Carcinogen treatment leads to mitotic defects and arrest in cancer and noncancer cells
- Published
- Accepted
- Subject Areas
- Cell Biology, Molecular Biology
- Keywords
- mitosis, cancer, lagging chromosomes, mitotic defects, carcinogen
- Copyright
- © 2018 Hartling et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2018. Carcinogen treatment leads to mitotic defects and arrest in cancer and noncancer cells. PeerJ Preprints 6:e27177v1 https://doi.org/10.7287/peerj.preprints.27177v1
Abstract
Genomic instability can manifest due to both chromosomal rearrangements and gain and loss of entire chromosomes. One mechanism by which a carcinogen acts is by increasing the rate of mitotic spindle defects during proliferation. These defects can lead to chromosomal instability that manifest as lagging chromosomes, anaphase bridges, or multipolar spindles. While several mechanisms exist to rectify these errors prior to completion of mitosis, some cells will escape repair, while others will prematurely exit mitosis. Here we examine the effects of two carcinogenic molecules: Fulvestrant, a chemotherapeutic that functions as a selective estrogen receptor degrader, and vinyl chloride, a hydrocarbon used to produce PVC. We exposed two cancer lines, A549 and UPCI:SCC103, and one noncancer line, GM03349, to increasing concentrations of the carcinogen for increasing durations, up to 48 hours exposure. We found that exposure to the carcinogen lowered the mitotic index in the cancer cell lines, while raising it in the noncancer line. Concurrently, we observed massive increases in the frequency of mitotic defects, with the most significant increases seen in prevalence of lagging chromosomes in prometaphase and metaphase and anaphase bridges. Live cell imaging showed that the occurrence of either of these defects had the strongest correlation with the likelihood that the cell would fail to complete mitosis. We also show that washing out the carcinogen decreases the frequency of mitotic defects in all three cell lines, but the mitotic index does not recover in the cancer cells. These findings demonstrate that carcinogen-induced mitotic defects have marked effects on the proliferative population of cells in terms of potential for contributing to chromosomal instability or removal from that population.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
A549 cell dividing normally, no discernable mitotic defect
A549 cell treated with 20 μM vinyl chloride for 24 hours before being labeled with SiR-DNA. 21 stacks in the Z plane were imaged every three minutes for a total duration of 1 hour, 24 minutes.
A549 cell with prometaphase/metaphase laging chromosome fails to complete mitosis
A549 cell treated with 20 μM vinyl chloride for 24 hours before being labeled with SiR-DNA. 21 stacks in the Z plane were imaged every three minutes for a total duration of 2 hours.
A549 cell with anaphase bridge that completes mitosis
A549 cell treated with 20 μM vinyl chloride for 24 hours before being labeled with SiR-DNA. 23 stacks in the Z plane were imaged every three minutes for a total duration of 4 hours 40 minutes.
UPCI:SCC103 cell with no discernable mitotic defect, fails to complete mitosis
UPCI:SCC103 cell treated with 20 μM vinyl chloride for 24 hours before being labeled with SiR-DNA. 22 stacks in the Z plane were imaged every three minutes for a total duration of 1 hour 54 minutes. The cell appears to eject chromosomal material after failing to proceed to anaphase.
UPCI:SCC103 cell with prometaphase lagging chromosome, fails to complete mitosis
UPCI:SCC103 cell treated with 20 μM vinyl chloride for 24 hours before being labeled with SiR-DNA. 24 stacks in the Z plane were imaged every three minutes for a total duration of 2 hours 36 minutes.
UPCI:SC103 cell with anaphase bridge that completes mitosis
UPCI:SCC103 cell treated with 20 μM vinyl chloride for 24 hours before being labeled with SiR-DNA. 21 stacks in the Z plane were imaged every three minutes for a total duration of 1 hour, 18 minutes.
Viability counts after treatment with Fulvestrant or vinyl chloride
Raw data for viability counts for A549, UPCI:SCC103, and GM03349 cells after treatment with increasing concentrations of Fulvestrant or Vinyl Chloride for 24, 36, or 48 hours. Viability was measured using flow cytometry. T-tests were performed comparing each experimental condition to control values individually, p-values are noted.
Mitotic defect frequency after Fulvestrant treatment
Raw data: A549, UPCI:SCC103 and GM03349 cells were treated with increasing concentrations of Fulvestrant for 24, 36, or 48 hours and scored for mitotic index. Cell counts and percentages are recorded. T-tests were performed comparing each experimental condition to control values individually, p-values are noted.
Mitotic Index after vinyl chloride treatment
Raw data: A549, UPCI:SCC103 and GM03349 cells were treated with increasing concentrations of vinyl chloride for 24, 36, or 48 hours and scored for mitotic index. Cell counts and percentages are recorded. T-tests were performed comparing each experimental condition to control values individually, p-values are noted.
Frequency of mitotic defects after Fulvestrant treatment
Raw data: A549, UPCI:SCC103 and GM03349 cells were treated with increasing concentrations of Fulvestrant for 24, 36, or 48 hours and scored for frequency of mitotic defects. Cell counts and percentages are recorded. T-tests were performed comparing each experimental condition to control values individually, p-values are noted.
Frequency of mitotic defects after vinyl chloride treatment
Raw data: A549, UPCI:SCC103 and GM03349 cells were treated with increasing concentrations of vinyl chloride for 24, 36, or 48 hours and scored for frequency of mitotic defects. Cell counts and percentages are recorded. T-tests were performed comparing each experimental condition to control values individually, p-values are noted.
Live Cell Imaging Outcomes
Raw Data: Summary of all movies generated. A549, UPCI:SCC103, and GM03349 cells were imaged (either with or without 24 hours exposure to 20 μM vinyl chloride) and outcomes recorded for both presence of mitotic defects and completion of mitosis. Movies were analyzed frame-by-frame for scoring purposes.
Mitotic Index after recovery from vinyl chloride treatment
Raw data: A549, UPCI:SCC103 and GM03349 cells were treated with 20 μM vinyl chloride for 24 hours before washout with fresh media. Cells were fixed at 0, 24, 48, 72, and 96 hours post-washout and scored for mitotic index. Cell counts and percentages are recorded. T-tests were performed comparing each experimental condition to control values individually, p-values are noted.
Frequency of mitotic defects after recovery from vinyl chloride treatment
Raw data: A549, UPCI:SCC103 and GM03349 cells were treated with 20 μM vinyl chloride for 24 hours before washout with fresh media. Cells were fixed at 0, 24, 48, 72, and 96 hours post-washout and scored for frequency of mitotic defects. Cell counts and percentages are recorded. T-tests were performed comparing each experimental condition to control values individually, p-values are noted.