Abstract
The HTLV-1 Tax oncoprotein rapidly induces cytogenetic damage which can be measured by a significant increase in the number of micronuclei (MN) in cells. Tax is thought to have both aneuploidogenic and clastogenic effects. To examine the cellular target for Tax which might mechanistically explain the clastogenic phenomenon, we tested the ability of Tax to induce MN in rodents cells genetically defective for either the Ku80 protein or the catalytic subunit of DNA protein kinase (DNAPKcs). We found that cells genetically mutated in Ku80 were refractory to Tax's induction of MN while cells knocked-out for DNAPKcs showed increased number of Tax-induced MN. Using a cytogenetic method termed FISHI (Fluorescent In Situ Hybridization and Incorporation) which measures the number of DNA-breaks in cells that contained unprotected 3'-OH ends, we observed that Tax increased the prevalence of unprotected DNA breaks in Ku80-intact cells, but not in Ku80-mutated cells. Taken together, our findings suggest Ku80 as a cellular factor targeted by Tax in engendering clastogenic DNA damage.
Highlights
We previously demonstrated that expression of the HTLVI Tax oncoprotein rapidly induces cytogenetic damage which is reflected in a significant increase in the prevalence of micronuclei (MN) in cells [1,2,3,4]
To check Tax's effect in cells impaired for non-homologous end-joining (NHEJ), we first monitored the ambient frequency of micronuclei in hamster xrs-6 cells which have a mutated Ku80 gene [10,17]
We found that DNA protein kinase catalytic subunit (DNAPKcs)-/- cells had a ten fold higher ambient frequency of MN when compared to wild type mouse embryo fibroblasts (MEFs) (DNA-PKcs+/+); and we saw that DNAPKcs heterozygous MEFs (DNAPKcs+/-) showed a five fold increase in MN over control MEFs (Fig. 2)
Summary
We previously demonstrated that expression of the HTLVI Tax oncoprotein rapidly induces cytogenetic damage which is reflected in a significant increase in the prevalence of micronuclei (MN) in cells [1,2,3,4]. To further characterize the phenomenon of Tax associated clastogenic DNA-damage, we wished to examine the status DNAbreaks in the nucleus and in MN in the presence or absence of Tax [4]. Using a cytogenetic method termed FISHI (Fluorescent In Situ Hybridization and Incorporation), DNA-breaks in the nucleus and in MN with centric or acentric DNA fragments could be characterized for the presence or absence of free 3'-OH ends. Free 3'-OH ends represent breaks which are accessible to the in situ addition of digoxigenin (DIG) -labeled dUTP using terminal deoxynucleotidyl transferase. An absence of accessible 3'-OH ends suggests that the breaks are protected and masked by a protein complex. In vivo, unprotected free 3'-OH ends may progress to larger lesions leading to increasingly serious chromosomal lesions which may eventually sow the seed for cellular transformation
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
