Asok Banerjee

Increased levels of tyrosinated α-, βIII-, and βIV-tubulin isotypes in paclitaxel-resistant MCF-7 breast cancer cells

Paclitaxel (PTX), the diterpene alkaloid, is a potent anti-cancer drug and is routinely used for the treatment of breast and ovarian cancers. The cellular targets of PTX are microtubules, which are composed of alpha- and beta-tubulin. Development of PTX resistance in patients has been a major problem associated with cancer chemotherapy. In an effort to get insight into this phenomenon of drug resistance, a PTX-resistant cell line from MCF-7 breast cancer cells has been generated. Western analysis of the cell extracts revealed that the resistant cells contain 2-fold higher amount of tyrosinated alpha-tubulin than those of the wild-type MCF-7 cells. Similar analyses of beta-tubulin with the isotype-specific monoclonal antibodies demonstrated that the PTX-resistant cells contain 2.5-fold higher amounts of beta(III) and 1.5-fold higher amount of beta(IV)-tubulin, while no difference was observed in the level of beta(I) isotype. These results demonstrate for the first time that PTX resistance is associated with an increase in the level of tyrosinated alpha-tubulin.

Tubulin structure and biochemistry.

Abstract In the past year, much has been learned about structure-function correlations in the tubulin molecule, and specifically about the nature and roles of post-translational modifications and tubulin isotypes. The interactions between tubulin and its ligands — both microtubule-associated proteins and anti-mitotic drugs — are becoming clearer at the molecular level.

Kinetics of association and dissociation of colchicine-tubulin complex from brain and renal tubulin Evidence for the existence of multiple isotypes of tubulin in brain with differential affinity to colchicine

The kinetics of colchicine binding to bovine brain tubulin have been reported to be biphasic under pseudo first order conditions [(1978) Biochemistry 17, 4466‐4472]. Unlike brain tubulin, the kinetics of colchicine binding to bovine renal tubulin are monophasic. The apparent on‐rate constant for the binding of colchicine to renal tubulin is found to be very close to that of the faster binding component in brain tubulin. Similarly, the dissociation of colchicine‐tubulin complex in the presence of iodide is biphasic for brain tubulin but monophasic for renal tubulin. Since brain and renal tubulin apparently differ in β‐tubulin, our results suggest that the biphasic nature of the kinetics for bovine brain tubulin could possibly originate from the existence of multiple isotypes of tubulin differing in drug binding affinity.

The Isotypes of Tubulin

The tubulin molecule is an α/β heterodimer. In most eukaryotes both α-and β-tubulin consist of isotypes encoded by different genes and differing in amino acid sequence. Differences among isotypes are often highly conserved in evolution, suggesting that they have functional significance. The complex isotype families in mammals, Drosophila and higher plants have been particularly well studied. Different isotypes often have different cellular and tissue distributions. In addition, purified isotypes display different properties including assembly, GTPase, conformation, dynamics, and ability to interact with anti-tumor drugs. The different cellular, tissue, and species distribution, as well as their primary structures and their in vitro properties give clues as to the possible functions of the different isotypes, which will be discussed in this chapter.

Roles of β-Tubulin Residues Ala428 and Thr429 in Microtubule Formation in Vivo

The C termini of β-tubulin isotypes are regions of high sequence variability that bind to microtubule-associated proteins and motors and undergo various post-translational modifications such as polyglutamylation and polyglycylation. Crystallographic analyses have been unsuccessful in resolving tubulin C termini. Here, we used a stepwise approach to study the role of this region in microtubule assembly. We generated a series of truncation mutants of human βI and βIII tubulin. Transient transfection of HeLa cells with the mutants shows that mutants with deletions of up to 22 residues from βIII and 16 from βI can assemble normally. Interestingly, removal of the next residue (Ala428) results in a complete loss of microtubule formation without affecting dimer formation. C-terminal tail switching of human βI and βIII tubulin suggests that C-terminal tails are functionally equivalent. In short, residues outside of 1–429 of human β-tubulins make no contribution to microtubule assembly. Ala428, in the C-terminal sequence motif N-QQYQDA428, lies at the end of helix H12 of β-tubulin. We hypothesize that this residue is important for maintaining helix H12 structure. Deletion of Ala428 may lead to unwinding of helix H12, resulting in tubulin dimers incapable of assembly. Thr429 plays a more complex role. In the βI isotype of tubulin, Thr429 is not at all necessary for assembly; however, in the βIII isotype, its presence strongly favors assembly. This result is consistent with a likely more complex function of βIII as well as with the observation that evolutionary conservation is total for Ala428 and frequent for Thr429.

The role of the B-ring of colchicine in the stability of the colchicine-tubulin complex

The binding of colchicine to tubulin is a slow, temperature-dependent and a poorly reversible process. Colchicine analogues modified in the B-ring of colchicine have been reported to bind to tubulin fairly rapidly (Ray, K., Bhattacharyya, B. and Biswas, B.B. (1981) J. Biol. Chem. 256, 6241-6244). In an effort to test the role of the B-ring in the reversibility of the colchicine-tubulin binding reaction, we have studied the kinetics of dissociation of the drug-tubulin complex for two B-ring-modified colchicine analogues under conditions in which the association reaction was blocked with a 40-fold excess of podophyllotoxin. In both cases, the dissociation was biphasic. The off-rate constants were determined and the results strongly suggest that the B-ring part of colchicine is responsible for the stability of the drug-tubulin complex. The dissociation data have been explained in terms of a binding model in which the binding of colchicine to tubulin involves a three-subdomain interaction rather than the previously suggested two-subdomain model (Andreu, J.M. and Timasheff, S.N. (1982) Biochemistry 21, 534-543).

Computational Design and Biological Testing of Highly Cytotoxic Colchicine Ring A Modifications

Microtubules are the primary target for many anti‐cancer drugs, the majority of which bind specifically to β‐tubulin. The existence of several β‐tubulin isotypes, coupled with their varied expression in normal and cancerous cells provides a platform upon which to construct selective chemotherapeutic agents. We have examined five prevalent human β‐tubulin isotypes and identified the colchicine‐binding site as the most promising for drug design based on specificity. Using this binding site as a template, we have designed several colchicine derivatives and computationally probed them for affinity to the β‐tubulin isotypes. These compounds were synthesized and subjected to cytotoxicity assays to determine their effectiveness against several cancerous cell lines. We observed a correlation between computational‐binding predictions and experimentally determined IC50 values, demonstrating the utility of computational screening in the design of more effective colchicine derivatives. The most promising derivative exhibited an IC50 approximately threefold lower than values previously reported for either colchicine or paclitaxel, demonstrating the utility of computational design and assessment of binding to tubulin.

Interaction of reduced glutathione with bovine brain tubulin

Incubation of phosphocellulose-purified tubulin with GSH at 30 degrees C results in an inhibition of colchicine binding activity. GSSG has a protective effect against the GSH-induced loss of colchicine-binding. Incubation of tubulin with GSH at 30 degrees C results in the formation of abnormal tubulin polymers which are insensitive to cold. Such aggregation is insensitive to antimicrotubular drugs. Aggregation is inhibited by GSSG but not by DTT or mercaptoethanol. GSH-induced aggregation is very sensitive to the ionic strength of the assembly medium; both the aggregation and colchicine binding inhibition induced by GSH are inhibited at higher ionic strength. These results indicate a very complex interaction of GSH with tubulin.

The Post-Translational Modifications of Tubulin

The tubulin molecule is unusual because of the number and nature of post-translational modifications that it undergoes. These modifications may be involved in regulating microtubule stability and interactions with microtubule-associated proteins, but they also may have as yet undiscovered functions. Certain of these modifications are found in many other proteins; these include phosphorylation of a serine residue in β-tubulin and acetylation of a lysine residue in α-tubulin. Other modifications occur exclusively, or almost exclusively in tubulin. Among these are the removal and addition of a tyrosine at the C-terminus of α and the addition of several glutamate or glycine residues to the γ-carboxyl group of glutamate residues in the C-terminal regions of both α and β. The identification of the mechanisms by which these modifications occur and of their roles in microtubule assembly and function are currently very active topics of research; they will be addressed in this chapter.

Effect of CH-35, a novel anti-tumor colchicine analogue, on breast cancer cells overexpressing the βIII isotype of tubulin.

SummaryThe subunit protein of microtubules is tubulin, which has been the target for some of the most successful and widely used anti-tumor drugs. Most of the drugs that target tubulin bind to the β subunit. There are many isotypes of β-tubulin and their distributions differ among different tissues. The βIII isotype is over-expressed in many tumors, particularly those that are aggressive, metastatic, and drug resistant. We have previously reported the design and synthesis of a series of compounds to fit the colchicine site on βIII but not on the other isotypes. In the current study, we tested the toxicity and the anti-tumor activity of one of these compounds, CH-35, on the human breast tumor MDA-MB-231 over-expressing βIII in a xenogeneic mouse model. We found that CH-35 was as toxic as Taxol® in vivo. Although the βIII-over-expressing cells developed into very fast-growing tumors, CH-35 was more effective against this tumor than was Taxol. Our results suggest that CH-35 is a promising candidate for future drug development.