Inhibition of Aurora kinases enhances chemosensitivity to temozolomide and causes radiosensitization in glioblastoma cells

Glioblastoma remains one of the most devastating human malignancies, and despite therapeutic advances, there are no drugs that significantly improve the patient survival. Altered expression of the Aurora kinases was found in different malignancies, and their inhibition has been studied in cancer therapy. In this study, we analyzed the expression of Aurora A and Aurora B in glioblastoma samples and also analyzed whether the effects of Aurora kinase inhibition were associated with temozolomide or not on cell lines and primary cultures of glioblastoma. RT-PCR assays were used to determine the mRNA expression in glioblastoma tumor samples and in the cell lines. Cell proliferation was measured by XTT assay, and apoptosis was determined by flow cytometry. Drug combination analyses were made based in Chou-Talalay method. Gamma radiation for clonogenic survival used the doses of 2, 4 and 6 Gy. Changes in Aurora B level were assessed by Western blot analysis. Aurora A and B were expressed in glioblastoma samples as well as in the glioblastoma cell lines (n = 6). Moreover, ZM447439, a selective Aurora kinase inhibitor, decreased the proliferation separately and synergistically with temozolomide in primary cultures and cell lines of glioblastoma. ZM also enhanced the effects of radiation on the two cell lines studied (U343 and U251), mainly when associated with TMZ in U343 cells. Treatment with ZM induced apoptotic cell death and diminished Aurora B protein level. These data suggest that Aurora kinase inhibition may be a target for glioblastoma treatment and could be used as adjuvant to chemo- and radiotherapy.

Nd bulk isotopes and 40Ar/39Ar ages of individual minerals from Aurora and Wilkes sub-glacial basin sediments

The Wilkes and Aurora basins are large, low-lying sub-glacial basins that may cause areas of weakness in the overlying East Antarctic ice sheet. Previous work based on ice-rafted debris (IRD) provenance analyses found evidence for massive iceberg discharges from these areas during the late Miocene and Pliocene. Here we characterize the sediments shed from the inferred areas of weakness along this margin (94°E to 165°E) by measuring40Ar/39Ar ages of 292 individual detrital hornblende grains from eight marine sediment core locations off East Antarctica and Nd isotopic compositions of the bulk fine fraction from the same sediments. We further expand the toolbox for Antarctic IRD provenance analyses by exploring the application of 40Ar/39Ar ages of detrital biotites; biotite as an IRD tracer eliminates lithological biases imposed by only analyzing hornblendes and allows for characterization of samples with low IRD concentrations. Our data quadruples the number of detrital 40Ar/39Ar ages from this margin of East Antarctica and leads to the following conclusions: (1) Four main sectors between the Ross Sea and Prydz Bay, separated by ice drainage divides, are distinguishable based upon the combination of 40Ar/39Ar ages of detrital hornblende and biotite grains and the e-Nd of the bulk fine fraction; (2) 40Ar/39Ar biotite ages can be used as a robust provenance tracer for this part of East Antarctica; and (3) sediments shed from the coastal areas of the Aurora and Wilkes sub-glacial basins can be clearly distinguished from one another based upon their isotopic fingerprints.

The proportion of individual lucerne plants resistant to Phytophthora medicaginis and Colletotrichum trifolii in Australian cultivars

Phytophthora root rot (Phytophthora medicaginis) and colletotrichum crown rot (Colletotrichum trifoli) are the 2 most serious pathogens of lucerne in eastern Australia. Work reported in this paper shows that in glasshouse tests of the 11 most commonly grown Australian lucerne cultivars, the proportion of individual plants with resistance to both pathogens ranges from 0 (Hunter River and Aurora) through to a maximum of 19.8% (Sequel HR). Within 9 of the cultivars, the proportion of individual plants resistant to the 2 pathogens was <7%. Since these 2 diseases are known to cause serious losses in eastern Australia, the results indicate further improvement in lucerne production can be obtained by increasing the proportion of individual plants in a cultivar resistant to both pathogens. This would be best achieved by identifying dominant sources of resistance and incorporating this into on-going lucerne breeding programs.

Mitotic regulation by Polo-like kinase 1 and the Chromosomal Passenger Complex

During mitosis, the duplicated genome must be accurately divided between two daughter cells. Polo-like kinase 1 (Plk1) and Aurora B kinase, together with its binding partners Incenp, Survivin and Borealin (chromosomal passenger complex, CPC), have key roles in coordinating mitotic events. The accuracy of cell division is safeguarded by a signaling cascade termed the mitotic spindle checkpoint (SC), which ensures that chromosomes are not physically separated before correct bipolar attachments have been formed between kinetochores and spindle microtubules (MT). An inhibitory “wait anaphase” signal, which delays chromosome separation (anaphase onset), is created at individual kinetochores and broadcasted throughout the cell in response to lack of kinetochore-microtubule (kMT) attachment or proper interkinetochore tension. It is believed that the fast turnover of SC molecules at kinetochores contributes to the cell’s ability to produce this signal and enables rapid responses to changing cellular conditions. Kinetochores that lack MT attachment and tension express a certain phosphoepitope called the 3F3/2 phosphoepitope, which has been linked to SC signaling. In the experimental part, we investigated the regulation of the 3F3/2 phosphoepitope, analyzed whether CPC molecules turn over at centromeres, and dissected the mitotic roles of the CPC using a microinjection technique that allowed precise temporal control over its function. We found that the kinetochore 3F3/2 phosphoepitope is created by Plk1, and that CPC proteins exhibit constant exchange at centromeres. Moreover, we found that CPC function is necessary in the regulation of chromatid movements and spindle morphology in anaphase. In summary, we identified new functions of key mitotic regulators Plk1 and CPC, and provided insighs into the coordination of mitotic events.

Functional characterization of proteins required for mitotic progression and the spindle assembly checkpoint

During mitotic cell division, the genetic material packed into chromosomes is divided equally between two daughter cells. Before the separation of the two copies of a chromosome (sister chromatids), each chromosome has to be properly connected with microtubules of the mitotic spindle apparatus and aligned to the centre of the cell. The spindle assembly checkpoint (SAC) monitors connections between microtubules and chromosomes as well as tension applied across the centromere. Microtubules connect to a chromosome via kinetochores, which are proteinaceous organelles assembled onto the centromeric region of the sister chromatids. Improper kinetochore-microtubule attachments activate the SAC and block chromosome segregation until errors are corrected and all chromosomes are connected to the mitotic spindle in a bipolar manner. The purpose of this surveillance mechanism is to prevent loss or gain of chromosomes in daughter cells that according to current understanding contributes to cancer formation. Numerous proteins participate in the regulation of mitotic progression. In this thesis, the mitotic tasks of three kinetochore proteins, Shugoshin 1 (Sgo1), INCENP, and p38 MAP kinase (p38 MAPK), were investigated. Sgo1 is a protector of centromeric cohesion. It is also described in the tension-sensing mechanism of the SAC and in the regulation of kinetochore-microtubule connections. Our results revealed a central role for Sgo1 in a novel branch of kinetochore assembly. INCENP constitutes part of the chromosomal passenger complex (CPC). The other members of the core complex are the Aurora B kinase, Survivin and Borealin. CPC is an important regulatory element of cell division having several roles at various stages of mitosis. Our results indicated that INCENP and Aurora B are highly dynamic proteins at the mitotic centromeres and suggested a new role for CPC in regulation of chromosome movements and spindle structure during late mitosis. The p38 MAPK has been implicated in G1 and G2 checkpoints during the cell cycle. However, its role in mitotic progression and control of SAC signaling has been controversial. In this thesis, we discovered a novel function for p38γ MAPK in chromosome orientation and spindle structure as well as in promotion of viability of mitotic cells.

A NOVEL FUNCTION FOR AURORA B KINASE IN THE REGULATION OF P53 BY PHOSPHORYLATION

The mitotic kinase Aurora B plays a pivotal role in mitosis and cytokinesis and governs the spindle assembly checkpoint which ensures correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in breast and other cancers and may be an important molecular target for chemotherapy. Tumor suppressor p53 is the guardian of the genome and an important negative regulator of the cell cycle. Previously, it was unknown whether Aurora B and p53 had mutual regulation during the cell cycle. A small molecule specific inhibitor of Aurora B, AZD1152, gave us an indication that Aurora B negatively impacted p53 during interphase and mitosis. Here, we show the antineoplastic activity of AZD1152 in six human breast cancer cell lines, three of which overexpress HER2. AZD1152 specifically inhibited Aurora B kinase activity, thereby causing mitotic catastrophe, polyploidy and apoptosis, which in turn led to apoptotic death. Further, AZD1152 administration efficiently suppressed tumor growth in orthotopic and metastatic breast cancer cell xenograft models. Notably, it was found that the protein level of Aurora B kinase declined after inhibition of Aurora B kinase activity. Investigation of the underlying mechanism suggested that AZD1152 accelerated the protein turnover of Aurora B by enhancing its ubiquitination. As a consequence of inhibition of Aurora B, p53 levels were increased in tissue culture and murine models. This hinted at a possible direct interaction between p53 and Aurora B. Indeed, it was found that p53 and Aurora B exist in complex and interact directly during interphase and at the centromere in mitosis. Further, Aurora B was shown to phosphorylate p53 at several serine/threonine residues in the DNA binding domain and these events caused downregulation of p53 levels via ubiquitination mediated by Mdm2. Importantly, phosphorylation of threonine 211 was shown to reduce p53’s transcriptional activity while other phosphorylation sites did not. On a functional level, Aurora B was shown to reduce p53’s capacity to mediate apoptosis in response to the DNA damaging agent, cisplatin. These results define a novel mechanism for p53 inactivation by Aurora B and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise p53’s tumor suppressor function.

Dissecting and resetting SETD2/H3K36ME3 deficiency in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is characterized by the presence of the BCR::ABL1 fusion gene, leading to a constitutively active tyrosine kinase that drives the disease. Genomic instability is a hallmark of CML, contributing to disease progression and treatment resistance. A study identified SETD2, a histone methyltransferase, as frequently dysfunctional in advanced-phase CML, resulting in reduced trimethylation of Histone H3 at lysine 36 (H3K36Me3). This loss is associated with poor prognosis and increased genetic instability. Investigations revealed that SETD2 dysfunction is caused by post-translational modifications mediated by Aurora kinase A and MDM2, leading to proteasome-mediated degradation. Aurora kinase A phosphorylates SETD2, while MDM2 ubiquitinates it, targeting it for degradation. Inhibition of MDM2 and Aurora kinase A restored SETD2 expression and activity, suggesting potential therapeutic targets. Loss of SETD2 and H3K36Me3 impairs DNA repair mechanisms, favoring error-prone repair pathways over faithful ones, exacerbating genetic instability. Reintroduction of SETD2 into deficient cells restored DNA repair pathways, preserving genomic integrity. Analysis of CD34+ progenitor cells from CML patients showed reduced SETD2 levels compared to healthy individuals, correlating with decreased clonogenic capacity. Notably, SETD2 loss is not detectable at diagnosis but emerges during disease progression, indicating its role as an early indicator of CML advancement. Therapeutically, inhibitors targeting Aurora kinase A, MDM2, and the proteasome showed efficacy in cells expressing SETD2, particularly in those with low SETD2 levels. Proteasome inhibitors induced apoptosis and DNA damage in SETD2-deficient cells, highlighting their potential for CML treatment. In conclusion, SETD2 acts as a tumor suppressor in CML, with its dysfunction contributing to genetic instability and disease progression. Targeting the mechanisms of SETD2 loss presents promising therapeutic avenues for controlling CML proliferation and restoring genomic integrity.

Reação de clones de umezeiro (Prunus mume Sieb. et Zucc.) e cultivares de pessegueiro [Prunus persica (L.) Batsch] ao nematóide anelado Mesocriconema xenoplax (Nemata: Criconematidae)

Com o objetivo de avaliar a reação de clones de umezeiro (Prunus mume Sieb. et Zucc.) e cultivares de pessegueiro [Prunus persica (L.) Batsch] ao nematóide anelado Mesocriconema xenoplax (Raski) Loof & de Grise, realizou-se o presente estudo em casa de vegetação do Departamento de Fitossanidade da FCAV/UNESP, Câmpus de Jaboticabal-SP. As plantas foram mantidas em vasos de cerâmica com 6 litros de capacidade, contendo uma mistura de solo e areia (1:1, v/v), previamente autoclavada a 121°C e 1kgf.cm-2 por 2 horas. Cada planta foi inoculada com 10mL de uma suspensão de 200 M. xenoplax por mL. Com os resultados obtidos, após 105 dias da inoculação, pode-se concluir que os Clones 05; 10 e 15 de umezeiro e as cultivares Okinawa e Aurora-1 de pessegueiro são suscetíveis a M. xenoplax. A cultivar Aurora-1 apresentou maior Fator de Reprodução (93,06).

Relação entre as características químicas e o valor dos pêssegos comercializados pelo sistema veiling frutas Holambra em Paranapanema-SP

O valor de comercialização do pêssego é reflexo da demanda e de sua apreciação pelo consumidor. A compreensão da diferença de valor entre os frutos das diferentes cultivares e da sua relação com as características que determinam o gosto do fruto, torna possível o estabelecimento de uma estratégia de comercialização visando ao aumento no consumo e na receita do produtor, além de dar subsídios aos programas de melhoramento genético. Neste trabalho, foram avaliadas as características de gosto de duas cultivares, 'Douradão' e 'Tropic Beauty', e de duas séries de cultivares, 'Aurora' e 'Dourado', que são as cultivares de pêssego mais produzidas no município de Paranapanema, maior produtor do Estado de São Paulo. Trabalhou-se com os valores de comercialização do leilão reverso, ou veiling, da Cooperativa Agroindustrial Holambra, no período de maior oferta do produto, entre 15 de outubro a 15 de novembro de 2004. A caracterização do gosto dos frutos foi feita através da determinação dos conteúdos de sólidos solúveis (SS) e de acidez titulável (AT), e da relação SS/AT dos frutos comercializados no Entreposto Terminal de São Paulo da CEAGESP. As médias dos conteúdos de sólidos solúveis (SS) não se mostraram significativamente diferentes. A acidez titulável (AT) e a relação SS/AT apresentaram valores médios significativamente diferentes. A cultivar 'Douradão' apresentou a maior relação SS/AT, seguida das séries varietais 'Dourado' e 'Aurora' e da cultivar Tropic Beauty. As diferenças na relação SS/AT não determinaram diferenças significativas no valor dos frutos do mesmo calibre, nos quatro materiais estudados. O valor de comercialização dos produtos mostrou-se significativamente afetado pelos calibres.

Produção e sazonalidade de pessegueiro e nectarineira sob florescimento espontâneo e com cianamida hidrogenada e óleo mineral

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Eficácia do carfentrazone-ethyl no controle de plantas aquáticas latifoliadas em caixas-d'água

Atualmente, alguns herbicidas estão sendo desenvolvidos para o controle de plantas daninhas aquáticas. O objetivo deste trabalho foi avaliar a eficácia do carfentrazone-ethyl em ambiente aquático para o controle pós-emergente de aguapé, alface-d'água e salvínia. O trabalho foi desenvolvido em caixas-d'água, no período de julho a setembro de 2004, no NUPAM - FCA/UNESP, em Botucatu. O delineamento experimental adotado foi o de blocos ao acaso, com sete tratamentos e quatro repetições, sendo as unidades experimentais constituídas pelas caixasd'água. Os tratamentos foram os seguintes: testemunha sem herbicida; Aurora 400 CE (75, 150 e 300 mL ha-1); Roundup (3,0 L ha-1), Aurora 400 CE + Roundup (75 mL + 3,0 L ha-1) e Aurora 400 CE + Arsenal N.A. (75 mL + 2,0 L ha-1). Observou-se que o tratamento Aurora 400 CE (300 mL ha-1) é altamente eficaz no controle de alface-d'água (Pistia stratiotes); o tratamento Roundup (3,0 L ha-1) é altamente eficaz no controle de aguapé (Eichhornia crassipes); o tratamento Aurora 400 CE + Roundup (75 mL + 3,0 L ha-1) é eficaz no controle de aguapé (E. crassipes), alface-d'água (P. stratiotes) e salvínia (Salvinia auriculata); e o tratamento Aurora 400 CE + Arsenal (75 mL + 2,0 L ha-1) é eficaz no controle de aguapé (E. crassipes) e alface-d'água (P. stratiotes). A mistura Aurora 400 CE + Roundup (75 mL + 3,0 L ha-1) apresentou-se viável e foi o único tratamento eficaz no controle das três espécies estudadas.

Composição acarina em diferentes cultivares de pessegueiros [Prunus persica (L.)], em Presidente Prudente, estado de São Paulo

Objetivou-se, nesta pesquisa, estudar a ocorrência natural de ácaros fitófagos e predadores em diferentes cultivares de pessegueiro, no município de Presidente Prudente-SP, Brasil. O estudo foi realizado no período de dezembro de 2002 a fevereiro de 2006. Amostras quinzenais de 72 folhas foram coletadas ao acaso, de pessegueiros das cultivares Talismã, Doçura 2, Dourado 2, Tropical, Aurora 1 e Aurora 2. Coletou-se um total de 2.594 ácaros, sendo 2.092 fitófagos, 403 predadores e 99 de hábitos alimentares pouco conhecidos, com 35 espécies de ácaros de 16 famílias. Aculus fockeui ocorreu de maneira esporádica, não causando danos visíveis às plantas. A família Phytoseiidae apresentou a maior abundância e o maior número de indivíduos. O predador Euseius citrifolius foi o mais abundante. Não houve preferência dos ácaros nas cultivares de pessegueiro avaliadas.

Frutificação, características físicas de frutos e produtividade em cultivares e seleções de pessegueiro em Vista Alegre do Alto-SP

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Infestación natural de Lonchaeidae (Diptera) en variedades de melocotón

Se estudió la ocurrencia de Lonchaeidae en variedades de melocotón, conducidos sobre los patrones ‘Okinawa’ y Umê: Tropical, Ouromel 3, Jóia 4, Régis, Talismã, Aurora 2, Aurora 1, Dourado 2 y Doçura 2. Se colectaron muestras de 30 frutos por planta en el Banco de Germoplasma en Presidente Prudente, Brasil. Se obtuvieron 633 especímenes de Lonchaeidae; 394 de ellos en frutos de variedades sobre ‘Okinawa’ y 239 sobre Umê. Se capturaron especies como Neosilba zadolicha, N. inesperata, N. pendula, N. certa y Neosilba spp. (hembras). N. zadolicha y N. inesperata, se observaron en 77,78% de las muestras de las variedades sobre ‘Okinawa’. Sobre el patrón Umê la mayor incidencia fue de N. zadolicha , N. inesperata y N. pendula (55,6%, 33,3% y 33,3% respectivamente). Las variedades Ouromel 3, Talismã, Doçura 2 y Aurora 2 presentaron mayores infestaciones por Lonchaeidae. N. certa tuvo una menor incidencia y sólo se observó en la variedad Doçura 2 sobre ‘Okinawa’. Plantas con mayor número de frutos presentaron mayor incidencia de moscas por fruto; sin embargo, no hubo correlación entre peso del fruto y número de moscas. No se observó diferencia para peso del fruto y número de moscas por fruto entre los dos patrones, ‘Okinawa’ y Umê. Melocotones crecidos en los patrones ‘Okinawa’ y Umê en Presidente Prudente están infestados por especies de Lonchaeidae que presentan preferencia independientemente del peso de los frutos. El aporte de este trabajo al conocimiento de la comunidad de especies de Lonchaeidae ofrece una base para su control.