A Ubiquitous β-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

Abstract

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the ␣-and ␤-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V ␤-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V ␤-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V ␤-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V ␤-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates. INTRODUCTIONMicrotubules are essential filamentous structures in eukaryotic cells where they are responsible for the directed movement of vesicles, the organization of the endoplasmic reticulum and Golgi apparatus in the cytoplasm, and the equipartitioning of chromosomes before cell division. The organelles are assembled from heterodimers of ␣-and ␤-tubulin, which polymerize in a head-to-tail manner to form linear protofilaments, and these associate laterally into tubular structures that normally consist of 13 protofilaments. Vertebrate ␣-and ␤-tubulins are each encoded by a 6-to 7-member multigene family that produces highly homologous and conserved gene products that differ most radically in their last 10 -15 amino acids (Sullivan, 1988;Luduena, 1998). These carboxyl-terminal sequences have been used to assign ␤-tubulin gene products to seven distinct classes (Lopata and Cleveland, 1987). Each of these classes (hereafter referred to as ␤1, ␤2, ␤3, ␤4a, ␤4b, ␤5, and ␤6) defines a ␤-tubulin isotype that differs significantly from other isotypes within the same organism, but differs very little from the same isotype in other vertebrate species.With the discovery that tubulin proteins in an organism are heterogeneous, a hypothesis was formulated suggesting that different tubulin proteins might perform different functions in the cell (Fulton and Simpson, 1976). In the intervening years, however, most of the experimental and genetic evidence has argued that different ␤-tubulins coassemble freely into all cellular microtubules (see Joshi and Cleveland, 1990; Luduena, 1998 for reviews). In gene replacement experiments using the fungal organism, Aspergillus nidulans, for example, May demonst...

Amino acid substitutions at proline 220 of β-tubulin confer resistance to paclitaxel and colcemid

“…Cells transfected with WT HAh1-tubulin cDNA (Fig. 3A) had normal cytoplasmic microtubule networks and mitotic spindles, consistent with previous studies (25,30,31). In the case of P220L and P220V (Fig.…”

Melanoma cell sensitivity to Docetaxel‐induced apoptosis is determined by class III β‐tubulin levels

“…Transfections were performed for 48-72 h as previously described [4] prior to each assay using either SiConTRol Non-targeting SiRNA pool (D-001206- [13][14][15][16][17][18][19][20] or the siGENOME SMARTpool for class III b-tubulin (TUBB3) siRNA (Dharmacon, Lafayette, CO).…”

“…Expression of mutant tubulin or differential expression of certain tubulin isotypes has been shown to correlate with the resistance profile of taxaneresistant cells [10][11][12]. In some cases, the induced expression of mutant tubulins [13] and b-tubulin isotypes classes III and V [14,15] were shown to confer resistance to paclitaxel. Purified mutant tubulins also have altered polymerisation characteristics [16] and increased expression of classes I, III, and IVa isotypes was reported in paclitaxel-resistant cells [10].…”

Ixabepilone: targeting βIII-tubulin expression in taxane-resistant malignancies

“…In mammals, the hI and hIVb isotypes are constitutively expressed in all tissues, whereas classes III, IVa, and II are primarily expressed in the brain and neurons (27 -29). Although expression of hV-tubulin was originally identified only in the chicken, the protein seems to be expressed at low levels in mammalian cells (30). Elevated expression of hV-tubulin has been shown to disrupt microtubule assembly and cause resistance to paclitaxel (30), suggesting that this protein may confer resistance to selected chemotherapeutic agents.…”