated fluorescence spectroscopy of porphyrin derivatives and aluminium phthalocyanine incorporated in vivo in a murine ascitic tumour model.

The effect of systemic administration on drug uptake at cellular level was evaluated using time-gated fluorescence spectroscopy performed on a murine ascitic tumour model. Mice bearing L1210 leukaemia were injected intraperitoneally or intravenously with 25 mg per kg body weight hematoporphyrin derivative (HpD), 12.5 mg per kg body weight photofrin II (PII), 25 or 5 mg per kg body weight disulphonated aluminium phthalocyanine (AlS2Pc). Every 2 h and for up to 22 or 30 h, mice were sacrificed, leukaemic cells extracted from the peritoneum, washed, and resuspended in buffer for fluorescence measurements. HpD and PII emission spectra were almost identical 12 h after intraperitoneal injection with main peaks at 630 nm and no appreciable changes afterwards. In the first 12 h, the PII fluorescence spectrum was constant, while in the case of HpD a shoulder at 615 nm was detectable. Similar fluorescence behaviour was observed after intravenous administration of porphyrin derivatives. These results seem to confirm that the tumour localizing fraction is the part actually retained by the cells. The AlS2Pc spectrum peaked at 685 nm and did not change in any of our experiments. AlS2Pc is incorporated more rapidly with respect to porphyrins, as was clearly observed in the case of intravenous administration, where the AlS2Pc fluorescence was readily detectable after 2 h, whereas the PII emission became apparent only after 4-6 h.

Cure with low-dose total-body irradiation of the hematological disorder induced in mice with the Friend virus: possible mechanism involving interferon-gamma and interleukin-2.

The effects of split low-dose total-body irradiation (TBI; 150 cGy) on production of interferon (IFN)-gamma and Interleukin-2 (IL-2) and on the growth characteristics of erythroid progenitor cells (BFU-E) have been assessed in normal mice, normal mice receiving TBI only, mice infected with the polycythemia-inducing strain of the Friend virus complex (FVC-P), and FVC-P infected mice receiving 150 cGy TBI on days 5 and 12. It was found that lymphocytes from the spleens of TBI-treated mice previously infected with FVC-P produced in response to phytohemagglutinin (PHA) and concanavalin A (Con A) stimulation up to 15 times greater amounts of IFN-gamma than cells from untreated FVC-P-infected mice. IL-2 production in Con A-stimulated spleen cell cultures also increased when cells were isolated from FVC-P-infected mice treated by low-dose TBI compared to untreated FVC-P-infected mice. TBI treatment was associated with greater than 99% ablation of "erythropoietin-independent" BFU-E colony formation. The results suggest that the cure of FVC-P-infected mice by low-dose TBI may result from activation of the IFN-gamma system and IL-2 production.

Effect of split low dose total body irradiation on SFFV mRNA, genomic DNA and protein expression in mice infected with the Friend virus complex.

DBA/2 mice infected with lethal dosages of Friend virus complex (FVC) can be 100% cured by split-dose total body irradiation (TBI) at 150 cGy, an effect associated with the restoration of the cellular immunity which is compromised by the virus. The exact mechanism underlying the curative effect is unknown, but it may involve the interferon (IFN) system and interleukin-2 (IL-2) production. Initial studies indicated that TBI did not directly inactivate the virus, suggesting that irradiation either acted on the target cells for virus replication or on other cells mediating the effect. We have now examined the effect of this relatively low dose TBI on replication, transcription, and protein expression of the Friend virus. Northern blot analysis revealed that in FVC infected mice treated with curative low dose TBI, no spleen focus-forming virus (SFFV)-specific mRNA species were detected. Southern blot analysis revealed that a 6.0 kb SFFV fragment could be detected in infected, untreated spleen cells, but not in cells from FVC-infected mice treated with TBI, or in uninfected spleen cells. Western blot analysis revealed that the SFFV envelope glycoprotein was expressed in the spleen cells from untreated FVC infected mice, but not in the cells from TBI treated FVC infected mice. These results, consistent with our previous findings of greatly reduced spleen focus forming units in mice with FVC which had been treated with this regimen of TBI, suggest the possibility of using such treatments in other retroviral associated disorders.

Induction of immune resistance against L1210 lymphatic leukemia in mice after lethal irradiation and reconstruction with fetal liver cells.

The immunohematopoietic potential of syngeneic fetal liver cells (SFLC) was examined and compared with syngeneic bone marrow cells (SBMC). SFLC generated about 3 times less 12th-day spleen colonies (CFU-S) than adult SBMC did. To test the SFLC ability for reconstitution of the immune system, mice were lethally total body irradiated (TBI) and transplanted i.v. with 3 x 10(7) SFLC or 1 x 10(7) SBMC. Thus, injected hematopoietic cells contained the same number of CFU-S. On days 28, 35, 42, and 49 after transplantation the mice were injected i.p. with 10(6) immunogenic L1210-Maf cells (L1210 leukemia cells treated in vitro with mafosfamide for inhibition of their growth in vivo) to test the ability for generation of immune response against L1210 leukemia. On day 56 the animals were challenged with 10(3) L1210 leukemia cells. Strong resistance against the leukemia was induced in TBI + SFLC and TBI + SBMC mice, suggesting that the SFLC similarly as SBMC are able to reconstitute immune system of the TBI host.

Induction of donor-type chimerism in murine recipients of bone marrow allografts by different radiation regimens currently used in treatment of leukemia patients.

Three radiation protocols currently used in treatment of leukemia patients before bone marrow transplantation (BMT) were investigated in a murine model (C57BL/6----C3H/HeJ) for BM allograft rejection. These include (a) a single dose of total body irradiation (8.5 Gy TBI delivered at a dose rate of 0.2 Gy/min), (b) fractionated TBI (12 Gy administered in six fractions, 2 Gy twice a day in 3 days, delivered at a dose rate of 0.1 Gy/min, and (c) hyperfractionated TBI (14.4 Gy administered in 12 fractions, 1.2 Gy three times a day in 3 days, delivered at a dose rate of 0.1 Gy/min). Donor-type chimerism 6 to 8 weeks after BMT and hematologic reconstitution on day 12 after BMT found in these groups were compared with results obtained in mice conditioned with 8 Gy TBI delivered at a dose rate of 0.67 Gy/min, routinely used in this murine model. The results in both parameters showed a marked advantage for the single dose 8.5 Gy TBI over all the other treatments. This advantage was found to be equivalent to three- to fourfold increment in the BM inoculum when compared with hyperfractionated radiation, which afforded the least favorable conditions for development of donor-type chimerism. The fractionated radiation protocol was equivalent in its efficacy to results obtained in mice irradiated by single-dose 8 Gy TBI, both of which afforded a smaller but not significant advantage over the hyperfractionated protocol. This model was also used to test the effect of radiation dose rate on the development of donor-type chimerism. A significant enhancement was found after an increase in dose rate from 0.1 to 0.7 Gy/min. Further enhancement could be achieved when the dose rate was increased to 1.3 Gy/min, but survival at this high dose rate was reduced. These results demonstrated indirectly that dose rate affects the expression of host-type pluripotent stem cells, the progeny of which appear 3 to 6 weeks after treatment with 8 Gy TBI delivered at a dose rate of 0.1 Gy/min, but which are eradicated if radiation is delivered at a dose rate of 1.3 Gy/min.

Yttrium-90-EDTMP: a radiotherapeutic agent in the treatment of leukaemias.

Yttrium-90 chelated by the tetraphosphonate EDTMP achieved a high uptake in bone and a rapid clearance from all soft tissues compared with 90Y nitrilotriacetate, citrate and acetate. The biological half-life of 90Y in the bone was greater than 72 h, but the quantity, and therefore dose, could be reduced by 50% using repeated, non-toxic chelation therapy with the calcium salt of DTPA. This treatment should be able to supplement current treatments for leukaemia where the dose of external beam radiation is associated with considerable morbidity.

Low dose total body irradiation: a potent anti-retroviral agent in vivo.

Immunosuppression characterizes many human diseases including leukemia and AIDS. Friend virus (FV)-induced murine leukemia is a useful model for studying both malignancy and immunosuppression. In a previous series of experiments, we have demonstrated that untreated FV-infected mice died within 40 days post-infection, whereas infected mice given 150 cGy total body irradiation (TBI) on days 5 and 12 exhibited long-term survival. In this report, we show that no leukemic cells or type C virus particles are found in the spleens of mice treated with TBI. In addition, both NK activity as well as bone marrow cell's proliferative responses to PHA and Con A were fully restored. This treatment produces long term control of FV-induced murine leukemia, and thus might have relevance for the treatment of a number of immunosuppressive diseases including AIDS.

Curative effect of split low dosage total-body irradiation on mice infected with the polycythemia-inducing strain of the Friend virus complex.

Split low dose total-body irradiation (TBI) with 150 cGy was assessed for its efficacy in modifying the disease induced in DBA/2 mice by the polycythemia-inducing strain of the Friend virus complex (FVC-P, composed of a Friend murine leukemia helper virus and a spleen focus-forming virus). All FVC-P injected mice were dead within 40 days; however, infected mice receiving TBI on days 5 and 12 exhibited long-term survival. FVC-P-injected mice receiving TBI treatment on days 5 and 12 had normal leukocyte counts, normal spleen weights, and no detectable spleen focus-forming virus. Although the FVC-P-infected mice had decreased proportions of L3T4+ cells and increased proportions of Lyt-2+ cells, these were returned to normal following TBI treatment. Apparently the time sequence of TBI treatments is important since one treatment with TBI on day 5, or two treatments with TBI on days 12 and 18, was not as efficacious. The inability of in vitro irradiation doses of up to 1000 cGy to inactivate FVC-P which was subsequently injected into murine hosts suggests that the effectiveness of the TBI treatment in vivo is not due to a direct radiation effect on the virus. These results indicate a possible relationship between L3T4+ and Lyt-2+ numbers or their ratio in the curative efficacy of TBI in FVC-P-infected mice.

Radiation sensitizing and radiation protective agents in experimental radiation therapy.

It appears that AB-132 potentiates radiation effects, and the local application of AET (MEG) protects the intestinal tract, the bladder and the skin against radiation toxicity. Combined use of selective radiation sensitizing and protecting agents may be considered for clinical studies.

Specific radioimmunotherapy using 90Y-labeled monoclonal antibody in erythroleukemic mice.

Radioimmunotherapy using 90Y-labeled diethylenetriamine pentaacetic acid-antibody conjugates was studied in Rauscher erythroleukemia virus-infected mice. Preliminary experiments showed that biodistribution profiles for nonrelevant mouse monoclonal antibody and polyclonal bovine immunoglobulin were identical in both normal and leukemic mice. Therefore, bovine immunoglobulin G was selected as the control immunoglobulin in order to permit comparison to current clinical trials of radioimmunotherapy regimens. Specific monoclonal antibody was two- to three-fold more potent than bovine immunoglobulin G in therapy, as assessed by reduction of splenomegaly (dose required for half-maximal effect, 9 microCi versus 16 to 27 microCi). Mice treated with 50 microCi 90Y-labeled control immunoglobulin had spleens which were twice the normal size and showed extensive areas of erythropoiesis indicative of the presence of tumor foci; in contrast, doses as low as 27 microCi 90Y-labeled specific antibody resulted in complete remission with no microscopic evidence of tumor foci in either spleen or liver. Although reversible marrow toxicity was observed it was not dose limiting. These results demonstrate that tumor-specific therapy is possible using 90Y-labeled antibody.