Date on Master's Thesis/Doctoral Dissertation

8-2022

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Wise, Sr., John P.

Committee Co-Chair (if applicable)

Beverly, Levi

Committee Member

Beverly, Levi

Committee Member

Hoyle, Gary W.

Committee Member

Kouokam, J. Calvin

Committee Member

Liu, Jilan

Committee Member

Wise, Sandra S.

Author's Keywords

Hexavalent chromium; centrosomes; chromosome instability; carcinogenesis

Abstract

Lung cancer is the deadliest form of cancer and resulted in 1.8 million deaths worldwide in 2020. While cigarette smoking is the most familiar cause of lung cancer, up to 25% of cases occur in non-smokers, thus other environmental agents are also causative. Hexavalent chromium [Cr(VI)] is a known lung carcinogen and poses occupational and environmental exposure risks relevant to humans, wildlife, and ecosystems. This dissertation considers the carcinogenic mechanisms of a highly potent, particulate, hexavalent chromium compound, zinc chromate. The molecular mechanism of carcinogenesis induced by Cr(VI) is not fully understood, but it is known that chromosome instability is a key effect. Chromosome instability refers to structural instability characterized by breaks and translocations, and numerical instability characterized by changing numbers of chromosomes. This dissertation focuses on how hexavalent chromium causes numerical chromosome instability in human lung cells and uses the One Environmental Health approach to gain insights into the associated mechanism using whale cells as a comparative model. The hypothesis of this project is: Prolonged hexavalent chromium exposure targets securin in human lung cells, leading to centrosome amplification and numerical chromosome instability, while the ability of whale cells to retain normal securin levels confers resistance to these effects. A main driver of numerical chromosome instability is centrosome amplification, defined in this study as a single cell with more than two centrosomes. We previously found Cr(VI) induces centrosome amplification, which increased with duration and concentration of exposure and correlated with Cr(VI)-induced aneuploidy. In Aim 1 we focused on a novel potential target of Cr(VI), securin. Securin is an important centrosome regulator because it is the canonical inhibitor of separase. Separase is the enzyme that causes centriole disengagement and permits centrosome duplication. Prolonged Cr(VI) exposure decreased securin protein levels in a dose-dependent manner. Securin protein loss was not due to changes in protein degradation, but rather a loss of securin mRNA. Three measures of securin function were analyzed to determine if decreased securin levels were sufficient to control separase activity. Separase cleaves itself, kendrin, and cohesin. Prolonged Cr(VI) exposure caused increased separase autocleavage, increased kendrin cleavage, and increased separation at centromeres caused by cohesin cleavage. Securin knockdown increased levels of aneuploidy after acute Cr(VI) exposure, in contrast to untransfected cells which retained normal background levels. Together these data showed Cr(VI) disrupts securin, a key protein in the maintenance of numerical chromosome stability. Aim 2 sought to uncover the mechanism of Cr(VI)-induced securin loss described in Aim 1. Transcription factors bind to gene promoter regions to enhance or inhibit gene transcription and have been shown to be disrupted by Cr(VI). Thus, we measured three securin-promoting transcription factors, Sp1, NF-YA, and E2F1, and two repressing transcription factors, KLF6 and p53. Cr(VI) elevated levels of securin-promoting factors in nuclear fractions, indicating Cr(VI) is not repressing their levels to decrease securin expression. E2F1 was previously shown to decrease with prolonged Cr(VI) exposure, but experimental knockdown revealed E2F1 is not a driving factor for Cr(VI)-induced securin loss. Securin expression loss was also not explained by p53 activation, as protein levels and phosphorylation did not correlate with securin loss. KLF6 nuclear protein levels were increased at prolonged timepoints, which may begin to explain reduction in securin levels. MicroRNA (miRNA) regulation is altered by Cr(VI) and is a possible mechanism for securin loss. Several miRNAs were found to be significantly up- and down-regulated at all tested concentrations and timepoints. Securin was not specifically targeted by any of the altered miRNAs. However, several centrosome-associated proteins are putative targets of Cr(VI)-induced miRNA alteration. The One Environmental Health approach acknowledges humans, wildlife, and ecosystems are connected by a shared environment and common toxicants such as Cr(VI). Aim 3 characterizes the effects of prolonged Cr(VI) exposure on whale cells. We measured securin levels and function in bowhead whale cells and found securin levels remained normal under all exposure conditions and neither premature centromere separation nor centrosome amplification were increased. We found Cr(VI) did not cause aneuploidy in sperm whale or bowhead whale cells. Interestingly, chromosome damage still occurred, indicating Cr(VI) is active inside whale cells. These data indicate maintenance of securin under Cr(VI) exposure may protect whale cells from developing centrosome amplification and numerical chromosome instability. Overall, this dissertation shows securin is targeted by prolonged Cr(VI) exposure in human lung cells and centrosome regulation pathways are central in the mechanism of Cr(VI) carcinogenesis. We showed whales are resistant to securin loss and the proposed downstream phenotypes, centrosome amplification and numerical chromosome instability. Together these data support the hypothesis Cr(VI)-induced securin loss leads to numerical chromosome instability.

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