Related Experiment Videos
Single step selection of cis-diamminedichloroplatinum(II) resistant mutants from a human ovarian carcinoma cell line
K McLaughlin1, I Stephens, N McMahon
1Department of Medical Oncology, CRC Beatson Laboratories, Bearsden, Glasgow, United Kingdom.
Abstract:
We have shown that cis diamminedichloroplatinum-(II) (DDP) resistant mutants can be isolated from the human ovarian carcinoma cell line A2780 using a single-step selection protocol with DDP. DDP resistant colonies were calculated to be present at a frequency of 1.7 x 10(-6)/viable cell using a fluctuation analysis. The mutational origin of these surviving colonies is inferred by the fact that their frequency is increased by treatment of the A2780 cells with the chemical mutagen ethyl methane-sulfonate, with a maximum frequency observed after a 3-day expression time. Independently isolated clones maintain, in the absence of selection, a DDP resistant phenotype up to 7-fold more resistant than the parental A2780 cells. The resistance modifiers aphidicolin and buthionine sulfoximine have no effect on the frequency of DDP resistant mutants. Therefore neither of these drugs appears to have an effect on increasing the sensitivity of DDP resistant mutants existing in a cell population prior to DDP exposure.
Insights
Researchers isolated cis diamminedichloroplatinum-(II) (DDP) resistant ovarian cancer cells. Their frequency increased with chemical mutagens, indicating a mutational origin for DDP resistance.
Area of Science:
- Cancer Research
- Molecular Biology
- Drug Resistance Mechanisms
Background:
- Cis diamminedichloroplatinum-(II) (DDP) is a key chemotherapeutic agent for ovarian cancer.
- Understanding the mechanisms of DDP resistance is crucial for improving treatment efficacy.
- Ovarian carcinoma cell lines provide a model for studying drug resistance.
Purpose of the Study:
- To isolate and characterize DDP-resistant mutants from the A2780 human ovarian carcinoma cell line.
- To investigate the mutational basis of DDP resistance.
- To determine the stability of the DDP-resistant phenotype.
Main Methods:
- Single-step selection protocol using cis diamminedichloroplatinum-(II) (DDP).
- Fluctuation analysis to determine the frequency of resistant mutants.
- Treatment with ethyl methane-sulfonate (a chemical mutagen) to assess mutational origin.
- Assessment of DDP resistance stability in isolated clones.
Main Results:
- DDP-resistant mutants were successfully isolated from the A2780 cell line at a frequency of 1.7 x 10(-6)/viable cell.
- The frequency of DDP-resistant mutants significantly increased after treatment with ethyl methane-sulfonate, confirming a mutational origin.
- Independently isolated resistant clones exhibited up to a 7-fold increase in DDP resistance compared to parental cells, maintaining this phenotype without selection.
- Resistance modifiers aphidicolin and buthionine sulfoximine did not affect the frequency of DDP-resistant mutant generation.
Conclusions:
- A robust method for isolating DDP-resistant mutants from ovarian cancer cells was established.
- The observed DDP resistance arises from genetic mutations.
- The DDP-resistant phenotype is stable and not influenced by the tested resistance modifiers in the generation phase.