Prognostic biomarkers for HNSCC using quantitative real-time PCR and microarray analysis: ß-tubulin isotypes and the p53 interactome.

In 2014, more than 40,000 people in the United States will be diagnosed with head and neck squamous cell cancer (HNSCC) and nearly 8400 people will die of the disease (www.cancer.org/acs/groups). Little is known regarding molecular targets that might lead to better therapies and improved outcomes for these patients. The incorporation of taxanes into the standard cisplatin/5-fluouracil initial chemotherapy for HNSCC has been associated with improved response rate and survival. Taxanes target the ß-subunit of the tubulin heterodimers, the major protein in microtubules, and halt cell division at G2/M phase. Both laboratory and clinical research suggest a link between ß-tubulin expression and cancer patient survival, indicating that patterns of expression for ß-tubulin isotypes along with activity of tumor suppressors such as p53 or micro-RNAs could be useful prognostic biomarkers and could suggest therapeutic targets. © 2014 Wiley Periodicals, Inc.

Regulation of tubulin expression by micro-RNAs: implications for drug resistance.

In this chapter, we provide an overview of methods for studying micro-RNA regulation of tubulin isotypes. In clinical studies, ß-tubulin isotypes were found to be biomarkers for tumor formation. In addition, because changes in the levels of specific ß-tubulin isotypes alter the stability of microtubules in mitotic spindles in vitro, it has been hypothesized that changes in microtubule protein levels could contribute to chemotherapy resistance. Over the past 15 years, micro-RNAs have been shown to target mRNAs in signaling pathways involved in tumor suppression, as well as tumorigenesis. Investigating micro-RNA regulation of tubulin isotypes will shed light on the mechanisms underlying the processes that implicate tubulin isotypes as biomarkers for aggressive tumors or chemotherapy resistance. The methods discussed in this chapter include the use of micro-RNA superarrays, next-generation sequencing, real-time PCR experiments, upregulation of micro-RNAs, and immunoprecipitation of RNA-induced silencing complex. We will show examples of data collected using these methods and how these data contribute to understanding paclitaxel resistance.

Coordinated regulation of ß-tubulin isotypes and epithelial-to-mesenchymal transition protein ZEB1 in breast cancer cells.

The regulation of ß-tubulin isotypes, the primary targets for antimitotic chemotherapeutic drugs like taxanes, has implications for drug response and drug resistance. Over the past 15 years, micro-RNAs have been studied widely as regulators of mRNA levels. For example, the tumor suppressor miR-200c was shown in cell culture to target mesenchymal genes, including ZEB1 [ Cochrane ( 2009 ) Mol. Cancer Ther. 8 ( 5 ), 1055 - 1066 ]. In that work, exogenous miR-200c was also shown to reduce ß-tubulin class III, one of its predicted targets. Furthermore, decreased miR-200c and increased ß-tubulin class III were associated with poor outcomes for ovarian cancer patients [ Cittelly , D.M. ( 2012 ) Mol. Cancer Ther. 11 ( 12 ), 2556 - 2565 ]. Because miR-200c targets the epithelial-to-mesenchymal inducer ZEB1, we wanted to know whether changes in ZEB1 parallel ß-tubulin isotype changes, implicating ß-tubulin isotypes in ZEB1-associated cell survival pathways. We found coordinated positive feedback regulation of mRNA for ZEB1 and ß-tubulin isotype classes I, III, and IVB in MDA-MB-231 breast cancer cells, commonly used as a model for triple-negative breast cancers. Low levels of paclitaxel (40 nM) were found to significantly reduce mRNA levels for these tubulin genes along with a 2-3-fold increase in miR-200c. ZEB1 silencing also reduced ß-tubulin isotype classes I, III, and IVB mRNA, whereas upregulation of ZEB1 was associated with increases in these isotype classes. Our work indicates that paclitaxel-induced reduction of ZEB1 and ß-tubulin isotypes are, in part, due to increased activity of miR-200c. These results suggest that in aggressive breast cancers, as modeled by MDA-MB-231 cells, ß-tubulin class III is a biomarker for cell survival mediated through ZEB1-induced tumor progression pathways.

Regulation of ß-tubulin isotypes by micro-RNA 100 in MCF7 breast cancer cells.

Antimitotic drugs are key components of combination chemotherapy protocols for hematological and solid tumors. The taxanes (e.g., paclitaxel) bind to the ß subunit of the tubulin heterodimer and reduce microtubule dynamics, leading to cell cycle arrest in G2/M. The effectiveness of combination chemotherapy is limited by tumor resistance to drugs initially or as a cumulative effect after several cycles of treatment. Because changes in the drug receptor may be linked to drug resistance, we investigated changes in ß-tubulin isotypes in response to paclitaxel treatment in MCF7 breast cancer cells. We found that paclitaxel induced a 2-3 fold increase in mRNA for ß-tubulin IIA and III genes, TUBB2A, and TUBB3. ß-Tubulin class III protein increased; however, ß-tubulin class II protein was not detected in these cells. Paclitaxel treatment following pretreatment with actinomycin D showed that the change in ß-tubulin class III was due to increased transcription and linked to G2/M arrest. The increase in ß-tubulin IIA mRNA was due to both enhanced stability and increased transcription, unassociated with G2/M arrest. We used micro-RNA superarrays to look for changes in families of micro-RNAs that might be linked to drug-induced changes in ß-tubulin isotype mRNA and/or protein. We found a significant decrease in the tumor suppressor, miR-100, in MCF7 cells in response to paclitaxel treatment. Transfection of MCF7 cells with miR-100 significantly reduced ß-tubulin I, IIA, IIB and V mRNA and prevented paclitaxel-induced increases in ß-tubulin isotypes. This is the first report of a micro-RNA that regulates these specific ß-tubulin isotype mRNAs.

Expression profiling of tubulin isotypes and microtubule-interacting proteins using real-time polymerase chain reaction.

Real-time polymerase chain reaction (PCR) has been used for quantification of intracellular mRNA levels in cell culture and tissue samples. It is an important tool for studying antimitotic drug effects on tubulin isotype and microtubule-interacting protein levels and for measuring differences in normal and tumor tissue samples that could have predictive or prognostic applications. Both quantitative and comparative methods are valuable approaches; however, the selection of either approach requires an understanding of their benefits and challenges. In this chapter, we provide detailed protocols for real-time PCR experiments, discuss issues to consider in selecting real-time PCR methodologies, and give examples utilizing either quantitative or comparative approaches.

Effect of noscapine and vincristine combination on demyelination and cell proliferation in vitro.

Peripheral neuropathy is a common, dose-limiting side effect of vincristine, a frontline therapy for acute lymphoblastic leukemia. Combination chemotherapy that reduces the neurotoxicity without compromising the efficacy of vincristine would improve patient outcomes. We performed in vitro studies using a combination of microtubule-binding antimitotics, noscapine and vincristine. In cell cultures containing neurons, astrocytes, and oligodendrocytes, vincristine caused demyelination as shown by transmission electron microscopy. A combination of vincristine and noscapine protected against demyelination. Human acute lymphoblastic and acute myelogenous leukemia cell lines CCRF-CEM and HL-60, respectively, were used to determine the antiproliferative effect of this novel drug combination. Vincristine and noscapine decreased cell proliferation with IC(50) concentrations of 1 nM and 20 microM, respectively. Analysis of dose-effect relationships using isobolograms and combination indices demonstrated that noscapine acts synergistically with vincristine. Thus, noscapine is a promising candidate for use with vincristine to decrease neurotoxicity and enhance antineoplastic effectiveness.

Pharmacokinetics, metabolites, and preclinical safety of vinflunine.

The novel microtubule inhibitor, vinflunine, has a unique mechanism of action that differs from other members of the vinca alkaloid class in terms of tubulin-binding affinity, microtubule dynamics, spiral formation, and intracellular accumulation. Vinflunine has shown significant activity in vivo, which involves its antimitotic, antiangiogenic, and antivascular properties. The promising preclinical activity of vinflunine has warranted further investigation in the clinical setting. This review explores the distinct interaction of vinflunine with its intracellular targets to gain insight into its mechanism of action and safety profile. The pharmacokinetic properties and metabolism of vinflunine in animals and in human subjects are also discussed, together with an analysis of potential drug-drug interactions and the influence of age, liver dysfunction, or renal dysfunction on the overall activity of vinflunine.

Exploring the mechanisms of action of the novel microtubule inhibitor vinflunine.

Microtubules have been identified as a suitable target for anticancer therapy, primarily based on their biological importance in coordinating chromosomal segregation at mitosis. Two main classes of microtubule-targeted agents, the taxanes and vinca alkaloids, suppress the dynamic behavior of spindle microtubules, inducing mitotic arrest and subsequent apoptotic cell death. Clinical activity of taxanes and first-generation vinca alkaloids in the treatment of solid tumors and hematologic malignancies, respectively, has prompted further research for novel analogs with improved clinical efficacy and safety. Such efforts have led to the development of vinflunine, a bifluorinated vinca alkaloid endowed with unique antitumor properties. Highlighted in this review are the key features of vinflunine that lead to effective suppression of microtubule dynamics and induction of cell death in cancer cells.

Beta-tubulin isotype classes II and V expression patterns in nonsmall cell lung carcinomas.

Previous studies suggest that beta-tubulin isotype protein levels could be useful as indicators of nonsmall cell lung cancer (NSCLC) aggressiveness. However, measurement of protein amounts in tissue samples by staining techniques is semiquantitative at best. Since technologies for measuring mRNA levels have become more efficient and quantitative, we wanted to determine whether beta-tubulin message levels may be useful as biomarkers. Quantitative real-time RT-PCR was used to measure the seven classes of beta-tubulin isotypes, stathmin and MAP4 mRNA levels in 64 NSCLC and 12 normal lung tissue samples. We found significantly higher fractions of beta-tubulin classes II and V mRNA compared to the other isotypes in all lung tumor samples (P < 0.05). In addition, the ratio of beta-tubulin classes II/V mRNA was significantly higher in NSCLCs compared to normal lung tissues (P < 0.001). The data suggest that the ratio of beta-tubulin classes II and V mRNA could be useful as a biomarker for NSCLC tumor differentiation and/or NSCLC aggressiveness. Furthermore, the ratio of MAP4 to stathmin mRNA was found to be higher in diseased lung tissues compared to normal lung tissues, suggesting this ratio might also be used as a clinically relevant biomarker for NSCLCs.

Murine leukemia P388 vinorelbine-resistant cell lines are sensitive to vinflunine.

The work presented here was initiated to explore the mechanisms underlying vinorelbine resistance in two previously established murine leukemia P388 cell lines (N.63 and N2.5). IC(50) measurements demonstrated that the vinorelbine-resistant cell line N.63 was sensitive to both vinblastine and vinflunine. In addition, vinorelbine-resistant cell line N2.5 retained sensitivity to vinflunine. We used flow cytometry with propidium iodide to measure G2/M arrest in response to drug treatment. Annexin V labeling was used as a marker of apoptosis and JC-1 dye labeling as a marker of mitochondrial membrane depolarization to explore differential responses that might help explain the absence of cross resistance to vinflunine. At equipotent (10X IC(50)) doses, after 8 h of drug treatment, vinflunine induced G2/M arrest in a significantly larger fraction of vinorelbine- resistant cells compared to vinorelbine. At the same drug doses, at 16 h after initiation of drug treatment, vinflunine induced a statistically significant greater apoptotic response and mitochondrial depolarization. The mitochondrial depolarization at 16 h was confirmed by Western blotting that showed release of cytochrome c. Comparison of apoptotic and mitochondrial depolarization responses in vinorelbine-resistant cells upon exposure to vinorelbine, vinblastine and vinflunine demonstrated the following pattern of drug activity: vinflunine > vinblastine > vinorelbine, confirming the importance of a antimitotic-induced mitochondria-mediated pathways in these P388 cell lines. We conclude that vinflunine may be preferred for treatment of specific cancers compared to other vinca alkaloids due to its enhanced effects on apoptotic pathways that follow G2/M arrest.

Methods for studying vinca alkaloid interactions with tubulin.

Vinca alkaloids play a vital role in chemotherapy protocols for a wide range of hematological and solid tumors. Studies of drug interactions with the drug target, tubulin or microtubules, have helped us to understand the cytotoxic and toxic effects. We present here in vivo and in vitro methods for studying vinca alkaloid interactions with tubulin. In vivo methods for examining drug effects on cell proliferation and intracellular tubulin or microtubules and direct visualization of drug effects by fluorescence microscopy are presented. In vitro methods for measuring drug affinity for tubulin by analytical ultracentrifugation, kinetics of drug binding by light scattering and drug effects on microtubules by turbidity are also presented.

The thermodynamics of vinca alkaloid-induced tubulin spirals formation.

Vinca alkaloids are antimitotic, anticancer agents that induce tubulin to form spiral polymers at physiological protein concentrations. We used sedimentation velocity to investigate the effects of six vinca alkaloids on tubulin spiraling. Fitting to a Wyman linkage model reveals a drug dependent change of over two orders of magnitude in spiraling potential, K(1)K(2). Thermodynamic analysis of LnK(1)K(2) data demonstrates large and positive DeltaS values, indicating that tubulin spiral formation is entropically-driven. From the curvature in van't Hoff plots of vinblastine data, we estimate DeltaC(p) for GTP and GDP conditions to be -439 and -396 cal/mol K. Partitioning of DeltaS into the hydrophobic effect, DeltaS(HE), change in rotational/translational freedom, DeltaS(RT) and change in protein conformation, DeltaS(other), demonstrates that the major driving force for tubulin spiral formation is burial of hydrophobic surfaces and that protein conformational changes do not make a significant contribution. Spiraling potential is an indicator of antimitotic activity in vivo, although turbidity studies indicate that there is no correlation between spiraling potential and microtubule inhibition in vitro. Mechanisms that explain this discrepancy are discussed.

Comparison of beta-tubulin mRNA and protein levels in 12 human cancer cell lines.

Antimitotic drugs are chemotherapeutic agents that bind tubulin and microtubules. Resistance to these drugs is a major clinical problem. One hypothesis is that the cellular composition of tubulin isotypes may predict the sensitivity of a tumor to antimitotics. Reliable and sensitive methods for measuring tubulin isotype levels in cells and tissues are needed to address this hypothesis. Quantitative measurements of tubulin isotypes have frequently relied upon inferring protein amounts from mRNA levels. To determine whether this approach is justified, protein and mRNA levels of beta-tubulin isotypes from 12 human cancer cell lines were measured. This work focused on only beta-tubulin isotypes because we had readily available monoclonal antibodies for quantitative immunoblots. The percentage of beta-tubulin isotype classes I, II, III, and IVa + IVb mRNA and protein were compared. For beta-tubulin class I that comprises >50% of the beta-tubulin protein in 10 of the 12 cell lines, there was good agreement between mRNA and protein percentages. Agreement between mRNA and protein was also found for beta-tubulin class III. For beta-tubulin classes IVa + IVb, we observed higher protein levels compared to mRNA levels.Beta-tubulin class II protein was found in only four cell lines and in very low abundance. We conclude that quantitative Western blotting is a reliable method for measuring tubulin isotype levels in human cancer cell lines. Inferring protein amounts from mRNA levels should be done with caution, since the correspondence is not one-to-one for all tubulin isotypes.

Analgesic effects of oral sucrose and pacifier during eye examinations for retinopathy of prematurity.

Oral sucrose reduces pain during heel sticks and venipunctures in preterm infants, but no studies have been done to determine the effectiveness of sucrose during eye examinations for retinopathy of prematurity. Therefore, the purpose of this study was to determine the effectiveness of local anesthetic eye drops and a pacifier, plus repeated doses of 24% sucrose, to relieve pain associated with eye examinations for retinopathy of prematurity. In this double-blind randomized controlled trial, 30 preterm infants were randomly assigned to one of two treatments, in which they received either local anesthetic eye drops, a pacifier, plus three doses of sterile water or local anesthetic eye drops, a pacifier, plus three doses of 24% sucrose during the eye examination. Treatment effectiveness was determined using a validated infant pain measure, the Premature Infant Pain Profile (PIPP), which includes measures of facial expressions, heart rate, and oxygen saturation and takes behavioral state and gestational age into consideration. Data were collected before, during, and following an examination of the left eye. Statistically significant differences in mean PIPP scores were found between the sucrose and water groups during the left eye examination. The mean PIPP score was 8.8 for the sucrose group and 11.4 for the water group ( t = 2.87, p = .008 two-tailed). No significant differences were found in PIPP scores immediately following the procedure. Sucrose and a pacifier may be beneficial for minimizing pain during eye examinations in preterm infants and should be considered as a part of evidence-based guidelines for relieving pain during this procedure.

Thyroid storm during pregnancy. A medical emergency.

Thyroid storm is the major risk to pregnant women with thyrotoxicosis. This life-threatening condition is more likely to occur with another precipitating factor such as labor and delivery, surgical delivery, infection, or trauma. Thyroid storm most often occurs in patients with undertreated or undiagnosed hyperthyroidism. As many as 20% to 30% of cases can end in maternal and fetal mortality. Therefore, critical care nurses must be able to recognize and initiate proper medical and nursing interventions promptly.

Beta class II tubulin predominates in normal and tumor breast tissues.

BACKGROUND: Antimitotic chemotherapeutic agents target tubulin, the major protein in mitotic spindles. Tubulin isotype composition is thought to be both diagnostic of tumor progression and a determinant of the cellular response to chemotherapy. This implies that there is a difference in isotype composition between normal and tumor tissues. METHODS: To determine whether such a difference occurs in breast tissues, total tubulin was fractionated from lysates of paired normal and tumor breast tissues, and the amounts of beta-tubulin classes I + IV, II, and III were measured by competitive enzyme-linked immunosorbent assay (ELISA). Only primary tumor tissues, before chemotherapy, were examined. Her2/neu protein amplification occurs in about 30% of breast tumors and is considered a marker for poor prognosis. To gain insight into whether tubulin isotype levels might be correlated with prognosis, ELISAs were used to quantify Her2/neu protein levels in these tissues. RESULTS: Beta-tubulin isotype distributions in normal and tumor breast tissues were similar. The most abundant beta-tubulin isotypes in these tissues were beta-tubulin classes II and I + IV. Her2/neu levels in tumor tissues were 5-30-fold those in normal tissues, although there was no correlation between the Her2/neu biomarker and tubulin isotype levels. CONCLUSION: These results suggest that tubulin isotype levels, alone or in combination with Her2/neu protein levels, might not be diagnostic of tumorigenesis in breast cancer. However, the presence of a broad distribution of these tubulin isotypes (for example, 40-75% beta-tubulin class II) in breast tissue, in conjunction with other factors, might still be relevant to disease progression and cellular response to antimitotic drugs.