Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia

Abstract

Chromosomal translocations involving the MLL gene occur in about 80% of infant leukemia. In the search for possible agents inducing infant leukemia, we identified bioflavonoids, natural substances in food as well as in dietary supplements, that cause site-specific DNA cleavage in the MLL breakpoint cluster region (BCR) in vivo. The MLL BCR DNA cleavage was shown in primary progenitor hematopoietic cells from healthy newborns and adults as well as in cell lines; it colocalized with the MLL BCR cleavage site induced by chemotherapeutic agents, such as etoposide (VP16) and doxorubicin (Dox). Both in vivo and additional in vitro experiments demonstrated topoisomerase II (topo II) as the target of bioflavonoids similar to VP16 and Dox. Based on 20 bioflavonoids tested, we identified a common structure essential for topo II-induced DNA cleavage. Reversibility experiments demonstrated a religation of the bioflavonoid as well as the VP16-induced MLL cleavage site. Our observations support a two-stage model of cellular processing of topo II inhibitors: The first and reversible stage of topo II-induced DNA cleavage results in DNA repair, but also rarely in chromosome translocations; whereas the second, nonreversible stage leads to cell death because of an accumulation of DNA damage. These results suggest that maternal ingestion of bioflavonoids may induce MLL breaks and potentially translocations in utero leading to infant and early childhood leukemia.

Figure 1

Diagram of the human genomic MLL. The top map represents the BamHI (B) DNA gene segments with the size (kb) indicated above (–24). The bottom map represents an enlargement of the 8.3-kb BamHI MLL BCR, which is involved in all MLL chromosome translocations. The entire MLL gene was analyzed for in vivo bioflavonoid- and VP16-induced cleavage sites, centromeric (cen) to telomeric (tel), by use of a series of MLL DNA probes from exons 1 to 34 and genomic fragments corresponding to each BamHI fragment (–24); these include three centromeric DNA probes (gray boxes) amplified from the cosmid COS20 using PCR at positions 18,652–19,825, 19,936–20,542, and 32,119–32,442 (24). The 7.0- and 1.3-kb cleavage fragments (below the map) representing the BCR were identified with the 0.74-kb cDNA probe (exons 5–11, indicated above map line). The black arrows above both maps represent the colocalizing bioflavonoid- and VP16-induced in vivo topo II cleavage site between nucleotides 6,800 and 7,000 and the DNase I HS site mapped previously (21).

Figure 2

Southern blots from BV173 (a and b) and human primary progenitor cells (c–e) incubated with bioflavonoids, ascorbic acid, and VP16. Above and from left to right: no addition of drugs (−), DMSO control (C), chemotherapeutic drug VP16 (VP), bioflavonoids as indicated, fisetin (F), genistein (G), quercetin (Q), dietary quercetin-supplement (Q*), and ascorbic acid (Asc). Concentrations (μM) tested are indicated below. Germline (8.3 kb) and the cleavage fragments (7.0 kb and 1.3 kb) are indicated to the left with arrowheads. The FACS results at the time of drug treatment from umbilical cord blood (CB) or peripheral blood (PB) CD34⁺ ex vivo expansion into myeloid progenitor (prog) cells were (c) 20% CD33⁺/15⁺; 51% CD11b⁺/15⁺; 14% CD11b⁺/14⁺, and 15% propidium iodide⁺ (PI); (d) 36% CD33⁺/15⁺; 33% CD11b⁺/15⁺; 26% CD11b⁺/14⁺, and 5% PI⁺; (e) from umbilical cord blood T lymphocyte progenitor cells ex vivo expansion: 27% CD4⁺/8⁺; 44% CD4⁺; 19% CD8⁺, and 10% PI⁺.

Figure 3

(a) Southern blot of BV173 cells incubated with bioflavonoids with similar ring substitutions, but different ring saturation or geometry (also see Table 1). Above and from left to right: DMSO (C), VP16 (VP), lanes A, B, and C: 4′,5,7-trihydroxyflavonoids (A, apigenin; B, naringenin; C, genistein); D and E: 3,3′4′,5,7-pentahydroxyflavonoids (D, quercetin; E, taxifolin); F and G: 5,7-dihydroxy-4′-methoxyflavonoids (F, acacetin; G, biochaninA). Concentrations (μM) are indicated below. Germline (8.3 kb) and the cleavage fragments (7.0 kb and 1.3 kb) are indicated to the left. (b) Southern blot shows the accumulative activity of bioflavonoids. Above and from left to right: DMSO (C), VP16 (VP), VP16 and quercetin (Q, VP), quercetin (Q), genistein (G), fisetin (F), quercetin, fisetin, and genistein (Q,F,G). Concentrations (μM) are indicated below. Germline and the cleavage fragments are indicated to the left. Below the Southern blot is a densitometry graph, which represents the bioflavonoid cleavage activity in the MLL BCR. Each bar represents the percentage (%) of cleavage and corresponds to the lane above.

Figure 4

Southern blots were hybridized with the 0.74-kb MLL BCR cDNA probe. (a) MLL cleavage induced by 25 μM or 50 μM quercetin and VP16 for 6 h can be reversed using two different methods: A, heat treatment of cells for 15 min at 60°C, immediately after the 6 h drug incubation; or B, an additional incubation of the drug-treated cells in drug-free media for 2 h at 37°C. (b) MLL BCR DNA cleavage induced by genistein and VP16 using an in nuclei topo II assay. Isolated BV173 nuclei were incubated with 2 units or 4 units of purified human topo II and 50 μM VP16 or genistein was added for 5 min at 30°C.

Figure 5

The common bioflavonoid minimal structures required for in vivo MLL BCR cleavage and in vitro topo II inhibition. The structure without the shaded side-group represents the flavones (2-phenyl-4H-1-benzopyran-4-one) and with the 3-OH, the flavonols. The benzopyran ring with 3-phenyl substitution (the shaded side-group) represents the isoflavones (3-phenyl-4H-1-benzopyran-4-one). The flavanones (2,3-dihydroflavones) or flavonoids with glycosidic or methoxy groups did not inhibit topo II activity and did not induce MLL BCR DNA breakage.