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Page 164 Elton et al. Cancer Drug Resist 2020;3:161-70 I http://dx.doi.org/10.20517/cdr.2019.117

through the DNA gate [39,40] . This intact DNA duplex then exits from the open C gate [39,40] , which comprises the
coiled-coil region (coiled domain) and the C-terminal domain from each monomer encoded by TOP2α
[37]
exons 28-35 [Figure 1B-i]. After T-segment strand passage and ATP hydrolysis, the G-segment double-
strand DNA break is resealed and free Tyr805 residues present in each TOP2α/170 subunit are regenerated.
Finally, the ATP gate is reopened, and this processive enzyme is reset for another round of catalytic
activity [39,40] . Given the complexity of this enzyme’s reaction cycle, truncated TOP2α isoforms, translated
as a result of alternative RNA splicing, may exhibit atypical TOP2α functionality and response to targeted
agents.

TOP2α/160 (INTRON 33 RETENTION) AND CHEMORESISTANCE
Several acquired and innate resistant models have been reported, which involve intron retention due to
[50]
alternative RNA processing of TOP2α mRNA [32-36] . Harker et al. generated a mitoxantrone resistant
human AML (HL-60) cell line designated HL-60/MX2 (35-fold resistant), by stepwise drug exposure from
1.7 to 170 nM. HL-60/MX2 cells were found to be cross-resistant to a number of TOP2α poisons including
[50]
etoposide, amsacrine, teniposide, daunorubicin, and doxorubicin . Compared to parental HL-60 cells, HL-
60/MX2 cells contained reduced TOP2α/170 protein levels and expressed a novel truncated TOP2α isoform
[51]
migrating at ~160 kDa (TOP2α/160) that localized predominantly to the cytoplasm . Interestingly,
TOP2α/160 (1436 aa and a calculated molecular weight of 164,052 Da) is the translation product of a
TOP2α mRNA (4550 nt) that harbors exons 1-33 and retains a processed intron 33 (125 nt) that contains
[32]
an in-frame stop codon and a consensus poly(A) site [Figure 1A-ii] . As a result of intron 33 retention
and processing, TOP2α/160 is missing the C-terminal 108 aa present in TOP2α/170 (1531 aa), which are
[32]
replaced by 14 unique aa encoded by translation of the exon 33/intron 33 “read-through” [Figure 1B-ii] .
Importantly, TOP2α/160 is missing the well-characterized nuclear localization signal (NLS) NLS1454-
1497 [46,47] [Figure 1B-ii]. This isoform is also missing a “chromatin tether” sequence, which interacts with
[52]
histone tails and anchors TOP2α/170 to nucleosomes . These deletions may account for the accumulation
[32]
of TOP2α/160 in the cytoplasm .
[53]
Similarly, Feldhoff et al. generated a resistant H209 small cell lung cancer cell line, designated H209/V6
(22-fold resistant), by stepwise selection in etoposide (from 0.2 to 6 µM). These investigators demonstrated
that, compared to parental H209 cells expressing TOP2α/170, H209/V6 cells only expressed a TOP2α/160
[53]
isoform . Additionally, it was shown by immunocytochemistry and cytoplasm/nuclear fractionation
[33]
[54]
studies that TOP2α/160 was primarily localized in the cytoplasm . Yu et al. subsequently characterized
a TOP2α mRNA splice variant (7090 nt) expressed in the etoposide resistant H209/V6 cell line that
harbored exons 1-33, the entire intron 33, and included exons 34 and 35 [see Figure 1A for orientation].
[32]
Although this mRNA is much longer than the 4550 nt transcript from HL60/MX2 cells , it is still
[32]
translated into the same TOP2α/160 (1436 aa, 164,052 Da) isoform described above by Harker et al.
[Figure 1A-ii] due to the in-frame stop codon present in retained intron 33, loss of the canonical NLS, and
consequent aberrant localization in the cytoplasm [33,53,54] .

[34]
Mo and Beck characterized TOP2α mRNA splice variants in TOP2α poison sensitive T-lineage tumor cell
lines (e.g., CEM, Jurkat, and H9). One of four TOP2α mRNA splice variants characterized in CEM cells was
[32]
identical to the transcript that was described above by Harker et al. with exons 1-33, followed by a retained
and processed intron 33 (4550 nt), and again encoded the identical TOP2α/160 (1436 aa, 164,052 Da). This
truncated TOP2α isoform and others generated from intron retention in T-cell lines were lacking the canonical
[34]
NLS and all were detected in cytoplasmic extracts . Interestingly, normal T-cells contained only TOP2α/170,
prompting these investigators to suggest that splice variants of TOP2α play a role in leukemogenesis, although
no further investigations to explore this possibility have been reported.

Together, these previous reports suggest that intron retention can play a role in generation of truncated
TOP2α isoforms secondary to alternative RNA processing. The production of truncated TOP2α isoforms
can be determinants of drug resistance and/or play a role tumor cell biology not yet characterized.