Proviral Integration Pattern Research Articles

441. Direct Comparison of EF1α and PGK Promoters Reveals Superior Performance of the PGK Promoter for Expression of the Common Gamma Chain in a Canine Model of In Vivo Foamy Virus Gene Therapy for Severe Combined Immunodeficiency

Foamy viruses (FV) are ideal vectors for in vivo gene therapy because FV infection is non-pathogenic in people, FV are more resistant to serum inactivation than lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and FV exhibit favorable proviral integration patterns compared to gammaretroviruses. In vivo gene therapy with a FV vector expressing the human common gamma chain (γC) under the short EF1α promoter results in a functional immune reconstitution in X-SCID dogs.

The Deletion of the Proline-Rich Region of HOXB4 Is Associated with Myeloid Leukemia in a Mouse Model

HOXB4 belongs to the family of homeobox transcription factors, which play a key role in hematopoietic development. The expression of HOXB4 induces a significant increase of long-term repopulating stem cells (SC) in human and mouse models, without inducing malignant transformation. So far the underlying mechanisms of the SC amplificatory impact of HOXB4 are poorly understood. In an attempt to understand the unique characteristics of HOXB4, we performed a mutational study by deleting its proline-rich region, which has been described to act as a transcriptional activation domain in many other proteins, like non-homeobox genes (e.g. p53, AP2) and other homeobox genes (e.g. HOXD4 and HOXA13). We performed in vitro and in vivo experiments transducing murine 5-FU enriched HSCs with the pMSCV-IRES-GFP based retroviral vector harbouring the HOXB4 wild-type (wt) and several mutants, including a Δproline HOXB4 mutant (ΔP), where the proline-rich sequence (50% P) between the amino acidic positions 71–120 in the exon 1 is deleted. In previous experiments, when HOXB4-ΔPro (n=14) was over expressed in 5-FU enriched progenitor cells from BM, we reported a significant decrease (75fold, p<0.05) of the 12 days Δ-CFU-S frequency, in comparison to the HOXB4-wt (n=5), while it still generated significantly more Δ-CFU-S in comparison to the GFP control (n=11) (35fold, p<0.0003). Furthermore, we performed the CRU assay by transplanting lethally irradiated C3H×C57Bl/PeB mice with serial dilutions of 5-FU isolated bone marrow progenitor cells, in order to evaluate the effect of the HOXB4-ΔPro on the competitive repopulating unit frequency. At the 16th week post transplantation we reported no significant difference in the CRU frequency between the mice receiving HOXB4wt (CRU 1/834, n=18) and the mice receiving HOXB4-Δpro (CRU 1/413, n=18) expressing transplants. However, in mice transplanted with HOXB4wt (n=12) 45.3 % of the circulating cells belonged to the transduced compartment compared to 19.2% in the HOXB4-Δpro group (n=13) (p<0.006), whereas the lineage distribution within the transduced compartment did not differ between both experimental arms 16 wks post transplant. Of note and in contrast to HOXB4wt, mice engrafted with HOXB4-ΔPro BM cells (n=9) developed myeloproliferation with a significant increase of Mac-1 and Gr-1 positive cells over time in the PB (29% Gr1 and 43% Mac1 wk 4–16 compared to 71.2% Gr1 and 86.6% Mac1 week 36–56 wk, p<0.004). The HOXB4-ΔPro mice developed acute myeloid leukemia without maturation, as confirmed by immunohistochemical analysis after a median latency time of 279 days (n=9), while the mice transplanted with HOXB4wt expressing BM cells did not develop disease after an observation for more than 466 days (n=5, p<0.05). The AML in HOXB4-ΔPro mice was readily transplantable (66.5 days for 2nd Tx, n=6; 43 days for 3rd Tx, n=4) (p<0.05 compared to 1st recipients). In order to investigate the proviral integration pattern in the transplanted mice, we performed LM-PCR. In more than five HOXB4-Δpro mice we did not find recurrent integration sites. Taken together our results demonstrate that the N-terminal proline-rich region of HOXB4 has an important function for the stem cell amplifying function of HOXB4 and that loss of this domain converts HOXB4 in a leukemogenic gene.

Stable Integration of Recombinant Adeno-Associated Virus Vector Genomes After Transduction of Murine Hematopoietic Stem Cells

We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.

Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions

Strategies to expand hematopoietic stem cells (HSCs) ex vivo are of key interest. The objective of this study was to resolve if ability of HOXB4, previously documented to induce a significant expansion of HSCs in culture, may extend to other HOX genes and also to further analyze the HOX sequence requirements to achieve this effect. To investigate the ability of Nucleoporin98-Homeobox fusion genes to stimulate HSC self-renewal, we evaluated their presence in 10- to 20-day cultures of transduced mouse bone marrow cells. Stem cell recovery was measured by limiting-dilution assay for long-term competitive repopulating cells (CRU Assay). These experiments revealed remarkable expansions of Nucleoporin98-Homeobox-transduced HSCs (1000-fold to 10,000-fold over input) in contrast to the expected decline of HSCs in control cultures. Nevertheless, the Nucleoporin98-Homeobox-expanded HSCs displayed no proliferative senescence and retained normal lympho-myeloid differentiation activity and a controlled pool size in vivo. Analysis of proviral integration patterns showed the cells regenerated in vivo were highly polyclonal, indicating they had derived from a large proportion of the initially targeted HSCs. Importantly, these effects were preserved when all HOX sequences flanking the homeodomain were removed, thus defining the homeodomain as a key and independent element in the fusion. These findings create new possibilities for investigating HSCs biochemically and genetically and for achieving clinically significant expansion of human HSCs.

Efficient and Rapid Induction of a Chronic Myelogenous Leukemia-Like Myeloproliferative Disease in Mice Receiving P210 bcr/abl-Transduced Bone Marrow

Expression of the 210-kD bcr/abl fusion oncoprotein can cause a chronic myelogenous leukemia (CML)-like disease in mice receiving bone marrow cells transduced by bcr/abl-encoding retroviruses. However, previous methods failed to yield this disease at a frequency sufficient enough to allow for its use in the study of CML pathogenesis. To overcome this limitation, we have developed an efficient and reproducible method for inducing a CML-like disease in mice receiving P210 bcr/abl-transduced bone marrow cells. All mice receiving P210 bcr/abl-transduced bone marrow cells succumb to a myeloproliferative disease between 3 and 5 weeks after bone marrow transplantation. The myeloproliferative disease recapitulates many of the hallmarks of human CML and is characterized by high white blood cell counts and extensive extramedullary hematopoiesis in the spleen, liver, bone marrow, and lungs. Use of a retroviral vector coexpressing P210 bcr/abl and green fluorescent protein shows that the vast majority of bcr/abl-expressing cells are myeloid. Analysis of the proviral integration pattern shows that, in some mice, the myeloproliferative disease is clonal. In multiple mice, the CML-like disease has been transplantable, inducing a similar myeloproliferative syndrome within 1 month of transfer to sublethally irradiated syngeneic recipients. The disease in many of these mice has progressed to the development of acute lymphoma/leukemia resembling blast crisis. These results demonstrate that murine CML recapitulates important features of human CML. As such, it should be an excellent model for addressing specific issues relating to the pathogenesis and treatment of this disease.

Efficient and Rapid Induction of a Chronic Myelogenous Leukemia-Like Myeloproliferative Disease in Mice Receiving P210 bcr/abl-Transduced Bone Marrow

Expression of the 210-kD bcr/abl fusion oncoprotein can cause a chronic myelogenous leukemia (CML)-like disease in mice receiving bone marrow cells transduced by bcr/abl-encoding retroviruses. However, previous methods failed to yield this disease at a frequency sufficient enough to allow for its use in the study of CML pathogenesis. To overcome this limitation, we have developed an efficient and reproducible method for inducing a CML-like disease in mice receiving P210 bcr/abl-transduced bone marrow cells. All mice receiving P210 bcr/abl-transduced bone marrow cells succumb to a myeloproliferative disease between 3 and 5 weeks after bone marrow transplantation. The myeloproliferative disease recapitulates many of the hallmarks of human CML and is characterized by high white blood cell counts and extensive extramedullary hematopoiesis in the spleen, liver, bone marrow, and lungs. Use of a retroviral vector coexpressing P210 bcr/abl and green fluorescent protein shows that the vast majority of bcr/abl-expressing cells are myeloid. Analysis of the proviral integration pattern shows that, in some mice, the myeloproliferative disease is clonal. In multiple mice, the CML-like disease has been transplantable, inducing a similar myeloproliferative syndrome within 1 month of transfer to sublethally irradiated syngeneic recipients. The disease in many of these mice has progressed to the development of acute lymphoma/leukemia resembling blast crisis. These results demonstrate that murine CML recapitulates important features of human CML. As such, it should be an excellent model for addressing specific issues relating to the pathogenesis and treatment of this disease.

Osteoprogenitor cells as targets for ex vivo gene transfer.

We transduced osteoprogenitor cells with recombinant retrovirus and analyzed proviral integration patterns into chromosomal DNA to detect for the first time the clonal and cellular fate of osteoprogenitor-derived progeny cells. Metaphyseal bone cells and diaphyseal stromal cells were isolated from the distal femurs of young rats, transduced with the vM5neolacZ recombinant retrovirus, and selected in the neomycin analog, G418. Following surgical marrow ablation of a femur in one leg of mature rats, retroviral-transduced metaphyseal or diaphyseal cells were injected into the ablated site. These rats were killed 5-6 days later. Metaphyseal and diaphyseal cells were isolated from distal femurs, selected in G418, and stained for beta-galactosidase (beta-gal+). The number and clonal origin of transduced progenitor cells were determined. High numbers of beta-galactosidase colonies with an osteoblast phenotype were obtained following metaphyseal transplants and detected in 100% of metaphyseal and none of diaphyseal specimens. In contrast, beta-galactosidase colonies derived from diaphyseal transplants were detected in 50% of specimens in both the metaphysis and diaphysis, and the absolute number of progenitor cell colonies was 60-fold less than metaphyseal transplants. Provirus was only detected in the ablated bones and not in the contralateral bone or other tissues. Proviral integration fragment analysis showed a single integration site for recovered metaphyseal cell clones, consistent with their origination from a common single progenitor. This is one of the first demonstrations of successful transplantation of clonal osteoprogenitors to their site of origin in bone. It may be possible to use these cells to target genes to bone for therapeutic use in skeletal and hematopoietic diseases.

Molecular Evidence That In Situ-Transduced Fetal Liver Hematopoietic Stem/Progenitor Cells Give Rise to Medullary Hematopoiesis in Adult Rats

We exploited the ability to transduce fetal liver hematopoietic stem/progenitor cells in situ with recombinant retrovirus, together with the ability to analyze proviral integration patterns into chromosomal DNA, to detect the cellular and organ fate of hematopoietic stem and progenitor-derived progeny in tissues and in the circulation of neonatal and adult rats. Two hundred seventeen fetuses were injected with retrovirus supernatant on day 16 of gestation, before the development of the bone marrow cavity. The progeny of 41 stem and progenitor cells from 97 liveborn rats were clonally identified. Pluripotent and lineage-restricted stem/progenitor clones derived from the fetal liver consistently gave rise to progeny in the marrow of newborn and adult rats. Patterns of differentiation of transduced stem and progenitor cells fell into distinct subsets. Blood cells derived from in situ transduced cells that originated in the fetal liver circulated throughout the life span of the adult animals. These data provide molecular evidence of the origin of medullary cavity hematopoiesis by cells derived from the fetal liver that were transduced in vivo, homed to the developing medullary cavity and proliferated in a normal medullary hematopoietic microenvironment.

Efficient Retroviral Gene Transfer to Purified Long-Term Repopulating Hematopoietic Stem Cells

Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient "compromised" bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor-derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (&gt; or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

Stem Cell Factor, Interleukin-3, and Interleukin-6 Promote Retroviral-Mediated Gene Transfer Into Murine Hematopoietic Stem Cells

The efficiency of retroviral-mediated gene transfer into hematopoietic stem cells (HSC) is dependent on the survival and self-renewal of HSC in vitro during retroviral infection. We have examined the effect of prestimulation of bone marrow with various cytokines, including the product of the Steel gene, Steel factor or stem cell factor (SCF) (the ligand for the c-kit receptor) on the efficiency of retroviral transduction of the human adenosine deaminase (hADA) cDNA into murine HSC. Bone marrow cells were prestimulated for 48 hours with hematopoietic growth factors, then cocultivated with the packaging cell line producing the ZipPGK-ADA simplified retrovirus for an additional 48 hours with continued growth factor exposure. Nonadherant cells from these cocultures were injected into lethally irradiated recipients. The content of day 12 colony-forming unit-spleen (CFU-S12) in SCF/interleukin 6 (IL-6)-prestimulated and cocultured bone marrow was more than threefold greater than that of IL-3/IL-6-prestimulated bone marrow cells. All mice receiving bone marrow cells infected with the PGK-ADA virus after prestimulation with IL-3/IL-6 or SCF/IL-6 demonstrated hADA expression in the peripheral blood after full hematopoietic reconstitution. While all recipients of IL-3/IL-6-prestimulated bone marrow expressed hADA at 4 months posttransplant, in three independent experiments examining a total of 33 mice, in most recipients of SCF/IL-6-prestimulated and infected bone marrow cells, the expression of human enzyme was higher than IL-3/IL-6 mice. Southern blot analysis of DNA from hematopoietic tissues from these same mice prepared at least 4 months posttransplantation also demonstrated a higher infection efficiency of HSC as measured by proviral integration patterns and genome copy number analysis. These results suggest that the higher level of hADA expression seen in mice receiving marrow prestimulated with SCF/IL-6 before retroviral infection is due to more efficient infection of reconstituting HSC. Other growth factor combinations were also studied; however, prestimulation with SCF/IL-6 or IL-3/IL-6 appeared optimal. Using retroviral-mediated gene transfer and viral integration patterns, Steel factor (SCF) in combination with IL-6 appears to increase the survival and self-renewal of reconstituting hematopoietic stem cells and proves useful in effecting expression of foreign genes in transplant recipients. Such pretreatment may also be useful in the application of retroviral transfer methods to human cells.

Stem cell factor, interleukin-3, and interleukin-6 promote retroviral- mediated gene transfer into murine hematopoietic stem cells

The efficiency of retroviral-mediated gene transfer into hematopoietic stem cells (HSC) is dependent on the survival and self-renewal of HSC in vitro during retroviral infection. We have examined the effect of prestimulation of bone marrow with various cytokines, including the product of the Steel gene, Steel factor or stem cell factor (SCF) (the ligand for the c-kit receptor) on the efficiency of retroviral transduction of the human adenosine deaminase (hADA) cDNA into murine HSC. Bone marrow cells were prestimulated for 48 hours with hematopoietic growth factors, then cocultivated with the packaging cell line producing the ZipPGK-ADA simplified retrovirus for an additional 48 hours with continued growth factor exposure. Nonadherant cells from these cocultures were injected into lethally irradiated recipients. The content of day 12 colony-forming unit-spleen (CFU-S12) in SCF/interleukin 6 (IL-6)-prestimulated and cocultured bone marrow was more than threefold greater than that of IL-3/IL-6-prestimulated bone marrow cells. All mice receiving bone marrow cells infected with the PGK-ADA virus after prestimulation with IL-3/IL-6 or SCF/IL-6 demonstrated hADA expression in the peripheral blood after full hematopoietic reconstitution. While all recipients of IL-3/IL-6- prestimulated bone marrow expressed hADA at 4 months posttransplant, in three independent experiments examining a total of 33 mice, in most recipients of SCF/IL-6-prestimulated and infected bone marrow cells, the expression of human enzyme was higher than IL-3/IL-6 mice. Southern blot analysis of DNA from hematopoietic tissues from these same mice prepared at least 4 months posttransplantation also demonstrated a higher infection efficiency of HSC as measured by proviral integration patterns and genome copy number analysis. These results suggest that the higher level of hADA expression seen in mice receiving marrow prestimulated with SCF/IL-6 before retroviral infection is due to more efficient infection of reconstituting HSC. Other growth factor combinations were also studied; however, prestimulation with SCF/IL-6 or IL-3/IL-6 appeared optimal. Using retroviral-mediated gene transfer and viral integration patterns, Steel factor (SCF) in combination with IL-6 appears to increase the survival and self-renewal of reconstituting hematopoietic stem cells and proves useful in effecting expression of foreign genes in transplant recipients. Such pretreatment may also be useful in the application of retroviral transfer methods to human cells.

Proviral DNA integration and transcriptional patterns of equine infectious anemia virus during persistent and cytopathic infections

The structure and integration patterns of equine infectious anemia virus (EIAV) proviral DNA and the patterns of viral transcription were examined in persistent and cytopathic infections of cultured cells. The results of Southern blot analyses indicated that, in persistently infected cells, about 30% of the EIAV provirus exists as randomly integrated DNA, while the remaining 70% is equally divided between unintegrated linear and closed circular forms. The cytopathic infection, in contrast, is characterized by levels of integrated provirus ranging from 65 to more than 90% of the total proviral DNA, depending on the extent of cytopathology exhibited by the virus strain employed. In both persistent and cytopathic infections, extensive Northern (RNA) blot analyses have revealed the presence of two major virus-specific transcripts, an 8.2-kilobase (kb) full-length genomic mRNA and a 3.5-kb single-spliced mRNA. A low-abundance 1.5-kb mRNA, presumably formed by a double-splicing event of the full-length RNA, was also detected in the cytopathic EIAV infection. The two major viral transcripts are present in approximately equal quantities in persistently infected cells, while the cytopathic infection reveals nearly a 30-fold higher level of viral transcripts in which the 3.5-kb species constitutes over 75% of the total viral mRNA. The relatively high proportion of proviral DNA integration and the simple pattern of viral transcription observed during EIAV infections appeared to be different from the generally observed patterns of predominantly unintegrated proviral DNA and multi-spliced viral mRNAs in cells infected with other lentiviruses such as visna virus or human immunodeficiency virus type 1. Moreover, the data suggested that the cytopathology of EIAV may be correlated in part with the degree of proviral DNA integration and levels of viral mRNA in infected cells, particularly that of the spliced 3.5-kb mRNA.

Characterization of cell markers in type B retroviral-induced thymic lymphomas—II. Surface antigen phenotype, karyotype and proviral integration pattern in cultured lymphoma cells and cloned lines

This study examined the surface antigen phenotype, karyotype and proviral integration patterns of cultured cells from murine thymic lymphomas induced by injecting neonatal mice with the type B leukemogenic retrovirus (DMBA-LV). Cells from the primary thymic lymphomas were established in mass culture and from these, clonal tumor cell lines were derived. During in vitro culture of lymphoma cells, Lyt 1 −2 + cells predominated with an apparent selection against cells of the Lyt 1 +2 − and Lyt 1 +2 + phenotypes. Of 21 cloned lines established, five had a diploid chromosome complement and expressed the Lyt 1 −2 + phenotype. The other 16 clones lines were characterized by trisomy of chromosome 15 and expressed the Lyt 1 +2 + or Lyt 1 −2 + phenotype. Cells characterized by either a diploid or trisomy chromosome complement were capable of growth in vivo. Southern blot analyses showed that during growth in culture, cells from the mass cultures and cloned lines continued to acquire low numbers of new integrated DMBA-LV proviral copies while maintaining the basic proviral integration pattern present in the DNA from cells of the primary lymphomas. These findings support the notion that the acquisition of new genetic information in cells from DMBA-LV-induced thymic lymphomas may contribute to the continual generation of tumor heterogeneity.

N-Methyl- N-nitrosourea-induced t-lymphomas of AKR/J mice contain somatically acquired ecotropic-like murine leukemia proviruses

We have studied somatically acquired murine leukemia proviral integrations in primary N-methyl- N-nitrosourea (MNU)-induced thymic lymphomas of AKR/1 mice. The majority of MNU-induced lymphomas contain newly acquired murine leukemia proviral sequences. In contrast to spontaneous AKRA lymphomas which contain multiple integrations of mink cell focus-forming recombinant proviruses, MNU-induced lymphomas contain ecotropic-related proviruses. This conclusion was based on the demonstration that EcoRI- and PvuII-digested DNA from MNU-induced lymphomas contains new 3′ proviral-cellular junction fragments that hybridize with the ecotropic-specific pAKV-4 and pAKV-5 hybridization probes. Also, EcoRI/ PstI double digests of DNA from MNU-induced lymphomas revealed that the acquired proviruses do not contain an internal 3′ EcoRI site characteristic of mink cell focus-forming recombinant viruses. The proviral integration patterns suggest that MNU-induced lymphomas are clonal or oligoclonal in nature. This conclusion is supported by comparison of proviral integration patterns in lymphomas obtained from thymus and spleen of individual mice, and by analyses of T-cell receptor β-chain gene rearrangements. The frequent occurrence of ecotropic-related proviral sequences in MNU-induced lymphomas suggests that these newly acquired proviruses may play a role in tumor development.

Cell transformation and tumor induction by Abelson murine leukemia virus in the absence of helper virus.

We investigated the role of the Moloney helper virus, Moloney murine leukemia virus (Mo-MuLV), in cell transformation and tumor induction by the defective Abelson murine leukemia virus (Ab-MuLV). A molecular clone of Ab-MuLV (P160 strain) was transfected into the psi 2 packaging cell line, and helper virus-free Ab-MuLV (psi 2) was harvested from the supernatant medium. Ab-MuLV (psi 2) was as efficient as helper virus-containing Ab-MuLV (Mo-MuLV) in the transformation of primary bone marrow cells in vitro. Inoculation of weanling BALB/c mice with Ab-MuLV (psi 2) induced nonthymic pre-B-cell lymphomas with high efficiency and short latency (28 days). Adult BALB/c mice were less sensitive to tumor induction by a factor of 100. Ab-MuLV (psi 2) did not induce tumors in weanling C57BL/6 mice, unlike Ab-MuLV (Mo-MuLV). Examination of the proviral integration pattern in Ab-MuLV (psi 2)-induced tumor cell DNA revealed that each of the tumors contained a single integrated provirus. Immunoprecipitation of viral-encoded proteins in helper virus-free tumor cell lines detected the P160 Ab-MuLV-transforming protein; however, no trace of the gag, pol, and env helper virus-encoded proteins was found. Our results indicate that integration and expression of a single Ab-MuLV genome is sufficient for efficient transformation of primary bone marrow cells by Ab-MuLV in vitro and tumor induction in susceptible mice. However, the helper virus may contribute to tumor induction in weanling resistant mice.

Tumor progression in murine leukemia virus-induced T-cell lymphomas: monitoring clonal selections with viral and cellular probes

Clonal selections occurring during the progression of Moloney murine leukemia virus (MuLV)-induced T-cell lymphomas in mice were examined in primary and transplanted tumors by monitoring various molecular markers: proviral integration patterns, MuLV insertions near c-myc and pim-1, and rearrangements of the immunoglobulin heavy chain and beta-chain T-cell receptor genes. The results were as follows. Moloney MuLV frequently induced oligoclonal tumors with proviral insertions near c-myc or pim-1 in the independent clones. Moloney MuLV acted as a highly efficient insertional mutagen, able to activate different (putative) oncogenes in one cell lineage. Clonal selections during tumor progression were frequently marked by the acquisition of new proviral integrations. Independent tumor cell clones exhibited a homing preference upon transplantation in syngeneic hosts and were differently affected by the route of transplantation.