A PMLRARalpha transgene initiates murine acute promyelocytic leukemia

Abstract

The malignant cells of acute promyelocytic leukemia (APL) contain a reciprocal chromosomal translocation that fuses the promyelocytic leukemia gene (PML) with the retinoic acid receptor alpha gene (RAR alpha). To test the hypothesis that the chimera PMLRAR alpha plays a role in leukemogenesis, we expressed a PMLRAR alpha cDNA in myeloid cells of transgenic mice. PMLRAR alpha transgenic mice exhibited impaired neutrophil maturation early in life, which progressed at a low frequency over the course of several months to overt APL. Both the preleukemic state and the leukemia could be transplanted to nontransgenic mice, and the transplanted preleukemia could progress to APL. The APL recapitulated features of the human disease, including a response to retinoic acid. Retinoic acid caused the leukemic cells to differentiate in vitro and in vivo, eliciting remissions of both the preleukemic state and APL in mice. Our results demonstrate that PMLRAR alpha impairs neutrophil differentiation and initiates the development of APL. The transgenic mice described here provide an apparently accurate model for human APL that includes clear evidence of tumor progression. The model should be useful for exploring the molecular pathogenesis of APL and the mechanisms of the therapeutic response to retinoic acid, as well as for preclinical studies of therapeutic regimens.

Figure 1

PMLRARα expression in bone marrow of transgenic mice. (a) Western blot. Total cell lysates from equal numbers of unfractionated bone marrow cells from the indicated hMRP8–PMLRARα transgenic lines or control cell lines were loaded into each lane. The blot was probed with antibodies against human Rarα. NB4 human APL cells (27) express PMLRARα (4), but HL-60 human myeloid leukemia cells do not (28). FVB/N samples were from nontransgenic mice. (b–e) Immunofluorescence. Bone marrow cells from healthy 8-week-old mice were cytospun and incubated with antiserum specific for human Pml (b and d) and the DNA-intercalating dye DAPI (c and e). (b and c) Control FVB/N. (d and e) Transgenic line 556. (×250.)

Figure 2

FACS analysis of bone marrow. Bone marrow cells were stained with Gr-1 and Mac-1 (murine Cd11b), markers for differentiation of murine myeloid cells. (a and b) Placebo or 5 mg ATRA pellets were implanted into 3-week-old FVB/N or line 556 mice; marrow was analyzed after 21 days. Each curve combines data from two mice. FVB/N–placebo, dotted line; line 556–placebo, thin line; FVB/N–ATRA, dashed line; line 556–ATRA, thick line. (a) Gr-1 antigen expression. (b) Side-scatter profile of Gr-1-positive cells from samples in a. (c–e) Murine APL responds to ATRA. (c) Age-matched FVB/N control. (d and e) Samples from second passage by transplantation of leukemia 935 (line 569). On days 14–21 after transplantation, mice were given placebo (d) or 1.35 mg ATRA (e) by intraperitoneal injection of oil suspension. Cells were isolated on day 28 after transplantation.

Figure 3

Murine APL. (a and b) Bone marrow isolates. (a) Control FVB/N. (b) Leukemia (mouse 1333, line 556). (c and d) Cytospins of 7-day methylcellulose cultures from passage 1 of leukemia 877 (line 553) cultured without (c) or with (d) 1 μM ATRA. (e and f) Bone marrow isolates from third passage of leukemia 877. On day 11 after transplantation, pellets containing placebo (e) or 5 mg ATRA (f) were implanted. Samples were prepared on day 16 after transplantation. Cells were stained with Wright’s Giemsa with Azure B. (×250.)