Novel insights into the pathogenesis of the Graffi murine leukemia retrovirus

Abstract

The Graffi murine leukemia virus (MuLV) was isolated in 1954 by Arnold Graffi, who characterized it as a myeloid leukemia-inducing retrovirus. He and his team, however, soon observed the intriguing phenomenon of hematological diversification, which corresponded to a decrease of myeloid leukemias and an increase of other types of leukemias. Recently, we derived two different molecular clones corresponding to ecotropic nondefective genomes that were named GV-1.2 and GV-1.4. The induced leukemias were classified as myeloid based on morphological analysis of blood smears. In this study, we further characterized the two variants of the Graffi murine retrovirus, GV-1.2 and GV-1.4, in three different strains of mice. We show that the Graffi MuLV is a multipotent retrovirus capable of inducing both lymphoid (T- and B-cell) and nonlymphoid (myeloid, erythroid, megakaryocytic) leukemia. Many of these are very complex with concomitant expression of different hematopoietic lineages. Interestingly, a high percentage of megakaryocytic leukemias, a type of leukemia rarely observed with MuLVs, arise in the FVB/n strain of mice. The genetic backgrounds of the different strains of mice influence greatly the results. Furthermore, the enhancer region, different for GV-1.2 and GV-1.4, plays a pivotal role in the disease specificity: GV-1.2 induces more lymphoid leukemias, and GV-1.4 induces more nonlymphoid ones.

FIG. 1.

Survival analysis of newborn BALB/c, NFS, and FVB/n mice injected with the molecular clones GV-1.4 (A) and GV-1.2 (B). The animals were sacrificed when they showed signs of advanced disease. The number of mice in each group is indicated in Table 1.

FIG. 2.

Flow cytometry analyses of several types of leukemias. (A) Flow cytometry of the most typical leukemias. Panels: a, T-cell leukemia; b: B-cell leukemia; c: myeloid leukemia; d: erythroid leukemia; e: megakaryocytic leukemia. (B) Flow cytometry of several atypical T-cell leukemias. The horizontal and/or vertical bars on each plot define the positive cells in comparison with isotype controls. N indicates the number of mice sharing the same phenotype (Table 1). The percentage of each cell population is indicated in each quadrant. The configuration of the TCRβ and J_H loci is indicated. Abbreviations: G, germ line; R, rearranged; FSC, forward side scatter.

FIG. 3.

Southern blot analysis of TCRβ (A) and immunoglobulin heavy chain (B) gene rearrangement. The type of leukemia is indicated above each lane. Abbreviations: B, B cell; M, myeloid; T, T cell; Mk, megakaryocytic; E, eryhthroid; C, controls. The molecular weights of the germ line fragments are indicated.

FIG. 4.

Northern blot analysis of some specific genes of the myeloid, erythroid, and megakaryocytic lineages. Total RNA was extracted from the spleens of leukemic and normal mice. (A) Analysis of the MPO gene. (B) Analysis of some genes of the erythroid and megakaryocytic lineages. Abbreviations: E, erythroid leukemias (Ter119⁺CD41⁻); EMk, erythroid-megakaryocytic leukemias (Ter119⁺CD41⁺); Mk, megakaryocytic leukemias (Ter119⁻CD41⁺); T, T-cell leukemias; B, B-cell leukemias; M, myeloid leukemias; C, control normal, BALB/c, NFS, and FVB/n mice (lanes 1 to 3 in panel A and 34 to 36 in panel B, respectively).

FIG. 5.

Agarose gel analysis of proviral enhancer PCR amplification in mature tumors. (A)T-cell leukemias induced with GV-1.2 (lanes 1, 2, 4, 6, 8, and 10) and GV-1.4 (lanes 3, 5, 7, 9, and 11) in BALB/c mice. (B) T-cell (lanes 1 to 6) and B-cell (lanes 7 to 11) leukemias induced with GV-1.2 (lanes 1 to 4) and GV-1.4 (lanes 5 to 11) in NFS mice. (C) Nonlymphoid leukemias induced with GV-1.4: erythroid leukemias (lanes 1 to 7) in BALB/c mice (lanes 1 to 2) and NFS mice (lanes 3 to 7); megakaryocytic leukemias in NFS mice (lanes 8 to 10); myeloid leukemias in NFS mice (lanes 11 to 13). Lane 14 is an erythroid leukemia induced with GV1.2 as a size control. (D) Nonlymphoid leukemias induced with GV-1.2: erythroid leukemias induced with GV-1.2 in NFS mice (lanes 2 to 4) and in FVB/n mice (lanes 5 to 12). Lane 1 is a leukemia induced by GV-1.4 in FVB mice as a size control.