Irradiated Blm-deficient mice are a highly tumor prone model for analysis of a broad spectrum of hematologic malignancies
Introduction
Human Bloom syndrome (BS) is an extremely rare autosomal recessive genetic disorder characterized by short stature, photosensitive skin rashes, immunodeficiency, reduced fertility, and predisposition to cancer [1], [2]. Individuals homozygous for the mutation exhibit frequent B cell non-Hodgkin lymphomas (NHL) and leukemia in their twenties and squamous cell carcinomas of the head and neck in their thirties [3], [4]. These tumors are similar to those occurring in the general population but are characterized by an earlier age of onset and the occurrence of multiple cancers in one individual [4].
The BLM protein is a member of the RecQ family of DNA helicases [5] and has an important functional role in the resolution of Holliday junctions that occur during the S phase of the cell cycle [6], [7] and prevention of excessive or inappropriate recombination [8], [9]. BLM-deficient cells attempt to repair certain types of DNA damage by excessive homologous recombination [10], which increases rates of loss of heterozygosity (LOH) resulting in elevated rates of tumorigenesis [11]. In a similar fashion to BS lymphocytes, Blm-deficient mouse embryonic stem (ES) cells also show a high rate of mitotic recombination. This feature has been utilized to generate homozygous mutant cells from cells with heterozygous mutations [12], [13]. A viable Blm-deficient mouse model (Blmm3/m3) has been engineered by gene targeting in mouse ES cells [14], providing a useful background for studying tumorigenesis due to elevated rates of mitotic recombination [12], [13]. Preliminary investigations of adult Blmm3/m3 mice monitored for 20 months indicated that the mice are tumor prone, developing lymphomas, carcinomas, and sarcomas [14]; however, a detailed analysis of lymphoma and leukemia development, their histopathologic subtypes, and associated genetic abnormalities has not yet been undertaken. These findings indicated that Blmm3/m3 mice could provide a useful model for the human disease with opportunities for understanding the genetic mechanisms underlying the variety of neoplasms that characterize this disorder. This prompted our laboratories and others to develop approaches for enhancing the utility of the model through (i) studies of proviral insertional mutagenesis to identify candidate oncogenes and tumor suppressor genes (TSG) and (ii) detailed histologic and immunophenotypic studies of the tumors to examine their relationship to tumors found in BS.
Hematopoietic neoplasms arise spontaneously in a number of strains of mice including those that express endogenous murine leukemia viruses (MuLV) at high levels [15], [16], [17], [18]. They can also be induced following exposure to irradiation [19] or chemicals, by exogenous infection with MuLV [20], [21], [22], or by genetic manipulation of the mouse genome [23], [24]. The tumor spectrum in each case varies and is generally restricted to lymphomas of certain types [24], [25], [26]. A prior study utilized proviral insertional mutagenesis screens to identify oncogenic networks operative in Blm-deficient strains that develop a high frequency of retrovirus-induced B cell lymphomas [27]. This high-throughput method has been developed for performing forward genetic screens to identify genes involved in pathogenesis of neoplasms in mice expressing high levels of MuLV. It is based on identification of proviral insertion sites that can activate proto oncogenes or inactivate tumor suppressor genes (TSG), a process sometimes referred to as “proviral tagging” [28], [29], [30]. Genomic regions that recurrently contain proviral integrations in independent tumors are referred to as common integration sites (CIS) and show substantial overlap with oncogenes or TSG involved in human cancers, indicating the power of this screening strategy [31], [32]. As noted above, however, this method tends to identify activating mutations in oncogenes rather than inactivating mutations in TSG. The Blm-deficient mouse model, due to the elevated rate of induced mitotic recombination [12], enhances the ability to identify recessive mutations associated with a particular phenotype and therefore may be valuable in identifying tumor suppressor genes involved in the development of hematopoietic neoplasms. Indeed, the fact that mitotic recombination between non-sister chromatids that are heterozygous for a mutation can produce daughter cells that have lost the wild-type allele and are then homozygous for the mutant allele [33]. This feature was exploited for identifying TSG at CIS in MuLV-expressing mice bearing the Blmm3/m3 mutation [27].
Because the stains selected for the insertional mutagenesis screen were methodologically strongly biased toward development of B cell lymphomas, it was unclear whether similar oncogenic networks might be involved in the development of the other hematopoietic and solid tumors shown to occur in humans and Blm-deficient mice. In addition, the long latencies for tumor development in the original Blmm3/m3 mutant mice [14] or those generated for the mutagenesis screens [27] greatly complicated the efficient analysis of a large number of animals in various studies including therapeutic efforts. The observations that patient-derived cell lines and cells from Blm-deficient mice exhibit genetic instability and have an activated double stranded DNA break response but are radioinsensitive suggested that Blm deficiency may be radiomimetic, possibly underlying the nontargeted effects of exposure to ionizing radiation [14], [34], [35], [36]. We therefore postulated that treatment of Blm-deficient mice with ionizing radiation might augment the contribution of Blm deficiency to transformation, resulting in the accelerated appearance without a change in the tissue distribution of malignancies.
To examine this possibility, we analyzed tumors arising in Blm-deficient mice with or without exposure to gamma irradiation. We analyzed the survival pattern, tumor incidence, and tumor spectrum to determine whether this system provides an accurate model of human Bloom syndrome and to assess whether the spectrum of tumors occurring in these mice is biased by Blm status or gamma irradiation. Our results revealed that irradiated Blm-deficient mice develop a broad spectrum of hematopoietic neoplasms that includes all lineages except megakaryocytic, dendritic, and NK cells, marking this as a particularly useful system for modeling human hematopoietic diseases. We present a detailed morphologic and immunohistochemical assessment of these tumors and validate a diagnostic panel of primary antibodies on tissue arrays of these tumors as a means to facilitate high-throughput immunohistochemical analysis of mouse hematologic malignancies.
Section snippets
Mice
Blmm3/+ mice [14] were bred and housed in specific pathogen-free (SPF) facilities conforming to the Home Office Code of Practice for the Housing and Care of Animals used in Scientific Procedures. The mice were maintained on a mixed 129S7 and C57BL/6Tyrc−Brd background (approximately 25% 129 and 75% C57BL/6Tyrc−Brd contribution). To generate homozygous Blmm3/m3 mice for this study, heterozygous mice were intercrossed, and homozygous mice were identified by molecular genotyping.
Irradiation
Four-week
Survival data
Over an observation period of 520 days, 217 irradiated mice (187 Blmm3/m3, 20 Blmm3/+, and 10 Blm+/+) and 15 nonirradiated mice (14 Blmm3/m3 and 1 Blm+/+) became moribund and were necropsied (Table 2). Within the group of irradiated mice, 233 primary pathologic lesions were identified, representing a cumulative incidence of 51% (208/409) in Blmm3/m3 mice, 13% (16/128) in Blmm3/+ mice, and 10% (9/88) in Blm+/+ mice. (Here the denominator represents the total number of mice within the group at
Discussion
In this study, we performed a detailed analysis of pathologic lesions, mainly tumors, arising in irradiated Blmm3/m3 mice, a model for human Bloom syndrome. The incidence and spectrum of tumors observed in mice homozygous for a null allele of Blm partly mimics those seen in individuals with Bloom syndrome. As in human Bloom syndrome [4], malignant lesions occurred more frequently than benign lesions, and lymphomas and leukemias predominated over carcinomas and sarcomas by a ratio of 3:1 [1].
Conflict of interest statement
The authors declare that they have no competing financial interests.
Acknowledgements
This work was supported by the Wellcome Trust and the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.
We thank C. Brandt for assistance with mouse necropsies and genotyping, and B. Haynes and the Sanger Atlas of Protein Expression group for technical assistance with histology and immunohistochemistry. Professors Jim Lupski and Ming-Qing Du provided useful comments on the manuscript. NIAID intramural editor B. R. Marshall assisted in preparation of
References (56)
Bloom's syndrome. XX. The first 100 cancers
Cancer Genet Cytogenet
(1997)- et al.
The Bloom's syndrome gene product is homologous to RecQ helicases
Cell
(1995) - et al.
Combined histologic and molecular features reveal previously unappreciated subsets of lymphoma in AKXD recombinant inbred mice
Leuk Res
(2001) - et al.
Accelerated appearance of multiple B cell lymphoma types in NFS/N mice congenic for ecotropic murine leukemia viruses
Lab Invest
(2000) - et al.
Retroviral insertion sites and cancer: fountain of all knowledge?
Cancer Cell
(2002) - et al.
Bethesda proposals for classification of lymphoid neoplasms in mice
Blood
(2002) - et al.
Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice
Blood
(2002) - et al.
Histologic and molecular characterizations of megakaryocytic leukemia in mice
Leuk Res
(2006) - et al.
Classification of lymphoid neoplasms: the microscope as a tool for disease discovery
Blood
(2008) - et al.
Splenic marginal zone lymphomas of mice
Am J Pathol
(1999)
Effects of ionizing radiation in targeted and nontargeted cells
Arch Biochem Biophys
Bloom syndrome: a Mendelian prototype of somatic mutational disease
Medicine (Baltimore)
Congenital telangiectatic erythema resembling lupus erythematosus in dwarfs; probably a syndrome entity
AMA Am J Dis Child
Chromosomal breakage and acute leukemia in congenital telangiectatic erythema and stunted growth
Ann Intern Med
The Bloom's syndrome gene product promotes branch migration of Holliday junctions
Proc Natl Acad Sci USA
The Bloom's syndrome helicase suppresses crossing over during homologous recombination
Nature
Defending genome integrity during DNA replication: a proposed role for RecQ family helicases
Bioessays
The human Bloom syndrome gene suppresses the DNA replication and repair defects of yeast dna2 mutants
Proc Natl Acad Sci USA
Possible anti-recombinogenic role of Bloom's syndrome helicase in double-strand break processing
Nucleic Acids Res
RecQ helicases: caretakers of the genome
Nat Rev Cancer
Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells
Nature
Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene
Nature
Cancer predisposition caused by elevated mitotic recombination in Bloom mice
Nat Genet
AKXD recombinant inbred strains: models for studying the molecular genetic basis of murine lymphomas
Mol Cell Biol
Lymphomas and high-level expression of murine leukemia viruses in CFW mice
J Virol
Spontaneous and induced leukemias of myeloid origin in recombinant inbred BXH mice
J Virol
Myeloid, B and T lymphoid and mixed lineage thymic lymphomas in the irradiated mouse
Carcinogenesis
Genetic study of lymphoma induction by AKR mink cell focus-inducing virus in AKR × NFS crosses
J Exp Med
Cited by (7)
Age of first cancer diagnosis and survival in Bloom syndrome
2022, Genetics in MedicineCitation Excerpt :A similar concern exists for radiation exposure in the diagnosis and treatment of people with Bloom syndrome. Irradiated Blm-deficient mice (Blmm3/m3) showed a 28-fold increased risk for tumors, compared with a 5-fold increased risk for wild-type and heterozygous (Blmm3/+) mice.17 Because of this concern, magnetic resonance imaging and ultrasonography are preferred over other modalities, such as computed tomography scans and radiographs that use ionizing radiation.
A Structural Guide to the Bloom Syndrome Complex
2021, StructureCitation Excerpt :Cancer predisposition and other phenotypes were first linked to defective DNA repair when chromosome instability was observed in Bloom syndrome patient cells (German and Crippa, 1966). Animal models of Bloom syndrome have demonstrated the vital link between accumulation of DNA damage and the phenotypes that recapitulate those seen in humans with the disorder (Holloway et al., 2010; Warren et al., 2010). As such, Bloom syndrome has informed us about the important role of DNA damage in both cancer initiation and its response to treatment.
Functions of BLM Helicase in Cells: Is It Acting Like a Double-Edged Sword?
2021, Frontiers in GeneticsIn vivo functional screening for systems-level integrative cancer genomics
2020, Nature Reviews CancerGuidelines for the Optimization and Validation of In Situ Hybridization
2020, Methods in Molecular BiologyRare hereditary diseases with defects in DNA-repair
2012, European Journal of Dermatology