Related Experiment Videos
High rate of multilocus deletion in a human tumor cell line
J Harwood1, A Tachibana, R Davis
1Clare Hall Laboratories, Hertfordshire, UK.
Abstract:
The nature of recessive mutations at the autosomal locus encoding the purine salvage enzyme adenine phosphoribosyl transferase (APRT) was analyzed in a highly malignant human tumor cell line (the colorectal carcinoma line SW620). Mutant strains resistant to the purine analog 8-azaadenine were obtained in two steps. The first step selection for partial drug resistance produced strains hemizygous for APRT as a result of high frequency loss of one allele. In the second step selection, low frequency base substitutions, small deletions, or insertions produced complete azaadenine resistance. Luria-Delbruck fluctuation analysis of each step of this process indicated that the rate of mutation resulting from allele loss was over 100-fold greater than the rate of mutation resulting from base substitution. There was no reproducible difference in the rate of loss of either of the two APRT alleles even though one maps to a rearranged chromosome. Similarly base substitution rates for the two alleles were not significantly different. Polymorphic loci surrounding APRT on chromosome 16 band q24 were lost together with the selected gene in all isolates while polymorphic loci on the short arm of the chromosome were retained. Thus the high frequency loss of APRT in SW620 appears to be the result of multilocus deletions. SW620 derivatives behaving as heterozygotes were also obtained in the first step selections, but these constituted only 5% of isolates.
Insights
Recessive mutations in adenine phosphoribosyl transferase (APRT) were studied in SW620 colorectal cancer cells. High-frequency allele loss, not base substitution, drove drug resistance, indicating multilocus deletions.
Area of Science:
- Molecular genetics
- Cancer cell biology
- Biochemistry
Background:
- Adenine phosphoribosyl transferase (APRT) is crucial for purine salvage.
- Recessive mutations in APRT can lead to drug resistance.
- Understanding mutation mechanisms in cancer cells is vital for therapeutic strategies.
Purpose of the Study:
- To analyze the nature of recessive mutations in the APRT gene in a human colorectal carcinoma cell line (SW620).
- To investigate the mechanisms driving drug resistance related to APRT mutations.
- To determine the relative frequencies of different mutation types, including allele loss and base substitutions.
Main Methods:
- Selection of mutant strains resistant to the purine analog 8-azaadenine in two steps.
- Luria-Delbruck fluctuation analysis to determine mutation rates.
- Analysis of polymorphic loci surrounding APRT to identify chromosomal alterations.
Main Results:
- High-frequency loss of one APRT allele occurred during initial drug selection, indicating hemizygosity.
- Subsequent selection revealed low-frequency base substitutions, deletions, or insertions leading to complete resistance.
- Mutation rate from allele loss was over 100-fold higher than from base substitution.
- Multilocus deletions involving the APRT locus on chromosome 16 band q24 were identified as the cause of high-frequency APRT loss.
Conclusions:
- The high frequency of recessive APRT mutations in SW620 cells is primarily driven by multilocus deletions, not base substitutions.
- Allele loss is a significantly more frequent mutational event than base substitution in this context.
- These findings highlight the role of large-scale genomic instability in generating drug resistance in cancer cells.