The Aftermath of Surviving Acute Radiation Hematopoietic Syndrome and its Mitigation.

Intensive research is underway to find new agents that can successfully mitigate the acute effects of radiation exposure. This is primarily in response to potential counterthreats of radiological terrorism and nuclear accidents but there is some hope that they might also be of value for cancer patients treated with radiation therapy. Research into mitigation countermeasures typically employs classic animal models of acute radiation syndromes (ARS) that develop after whole-body irradiation (WBI). While agents are available that successfully mitigate ARS when given after radiation exposure, their success raises questions as to whether they simply delay lethality or unmask potentially lethal radiation pathologies that may appear later in time. Life shortening is a well-known consequence of WBI in humans and experimental animals, but it is not often examined in a mitigation setting and its causes, other than cancer, are not well-defined. This is in large part because delayed effects of acute radiation exposure (DEARE) do not follow the strict time-dose phenomena associated with ARS and present as a diverse range of symptoms and pathologies with low mortality rates that can be evaluated only with the use of large cohorts of subjects, as in this study. Here, we describe chronically increased mortality rates up to 660 days in large numbers of mice given LD70/30 doses of WBI. Systemic myeloid cell activation after WBI persists in some mice and is associated with late immunophenotypic changes and hematopoietic imbalance. Histopathological changes are largely of a chronic inflammatory nature and variable incidence, as are the clinical symptoms, including late diarrhea that correlates temporally with changes in the content of the microbiome. We also describe the acute and long-term consequences of mitigating hematopoietic ARS (H-ARS) lethality after LD70/30 doses of WBI in multiple cohorts of mice treated uniformly with radiation mitigators that have a common 4-nitro-phenylsulfonamide (NPS) pharmacophore. Effective NPS mitigators dramatically decrease ARS mortality. There is slightly increased subacute mortality, but the rate of late mortalities is slowed, allowing some mice to live a normal life span, which is not the case for WBI controls. The study has broad relevance to radiation late effects and their potential mitigation and epitomizes the complex interaction between radiation-damaged tissues and immune homeostasis.

Strong selection for cells containing new ecotropic recombinant murine leukemia virus provirus after propagation of C57BL/6 radiation-induced thymoma cells in vitro or in vivo.

Using the Southern procedure, we have studied the presence of ecotropic-specific murine leukemia viral sequences in genomic DNA isolated from primary X-ray-induced thymomas, from lymphoid cell lines established from them, or from secondary tumors passaged in vivo. We found that primary radiation-induced thymomas and infiltrated spleens do not harbor newly acquired ecotropic provirus. However, additional ecotropic proviruses (which appear recombinant in the gagpol region) could be detected in most of the tumorigenic cell lines established in vitro from them and in tumors arising from subcutaneous transplantation of the primary thymomas. These results suggest that primary radiation-induced thymomas may not be clonal. They also indicate a strong correlation between the presence of ecotropic recombinant proviruses in the genome and the growth ability, both in vitro and in vivo, of specific cells within these thymomas, suggesting a possible mitogenic function for murine leukemia virus.

Induction of lymphoma and associated xenotropic type C virus in C57L mice by whole-body irradiation.

Adult C57L mice received sublethal whole-body X-irradiation. Between 3 and 11 months later, 5 of the 7 exposed mice developed histopathologically confirmed malignant lymphomas (lymphocytic type) that primarily involved the thymus. The lymphomas were readily transplantable to other C57L mice of any age, which developed fetal lymphomatous involvement; the tumor cells could also be propagated in tissue culture. A xenotropic murine type C virus (MuX) was isolated from the cultured lymphoma cells after cocultivation with a permissive dog line. MuX activity was demonstrated by electron microscopy, complement fixation, indirect fluorescent antibody, infectivity, and genome rescue.

Provirus integration and myc amplification in 90Sr induced osteosarcomas of CF1 mice.

In mice, endogenous retroviruses are known to be activated during the course of radiation osteosarcomagenesis. Using the Southern blotting procedure, we have studied the presence of somatically acquired proviruses in genomic DNA isolated from seven primary 90Sr induced osteosarcomas and one osteosarcoma cell line, 0-127a1, of the CF1 mouse strain. Specific hybridization probes demonstrated the presence of newly integrated ecotropic proviruses in four primary tumors. Probably, clonally integrated proviruses were present at distinct locations in different subpopulations of tumor cells, reflecting tumor heterogeneity. Genomic DNA isolated from cultured osteosarcoma cells contained different additional MCF-related proviruses. No proviruses were found integrated in the vicinity of c-myc, but a large domain containing the complete c-myc gene was found amplified in one primary tumor (greater than 22 kbp) and in 0-127a1 cells (greater than 39 kbp). Our data suggest that activated retroviruses are not essential for the development of radiogenic osteosarcomas in CF1 mice, but they might be responsible for the deregulated expression of a growth promoting gene in some bone tumor cells.

Identification of malignant cell clones in radio-induced murine thymic lymphomas by viral and cellular probes.

The role of B ecotropic recombinant retroviruses in the emergence and the progression of radio-induced thymic lymphomas was evaluated by analyzing the cell populations present in nine primary and in in vivo propagated tumors. For this, tumor DNAs were analyzed by the Southern method using probes specific for newly acquired proviral sequences, T-cell receptor beta-chain, and immunoglobulin heavy chain genes. Our results show that primary radio-induced tumors are composed of several tumoral cell clones but do not support that malignant cell transformation and proliferation are conferred, solely, by the newly acquired ecotropic recombinant retroviral sequences.

Measurement of binding specificity between T cell lymphomas and murine leukemia viruses.

We have previously reported the presence of receptors on radiation leukemia virus (RadLV)-induced thymomas and malignant thymocytes from AKR mice which specifically bind retrovirus produced by these T cell clones. These receptors have been shown to have specificity for virus reminiscent of an immune-specific receptor. Previous studies on T cell lymphoma binding to retroviruses have involved measurement of the interaction of labelled virus with cells using fluorescence-activated cell sorter (FACS) analysis (McGrath et al., J. Virol. (1978) 25, 923; McGrath and Weissman, Cell (1979) 17, 65; Weissman and McGrath, Curr. Top. Microbiol. Immunol. (1982) 98, 103). Here we report development of an assay for measuring lymphoma binding to virus, prepared as an immunoabsorbent adhered to a microtiter plate. Using this assay, we have shown that only T and not B cell lymphomas can bind to T cell-tropic viruses, and some cell lines have greatest specificity for homologous virus. The AKR-derived T cell lymphomas, SL3 and KKT-2, show greater specificity for leukemogenic AKR viruses, than for an AKR xenotropic virus or the recombinant AKR virus, MCF247. The RadLV-induced T cell lines, C6VL/1 and BL/VL3, have been found to bind cross-reactively to several different murine leukemia viruses (MuLVs). RadLV-induced T cell lymphomas do have greater specificity for their cognate retroviruses since free, homologous retrovirus can best block the interaction between cells and virus adhered to the wells of a microtiter plate. Cross-reactive interactions are more easily demonstrated by this assay, probably because low avidity interactions are stabilized as a result of the mode of virus presentation. Binding specificity for retroviral envelope determinants has been demonstrated using a rat anti-retroviral antiserum prepared as an F(ab)1 fragment. This antiserum can inhibit the interaction between the C6VL/1 thymoma and its RadLV virus. Specificity of this antibody for a gp70-like protein was confirmed by NaDodSO4-polyacrylamide gel electrophoresis (PAGE) and by loss of this activity after absorption of antibody on virus. Antibodies specific for RadLV/VL3 gp70 determinants can inhibit the interaction of C6VL/1 with RadLV/VL3 suggesting that cross-reactive binding to heterologous virus is also specific for a gp70 viral env determinant.

Patterns of thymocyte differentiation markers on virus and radiation induced lymphomas of C57BL/Ka mice.

To better understand the biology of tumorigenesis in virus and radiation lymphomas of C57Bl/Ka mice, we have examined the cell surface phenotypes of a large series of primary tumors induced by both agents. Data derived using flow cytometry and recently available monoclonal antibodies to thymocyte differentiation antigens supports three major conclusions. First, tumor cell populations are unimodal for staining with most antibodies and are probably of clonal origin. Second, many, but not all, tumor cells show surface phenotypes similar to those of previously defined subpopulations of normal thymocytes. Third, at the cell surface level, no major differences between virus- and radiation-induced lymphomas can be discerned. Our data thus further define the relationship between thymomas induced by these two agents.

Involvement of peritoneal macrophages and spleen stromal cells in X-irradiation-induced reticulum cell neoplasms in C57BL/6 mice.

Some correlation was observed between the occurrence of FA-positive PE-MO and spleen stromal cells (removed from X-irradiated RCN-bearing old-adult B6 mice) and the generation of RCN. No significant correlation was found between the viral content of lymphoid organs from the same mice and the occurrence of RCN. The main viral particle detected in lymphoid organs from radiation-induced RCN-bearing mice was the xenotropic virus. Ecotropic viruses were detected in a few spleens and Payer patches from such mice. These ecotropic viruses showed very poor lymphomagenic activity and required 400R X-ray as a cofactor. No dualtropic viruses were detected. However, inoculation of ecotropic (SFA2) helper virus to X-irradiated old-adult B6 mice, resulted in an efficient rescue of lymphomagenic viruses, enriched with phenotypically mixed, dualtropic viruses. Some of these DT viral preparations were cloned and seemed to consist mainly of xenotropic sequences. Thus, inoculation of helper viruses influenced the generation and selection of DT viruses. Such viral preparations, enriched with DT viruses, had a better lymphomagenic activity compared to endogenous ecotropic viruses, isolated from radiation-induced RCN-bearing mice. Indirect evidence suggested the involvement of a defective (xenotropic and possibly adjacent cellular genes) particle in lymphoma induction. To conclude, a possible mechanism for the development of radiation-induced RCN is suggested, emphasizing the role of MO in such a process.

Structure of endogenous retroviruses expressed in radiation-induced and spontaneous murine bone tumours.

The molecular structure of murine retroviruses expressed in spontaneous and radiation-induced bone tumours was studied. These viruses induce osteomas, lymphomas and osteopetrosis in mice of the NMRI strain. RNase T1 fingerprint analysis indicates the presence of mixed virus populations in the tumours, with major components showing close relationship to Akv MuLV. Cloned viruses, closely related to Akv MuLV, have the same oncogenic properties as the original mixtures. In its nucleotide sequence of the repeat segments of the transcriptional enhancer in the LTR, one cloned virus analysed was distinct from Akv MuLV, but closely related to a spontaneous bone tumour virus isolate, FBJ MuLV.

Common proviral integration sites in C57BL mouse lymphomas induced by radiation leukemia virus and absence of novel virus-related sequences in radiogenic lymphoma DNA.

Three C57BL/Ka mice were inoculated with RadLV/VL3, a thymotropic and leukemogenic virus population released by the permanent BL/VL3 cell line, which was derived from a C57BL/Ka lymphoma induced by radiation leukemia virus (RadLV). The neoplastic thymus and bone marrow cells from these mice were grown until the cultures became permanently established, and their DNAs were examined for the presence of virus-related sequences by restriction enzyme analysis. All six cell lines displayed identical EcoRI and BamHI restriction fragments, not found in control C57BL/Ka DNA and accounting for the presence of more than one novel provirus. The primary and secondary tumors were thus clonal and, even though they were obtained from different animals, possessed identical integration sites. The BL/VL3 cell line also displayed the clonal appearance of novel proviral sequences, partly identical, with respect to location and BamHI restriction pattern, to those found in the RadL/VL3-induced tumor cell lines. Neither radiation-induced tumors, nor cloned cell lines derived therefrom, whether producing leukemogenic virus (BL/RL12-P) or not (BL/RL12-NP), displayed the presence of novel virus-related sequences when compared with control tissues. Our results strongly suggest not only that, as described in the case of avian leukosis virus-induced tumors of the chicken, RadLV-induced leukemogenesis might be a consequence of cellular gene activation by promotor insertion, but also that more than one integration site might be involved. Radiation-induced tumorigenesis might be initiated by a comparable mechanism, not requiring the participation of a virus.

Evidence of recombinant ecotropic provirus integration in thymic lymphomas induced by direct or indirect radiation effects.

Several investigators described the occurrence of ecotropic recombinant proviruses in the DNA of in-vivo or in-vitro propagated radio-induced lymphomas, but such proviruses were never detected in primary tumors. To assess their biological significance in the tumorigenic process, we reinvestigated the presence of new proviruses chiefly in primary radio-induced tumors and in models of radioleukemogenesis which could give additional support for their role. Such models included thymic lymphomas originating after (i) graft of non-irradiated thymuses in thymectomized irradiated mice and (ii) the injection of a B-ecotropic retrovirus (T1223/B) in association with a subleukemogenic dose of irradiation. We report for the first time that new ecotropic proviral sequences are encountered in a significant number (30%) of primary lymphomas induced directly by irradiation or indirectly in non-irradiated thymuses grafted in irradiated hosts. The existence of a 3.5-kbp Kpn1 restriction fragment with ecotropic sequences in the digested DNA of these tumor cells indicates that these new sequences belong to an ecotropic provirus recombinant in the gag-pol region. We observed that most of the primary radio-induced tumors in which novel recombinant provirus could be detected, displayed the integration at a single or at a few sites, demonstrating their clonality with respect to viral integration. The same was observed in thymic lymphomas arising after T1223/B virus injection and irradiation and in in-vivo or in-vitro propagated tumors. Altogether, these data bring the first evidence of the integration of ecotropic recombinant proviral genomes in a significant number of primary radiation induced thymic lymphomas and of their possible role in view of their frequent occurrence in grafted thymomas.

Activation and biological properties of endogenous retroviruses in radiation osteosarcomagenesis.

The activation of endogenous retroviruses (MuLV) by internal irradiation and the presence of activated retroviruses in radiation-induced murine osteosarcomas as well as their biological properties in vivo and in vitro were studied. Ecotropic and xenotropic MuLV were expressed dependent on the radiation dose in spleen, bone marrow and bone tissues of C57Bl/6 mice after 224Ra treatment. Radiation-induced osteosarcomas of BALB/c, C57Bl/6 and C3H X 101/F1 mice harboured infectious ecotropic and/or xenotropic viruses whereas in osteosarcomas of NMRI mice predominantly virus protein could be detected. In about 50% of the radiation-induced osteosarcomas of BALB/c mice an amplification of ecotropic proviruses could be detected. This was not found in clonally grown cells from non-tumorous tissues. MuLV from radiation-induced osteosarcomas induced osteopetrosis, osteomas and lymphomas after infection of newborn NMRI mice. In differentiating bone tissue the viruses were found to infect predominantly osteoblast precursor cells suggesting that virus infection results in increased growth and metabolic activity of these cells, which may be a possible mechanism for their pathogenic action in bone tissues.

Establishment and characterization of C-type RNA virus-producing cell lines from radiation-induced murine osteosarcomas.

Eight cell lines were established from murine osteosarcomas induced in vivo with the radionuclides 224Ra and 227Th. They have been compared by light and electron microscopy, by karyology, and by their growth properties. The morphology, the growth pattern, and the ability to induce tumors in mice indicate that five of them are tumor cell lines. Chromosome studies demonstrated that the five cell lines have marker chromosomes. The other cell lines only showed some criteria generally used to score for transformation of fibroblasts and they may be derived from stromal cells. All cell lines release virus particles in the culture fluid which have the typical properties of RNA tumor viruses. They possess C-type morphology, a density of 1.16--1.18 g/cm3, a 60--70 S RNA, a RNA dependent DNA polymerase and they induce syncytia in rat XC cells. The possible significance of these virus particles in radiation osteosarcomagenesis is discussed.

Lack of evidence for the involvement of type-C and type-B retroviruses in radiation leukemogenesis of NFS mice.

Southern blot analysis revealed no difference between the DNA from radiation-induced thymic lymphomas and DNA from normal NFS mice. The probes used in the Southern blot analyses used a murine leukemia virus (MuLV) env DNA probe (pXenv), which specifically hybridizes with xenotropic and recombinant viral env genes, and mouse mammary tumor virus (MMTV) DNA probes (MMTV gag-pol, MMTV env, and MMTV LTR). This suggests that radiation leukemogenesis was not associated with gross alteration of the organization of these retroviral genomes. In DNA from radiation-induced thymic lymphoma, there was no indication of gross rearrangement in the common integration site of MuLV, pim-1, or in the common integration sites of MMTV, int-1 and int-2. Dot blot analysis of RNA from radiation-induced thymic lymphomas and normal thymuses demonstrated that there was no substantial difference between them in the expression of retroviral sequences, pim-1, pvt-1, int-1, or int-2, although transcripts that could be hybridized to the retroviral sequences were slightly elevated in some radiation-induced thymic lymphomas. These results show that radiation leukemogenesis does not appear to involve the activation of endogenous type-C and type-B retroviruses.

Pathogenesis of radiation and virus-induced bone tumors.

Bone cancer can be induced by radionuclides that localize in the skeleton. Histologically, these experimentally induced tumors resemble those found naturally in man; they range from densely ossified osteogenic sarcomas to osteolytic tumors with giant cells and only a small osteoid component. Fibrosarcomas and hemangiosarcomas also can occur in some species. It has not been possible to determine the dose in terms of absorbed energy necessary for bone-tumor induction because radionuclides are not deposited uniformly, and they diminish in amount with time. Also, the precise time when irreversible noeplastic change occurs is not known. With X-rays, however, 500 rads delivered to the endosteal surface of a mouse femur has been shown to cause osteogenic sarcoma. Bone tumors can be induced in mice by viruses. FBJ osteosarcoma virus and RFB osteoma virus were obtained from spontaneous tumors; FBR osteosarcoma virus came from a radiation-induced tumor. All three are RNA viruses with C-type particle morphology, and they are propagated by injecting cell-free extracts of virus-induced tumor. All three are RNA viruses with C-type particle morphology, and they are propagated by injecting cell-free extracts of virus-induced tumor into newborn mice. Interaction studies with bone-seeking radionuclides and these viruses have led to the hypothesis that radiation produces cancer by inactivating a viral inhibitor. There is also evidence of a bone tumor virus in the human disease. The injection of cell-free extracts of human bone cancer into newborn Syrian hamsters has induced a variety of mesenchymal tumors at a rate significantly higher than in the control hamsters. Sixty tumors of this type, including 20 osteosarcomas, 11 fibrosarcomas, and 9 osteomas, have been diagnosed so far in experimental animals; in control hamsters there has been only one, a fibrosarcoma. Immunofluorescence assays and cytotoxicity studies indicated that these hamster tumors carried a human antigen.

Amplification of endogenous proviral MuLV sequences in radiation-induced osteosarcomas.

The endogenous ecotropic provirus of BALB/c mice was found to be amplified in 17 out of 29 radiation-induced osteosarcomas. In contrast, 19 clonal cell lines established from bone-marrow cells of a tumor-bearing mouse, which were used as controls, did not reveal newly acquired ecotropic proviruses. Ecotropic viral RNA was expressed in tumors that showed reintegrated proviruses. DNA probes from 2 tumors, derived from cellular sequences flanking the newly integrated proviruses, did not detect DNA rearrangements in any of the other tumors. The possible role of activated endogenous retroviruses in the development of radiation-induced osteosarcomas is discussed.

[Isolation and characterization of retroviruses expressed in murine osteosarcomas induced by 90Sr]. / Isolement et caractérisation de rétrovirus exprimés dans les ostéosarcomes murins induits par le 90Sr.

The induction of osteosarcomas with 90Sr in CF1 mice is associated with the expression of ecotropic type-C RNA viruses devoid of sarcomatogenic activity. In contrast, the FBR murine osteosarcoma virus complex, isolated from a 90 Sr-induced osteosarcoma of a X/Gf mouse [M. Finkel et al. (1)], causes the rapid appearance of osteosarcomas in newborn mice and transforms fibroblasts in vitro. The transforming capacity of FBR murine sarcoma virus has been associated as an oncogene homologous to v-fos.

Viruses in osteosarcomas induced by 226Ra. A study of the induction of bone tumours in mice.

Three groups (15 in each) of 3-month-old mice were injected intraperitoneally with 0-5 muCi 226Ra. They were male and female C3H/H and female CBA/H. The groups were matched by similar controls that were not injected. Osteosarcomas were found in 14/15 of the female C3H/H mice--animals known to carry mammary-tumour viruses in milk--compared with 6/15 in their male counterparts. In CBA/H female mice, which have a very low incidence of both mammary tumours and leukaemia, osteo-sarcomas developed in 7/15 of the animals. The CBA/H mice lived about 5-8 months longer and revealed their osteosarcomas at a correspondingly later time than C3H/H mice. Virus particles were observed in each of the three mammary tumours and in 15 out of the 17 osteosarcomas examined by electron microscopy. No definitive statement can be made from this experiment whether or not osteosarcoma was caused by a virus; but the results suggest that viruses may be a contributory factor in the development of osteosarcoma in these animals.

Implication of human papillomavirus in postirradiation dysplasia.

The presence of human papillomavirus (HPV) DNA and association of condylomata acuminata (CA) in the biopsy tissues of postirradiation dysplasia (PRD) of the cervix and/or vagina from 17 patients who previously had radiation therapy for malignancies of the uterine cervix, vagina, and endometrium were evaluated with DNA in situ hybridization. Eight of 17 patients (47.1%) had HPV DNA identified in the lesions of postirradiation dysplasia (PRD). Five of eight cases (62.5%) contained HPV DNA of more than one type. Type 16 HPV DNA (HPV-16) was the most frequently identified type. Several PRD lesions also contained HPV-6, HPV-18, HPV-31, and/or HPV-33 DNA. Eleven patients (64.7%) showed CA in the vicinity of PRD. In two cases, different types of HPV were found in the lesions of PRD and contiguous CA. The frequency of the cases containing HPV DNA, the types of HPV, and the distribution pattern of silver grains in the preparations of in situ hybridization over the nuclei of cells of PRD were very similar to those found in naturally occurring dysplasia. Based on these findings, persistent or repeat HPV infection was the most likely etiologic factor of PRD, which might be facilitated by immunosuppression due to pelvic irradiation.