β-Tubulin class II predominates in breast tissues
This is the first quantitative comparison of tubulin isotype protein levels in human tissues. Previous work has indicated that vertebrate tissues vary in their tubulin isotype composition. β-Tubulin class II predominates in brain tissues and has been found in smaller amounts in other tissues [
19,
45]. Classes III and IVa are primarily found in neurons, whereas classes I and IVb are found in many tissues [
10,
19]. β-Tubulin class V is present in small amounts in many cell types [
11] and class VI is found in blood cells and hematopoietic tissues [
49,
50]. Much of this quantitative work was done by comparing mRNA levels. However, mRNA and protein levels are not correlated for many human and yeast proteins, especially those that are regulated by post-transcriptional processes [
51‐
53], although there is controversy regarding correct statistical analysis and interpretation of some of these data [
54]. Intracellular tubulin levels are under both transcriptional and post-transcriptional control. Fine-tuning of tubulin levels during the cell cycle occurs by autoregulation. This process alters mRNA stability and requires both an essential sequence on polysomal β-tubulin mRNA and an amino-terminal Met-Arg-Glu-Ile sequence on the nascent tubulin polypeptide [
55‐
57]. Furthermore, α-tubulin mRNA levels remain high even when protein synthesis is repressed [
58]. In fact, α-tubulin synthesis in CHO cells seems to have a role in regulating β-tubulin protein levels.
This phenomenon of tubulin post-transcriptional autoregu-lation was demonstrated in a wide variety of vertebrate and invertebrate cell types. It suggests that individual tubulin isotype protein levels are all similarly regulated, although this has not been demonstrated. Because the total amount of intracellular tubulin depends on both mRNA stability and protein half-life, these data together indicate that high levels of tubulin mRNA might be associated with low levels of free tubulin subunits. This is supported by the recent mass spectrometry and isoelectric focusing work that demonstrated a lack of correlation between tubulin isotype protein levels and mRNA levels in paclitaxel-resistant cell lines [
59]. However, tubulin mRNA levels are currently interpreted by most investigators as directly representing the total intracellular tubulin protein pool. One alternative to mRNA quantification is immunostaining. Although immunostaining of cells is useful for localizing proteins in tissues and cells, accurate quantification by this method is difficult [
41]. Nevertheless, quantitative measurement of protein levels is crucial for understanding intracellular events. In our studies, quantitative real-time PCR data from selected breast tissue samples revealed that β-tubulin mRNA and protein levels are not correlated. These results indicate that quantitative studies of β-tubulin isotypes that are done exclusively with PCR techniques should be interpreted with caution because functional protein levels cannot be inferred.
Consistent with our results is the finding of both β-tubulin classes I and II by immunostaining in many human tissues and cell types [
60]. To understand the relationship between the drug target levels and the development of drug resistance, it is essential to know the actual protein amounts in tissues and cells. We demonstrate here by a quantitative ELISA assay that the β-tubulin class II protein is on average the most abundant isotype in human breast tissues. We found a distribution of β-tubulin class II levels, ranging from 0.41 to 0.76. This wide distribution was not expected, because our study of tubulin isotypes in pig brains showed very little brain-to-brain variation. To evaluate the significance of the distribution of β-tubulin classes II and I + IV in breast tissues, we measured the tissue levels of Her2/neu protein, known to be amplified in 30% of all breast tumors and associated with poor prognosis [
48]. All tumor tissues expressed higher levels of Her2/neu relative to normal tissues. However, there was no correlation between this biomarker and levels of β-tubulin classes II or I + IV isotype levels. Thus the significance of the range of isotype levels in breast tissues remains unknown. Although it is possible that β-tubulin class II levels might be correlated with a prognostic bio-marker not yet examined [
61], these results suggest that a normal patient-to-patient variation might exist.
The ELISA method described here involves total extraction of tubulin and does not differentiate between polymeriz-able and nonpolymerizable tubulin fractions. There are reports of tubulin isotypes that are differentially incorporated into microtubule structures [
62]. However, it is not possible to fractionate microtubules from total cytoplasmic tubulin with quick-frozen human tissue samples. Current experiments are exploring this aspect of tubulin isotype utilization in cell culture studies. One complexity in our work is the sampling of breast tissue. It is possible that samples are composed of different amounts of heterogeneous cell types. However, because our data show similar isotype amounts in normal and tumor breast tissues, differential tissue sampling seems not to be an issue in these experiments. Our method is based in part on an ELISA assay developed by Thrower and colleagues [
63] for tubulin extracts. This work fulfills their suggestion to quantify changes in tubulin isotype levels with the appropriate antibodies, and establishes a sensitive and accurate assay to complement mRNA assays and studies of the response of cultured cells to antimitotic drugs.
The patient diagnoses for these tumor samples were most commonly infiltrating ductal carcinoma (12 of 20 samples). We found no correlations between tubulin isotype levels and any of the variables given in the pathology reports (estrogen receptor, progesterone receptor, age, race, or histology grade). It should be noted that the 'normal' breast tissues examined in this study were excised from a site distal to the tumor site but in the same breast. Histological examination of the 'normal' tissues indicates the presence of fibrocystic changes in many of the samples and therefore, although these changes are not uncommon in normal aging, they cannot be classified as truly 'normal' breast tissue. However, independent pathology reports determined that the 'normal' tissues were 'non-tumor' tissues with the exception of N1, N12, and N13, in which tumor cells were noted. N1 and N13 were estimated to contain more than 5% tumor cells. N12, however, had tumor cells throughout the breast tissue and therefore although the 'normal' sample was taken distal to the tumor it contained significant numbers of tumor cells. N12 was also shown in our ELISA studies to have relatively high levels of Her2/neu protein. Thus, the Her2/neu ELISAs provided a second means of grossly differentiating between 'tumor' and 'normal' tissues for these patients.
It is important to note that many tumor tissues do not express Her2/neu; it is therefore not a generally useful marker for differentiating between tumor and normal tissues. In addition, our work demonstrates that although β-tubulin class III is a marker for aggressive neuroendocrine tumors [
36,
64], it is not a significant biomarker in breast tumors. We speculate that this is because breast tissue rarely dysregulates to neuroendocrine cell types.
Tumor and normal breast tissues have similar tubulin isotype levels
Over the past decade paclitaxel and docetaxel have become important chemotherapeutic agents for both ovarian and breast cancers [
1,
2]. Considerable interest has focused on the target receptor – tubulin – and mechanisms underlying efficacy, toxicity, and drug resistance [
7,
8,
23,
24,
27,
28]. Whether tubulin isotype levels might determine cellular responses to antimitotic agents remains controversial. For example, paclitaxel-resistant lung cancer cells and ovarian tumors were shown to have increased levels of tubulin isotypes, particularly β-tubulin classes III and IVa [
28]. Furthermore, when paclitaxel-resistant lung cancer cells were treated with antisense oligonucleotides for β-tubulin class III, the drug resistance was partly reversed, coincident with decreased protein levels of β-tubulin class III [
65]. However, it should be noted that β-tubulin classes I, II, III, and IVa all increased significantly in paclitaxel-resistant lung cancer cells, indicating that the drug effect did not uniquely involve a single tubulin isotype. In other work, β-tubulin classes III and IVa were shown to increase in paclitaxel-resistant prostate cancer cells [
29]. However, these two isotypes continued to be expressed in the lowest amounts.
In ovarian cancer cells, mutations in residues near the putative paclitaxel-binding site of β-tubulin class I were found in paclitaxel-resistant ovarian cancer cells [
23]. Other work suggests that changes in tubulin isotypes alone cannot confer resistance to antimitotics. It was demonstrated that one group of antimitotic agents, vinca alkaloids, bind with similar affinity to all β-tubulin isotypes [
12], thus suggesting for therapy with vinorelbine, vinblas-tine, or vincristine that tubulin isotype levels are incidental for efficacy. Furthermore, transfection of CHO cells with β-tubulin classes I, II, or IVb did not produce resistance to paclitaxel [
32]. The alternative hypothesis, derived from the work of Cabral and colleagues, is that rather than the tubulin isotype levels, the amount of polymerized tubulin is the critical factor that regulates response to antimitotic drugs. For example, total amounts of polymerizable tubulin are decreased in paclitaxel-resistant cells, whereas col-cemid-resistant tubulin or vinca alkaloid-resistant cells have increased levels of assembled tubulin [
34,
66,
67]. Furthermore, when cells were transfected with β-tubulin class III, weak resistance to paclitaxel was found, and this resistance was associated with decreased microtubule stability [
68]. This is consistent with the different modes of action of these drugs: paclitaxel stabilizes microtubules and colcemid inhibits tubulin assembly. In support of this hypothesis, tubulin mutations consistent with altered assembly properties have been identified in paclitaxel-resistant and vinca alkaloid-resistant cells [
33,
66,
69].