The HTLV-I Tax oncoprotein induces rapid cytogenetic damage which can be measured by a significant increase in the number of micronuclei (MN) and unstabilized DNA breaks in cells [1–4]. Tax is thought to have both aneuploidogenic and clastogenic effects [3–7]. We previously characterized the phenomenon of Tax-associated clastogenic DNA damage by examining the status of DNA breaks in the nucleus and in the MN in the presence or absence of Tax [4–6]. In particular, we characterized DNA breaks for the presence or absence of free 3′-OH ends . Free 3′-OH ends represent breaks accessible to the in situ addition of digoxigenin (DIG)-labeled dUTP using terminal deoxynucleotidyl transferase. On the other hand, an absence of accessible 3′-OH ends suggests that the breaks maybe protected by a protein complex(es).
Unprotected free 3′-OH ends can progress to larger lesions leading to increasingly serious chromosomal defects which may sow the seed for cellular transformation [4–6]. Previously, we were interested to examine the cellular target for Tax in an attempt to explain mechanistically its clastogenic phenomenon. Accordingly, we tested the ability of Tax to induce MN and unstabilized DNA breaks in rodent cells genetically defective for either the Ku80 protein or the catalytic subunit of DNA protein kinase (DNA PKcs) . We found that the Ku80 mutant cells were refractory to the induction of MN by Tax while cells knocked out for DNA PKcs remained responsive to Tax induction of increased MN . Moreover, Tax expression increased the prevalence of unprotected DNA breaks in Ku80-intact cells, but not in Ku80-mutated cells , implicating Ku80 as a necessary cellular factor targeted by Tax for engendering clastogenic DNA damage .
In the earlier experiments, we studied the frequency of MN and the prevalence of unstable DNA breaks after transfection of an entire cell population with a Tax-expression plasmid, evaluating the bulk cytogenetic damage on all the “transfected” cells without segregating those specifically expressing Tax from those that did not express Tax [1–6]. In the present work, we have focused the analysis to studying the frequency of MN and unstable DNA breaks in cells specifically identified to express GFP-Tax that has been shown in many publications to reflect the activities of wild type Tax protein. Moreover, we have extended the analyses of Tax effects beyond the Ku80 and PKcs proteins  to also include the XRCC4 and H2AX proteins. En toto, Ku80, PKcs, XRCC4 and H2AX are proteins directly or indirectly involved in NHEJ repair [8–13]. Ku80, PKcs, and XRCC4 function sequentially in the NHEJ pathway which repairs DNA double strand breaks [8, 9]. Ku80 substantially protects the breaks  allowing subsequent intervention by PKcs  which, in turn, appears to be vital for the recruitment of the XRCC4/ligase IV proteins to religate DNA breaks thus completing repair [8, 12, 13]. The NHEJ system is influenced by the histone H2AX which marks damaged DNA and undergoes various types of modifications in response to double-strand DNA breaks [14, 15].
Here, we have employed wild type and Ku80- [16, 17], PKcs- , XRCC4- [8, 19] or H2AX-  mutant cells to examine the induction of MN and the prevalence of unstabilized DNA breaks in cells without or with the expression of Tax. In all the cells, DNA breaks were assessed for their frequency and also for their signal strength produced by DIG-dUTP incorporation. To interpret the latter readout, we compared Tax-induced DNA signal-size with corresponding signal-size of breaks induced by etoposide. Etoposide is known to elicit DNA scission . It interferes with the protective action of Ku proteins leaving unstabilized topoisomerase-induced breaks . Because DIG-dUTP signal strength is expected to reflect the size of the DNA lesion at DNA breaks, our approach allows us to quantify the extent of DNA-damage in the various mutant cells by the size of the DIG-dUTP incorporation signals in the presence of Tax or etoposide. Thus, we could compare the relative contribution of loss of Ku80, PKcs, XRCC4, or H2AX to Tax-induced DNA damage.