| Abstract |
Microtubules are composed of α,β-tubulin heterodimers and are involved in the
organization and function of eukaryotic cells. Although tubulin is considered as a
cytoplasmic protein, there are reports supporting the presense of tubulin in nuclei of cultured
animal cells. Earlier immunocytochemical studies suggest that βΙΙ-tubulin is the main nuclear
isoform. We therefore investigated, using different techniques, the mechanism that regulates
the sub-cellular localization of tubulin in human carcinoma cell lines. We examined the
localization of either endogenous soluble α,β-tubulin or GFP-tagged βΙΙ-, ΔβΙΙ (deletion from
the sequence of βΙΙ-tubulin of amino acids 232-266 corresponding to three putative NESs)
and βΙV-tubulin and we found that were located in both nucleus and cytoplasm. The
abundance of tubulin in the nucleus was greater in the case of GFP-ΔβΙΙ-tubulin. The
accumulation in the nucleus of GFP-tagged isoforms βΙΙ- and βIV-tubulin was increased
when cells have been treated with leptomycin B. Moreover, endogenous soluble tubulin was
accumulated in the nucleus under depolymerization conditions of microtubules and low
temperature, whereas its nuclear export was partially inhibited by leptomycin B. These
results suggest that soluble tubulin a) enters the nucleus by passive diffusion, b) is
accumulated in the nucleus by its interaction with nuclear components, probably H3 and c) is
exported in the cytoplasm by an active mechanism, which is partially dependent on exportin-
1.
We have previously shown that binding of soluble tubulin to the nuclear envelope /
peripheral heterochromatin inhibits the association of heterochromatin protein 1 (HP1) with
the nuclear envelope. Therefore, we investigated the interaction of tubulin with nuclear
envelope proteins and we found that tubulin binds in a specific and dose-dependent manner
to histone H3. Post-translational modifications in the amino-terminal domain of H3 and
polyglutamic stretches in the carboxy-terminal domain of tubulin are not required for this
interaction. Moreover, tubulin inhibits binding of H3 to all HP1 variants and LBR, prevents
formation of the multi-component complex consisting of LBR, HP1 and core histones and,
therefore, might act as a regulator for the association/dissociation of HP1-enriched
heterochromatin to the nuclear envelope.
Paclitaxel (taxol) is the most important anticancer drug that is widely used in the
treatment of human carcinomas. A disadvantage of this drug is the low solubility in water
and in most pharmaceutically acceptable solvents. In this work we used analogues in which
multiple copies of paclitaxel were covalently bound to synthetic carriers (Ac-[Lys-Aib-
Cys(CH2CO-2’-paclitaxel)]n-NH2, n=2, 3, 4) aiming the improvement of the water solubility.
We found that Ac-[Lys-Aib-Cys(CH2CO-2’-paclitaxel)]4-NH2 inhibited the proliferation of
cancer cells more potently than other synthetic analogues and paclitaxel. Moreover, the
effectiveness of Ac-[Lys-Aib-Cys(CH2CO-2’-paclitaxel)]4-NH2 on cell cycle arrest at G2/M
phase and the production of multinucleated cells which often presented nuclear envelope
discontinuity, was greater compared to paclitaxel. In conclusion, our results indicate an
improved biological activity of the synthetic analogue, compared to paclitaxel.
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