Vulnerabilidades específicas de células malignas humanas dependentes de Ras oncogênico: FGF2 e PMA como supressores de tumor / Specific vulnerabilities of human malignant cells dependent on oncogenic Ras: FGF2 and PMA as tumor suppressors

Um passo limitante no desenvolvimento de fármacos para terapias do câncer está na descoberta de vulnerabilidades específicas de células tumorais que sirvam à identificação de alvos moleculares apropriados à intervenção farmacológica. Esta é a motivação central desta tese, cuja abordagem experimental focaliza a ação oncogênica das proteínas Ras. Amplificação ou mutação ativadora nos proto-oncogenes ras estão entre as alterações genéticas mais frequentes em cânceres. Essas lesões genéticas aparecem na origem etiológica de múltiplas formas de fenótipos malignos. Mas, essas lesões oncogênicas também conferem susceptibilidades letais às células malignamente transformadas frente a determinados agentes que não interferem significativamente nas funções vitais de células normais. Nos últimos anos nosso laboratório vem estudando os mecanismos moleculares da ação antiproliferativa do fator de crescimento FGF2 (Fibroblast Growth Factor2) e do éster de forbol PMA (Phorbol-12-Myristate-13-Acetate) em linhagens de células murinas malignas dependentes de ras oncogênico. Nesta tese investigamos quanto de nossas observações anteriores com células murinas são aplicáveis a células humanas. Nesse sentido focalizamos a linhagem HaCaT de queratinócitos humanos imortalizados e seus subclones malignizados por expressão ectópica de H-RasV12; além disso, numa triagem inicial também examinamos treze linhagens celulares humanas derivadas de tumores naturais portadores de mutação ativadora em H-Ras, N-Ras ou K-Ras. Nossos resultados mostram que os queratinócitos da linhagem parental HaCaT expressam receptores de FGFs e respondem mitogenicamente tanto a FGF2 como a PMA; portanto, ambos FGF2 e PMA são benéficos aos queratinócitos HaCaT. Por outro lado, o FGF2 mostrou-se citotóxico para subclones HaCaT que expressam H-RasV12 induzível, mas sublinhagens HaCaT com expressão constitutiva de H-RasV12 mostraram-se resistentes à ação citotóxica de FGF2. Diferentemente de FGF2, PMA bloqueou a proliferação de sublinhagens clonais HaCaT-H-RasV12 em ambos substrato sólido e suspensão de agarose e, também, reduziu a estratificação dos queratinócitos HaCaT-H-RasV12 em culturas organotípicas. PMA foi citotóxico e não citostático, pois induziu morte apoptótica sem causar arresto em nenhuma fase específica do ciclo celular. Em HaCaT parental, PMA induziu aumento transitório dos níveis intracelulares de espécies reativas de oxigênio (ROS), mas nos queratinócitos HaCaT-H-RasV12, PMA causou aumentos mais altos e persistentes de ROS, o que promove forte estresse oxidativo, provavelmente responsável pela toxidez deste ester de forbol. Entre as treze linhagens celulares humanas malignas com H-Ras, N-Ras ou K-Ras mutados, onze foram vulneráveis à ação citotóxica de PMA; mas apenas uma delas, a linhagem de tumor urotelial UM-UC-3, foi sensível ao efeito anti-proliferativo de FGF2. Em conclusão, células malignas humanas com Ras mutado parecem superar rapidamente uma possível toxidez de FGF2, mas não ultrapassam a toxidez causada por PMA

A challenge in drug development for cancer therapy is the discovering of molecular targets suitable for pharmacological interference. This challenge was the main motivation of the present thesis. Amplification or activating mutation in ras proto-oncogenes are among the most frequent genetic lesions in human cancer. Actually, mutated Ras onco-proteins are in the etiological roots of multiple malignant phenotypes; however these onco-proteins also cause specific lethal vulnerabilities even in robust malignant cells. Recently, our laboratory reported that malignant murine cell lines dependent on oncogenic Ras are prone to toxicity initiated by FGF2 (Fibroblast Growth Factor 2) and PMA (Phorbol-12-Myristate-13-Acetate), which are not harmful to normal cells. This cytotoxicity of FGF2 and PMA very likely follows different molecular mechanisms, which, however, are not yet completely understood. The aim of this thesis was to investigate whether these vulnerabilities found in murine malignant cells were also valid for human malignant cell lines dependent on oncogenic Ras. To this end the experimental approach was focused on the HaCaT cell line of immortalized human keratinocytes and its sublines transformed by H-RasV12 ectopic expression. In addition thirteen human cell lines derived from natural tumor carrying mutated H-Ras, N-Ras or K-Ras oncogenes were also screened. The results showed that HaCaT keratinocytes express FGF receptors and respond mitogenically to both FGF2 and PMA. On the other hand, FGF2 was cytotoxic to HaCaT subclones expressing inducible H-RasV12. But, HaCaT sublines constitutively expressing H-RasV12 were resistant to FGF2 toxicity. However, PMA was toxic to all HaCaT-H-RasV12 sublines, inhibiting proliferation in both solid substrate and agarose suspension cultures and, also reducing stratification in organotypic cultures. Furthermore, in HaCaT-H-RasV12 sublines, but not in the parental HaCaT line, PMA caused a persistently high increase in intracellular levels of reactive oxygen species (ROS) and concomitantly induced apoptosis. Moreover, eleven of the thirteen human tumor cell lines with mutated H-Ras, N-Ras or K-Ras, were growth inhibited by PMA, whereas only one of them was inhibited by FGF2, the urothelial tumor cell line UM-UC-3. In conclusion, human malignant cells driven by Ras oncogenes very likely rapidly overcome FGF2 toxicity, whereas they remain stably vulnerable to PMA cytotoxicity

Increased efficiency for performing colony formation assays in 96-well plates: novel applications to combination therapies and high-throughput screening.

The colony formation assay (CFA) is the gold standard for measuring the effects of cytotoxic agents on cancer cells in vitro; however, in its traditional 6-well format, it is a time-consuming assay, particularly when evaluating combination therapies. In the interest of increased efficiency, the 6-well CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation therapy on the FaDu (human hypopharyngeal squamous cell) and A549 (human lung) cancer cell lines. Its ability to evaluate combination therapies was investigated by the generation of dose-response curves for the combination of cisplatin and radiation therapy on FaDu and A549 cells. The 96-well CFA was then transferred to a robotic platform for evaluating its potential as a high-throughput screening (HTS) readout. The LOPAC1280 library was screened against FaDu cells, and eight putative hits were identified. Using the 96-well CFA to validate the eight putative chemicals, six of the eight were confirmed, resulting in a positive hit rate of 75%. These data indicate that the 96-well CFA can be adopted as an efficient alternative assay to the 6-well CFA in evaluating single and combination therapies in vitro, providing a possible readout that could be used on a HTS platform.

[A method used in myeloma cell cloning spot image segmentation].

A new interactive image segmentation method used in the multiple myeloma cloning spots image segmentation is presented in the paper. Based on the theory of graph cuts, some pixels are selected as the front object and the background seeds, and the other parts are treated as the unknown region. Then, an energy function is constructed and initialized through K-means, and the minimum cut method is used in the segmentation by energy minimization. Last, the image is eroded and dilated, and the cloning separate parts could be got effectively. For the pixels which may be partitioned wrongly, we use a tool similar to a brush to re-mark the front object or the background, and divide once again. Both subjective the evaluation criteria and the RUMA, evaluation criteria are used to evaluate the method, and the experiment results are satisfactory.

Efecto reductor de la lactona sesquiterpénica "Helenalina" y sus derivados metálicos en la formación del complejo estradiol receptor en tejido tumoral mamario / Reductive effect of sesquiterpenic lactone \"Helenaline\" and its metalic derivates in complex estradiol-receptor formation in breast tumoral tissue

Dentro de la terapia sistémica empleada para el tratamiento de cáncer mamario, ha sido extensamente utilizada la quimioterapia, la cual ha sido apoyada por muy diversos compuestos en cuanto a origen y composición química. Sin embargo, todos ellos, producen diversos efectos colaterales adeversos, dignos de tomarse en cuenta. Por este hecho, precisa estudiar nuevas posibilidades en donde el fármaco aplicado, actúa selectivamente sobre célula tumoral, sin lesionar tejido sano. Para su efecto, se estudió una gamma lactona llamada "Helenalina" y sus derivados metálicos He-Co, He-Hg y He-Cu, cuya composición química les permite reaccionar con residuos -SH presentes en el receptor de la célula tumoral, los cuales al intercalarse por una reacción previa, podría modificar su composición estructural y finalemente su afinidad por la hormona. Se investigó el efecto de inhibición para la formación del complejo estradiol-receptor en el citosol de tejido tumoral mamario empleando Helenalina a 12 n M y 126 n M, obteniéndose un efecto de inhibición de 14 por ciento y 56 por ciento respectivamente. Cuando se estudió He-Co, He-Hg y He-Cu este efecto se vió aumentado, obteniéndose 11 por ciento, 10.5 por ciento y 60 por ciento con 12 n m y 44.5 por ciento, 74.5 por ciento y 86 por ciento con 126 n M respectivamente

Effect of medium exchange rate on colony growth of the human tumor cell line MDA-231 cloned within the perfused capillary cloning system.

The conventional human tumor stem cell assay is limited by the lack of flexibility for drug scheduling with single agents and its inability to test drug combinations. Recently, we described the cloning of tumor cell lines within porous glass capillary tubes which allow free exchange of substances. The present study describes the influence of various perfusion modalities on the colony growth of the human tumor cell line MDA-231 cloned within the perfused capillary cloning system (PCCS). Colony growth of tumor cells within the PCCS is strongly dependent on perfusion tube volume, flow rate and duration of perfusion with growth medium. Best colony growth was achieved using a perfusion tube volume of 12 ml resulting in a cloning efficiency of 36.3%. Continuous perfusion with fresh medium did not improve the cloning efficiency; in fact, colony growth was hampered compared to colony growth within unperfused porous capillaries. However, cloning efficiency was acceptable when continuous perfusion was started at day 6 (26.4%) instead of day 0 (17.2%), or when a short perfusion with high volume (12 ml/h) was discontinued after 1 h at day 0. In contrast to the conventional capillary cloning system the PCCS has the potential for investigating secretion and kinetics of tumor-specific factors and the effect of growth-stimulating or growth-inhibiting drugs.

Tumor cell detection method using complement-mediated cytolytic reaction and imaging sensor system.

A novel tumor-detection system consisting of complement-mediated cytolytic reaction and an image processing system was developed for the simple and rapid determination of tumor cells. The present system consists of a CCD image sensor, image memory board, personal computer, and microscope. When monoclonal antibody 3C4, which is specific to the guinea pig hepatoma L-10, was added to cell suspension, only L-10 cytolysis occurred. Cytolysis caused a decrease in brightness of the cells observed by phase-contrast microscopy. The phase contrast image of the cells before cytolysis was converted to a digitalized signal and stored in computer memory. After cytolysis, a brightness threshold above that of lysed cells was subtracted from the digitalized signal and compared to the signal stored before reaction. L-10 cells in mixed cell suspension were determined specifically by the system. Measurement time was only 2 sec and overall time, including reaction time, was approximately 30 min. Since this method does not require a cell washing process, automation of the whole system is possible.

Clinical praxis and laboratory procedures in subrenal capsule assay (SRCA).

Subrenal capsule assay (SRCA) is a promising method in cancer research and in the selection of individual chemotherapy for cancer patients. The laboratory procedures of SRCA are as follows: on day zero 1 X 1 X 1 mm pieces of fresh human tumour are carefully prepared. The pieces are transferred by a trocar under the outer capsule of normal immunocompetent mice, one piece to each. The mice (about 30-40/test) are divided randomly into groups of at least five. The size of each piece (initial size) is measured in situ by a stereomicroscope fitted with an ocular micrometer. On days 1-5 cytostatics are administered to the animals. On day 6 the animals are killed and the final tumour size is measured. The difference between the final and initial tumour size describes the response of the tumour to the treatments. The critical point of the assay is the selection of the tumour fragment which has to be representative, as homogeneous as possible and contain living tumour cells. When the sample is in pieces of about 3 X 3 mm (4-5 tumour fragments are sufficient for the assay) in the culture medium, it may be stored in an ice bath or at room temperature for one day. During this time, though as rapidly as possible, the sample has to be delivered to a research laboratory where the SRCA is carried out.