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including insensitivity of Epo B-induced cell kill to caspase inhibition, which had
been previously observed in HeLa cells by McDaid and Horwitz.
55
As is generally the case for anticancer drugs, the cellular response to micro-
tubule-stabilizing agents can be modulated by adaptive changes of the cell that lead to
acquired drug resistance. Alternatively, cells may be inherently protected from the
antiproliferative effects of cytotoxic agents by a variety of mechanisms. In contrast
to paclitaxel (as well as other standard cytotoxic anticancer agents), Epo A/B are
not susceptible to phosphoglyco-protein-170 (P-gp)-mediated drug efflux and thus
retain full antiproliferative activity against the corresponding multidrug-resistant
cell lines in vitro (Table 1-1).
11,19,20,45
This characteristic may provide a distinctive
advantage of epothilones over current taxane-based therapy, but the clinical signif-
icance of P-gp-mediated drug resistance is a matter of significant debate.
56
On the
other hand, recent discoveries from various laboratories demonstrate that cancer
cells can become resistant to epothilones through alternative mechanisms, such
as tubulin mutations. For example, Wartmann and Altmann have isolated an
epothilone-resistant subline of the KB-31 epidermoid carcinoma cell line (termed
KB-31/C5), which carries a single point-mutation (Thr274 Pro) in the HM40 tubulin
gene (the major b-tubulin isoform expressed in these cells).
37
Similar findings have
been independently reported by Giannakakou et al., who have produced an Epo
A-resistant cell line, 1A9/A8, in which Thr274 is mutated to Ile rather than to Pro.
57
Thr274 maps to the taxane-binding site on b-tubulin,
58
which based on competition
studies, is likely to be targeted also by epothilones (vide supra). Consistent with the
notion of a shared binding site between epothilones and paclitaxel, both KB-31/C5
as well as 1A9/A8 cells are cross-resistant to paclitaxel, albeit to varying degrees.
More recently, He et al. have generated three different Epo-resistant cell lines,
A549B40, HeLa.EpoA9, and HeLa.EpoB1.8, each of which is characterized by a
specific b-tubulin mutation, namely Gln292Glu in A549B40 cells, Pro173Ala in
HeLa.EpoA9 cells, and Tyr422Cys in HeLa.EpoB1.8 cells.
59
The highest degree of
resistance is associated with A549.EpoB40 cells, which are 95-fold resistant to Epo B
and exhibit marked cross-resistance with other microtubule-stabilizing agents, with the
notable exception of discodermolide. The b-tubulin mutations identified by the Horwitz
laboratory map to sites on b-tubulin that have been suggested to be involved in paclitaxel
binding and lateral protofilament interaction (Gln292), GTP hydrolysis (Pro173), and
binding of MAPs (Tyr422), respectively.
The above Gln292Glu mutation in combination with a second mutation at posi-
tion 231 of b-tubulin (Thr
!
Ala) was also identified by Verrills et al.
60
in a highly
resistant subline of the human T-cell acute leukemia cell line CCRF-CEM (termed
dEpoB300), which had been selected with Epo D (deoxyEpo B). dEpo300 cells are
307-fold resistant to the selecting agent Epo D and exhibit 77-fold and 467-fold
cross-resistance with Epo B and paclitaxel, respectively. Epo D failed to induce
any measurable tubulin polymerization in cell lysates prepared from dEpoB300
cells at 8 mM compound concentration, which thus illustrates that impaired growth
inhibition is indeed paralleled by diminished effects on tubulin polymerization.
In summary, all tubulin mutations identified to date in epothilone-resistant cells
are found in regions of the tubulin structure, which are predicted to be important for
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