Genomic Instability Is an Early Event in Aluminium-Induced Tumorigenesis

Abstract

Genomic instability is generally considered as a hallmark of tumorigenesis and a prerequisite condition for malignant transformation. Aluminium salts are suspected environmental carcinogens that transform mammary epithelial cells in vitro through unknown mechanisms. We report here that long-term culture in the presence of aluminium chloride (AlCl₃) enables HC11 normal mouse mammary epithelial cells to form tumours and metastases when injected into the syngeneic and immunocompetent BALB/cByJ strain. We demonstrate that AlCl₃ rapidly increases chromosomal structural abnormalities in mammary epithelial cells, while we failed to detect direct modulation of specific mRNA pathways. Our observations provide evidence that clastogenic activity-a well-recognized inducer of genomic instability-might account in part for the transforming abilities of aluminium in mammary epithelial cells.

Keywords: aluminium; breast cancer; carcinogenesis; clastogens; genomic instability.

Figure 1

AlCl₃ transforms HC11 cells in vitro. (A) HC11 cells cultured for 70–73 weeks in the presence of the indicated concentrations of AlCl₃ or the same dilution (1/1000) of solvent (H₂O) alone were seeded in triplicate in ultra-low attachment 6-well plates at the density of 3 × 10⁴ cells/well and grown in complete medium—in the absence of AlCl₃—for 6 days. At the end of the incubation, cell proliferation was measured as reported in Materials and Methods. Data presented in the graph are means +/− SEM from three independent experiments. * p-value AlCl₃ vs. H₂O < 0.0001 (Dunnett’s multiple comparisons test) for both AlCl₃ concentrations. (B) HC11 cells cultured for 71 weeks in the presence of the indicated concentrations of AlCl₃, or the same volume (1/1000) of solvent (H₂O) alone were injected subcutaneously into the flank of 6-week-old BALB/cByJ female mice. Mice were inspected twice a week for tumour formation and sacrificed five months after injection. The graph shows the weight of each individual tumour at the time of dissection. A total of ten mice/condition were used, in two independent experiments. (C) HC11 cells cultured for 71 weeks in the presence of AlCl₃ 10 μM, or the same volume (1/1000) of solvent (H₂O) alone were injected into the mammary fat pad of the right ventral nipple of 6 weeks old BALB/cByJ female mice. Mice were sacrificed five months after injection. The graph shows the weight of each individual tumour at the time of dissection. Five mice or four mice were used for the H₂O or AlCl₃ 10 μM conditions, respectively.

Figure 2

AlCl₃ promotes HC11 cell tumour metastasis in syngeneic immunocompetent mice. (A) Whole lungs at the time of dissection or (B) hematoxylin/eosin (HE) staining of a lung metastasis found in a BALB/cByJ female mouse injected subcutaneously with HC11 cells transformed in vitro by AlCl₃ 10 μM (see Figure 1B). (C) HE staining of a hepatic metastasis in a Balb/cByJ female mouse injected orthotopically with HC11 cells transformed in vitro by AlCl₃ 10 μM (see Figure 1C). Bar = (B) 50 μm; (C) 100 μm.

Figure 3

Increased chromosomal rearrangements in HC11 cells transformed in vitro by AlCl₃. HC11 cells cultured for 71 weeks in the presence of the indicated concentrations of AlCl₃, or the same volume (1/1000) of solvent (H₂O) alone were analysed for the presence of chromosomal rearrangements by MFISH. The photos shown are representative examples of the results obtained. Arrows indicate chromosomal rearrangements observed uniquely in the specific condition considered. Fragments at the bottom left of the AlCl₃ 100 μM image are pericentromeric repetitive DNA markers.

Figure 4

Increased chromosomal rearrangements in NMuMG cells transformed in vitro by AlCl₃. NMuMG cells cultured for 38 weeks in the presence of AlCl₃ 100 μM or the same volume (1/1000) of solvent (H₂O) alone (NMuMG Series I, see text) were analysed for the presence of chromosomal rearrangements by MFISH. The photos shown are representative examples of the results obtained. Arrows indicate chromosomal rearrangements observed uniquely in the specific condition considered.

Figure 5

Short exposure to AlCl₃ increases chromosomal abnormalities and γ-H2AX nuclear foci in HC11 cells. (A) Quantification of cytogenetically abnormal cells in HC11 parental cells incubated for 24 h in the presence of AlCl₃ 100 μM or the same dilution (1/1000) of solvent (H₂O) alone. The graph shows the percentage of DAPI-stained metaphases containing at least one cytogenetic abnormality. Data (mean +/− SEM) are from three independent experiments where at least 100 metaphases per condition and per experiment were evaluated blind. * p < 0.0001 (chi-square test); * p< 0.0001 (logistic regression). (B) Quantification of γ-H2AX immunostaining in HC11 cells incubated for 24 h in the presence of AlCl₃ 100 μM or the same dilution (1/1000) of solvent (H₂O) alone. The graph shows the percentage of cells with at least 10 γ-H2AX nuclear foci/nucleus. Data (mean +/− SEM) are from three independent experiments where at least 450 cells per condition and per experiment were counted. * p< 0.0001 (chi-square test). (C) Detail of chromosomal abnormalities presented in (A). Key: 1 = radials, 2 = DSB, 3 = premature chromosome condensation, 4 = fragmentation, 5 = telofusion, 6 = premature chromatid separation. * p < 0.01 (chi-square test); ** p < 0.05 (chi-square test).