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Purpose Quantitation of β-tubulin isotype expression in taxane resistant human tumor tissue has been difficult to achieve because of the limited availability of validated antibodies. Here we present a label-free MS method to quantitate relative expression levels of β-tubulin isotypes. Experimental design Using isotype-specific reporter peptides, we determined relative β-tubulin isotype expression levels in human lung tumor tissue. Results Four reporter peptides were chosen to quantitate the βI/βII, βIV, βIII, and βV tubulin isotypes. These peptides were validated using human cancer cell lines. The label-free method was then used to determine β-tubulin isotype expression in nine human lung tumor samples, which had been described as high or low βIII-tubulin expressing using immunohistochemistry. It was found that βI/βII (accounting for 18.7–65.7% of total β-tubulin) and βIVa/βIVb (26.3–79.1%) were the most abundant isotypes and that the βIII (0–8.9%) and βV (1.0–10.4%) were less abundant in the tissue. We also categorized the samples as high or low βIII-tubulin expressing. Conclusion and clinical relevance With this method we can determine the relative expression levels of β-tubulin isotypes in human tumor tissue. This method will facilitate studies assessing the use of tubulin isotypes as biomarkers of taxane resistance.

BACKGROUND Paclitaxel is an antimitotic agent isolated from the Pacific yew tree. It has demonstrated antitumor activity in several cancers and is the first of a new class of antineoplastic agents containing a taxane ring system. Its levels in serum and urine have been measured previously by high performance liquid chromatography (HPLC). In this study, the authors developed two competitive radioimmunoassay methods to determine whether they could reliably be used to measure levels of paclitaxel in sera and in cerebrospinal, ascitic, and pleural fluids. METHODS A monoclonal antibody prepared against paclitaxel was employed in an immunoradiometric assay (IRMA), in which 125I-labeled antibody was used, and in a more conventional tritiated radioimmunoassay (RIA),in which 3H-paclitaxel was used. RESULTS Both radioimmunoassays detected levels of paclitaxel in sera that were comparable to those observed with HPLC. However, the IRMA was the most sensitive. Only the IRMA was able to detect low levels of paclitaxel in cerebrospinal fluid after paclitaxel infusion and in sera 3 weeks after infusion. Both the IRMA and RIA methods were able to detect paclitaxel in ascitic and pleural fluids. CONCLUSIONS Monitoring paclitaxel levels reliably in sera and other bodily fluids is possible with these radioimmunoassays and may be of value in predicting and preventing toxicity and optimizing paclitaxel treatments. Cancer 1997; 79:1022-30. © 1997 American Cancer Society.

Taxol, a natural product with significant anti-tumor activity, stabilizes microtubules and arrests cells in the G2/M phase of the cell cycle. It has been reported that taxol has additional effects in cells, including an increase in tyrosine phosphorylation of proteins and activation of MAP kinase. We investigated a possible effect of taxol on tyrosine phosphorylation of Shc and on formation of the Shc/Grb-2 complex in the murine macrophage-like cell line RAW 264.7. Shc, an SH2 domain containing adaptor protein, was immunoprecipitated from lysates of taxol-treated cells with anti-phosphotyrosine antibody and its identity determined by Western blotting with anti-Shc antibody. Non-denatured Shc containing protein complexes were immunoprecipitated with anti-Shc antibody, and analysis with an anti-Grb2 antibody revealed the presence of the 24-kDa Grb2 protein. Taxol also activated Raf-1 kinase and ERK1/ERK2 MAP kinases in these cells. These results demonstrate that taxol affects tyrosine phosphorylation of Shc and this may result in the activation of the Raf-1/MAPK cascade.

Taxol is a structurally complex natural plant product with a novel mechanism of action. The supply of this drug is limited by its low abundance in the bark of the slow-growing yew tree from which it is extracted. The chemical complexity of taxol has hampered the development of a feasible process to synthesize large quantities. Analogues are being made from a precursor found in the needles of the yew tree. However, there is a need to develop a more efficient method to provide adequate supplies of this drug. This review article summarizes the preclinical and clinical studies of taxol in ovarian cancer. Phase I studies have identified the drug's toxicities. Neutropenia has been the dose-limiting toxicity in most trials, and premedications and longer infusion schedules have been used to reduce the incidence and severity of hypersensitivity reactions. The intraperitoneal administration of taxol in Phase I studies showed a pharmacologic advantage with acceptable toxicity. Its activity in ovarian cancer was noticed first in Phase I trials at the Albert Einstein College of Medicine and Johns Hopkins University. These observations led to Phase II testing, which documented response rates of 20-35% in patients with relapsed or refractory ovarian cancer. Phase III trials of taxol and cisplatin versus cyclophosphamide and cisplatin in untreated patients with ovarian cancer are in progress. Studies combining taxol with colony-stimulating factors and cisplatin are ongoing. Taxol is an important new drug in ovarian cancer. Its unique mechanism of action and toxicities make it an attractive agent to use in combination with currently active drugs. Future studies will determine the role of taxol in the management of this disease, but the widespread availability of this drug will depend on the development of a feasible synthetic process.

Multi-drug-resistant cells overproduce a 130-180-kDa integral membrane phosphoglycoprotein known as P-glycoprotein which acts as an energy-dependent drug efflux pump. While P-glycoprotein has been shown to transport hydrophobic anti-tumor drugs out of multi-drug-resistant cells in tissue culture, its endogenous substrates remain unknown. This report shows that 3H-corticosterone can specifically photoaffinity label P-glycoprotein. Furthermore, corticosterone is effluxed from multi-drug-resistant cells by P-glycoprotein. These data suggest that corticosterone may be an endogenous substrate for P-glycoprotein.

Development of resistance to colchicine in the mouse macrophage-like cell line J774.2 coincides with the expression of a variety of phenotypic traits. A cloned subline (J7/CLC-20), maintained in 20 microM colchicine, exhibits reduced steady-state association with drug, increased presence of a 140,000-145,000 dalton (140-145 kD) phosphoglycoprotein associated with the plasma membrane, double minute chromosomes and cross-resistance to other drugs. While similar phenotypic traits are observed in J774.2 cells resistant to taxol and vinblastine, differences in the electrophoretic mobilities of the resistance-specific glycoproteins in each of the three sublines suggest that multi-drug resistant sublines exhibit specificity for individual drugs. In an attempt to elucidate the relationships between the phenotypic traits associated with colchicine resistance, the degree of colchicine resistance in J7/CLC-20 cells was modulated and the levels of expression of the phenotypic traits were quantitated. In the absence of colchicine in the growth medium, J7/CLC-20 cells reverted to drug sensitivity within 35 days. A decrease in the level of resistance coincided with coordinate changes in both the quantity of the resistance-specific glycoprotein and the average number of double minute chromosomes. We propose that the emergence and disappearance of the resistance-specific glycoprotein and double minute chromosomes may be closely linked. However, J7/CLC-20 cells which had regained their drug sensitivity after growth in drug-free medium maintained a reduced level of steady-state drug association. The persistence of reduced drug association in cells that have reverted to a drug-sensitive state suggests that this phenomenon, although related to colchicine resistance, need not be the primary or only mechanism of drug resistance.

There are 3 members of the multidrug-resitance gene family expressed in mouse. Only one of these, mdr lb, and its gene product P-glycoprotein are induced to high levels in the mouse endometrium during pregnancy. It is shown here that P-glycoprotein in the gravid uterus is significantly larger (Mr 155000) compared to P-glycoprotein encoded by mdr lb in a murine multidrug-resistant cell line (Mr 140000). However, both species co-migrate after enzymatic removal of N-linked sugars (Mr 125000). These results demonstrate that differential glycosylation of the mdr lb gene product contributes to molecular heterogeneity found in P-glycoprotein from normal and multidrug-resistant cells.

The oxidation-reduction potential of Cu(II)-bleomycin was measured at 25 °C, pH 7.0. With dithionite as reductant, the potential was −105 ± 9 mV with an n value near unity. With cysteine as reductant, the potential was −49 ± 10 mV, with an n value substantially greater than unity. The potentials are within a range that would allow the reduction of Cu(II)-bleomycin to take place in a cell.