Hexavalent Chromium Targets Securin to Drive Numerical Chromosome Instability in Human Lung Cells

Abstract

Hexavalent chromium [Cr(VI)] is a known human lung carcinogen with widespread exposure in environmental and occupational settings. Despite well-known cancer risks, the molecular mechanisms of Cr(VI)-induced carcinogenesis are not well understood, but a major driver of Cr(VI) carcinogenesis is chromosome instability. Previously, we reported Cr(VI) induced numerical chromosome instability, premature centriole disengagement, centrosome amplification, premature centromere division, and spindle assembly checkpoint bypass. A key regulator of these events is securin, which acts by regulating the cleavage ability of separase. Thus, in this study we investigated securin disruption by Cr(VI) exposure. We exposed human lung cells to a particulate Cr(VI) compound, zinc chromate, for acute (24 h) and prolonged (120 h) time points. We found prolonged Cr(VI) exposure caused marked decrease in securin levels and function. After prolonged exposure at the highest concentration, securin protein levels were decreased to 15.3% of control cells, while securin mRNA quantification was 7.9% relative to control cells. Additionally, loss of securin function led to increased separase activity manifested as enhanced cleavage of separase substrates; separase, kendrin, and SCC1. These data show securin is targeted by prolonged Cr(VI) exposure in human lung cells. Thus, a new mechanistic model for Cr(VI)-induced carcinogenesis emerges with centrosome and centromere disruption as key components of numerical chromosome instability, a key driver in Cr(VI) carcinogenesis.

Keywords: centrosome amplification; chromosome instability; hexavalent chromium; securin.

Figure 1

Prolonged Cr(VI) exposure decreases securin protein levels. (A) Representative Western blot for securin. Alpha-tubulin was used as a loading control. (B) Securin whole cell protein levels decreased after 120 h Cr(VI) exposure. Data are expressed as percent of untreated control cells and reflect the mean of three independent experiments. Error bar = standard error of the mean. * Significantly different from the control group (p < 0.05).

Figure 2

Cr(VI) does not change rates of securin degradation. (A–C) Representative Western blots for securin after cycloheximide (CHX) treatment. Alpha-tubulin was used as a loading control. (A) Securin protein after 24 h Cr(VI). (B) Securin protein after 72 h Cr(VI). (C) Securin protein after 120 h Cr(VI). (D) Representative plot of securin protein degradation over time on log base 2 scale (120 h zinc chromate exposure example). Solid lines represent the securin signal and dotted lines represent the best fit line used for calculating the protein half-life. (E) Securin half-life did not significantly change after zinc chromate exposure. Data reflect the mean of three independent experiments. Error bar = standard error of the mean. No condition was significantly different from the untreated control group.

Figure 3

Cr(VI) exposure decreases securin mRNA after 24 and 120 h. Data are expressed as relative expression compared to untreated control cells and reflect the mean of three independent experiments with three technical replicates each. Error bar = standard error of the mean. * Significantly different from the control group (p < 0.05).

Figure 4

Prolonged Cr(VI) exposure induced separase cleavage and decreased separase mRNA expression. (A) Representative Western blot for separase. Alpha-tubulin was used as a loading control. (B) Separase full-length protein levels increased slightly after 120 h Cr(VI). (C,D) Cleaved separase protein levels greatly increased after 120 h Cr(VI). (E) Separase mRNA levels decreased after 24 and 120 h zinc chromate exposure. Data are expressed as percent of untreated control cells and reflect the mean of three independent experiments. Error bar = standard error of the mean. * Significantly different from the control group (p < 0.05).

Figure 5

Prolonged Cr(VI) exposure induced kendrin cleavage. (A) Representative Western blot showing full-length and cleaved kendrin bands. Alpha-tubulin was used as a loading control. (B) Full-length kendrin levels decreased after 120 h Cr(VI). (C) Cleaved kendrin protein levels increased after 120 h. Data are expressed as percent of untreated control cells and reflect the mean of three independent experiments. Error bar = standard error of the mean. * Significantly different from the control group (p < 0.05).

Figure 6

Prolonged Cr(VI) exposure increases nuclear SCC1 cleavage. (A) Representative Western blot showing full-length and cleaved SCC1 bands from nuclear extract. Lamin B1 was used as a loading control. (B) The ratio of cleaved/full-length nuclear SCC1 was not significantly altered but showed an increasing trend, indicating increased cohesin cleavage. Data are expressed as percentage of untreated control cells and reflect the mean of two independent experiments. Error bars = standard error of the mean. No condition was significantly different from the control group.

Figure 7

Prolonged Cr(VI) exposure decreased cyclin B1 levels. (A) Representative Western blot for cyclin B1. GAPDH was used as a loading control. (B) Cyclin B1 whole cell protein levels decreased after 120 h Cr(VI) exposure. Data are expressed as percent of untreated control cells and reflect the mean of three independent experiments. Error bar = standard error of the mean. * Significantly different from the control group (p < 0.05).

Figure 8

Cr(VI) decreased the percentage of cells in G1 and increased the percentage of cells in G2/M. Data are expressed as percent of untreated control cells and reflect the mean of three or four independent experiments. Error bar = standard error of the mean. ** Significantly different from the control group (p < 0.001).

Figure 9

Mechanistic model of Cr(VI)-induced numerical chromosome instability.