Role of promyelocytic leukemia (PML) protein in tumor suppression

Abstract

The promyelocytic leukemia (PML) gene encodes a putative tumor suppressor gene involved in the control of apoptosis, which is fused to the retinoic acid receptor alpha (RARalpha) gene in the vast majority of acute promyelocytic leukemia (APL) patients as a consequence of chromosomal translocations. The PMLRARalpha oncoprotein is thought to antagonize the function of PML through its ability to heterodimerize with and delocalize PML from the nuclear body. In APL, this may be facilitated by the reduction to heterozygosity of the normal PML allele. To determine whether PML acts as a tumor suppressor in vivo and what the consequences of deregulated programmed cell death in leukemia and epithelial cancer pathogenesis are, we crossed PML(-/-) mice with human cathepsin G (hCG)-PMLRARalpha or mammary tumor virus (MMTV)/neu transgenic mice (TM), models of leukemia and breast cancer, respectively. The progressive reduction of the dose of PML resulted in a dramatic increase in the incidence of leukemia, and in an acceleration of leukemia onset in PMLRARalpha TM. By contrast, PML inactivation did not affect neu-induced tumorigenesis. In hemopoietic cells from PMLRARalpha TM, PML inactivation resulted in impaired response to differentiating agents such as RA and vitamin D3 as well as in a marked survival advantage upon proapoptotic stimuli. These results demonstrate that: (a) PML acts in vivo as a tumor suppressor by rendering the cells resistant to proapoptotic and differentiating stimuli; (b) PML haploinsufficiency and the functional impairment of PML by PMLRARalpha are critical events in APL pathogenesis; and (c) aberrant control of programmed cell death plays a differential role in solid tumor and leukemia pathogenesis.

Figure 1

PML inactivation leads to an increase in frequency and an earlier leukemia onset in hCG-PMLRARα TM. (a–d) hCG-PMLRARα TM were mated with mice in which the PML gene was disrupted by homologous recombination. The various genotypes and the numbers of mice analyzed are indicated. LFS (in days) was significantly shorter and leukemia frequency higher in hCG-PMLRARα^+/−PML^−/− and hCG-PMLRARα^+/−PML^+/− mice compared with hCG-PMLRARα^+/−PML^+/+ mice (a). The leukemia frequency was also increased in hCG-PMLRARα^+/+ mice compared with hCG-PMLRARα^+/− mice either in PML^+/+ (b), PML^+/− (c), or PML^−/− (d) backgrounds. (e) Leukemic cells in all the studied groups exhibited similar features. PB smears and BM cytospin preparations from leukemic hCG-PMLRARα^+/−PML^−/− and hCG-PMLRARα^+/−PML^+/+ mice were stained with Wright-Giemsa stain. The arrows indicate the leukemic cells. Original magnification: ×1,000.

Figure 1

PML inactivation leads to an increase in frequency and an earlier leukemia onset in hCG-PMLRARα TM. (a–d) hCG-PMLRARα TM were mated with mice in which the PML gene was disrupted by homologous recombination. The various genotypes and the numbers of mice analyzed are indicated. LFS (in days) was significantly shorter and leukemia frequency higher in hCG-PMLRARα^+/−PML^−/− and hCG-PMLRARα^+/−PML^+/− mice compared with hCG-PMLRARα^+/−PML^+/+ mice (a). The leukemia frequency was also increased in hCG-PMLRARα^+/+ mice compared with hCG-PMLRARα^+/− mice either in PML^+/+ (b), PML^+/− (c), or PML^−/− (d) backgrounds. (e) Leukemic cells in all the studied groups exhibited similar features. PB smears and BM cytospin preparations from leukemic hCG-PMLRARα^+/−PML^−/− and hCG-PMLRARα^+/−PML^+/+ mice were stained with Wright-Giemsa stain. The arrows indicate the leukemic cells. Original magnification: ×1,000.

Figure 2

PML inactivation does not affect tumorigenesis in MMTV/neu TM. (a) MMTV/neu TM were mated with PML^−/− mice and their progenies monitored for the incidence of breast tumors. The various genotypes and the numbers of mice analyzed are indicated (PML^+/+, red line; PML^+/−, black line; PML^−/−, green line). A similar frequency and TFS was observed in the MMTV/neu TM regardless of the PML genotype. (b and c) Assessment of apoptosis (TUNEL) and proliferation (Ki67 staining) in breast tumors from MMTV/neu/PML^+/+ and MMTV/neu/PML^−/− mice. Apoptotic cells are virtually absent in these tumors (<1/400 cells scored in both genotypes: see indents) whereas numerous proliferating cells are detected in comparable numbers regardless of the PML background. Arrows show examples of positively stained cells. (d) PML is expressed in breast tumors from MMTV/neu TM. Immunostaining with an anti-PML Ab of breast tumor frozen sections from MMTV/neu TM shows the characteristic PML nuclear speckled pattern. Original magnification: ×400.

Figure 2

PML inactivation does not affect tumorigenesis in MMTV/neu TM. (a) MMTV/neu TM were mated with PML^−/− mice and their progenies monitored for the incidence of breast tumors. The various genotypes and the numbers of mice analyzed are indicated (PML^+/+, red line; PML^+/−, black line; PML^−/−, green line). A similar frequency and TFS was observed in the MMTV/neu TM regardless of the PML genotype. (b and c) Assessment of apoptosis (TUNEL) and proliferation (Ki67 staining) in breast tumors from MMTV/neu/PML^+/+ and MMTV/neu/PML^−/− mice. Apoptotic cells are virtually absent in these tumors (<1/400 cells scored in both genotypes: see indents) whereas numerous proliferating cells are detected in comparable numbers regardless of the PML background. Arrows show examples of positively stained cells. (d) PML is expressed in breast tumors from MMTV/neu TM. Immunostaining with an anti-PML Ab of breast tumor frozen sections from MMTV/neu TM shows the characteristic PML nuclear speckled pattern. Original magnification: ×400.

Figure 3

PML inactivation impairs the growth inhibitory and differentiating activities of vitamin D₃ (Vit. D) and RA in BM cells from hCG-PMLRARα^+/− TM. Hemopoietic cells from WT (white bars), PML^−/− (dotted bar), hCG-PMLRARα^+/−PML^+/+ (black bar), and hCG-PMLRARα^+/−PML^−/− (gray bar) mice were cultured in semisolid media (a and c) or in liquid media (b and d) in the presence or absence of vitamin D₃ (10⁻⁹ M) or RA (10⁻⁷ M). The number of myeloid colonies (CFU-GM) formed in methylcellulose (a and c) and the expression of CD11b in liquid cultures (b and d) were analyzed after 7 and 3 d, respectively. The results are reported as percentages observed in treated samples relative to controls. For either a, b, c, or d, one experiment performed in triplicate, out of three independent experiments with similar results, is shown (Materials and Methods).

Figure 4

PML inactivation enhances the protection from Fas-induced apoptosis in BM cells from hCG-PMLRARα^+/− TM. BM cells from mice of indicated genotypes were cultured in liquid media (a) or semisolid media (b) in the presence or absence of 100 ng of anti-Fas Ab. (a) The number of apoptotic cells in the liquid culture assay was determined after 24 h by staining with propidium iodide (PI) and annexin V. Histogram bars represent the percentage of apoptotic cells induced by anti-Fas treatment. The mean percentage of apoptotic cells in WT cultures is presented as 100%. Illustrative dot plots from flow cytometric analyses of Fas-stimulated BM cultures from mice of indicated genotypes are shown in the right panel. (b) CFU-GM from in vitro methylcellulose colony assays were scored at day 7. The results are reported as the percentage of myeloid colonies observed in treated samples relative to controls. For both a and b, one experiment performed in triplicate, out of three independent experiments with similar results, is shown (Materials and Methods).