Enrichment of hepatic glycogen and plasma glucose from H218 O informs gluconeogenic and indirect pathway fluxes in naturally feeding mice.

Deuterated water (2 H2 O) is a widely used tracer of carbohydrate biosynthesis in both preclinical and clinical settings, but the significant kinetic isotope effects (KIE) of 2 H can distort metabolic information and mediate toxicity. 18 O-water (H2 18 O) has no significant KIE and is incorporated into specific carbohydrate oxygens via well-defined mechanisms, but to date it has not been evaluated in any animal model. Mice were given H2 18 O during overnight feeding and 18 O-enrichments of liver glycogen, triglyceride glycerol (TG), and blood glucose were quantified by 13 C NMR and mass spectrometry (MS). Enrichment of oxygens 5 and 6 relative to body water informed indirect pathway contributions from the Krebs cycle and triose phosphate sources. Compared with mice fed normal chow (NC), mice whose NC was supplemented with a fructose/glucose mix (i.e., a high sugar [HS] diet) had significantly higher indirect pathway contributions from triose phosphate sources, consistent with fructose glycogenesis. Blood glucose and liver TG 18 O-enrichments were quantified by MS. Blood glucose 18 O-enrichment was significantly higher for HS versus NC mice and was consistent with gluconeogenic fructose metabolism. TG 18 O-enrichment was extensive for both NC and HS mice, indicating a high turnover of liver triglyceride, independent of diet. Thus H2 18 O informs hepatic carbohydrate biosynthesis in similar detail to 2 H2 O but without KIE-associated risks.

Caferana seeds (Bunchosia glandulifera) as a new source of nutrients: Evaluation of the proximal composition, solvent extraction, bioactive compounds, and δ-lactam isolation.

The proximal composition, amino acid, carbohydrate, and volatile profiles of caferana (Bunchosia glandulifera) seeds flour were here assessed. Seeds were also subjected to the following extraction processes: one with pressurized ethanol (PLE) and two with ethanol + supercritical CO2 mixture at different temperatures and pressures (SC1 and SC2). Extracts were characterized in terms of caffeine, total phenolic, and δ-lactam. The characterization of caferana seed and its extracts is unprecedented in terms of carbohydrate and volatiles profiles, besides the δ-lactam identification/isolation. SC2 extract exhibited a higher caffeine (9.3 mg/g) and δ-lactam (29.4 mg/g) content, whereas the PLE extract contained a higher total phenolic amount (3.0 mgGAE/g). Caferana is regionally associated to protective effects on mental health. Its byproduct (seed) revealed to be a promising source of bioactive compounds, and a potential raw material of nutritive extracts and flours that can be incorporated into pharmaceutical, nutraceutical, cosmetic, and food products.

Enfermagem e a educação em saúde / Nursing and health education

Introdução: A educação em saúde promove uma estratégia que potencializa o cuidado de enfermagem ao envolver atividades educativas na assistência ao paciente. Objetivo: Refletir sobre as ações de educação em saúde realizadas pela enfermagem e, a construção do vínculo para transferência de conhecimento científico voltado para a área. Método: O texto é uma revisão narrativa realizada com base em periódicos nacionais e internacionais. As bases consultadas foram: Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS), Biblioteca Regional de Medicina (BIREME) e Biblioteca Virtual de Saúde (BVS) utilizando os descritores: "Educação em Saúde"; "Enfermagem" e "Paciente", em idiomas português e inglês. Resultados: Foram agrupados em duas categorias temáticas: "Ações de educação em saúde e enfermagem" e "Educação em saúde e a transformação do conhecimento pelos usuários", de modo a permitir uma compreensão dos dados encontrados. Considerações Finais: O processo pedagógico na realização de uma atividade educativa em enfermagem pode apresentar melhor resultado, quando aplicado com a confiança de um bom atendimento para um fácil aprendizado

Introduction: Health education promotes a strategy that enhances nursing care by involving educational activities in patient care. Objective: Reflect on the health education actions carried out by nursing and the construction of the link for the transfer of scientific knowledge aimed at the area. Method: The text is a narrative review based on national and international journals. The bases consulted were: Latin American and Caribbean Literature in Health Sciences (LILACS), Regional Library of Medicine (BIREME) and Virtual Health Library (VHL) using the descriptors: "Health Education"; "Nursing" and "Patient", in Portuguese and English. Results: They were grouped into two thematic categories: "Health and nursing education actions" and "Health education and the transformation of knowledge by users", in order to allow an understanding of the data found. Final considerations: The pedagogical process in carrying out an educational activity in nursing can present a better result, when applied with the confidence of good service for easy learning

Femoral torsion evaluation by computed tomography in a young Brazilian population with hip pain and femoroacetabular impingement.

INTRODUCTION: The aim of the study was to evaluate femoral torsion (FT) by computed tomography (CT) in young patients with hip pain and femoroacetabular impingement (FAI) in a Brazilian population. After the diagnosis of impingement, the complete analysis from the hip biomechanics and morphology has become essential. METHODS: Forty-one patients from 18 to 45 years presenting hip pain without arthrosis (Tönnis<2) were evaluated by CT scan from February 2017 to February 2018. All patients presented hip pain for at least 3 months and FAI. They have followed the same radiographic protocol and modified Harris Hip Score (mHHS) evaluation. Statistical analyses by software R version 3.4.4. with significance p < 0,05. RESULTS: After exclusion criterias, twenty-six patients (9 bilateral) were included, mostly man (73%). The average age was 35 years for both genders. BMI was 19 kg/m2 for women and 24 kg/m2 for men and the mean modified Harris Hip Score was 67 points. We have found femoral torsion changes in 11 hips with pain (31%) and high variability (60%). The mean FT was 14,5°, ranging from 0 to 39°. Patients with hip pain, CAM impingement and altered FT had no correlation when compared to controls (without pain) (p = 0.234), neither Mixed impingement (p = 0.314). Patients with Pincer impingement and painful hips had higher FT (16,63°) than controls (11,77°) (p = 0.045). Conclusion: The presence of torsional alterations in almost 1/3 of the patients with FAI and the high variability reveal the importance of measuring FT at this disease.

p-Aminobenzoic acid as an alternative chemical biopsy agent for human hepatic UDP-glucose.

p-Aminobenzoic acid (PABA) was evaluated for noninvasive sampling of UDP-glucose in the liver. Six healthy subjects ingested 550 mg PABA during a breakfast meal. Urine was collected 0-2 and 2-4 h after PABA ingestion. N-acetyl PABA glucuronide (NAPG) was identified with 522 ± 212 µmol recovered in the 2-4 h urines. One of the subjects ingested 2 g of 98% [U-2H7]glucose alongside PABA and the NAPG was analyzed for positional 2H-enrichment by 2H NMR following derivatization to 5-O-acetyl monoacetone glucuronolactone. In conclusion, PABA is an effective agent for the chemical biopsy of hepatic UDP-glucose in humans.

Reflexão sobre a classificação de risco como tendência para o pronto-socorro infantil / Reflection on risk classification as a trend for the infant emergency room / La reflexión sobre la calificación de riesgo como una tendencia para la emergencia del niño

Objetivo: explanar acerca da classificação de risco como uma tendência para o serviço de pronto-socorro infantil. Método: trata-se de um ensaio teórico-reflexivo, realizado no período de maio de 2017 a outubro de 2018, fundamentando na literatura encontrada nas bases de dados: PubMed, CINAHL e LILACS. Os descritores utilizados foram: "Medição de Risco", "Serviços Médicos de Emergência", "Serviço Hospitalar de Emergência", "Pediatria", "Identificação da Emergência"; "Triagem" e "Superlotação". Foram utilizados como critérios de inclusão: publicações em português, inglês e espanhol, no período de 2000 a 2016, com textos completos disponíveis gratuitamente. Excluídas as publicações que não se relacionavam à temática do estudo e que apresentassem duplicidade. Resultados: a classificação de risco mostra-se como tendência, com possibilidade de avançar, uma vez que a solução de muitos dos fatores que contribuem para a superlotação dos prontossocorros acarretaria maiores custos financeiros do que a implantação da classificação de risco infantil. Considerações Finais: a classificação de risco pode contribuir para a organização dos atendimentos ao cliente pediátrico que, procura por atendimento em prontos-socorros sem critérios clínicos.(AU)

Objective: to explain about the classification of risk as a tendency for the emergency room service for children. Method: it is a theoretical-reflective essay, carried out from May 2017 to October 2018, based on the literature found in the databases: PubMed, CINAHL and LILACS. The descriptors used were: "Risk Measurement", "Emergency Medical Services", "Emergency Hospital Service", "Pediatrics", "Emergency Identification", "Screening" and "Overcrowding". Inclusion criteria were: publications in Portuguese, English and Spanish, from 2000 to 2016, with full texts available free of charge. Publications that were not related to the subject of the study and which presented duplicity were excluded. Results: the risk classification is a trend with the possibility of advancing, since the solution of many of the factors that contribute to the overcrowding of the emergency rooms would entail higher financial costs than the implantation of the classification of child risk. Final Considerations: risk classification may contribute to the organization of pediatric care to the patient that seeks care in emergency rooms without clinical criteria.(AU)

Objetivo: explicar acerca de la clasificación de riesgo como una tendencia al servicio de socorro infantil. Método: se trata de un ensayo teórico-reflexivo, realizado en el período de mayo de 2017 a octubre de 2018, fundamentando en la literatura encontrada en las bases de datos: PubMed, CINAHL y LILACS. Los descriptores utilizados fueron: "Medición de Riesgo", "Servicios Médicos de Emergencia", "Servicio Hospitalario de Emergencia", "Pediatría", "Identificación de la Emergencia"; "Triage" y "Superlotación". Fueron utilizados como criterios de inclusión: publicaciones en portugués, Inglés y Español, de 2000 a 2016, con el texto completo de libre acceso. Excluidas las publicaciones que no se relacionaban a la temática del estudio y que presentasen duplicidad. Resultados: la clasificación de riesgo se muestra como tendencia, con posibilidad de avanzar, ya que la solución de muchos de los factores que contribuyen a la superpoblación de los prontos de socorro acarrearía mayores costos financieros que la implantación de la clasificación de riesgo infantil. Consideraciones finales: la clasificación de riesgo puede contribuir a la organización de las atenciones al cliente pediátrico que, busca por atención en prontos de socorro sin criterios clínicos.(AU)

Avaliação da torção femoral por tomografia computadorizada em pacientes jovens sem artrose e com dor no quadril por impacto fêmoro-acetabular / Femoral torsion evaluation by computed tomography in young patients without arthrosis with hip pain and femoral-acetabular impingement

Com a descoberta do impacto fêmoro-acetabular (IFA) que corresponde a maior causa de artrose do quadril, a análise biomecânica do quadril tornou-se fundamental. O objetivo do trabalho foi avaliar por tomografia computadorizada a torção femoral em pacientes jovens sem artrose com dor no quadril por IFA. No período entre janeiro de 2017 e março de 2018 foram avaliados 26 pacientes (52 quadris) entre 18 a 45 anos com dor no quadril e sem artrose. Todos os pacientes apresentavam dor e IFA (9 bilaterais) totalizando 35 quadris, sendo a maioria homens (73%). A idade média foi de 35 anos para ambos os sexos. O índice de massa corporal médio foi de 19 kg/m2 para mulheres, 24 kg/m2 para homens e a escala de Harris média foi de 67 pontos. Foram encontradas alterações da torção femoral em 11 quadris com dor (31%). A torção média encontrada foi de 14,5 graus, variando 0 a 39 graus (coeficiente de variabilidade= 60%). Pacientes com dor no quadril, torção femoral alterada e impacto do tipo CAM não apresentaram diferença quando comparados aos quadris sem dor (controle) (p= 0,234), como também no impacto tipo Misto (p= 0,314). No impacto tipo Pincer, a torção média dos quadris dolorosos foi de 16,63 graus e a torção média dos controles foi de 11,77 graus (p= 0,045). A presença de alterações na torcionais em quase 1/3 dos pacientes com IFA e a alta variabilidade encontrada revelaram a importância da medida da torção femoral nesta doença

The aim of the study was to evaluate femoral torsion in young patients with hip pain and femoral acetabular impingement (FAI). After the discovery of impingement, the complete analysis from the hip biomechanics has become essential. Twenty-six patients from 18 to 45 years presenting hip pain without arthrosis (Tonnis< 2) were evaluated. All patients presented both pain and FAI (9 bilateral), mostly man (73%). The average age was 35 years for both genders. BMI was 19 kg/m2 for women and 24 kg/m2 for men and the mean modified Harris Hip Score was 67 points. We have found femoral torsion changes in 11 hips with pain (31%) and high variability (60%). The mean femoral torsion was 14,5 degrees, ranging from 0 to 39 degrees. Patients with hip pain, CAM impingement and altered femoral torsion had no correlation when compared to controls (without pain) (p= 0.234), neither Mixed impingement (p= 0,314). Patients with Pincer impingement and painful hips had higher femoral torsion (16,63 degrees) than controls (11,77) (p= 0.045). The presence of torsional alterations in almost 1/3 of the patients with FAI and the high variability reveal the importance of measuring femoral torsion at this disease

Peloruside A is a microtubule-stabilizing agent with exceptional anti-migratory properties in human endothelial cells.

Peloruside A is a novel antimitotic drug originally isolated from the marine sponge Mycale hentschieli. Previous studies showed that peloruside A stabilizes microtubules by binding to a site on tubulin distinct from paclitaxel, another microtubule stabilizing drug. Peloruside A blocks mitosis, but little is known about the effects on other cellular activities. Here we report that peloruside A is the most potent microtubule inhibitor yet tested for its ability to block endothelial cell migration. Quantitative analysis indicated that it inhibits microtubule dynamics and endothelial cell migration at 1/200(th) of the concentration needed to inhibit cell division (the cytotoxic concentration), indicating that it could potentially have a large margin of safety when used to specifically target angiogenesis. By comparison, paclitaxel, a well-known cancer therapeutic drug, suppresses cell migration at 1/13(th) of its cytotoxic concentration; and vinblastine suppresses cell migration at just slightly below its cytotoxic antimitotic concentration. Thus, different microtubule targeted drugs have varying relative potencies for inhibition of cell migration versus cell division. The results suggest that peloruside A may be an especially useful agent for anti-angiogenesis therapy and point to the likelihood that other antimitotic drugs might be found with an even larger potential margin of safety.

Resistance to peloruside A and laulimalide: functional significance of acquired ßI-tubulin mutations at sites important for drug-tubulin binding.

Cancer cell lines selected for resistance to microtubule targeting agents (MTA) often have acquired mutations in the ß-tubulin binding sites for these agents. Despite strong correlational evidence, the functional and quantitative significance of such mutations in the resistance to MTA remains unknown. We recently showed that peloruside A (PLA) and laulimalide (LAU)-resistant cancer cell lines, 1A9-R1 (R1) and 1A9-L4 (L4), generated through multi-step selection of human 1A9 ovarian cancer cells with high concentrations of either PLA (for R1) or LAU (for L4) have single distinct mutations in their ßI-tubulin gene. The R1 cells have a mutation at amino acid position 296 (A296T), and the L4 cells have a mutation at position 306 (R306H/C), both of which lie at the putative binding sites of PLA and LAU. To gain insights on the functional role of these mutations in the resistance phenotype, R1 and L4 cells were transfected with wild type ßI-tubulin. MTT cell proliferation assays revealed that restoration of wild type ßI-tubulin expression partially sensitized the R1 and L4 cells to PLA and LAU. Cell cycle analysis and intracellular tubulin polymerization assays demonstrated that the increased sensitivity was correlated with an increased ability of PLA and LAU to induce G2-M arrest and tubulin polymerization in the cells. Unlike paclitaxel-selected clones of 1A9 cells, both R1 and L4 cells exhibited a functional p53 gene, and the abundance of the mismatch repair gene hMSH2 (human mutS homolog 2) was comparable to the parental 1A9 cells. This study provides the first direct evidence that A296 and R306 of ßI-tubulin are important determinants of the PLA and LAU response in cancer cells.

Paclitaxel resistance by random mutagenesis of α-tubulin.

Many mammalian ß-tubulin mutations that confer paclitaxel resistance have been characterized, but little is currently known about the role of α-tubulin mutations in drug resistance. Previous studies using two-dimensional gel electrophoresis showed that α-tubulin mutations occur with a frequency equal to ß-tubulin mutations among CHO cells selected for resistance to paclitaxel but the identities of those mutations are largely unknown. We have now sequenced the major α-tubulin gene in several paclitaxel resistant CHO cell lines with lesions in genomic DNA and identified five mutations that predominately affect the amino terminal part of the protein. We also used random mutagenesis and transfection of α-tubulin cDNA to select further paclitaxel resistant mutants in an effort to remove genomic constraints that may limit the diversity of mutations. This approach led to the identification of 16 additional mutations that were distributed throughout the α-tubulin sequence. The mutations were confirmed as sufficient to confer resistance by site-directed mutagenesis, and they acted by a mechanism that involved reductions in microtubule assembly. One mutation prevented the acetylation of α-tubulin but otherwise produced a phenotype similar to the other mutations. A scan of the literature revealed that a significant number of drug resistance mutations overlap or lie close to lesions that have been reported in patients with brain disorders suggesting that alterations in microtubule assembly underlie both cellular resistance and developmental defects.

Microtubule dynamics control tail retraction in migrating vascular endothelial cells.

Drugs that target microtubules are potent inhibitors of angiogenesis, but their mechanism of action is not well understood. To explore this, we treated human umbilical vein endothelial cells with paclitaxel, vinblastine, and colchicine and measured the effects on microtubule dynamics and cell motility. In general, lower drug concentrations suppressed microtubule dynamics and inhibited cell migration whereas higher concentrations were needed to inhibit cell division; however, surprisingly, large drug-dependent differences were seen in the relative concentrations needed to inhibit these two processes. Suppression of microtubule dynamics did not significantly affect excursions of lamellipodia away from the nucleus or prevent cells from elongating; but, it did inhibit retraction of the trailing edges that are normally enriched in dynamic microtubules, thereby limiting cell locomotion. Complete removal of microtubules with a high vinblastine concentration caused a loss of polarity that resulted in roundish, rather than elongated, cells, rapid but nondirectional membrane activity, and little cell movement. The results are consistent with a model in which more static microtubules stabilize the leading edge of migrating cells, whereas more dynamic microtubules locate to the rear where they can remodel and allow tail retraction. Suppressing microtubule dynamics interferes with tail retraction, but removal of microtubules destroys the asymmetry needed for cell elongation and directional motility. The prediction that suppressing microtubule dynamics might be sufficient to prevent angiogenesis was supported by showing that low concentrations of paclitaxel could prevent the formation of capillary-like structures in an in vitro tube formation assay.

Detection and quantification of microtubule detachment from centrosomes and spindle poles.

Microtubule detachment from microtubule organizing centers is an important cellular process required for normal cell proliferation. When cells enter mitosis, microtubule turnover increases along with a concurrent increase in microtubule detachment. MCAK, a kinesin-related protein whose abundance is highest during the early stages of mitosis, has been shown to regulate microtubule detachment. Abnormal increases or decreases in the frequency of detachment interfere with spindle function and inhibit cell division. It has been shown that drugs able to promote microtubule assembly (e.g., paclitaxel, epothilones) prevent cell division by suppressing microtubule detachment from centrosomes. Conversely, cytotoxic concentrations of microtubule destabilizing drugs (e.g., vinblastine, nocodazole), tubulin mutations that cause paclitaxel resistance, and specific ß-tubulin isotypes increase the frequency of microtubule detachment. In this chapter, we describe a method to calculate the frequency of microtubule detachment by transfecting cells with EGFP-MAP4 and directly observing detachment by live cell imaging.

The role of microtubules and their dynamics in cell migration.

Although microtubules have long been implicated in cell locomotion, the mechanism of their involvement remains controversial. Most studies have concluded that microtubules play a positive role by regulating actin polymerization, transporting membrane vesicles to the leading edge, and/or facilitating the turnover of adhesion plaques. Here we used wild-type and mutant CHO cell lines with alterations in tubulin to demonstrate that microtubules can also act to restrain cell motility. Tubulin mutations or low concentrations of drugs that suppress microtubule dynamics without affecting the amount of microtubule polymer inhibited the rate of migration by preventing microtubule reorganization in the trailing portion of the cells where the more dynamic microtubules are normally found. Under these conditions, cells along the edge of a wound still extended lamellipodia and elongated toward the wound but were inhibited in their ability to retract their tails, thus retarding forward progress. The idea that microtubules normally act to restrain cell locomotion was confirmed by treating cells with high concentrations of nocodazole to depolymerize the microtubule network. In the absence of microtubules, wild-type CHO and HeLa cells could still move at near normal speeds, but the movement became more random. We conclude that microtubules act both to restrain cell movement and to establish directionality.

Random mutagenesis of ß-tubulin defines a set of dispersed mutations that confer paclitaxel resistance.

PURPOSE: Previous research showed that mutations in ß1-tubulin are frequently involved in paclitaxel resistance but the question of whether the mutations are restricted by cell-type specific differences remains obscure. METHODS: To circumvent cellular constraints, we randomly mutagenized ß-tubulin cDNA, transfected it into CHO cells, and selected for paclitaxel resistance. RESULTS: A total of 26 ß1-tubulin mutations scattered throughout the sequence were identified and a randomly chosen subset were confirmed to confer paclitaxel resistance using site-directed mutagenesis of ß-tubulin cDNA and transfection into wild-type cells. Immunofluorescence microscopy and biochemical fractionation studies indicated that cells expressing mutant tubulin had decreased microtubule polymer and frequently suffered mitotic defects that led to the formation of large multinucleated cells, suggesting a resistance mechanism that involves destabilization of the microtubule network. Consistent with this conclusion, the mutations were predominantly located in regions that are likely to be involved in lateral or longitudinal subunit interactions. Notably, fourteen of the new mutations overlapped previously reported mutations in drug resistant cells or in patients with developmental brain abnormalities. CONCLUSIONS: A random mutagenesis approach allowed isolation of a wider array of drug resistance mutations and demonstrated that similar mutations can cause paclitaxel resistance and human neuronal abnormalities.

Control of MCAK degradation and removal from centromeres.

Mitotic centromere associated kinesin (MCAK) is a kinesin related protein with the ability to stimulate microtubule depolymerization. It is found at spindle poles, where it may be involved in poleward microtubule flux, and at kinetochores and centromeres where it plays a role in correcting chromosome alignment errors. Its microtubule depolymerase activity and recruitment to centromeres is regulated by phosphorylation, but little is known about how MCAK is maintained at appropriate levels. We previously reported that MCAK accumulates during the cell cycle and is then degraded during mitosis. Using proteomic analysis, we have now identified a new phosphorylation site on MCAK that is responsible for its degradation. Mutation of the site to prevent phosphorylation prolonged the stability of the protein beyond the metaphase to anaphase transition and into the subsequent cell cycle whereas a phosphomimetic mutation accelerated degradation. Unexpectedly, the mutation that prevented phosphorylation also inhibited the removal of MCAK from centromeres causing it to remain attached throughout the cell cycle. Even low expression of phosphorylation-resistant MCAK delayed mitosis and interfered with cell division. Mitotic defects were also observed by overexpressing a green fluorescent protein-tagged version of wild-type MCAK that similarly escaped degradation and accumulated to toxic levels, but did not remain associated with kinetochores during interphase. The results demonstrate that degradation is an important mechanism for controlling the activity of MCAK.

Mitotic centromere-associated kinesin (MCAK) mediates paclitaxel resistance.

Paclitaxel has powerful anticancer activity, but some tumors are inherently resistant to the drug, whereas others are initially sensitive but acquire resistance during treatment. To deal with this problem, it will be necessary to understand the mechanisms of drug action and resistance. Recent studies indicate that paclitaxel blocks cell division by inhibiting the detachment of microtubules from centrosomes. Here, we demonstrate that mitotic centromere-associated kinesin (MCAK), a kinesin-related protein that destabilizes microtubules, plays an important role in microtubule detachment. Depletion of MCAK altered mitotic spindle morphology, increased the frequency of lagging chromosomes, and inhibited the proliferation of WT CHO cells, confirming that it is an essential protein for cell division. In contrast, MCAK depletion rescued the proliferation of mutant paclitaxel-dependent cell lines that are unable to divide because of defective spindle function resulting from altered α-tubulin or class III ß-tubulin overexpression. In concert with the correction of mitotic defects, loss of MCAK reversed an aberrantly high frequency of microtubule detachment in the mutant cells and increased their sensitivity to paclitaxel. The results indicate that MCAK affects cell sensitivity to mitotic inhibitors by modulating the frequency of microtubule detachment, and they demonstrate that changes in a microtubule-interacting protein can reverse the effects of mutant tubulin expression.

New insights into mechanisms of resistance to microtubule inhibitors.

Mechanisms to explain tumor cell resistance to drugs that target the microtubule cytoskeleton have relied on the assumption that the drugs act either to suppress microtubule dynamics or to perturb the balance between assembled and nonassembled tubulin. Recently, however, it was found that these drugs also alter the stability of microtubule attachment to centrosomes, and do so at the same concentrations that are needed to inhibit cell division. Based on this new information, a new model is presented that explains resistance resulting from a variety of molecular changes that have been reported in the literature. The improved understanding of drug action and resistance has important implications for chemotherapy with these agents.

Class III ß-tubulin counteracts the ability of paclitaxel to inhibit cell migration.

Class III ß-tubulin (ß3) is associated with tumor aggressiveness, resistance to therapy, and patient relapse. To elucidate its action, we tested ß3's effect on cell migration. Expression of ß3 in HeLa and MCF-7 did not alter the intrinsic rate of cell migration, but it prevented the inhibition of migration by low, nontoxic concentrations of paclitaxel. The effects on cell motility were confirmed in CHO cells with tetracycline regulated expression of ß3. Cell migration and microtubule dynamics were inhibited by similar concentrations of paclitaxel, but required a 5-10 fold higher drug concentration when ß3 was expressed. The directionality of migration was normal in paclitaxel, but cells spent more time in a "paused" state during which there was no net movement. These studies support a model in which paclitaxel inhibits cell migration by suppressing microtubule dynamics and ß3-tubulin counteracts paclitaxel action by maintaining microtubule dynamic activity. The results provide a potential explanation for the aggressiveness of ß3-expressing tumors.

Overexpression of mitotic centromere-associated Kinesin stimulates microtubule detachment and confers resistance to paclitaxel.

Numerous studies have implicated mutations in tubulin or the overexpression of specific tubulin genes in resistance to microtubule-targeted drugs. Much less is known about the role of accessory proteins that modulate microtubule behavior in the genesis of drug resistance. Here, we examine mitotic centromere-associated kinesin (MCAK), a member of the kinesin family of microtubule motor proteins that has the ability to stimulate microtubule depolymerization, and show that overexpressing the protein confers resistance to paclitaxel and epothilone A, but increases sensitivity to colcemid. Cells transfected with FLAG-tagged MCAK cDNA using a tet-off-regulated expression system had a disrupted microtubule cytoskeleton and were able to survive a toxic concentration of paclitaxel in the absence, but not in the presence of tetracycline, showing that drug resistance was caused by ectopic MCAK production. Moreover, a population that was heterogeneous with respect to FLAG-MCAK expression became enriched with cells that produced the ectopic protein when it was placed under paclitaxel selection. Similar to previously isolated mutants with altered tubulin, paclitaxel resistant cells resulting from MCAK overexpression were found to have decreased microtubule polymer and a seven-fold increase in the frequency of microtubule detachment from centrosomes. These data are consistent with a model for paclitaxel resistance that is based on stability of the attachment of microtubules to their nucleating centers, and they implicate MCAK in the mechanism of microtubule detachment.