Abstract In this work the extent of apoptosis was studied in a set of 504 benign and malignant breast lesions to elucidate its role in breast tumor development and progression. Also the correlation of apoptosis with estrogen and progesterone receptor positivity, cell proliferation and patients' prognosis was studied. The breast lesions were also analyzed immunohistochemically with antibodies to apoptosis regulating proteins bcl-2 and bax, and caspases 3, 6 and 8. In addition, the immunohistochemical expression of NO• synthesizing enzyme iNOS in relation to apoptosis and angiogenesis was studied. Furthermore, the expression of the antioxidative enzyme MnSOD was studied in relation to apoptosis and cell proliferation. According to the results, the apoptotic index was lowest in benign breast lesions. It was higher in in situ carcinomas, where a gradual increase in the extent of apoptosis from grade I to III in situ carcinoma was seen. The apoptotic index in invasive carcinomas was higher than in in situ carcinomas, and also in invasive carcinomas there was a gradual increase in apoptosis from grade I to III carcinomas. The apoptotic index was highest in recurrent carcinomas. Strong bcl-2 expression was usually found in benign breast lesions but the immunoreactivity decreased in in situ and invasive carcinomas. There was a significant inverse association between bcl-2 immunoreactivity and the extent of apoptosis. Low bcl-2 immunoreactivity also associated with estrogen- or progesterone receptor negativity. In contrast, bax expression did not show any significant association with apoptosis, hormone receptors or the histologic types of tumors. Strong cytoplasmic caspase 3, 6 and 8 immunoreactivity was found in most carcinomas. It was weaker in in situ carcinomas and only weak immunoreactivity could be seen in benign breast lesions. There was a significant association between the extent of apoptosis and caspase immunoreactivity. iNOS expression was found in both tumor and stromal cells. iNOS expression in tumor cells was more frequently found in invasive than in in situ carcinomas. Its expression correlated significantly with a high apoptotic index and high vascularization of the lesion. There was significantly less MnSOD immunoreactivity in invasive breast carcinomas compared to in situ carcinomas or benign hyperplasias. MnSOD immunoreactivity did not associate with the extent of apoptosis, but there was a marginal inverse association between cell proliferation and MnSOD expression. Increased apoptosis was significantly associated with a high cell proliferation, and inversely associated with a positive estrogen status. A high apoptotic index (< 0.50%) was associated with a decreased survival of the patients. The results of this study show that apoptosis plays a decisive role in the development and progression of breast carcinoma. It is influenced not only by apoptosis regulating proteins, such as bcl-2 and caspases, but also by the estrogen receptor status. Apoptosis was also associated with iNOS positivity, the effect of which is mediated through increased NO• production. In line with the suggested role of MnSOD as a tumor suppressor gene, its expression was downregulated in invasive breast carcinoma. In conclusion, the association of apoptosis with patient survival in breast carcinoma may be secondary to its association with tumor cell proliferation and high tumor grade, not necessarily suggesting any causal association between apoptosis and survival.

Amyloidoses comprise a group of diseases where normal or mutated protein precipitates into amyloid fibrils. The deposition of fibrils causes dysfunction of organs and toxicity to nervous tissue. Up to date, 24 different proteins and peptides are known to be able to form amyloid fibrils. The most well known are Amyloid beta peptide and Prione protein causing Alzheimer’s disease and Creutzfeld Jacob’s disease respectively. The aims of this thesis were to investigate the structural properties of cytotoxic amyloid and examine the mechanisms involved. The model protein mostly used in the studies was the plasma protein transthyretin (TTR). Familial Amyloidotic Polyneuropathy (FAP) is a hereditary, autosomal-dominant neurodegenerative disease caused by point mutations in the TTR gene. One of the most common variants of FAP is a mutation in position 30 where alanine is exchanged for methonine. This gives rise to “Skellefteåsjukan” in Sweden. TTR is secreted into the plasma as a tetramer. Point mutations destabilize the tetramer leading to disassembled monomers, which undergo partial denaturation as an initiation step to aggregation and amyloid fibril formation. In vivo amyloidogenesis takes a long time and does not occur until late in adult life. Most of the clinical TTR mutations do not form amyloid in vitro under physiological conditions. We have created amyloidogenic TTR mutants that are prone to aggregate and form fibrils under physiological conditions. This provides us with a model system on the cellular level for studies of the mechanisms of amyloid associated cytotoxicity as we can control the aggregation process and capture defined stages in the TTR amyloidogenic pathway. We used Atomic Force Microscopy (AFM) to follow the morphology of aggregates during fibril formation. Initially, amorphous aggregates were formed that subsequently matured into fibrillar structures, denoted protofilaments. This observation was interpreted as an optimisation of ß-strand registers. In addition we identified a correlation between the presence of early-formed aggregates of TTR and cytotoxicity. The toxic response was mediated via an apoptotic mechanism. We were not able to more carefully determine the structure and size of the toxic TTR species. To address this problem we turned to another amyloidogenic protein, equine lysozyme (EL). Intermediate samples corresponding to the aggregation and growth phase of amyloid fibrils of EL were collected. These samples were subjected to cytotoxicity assays as well as monomeric starting material and mature amyloid fibrillar species. The results clearly showed that the soluble oligomers were cytotoxic in contrast to the monomers and fibrils. Our data indicate that the toxic properties of the oligomers are size dependent. In this thesis we asked the question whether all mutated forms of TTR can be expressed and secreted or if there is a selection against the most aggressive mutations in vivo? We transfected hematopoetic K562 cells with wild type or mutant TTR, with or without the N-terminal signal peptide, responsible for secretion, to generate both extra- and intracellular TTR. We show that the post-translational quality control of the cells does not allow intracellular mutant TTR outside the secretory pathway, possibly due to the cytotoxic effects, while translocated to the secretory pathway made it escape the quality control permitting secretion and amyloid formation outside the cells. We have further analyzed the cytotoxic mechanisms induced by TTR oligomers with a focus on intracellular apoptotic signalling pathways. We show that TTR oligomers bind to the surface of the target cells but are not taken up, that is in contrast to mature fibrils that do not bind them at all. The apoptotic response occurred in a caspase-independent and a free radical dependent way.

La apoptosis es una forma de muerte controlada por unas proteasas llamadas caspasas. La activación de las caspasas lleva a la desintegración controlada de la célula y a su fagocitación por parte de macrófagos. La necrosis es una forma de muerte que no depende de caspasas, pero que sí que puede tener un control y ser homeostático. Buena parte de los tumores presentan la característica de tener un metabolismo glicolítico superior al de la mayoría de los tejidos. Además, esta gran dependencia en la glucosa hace a las células bastante sensibles tanto a su ausencia como a la inhibición del metabolismo glicolítico. Esta característica es usada tanto en diagnóstico como en tratamientos en pruebas: la 2-deoxiglucosa se está utilizando en ensayos clínicos. Otra característica de las células tumorales es la de ser resistentes a muchos inductores de muerte, tanto de apoptosis como de necrosis mediante la inducción de proteínas anti-apoptóticas de la familia Bcl-2 o la inhibición de las proapoptóticas BH3. Además, muchos tumores sólidos pueden sufrir ambientes con escasez de nutrientes. Se da la coincidencia de que muchos oncogenes están a su vez relacionados con el metabolismo. Todos estos datos hacen interesante el estudio del efecto de la privación de glucosa sobre varios tipos celulares. Por un lado, caracterizar los efectos de la privación de glucosa en células que tienen bloqueada la vía mitocondrial de la apoptosis al ser deficientes en las proteínas Bax y Bak (DKO). Por otro, estudiar los efectos de la privación de glucosa en células tumorales humanas. Dentro de esta caracterización, es importante averiguar si se mueren por apoptosis o necrosis. Qué elementos reguladores están implicados, protectores o sensibilizadores. Si es apoptosis, y la muerte depende de caspasas, averiguar qué caspasas son las iniciadoras y cómo se están activando. Y cuál es el papel del metabolismo en general y de la autofagia en particular en esta muerte. En esta tesis se muestra cómo la privación de glucosa induce actividad caspasa en células deficientes en Bax y Bak. Y que esta actividad caspasa es dependiente de la caspasa iniciadora 8, no sólo en fibroblastos DKO, sino también en células HeLa. Aquí enseñamos que la apoptosis inducida por la privación de glucosa en células DKO induce actividad caspasa, degradación del ADN, condensación atípica de la cromatina, externalización de la fosfatidilserina y demás rasgos típicos de apoptosis como el corte de sustratos específicos de caspasas. Todo esto, excepto la condensación de la cromatina es inhibido por inhibidores de caspasas. Nosotros hemos demostrado que la privación de glucosa induce apoptosis independientemente de la interacción de ligandos y receptores de muerte. Así mismo, hemos demostrado que la activación de caspasa-8 no se inhibe por FLIP, y probablemente FADD no es la molécula adaptadora o reclutadora de caspasa-8. Hemos intentado ver si otros mecanismos de activación de la caspasa-8 que han sido publicados en los últimos años podrían ser la causa. FLIP no está implicados en la muerte inducida por privación de glucosa. Un posible reclutamiento de la caspasa-8 al complejo formado por RIPK1 no es el responsable, ni tampoco la actividad kinasa de RIPK1 ya que la necrostatina no protege de la muerte. El estudio del papel de la autofagia da resultados contradictorios. Por un lado, la autofagia sería un proceso que sensibilizaría a las células a la privación de glucosa, ya que la 3-metil-adenina protege, y las células deficientes en Atg7 están también protegidas. Pero por otro lado, la autofagia no tendría un papel importante, porque la presencia de cloroquina no afecta a la privación de glucosa. La privación de glucosa induce una acumulación de p62, que no parece ser la causa de la activación de la caspasa-8 ya que su silenciamiento no protege. / Apoptosis induced by most stimuli proceeds through the mitochondrial pathway. One such stimulus is nutrient deprivation. In this study we studied death induced by glucose deprivation in cells deficient in Bax and Bak. These cells cannot undergo mitochondrial outer membrane permeabilization (MOMP) during apoptosis, but they undergo necrosis when treated with MOMP-dependent apoptotic stimuli. We find in these cells that glucose deprivation, rather than inducing necrosis, triggered apoptosis. Cell death required caspase activation as inhibition of caspases with peptidic inhibitors prevented death. Glucose deprivation-induced death displayed many hallmarks of apoptosis, such as caspase cleavage and activity, phosphatidyl-serine exposure and cleavage of caspase substrates. Neither overexpression of Bcl-xL nor knockdown of caspase-9 prevented death. However, transient or stable knockdown of caspase-8 or overexpression of CrmA inhibited apoptosis. Cell death was not inhibited by preventing death receptor-ligand interactions, by overexpression of c-FLIP or by knockdown of RIPK1. Glucose deprivation induced apoptosis in the human tumor cell line HeLa, which was prevented by knockdown of caspase-8. Thus, we have found that glucose deprivation can induce a death receptor-independent, caspase-8-driven apoptosis, which is engaged to kill cells that cannot undergo MOMP.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 106-117

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 117-138).

Made available in DSpace on 2016-04-07T13:20:28Z (GMT). No. of bitstreams: 2 desio_oliveira_ioc_dout_2011.pdf: 1175865 bytes, checksum: f838717ed871f60759465b1b71ebee3c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A infecção pelo Trypanosoma cruzi promove alterações em órgãos linfóides. O timo apresenta-se atrofiado com depleção de células CD4+CD8+ (DP) na fase aguda. Em órgãos linfóides periféricos, os linfonodos subcutâneos (LSC) e o baço apresentam-se hipertrofiados com aumento de células T e B, enquanto os linfonodos mesentéricos (LM) apresentam-se atrofiados devido à morte dessas células. Nesse trabalho estudamos os efeitos da infecção pelo T. cruzi sobre células epiteliais tímicas (TEC) e timócitos, bem como o papel do timo no comportamento de órgãos linfóides periféricos. Em experimentos de infecção in vitro, avaliamos TEC, abordando a expressão de ligantes e receptores da matriz extracelular (ECM), e ainda, as possíveis conseqüências desse aumento de ECM sobre a interação de TEC/timócitos ou TEC/parasita. Nossos resultados demonstram que a infecção promove diminuição do número de TEC, alterações morfológicas e aumento de ECM em culturas infectadas. Observamos também que componentes da ECM são requeridos na interação entre TEC/parasita. Curiosamente, o menor crescimento de TEC em culturas infectadas está associada com a inibição do ciclo celular e não com apoptose No que se refere à interação TEC/timócitos, observamos que a adesão foi maior nas culturas infectadas, especialmente sobre TEC parasitadas. Esse aumento de adesão entre TEC/timócitos corrobora a hipótese descrita anteriormente pelo nosso grupo que a infecção favorece a migração de timócitos para periferia. Estendendo nossa análise ao compartimento linfóide do timo, investigamos a apoptose de timócitos na infecção. Observamos que a celularidade do timo diminui viii durante a infecção, juntamente com aumento de apoptose de timócitos CD4-CD8- (DN), DP, CD4 e CD8. Procurando entender a via envolvida na apoptose desses timócitos, demonstramos que a atividade de caspases total, caspase 8, caspase 9 e caspase 3 estão aumentadas na infecção. Observamos que ambas caspases iniciadoras caspase 8 (via extrínseca, Fas, TNF, TRAIL) e caspase 9 (via intrínseca, privação de fatores) parecem estar envolvidas na depleção desses timócitos Esse dado foi confirmado nos experimentos de bloqueio de morte com inibidores de caspases, onde observamos que o tratamento in vivo (injeção intratímica) e in vitro com zVAD (inibidor geral de caspases) em timócitos de animais infectados foi mais eficaz no bloqueio da morte do que os inibidores de caspase-8 (zIETD) ou caspase-9 (zLEHD). Além disso, animais infectados e tratados com zVAD apresentaram a celularidade do timo parcialmente recuperada, mais especificamente em timócitos DN e DP. Finalmente, procurando entender o papel do timo na resposta imune regional de órgãos linfóides periféricos na infecção, camundongos foram timectomizados antes da infecção para avaliação da celularidade dos LSC, LM e baço. Nossos dados demonstram que, mesmo com a ausência do timo, a hipertrofia dos LSC e a atrofia dos LM permaneciam inalterados, entretanto, encontramos significativo acúmulo de linfócitos T e B no baço de animais infectados. Em conjunto, nossos resultados demonstram que as alterações observadas no componente epitelial do microambiente tímico, assim como em timócitos de animais infectados favorecem a migração e morte destes timócitos e a atrofia desse tecido. Além disso, demonstramos que células do timo possuem papel imunoregulatório no baço durante a infecção / Trypanosoma cruzi infection promotes lymphoid organ alterations. The thymus is atroph ied with CD4 + CD8 + (DP) thymocyte depletion in the acute phase of infection. In p eripheral lymphoid organs, subcuta neous lymph nodes (LSC) and spleen present hypertrophy, with increase of T and B cells, whereas mesenteric lymph nodes (LM) are atrophied due to the death of these cells . In this work, we studied the effects of T. cruzi infec tion in thymic epithelial cells (TEC) and thymocytes , as well as the role of thymus in the behavior of peripheral lymphoid organs . In experiments of in vitro infection, we evaluated TEC, concerning the expression of extracellular matrix (ECM) ligands and receptor s , and also the possible conseq uences of ECM increase in TEC /thymocyte or TEC/parasit e intera ctions . Our data demonstrate that T. cruzi infection promotes a decrease of TEC number , morphological alterations and ECM increase in infected cultures . We observed that ECM components ar e required in the interaction between TEC and parasit es . Curiously, the decrease in TE C number in infected cultures is associated to cell cycle inhibition but not to apoptos is . Concerning TEC/thymocyte interactions, we observed that adhesion was greater in infected cultures, especially on parasitized TEC . This increase in TEC/thymocyte adhesion corroborates the hypothesis previously described by our group that the infection favors migration of thymocyte s to the periphery . Extending our analysis to th ymic ly mphoid compartment, we investigated thymocyte apoptosis following infection . We observed that thymus cellulari ty decreases during infection, together with the increase of apopt osis in CD4 - CD8 - (DN), DP, CD4 and CD8 thymocyte s. Searching to understand what death pathway is involved in thymocyte apoptosis, we demonstrated that the activity of total cas pases, caspase - 8, caspase - 9 and caspase-3 (ef f e c tor caspase ) are increased in infection . We observ ed that both initiator ca spase - 8 (extrinsic pathway, Fas, TNF , TRAIL) and caspase - 9 (intrinsic pathway, factor deprivation) seem to be involved in thymocyte deple tion. These data were confirmed in death blocking experiments with caspase inhibitors , in which w e showed that in vivo (intrat hy mic inje ction ) and in vitro treatment s with zVAD ( general caspase in hibitor ) were more effective in blocking thymocyte death than the in hibitors of caspase - 8 (zIETD) or caspase - 9 (zLEHD) separately . Moreover , infected animals treated with zVAD showed a partial recovery of thymus cellularity , more specifica lly in DN and DP thymocytes . Finally, searching to understand the role of the thymus in the regional immune response of peripheral lymphoid organs in infection , mice were t hy mectomiz e d prior to infection to evaluation of LSC, LM and spleen cellularity. Our data demonstrate d that, even in the absence of the thymus, LSC hypertrophy and LM atrophy were not altered ; however , we found a significant accumulation of T and B lymphocytes in the spleen of infected animals. Conjointl y , our results show that the alterations observed in the epithelial component of the thymus microenvironment, as well as in thymocyte s of infected animals favor the migration and death of thymocyte s and the atrophy of this tissue . Besides that , we demonstr at ed that thymic cells have an immunoregulatory role in the spleen during infection .

LIMA, Bruno Bezerra. Efeitos das toxinas A e B do Clostridium difficile sobre a via de WNT/Beta-catenina em células epiteliais intestinais. 2014. 79 f. Tese (Doutorado em Farmacologia) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2014. / Submitted by denise santos (denise.santos@ufc.br) on 2015-05-19T12:12:30Z No. of bitstreams: 1 2014_tese_bblima.pdf: 2558018 bytes, checksum: 9b5ba19b7dbb3fd33020f7fcd72564f7 (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2015-05-19T12:37:54Z (GMT) No. of bitstreams: 1 2014_tese_bblima.pdf: 2558018 bytes, checksum: 9b5ba19b7dbb3fd33020f7fcd72564f7 (MD5) / Made available in DSpace on 2015-05-19T12:37:54Z (GMT). No. of bitstreams: 1 2014_tese_bblima.pdf: 2558018 bytes, checksum: 9b5ba19b7dbb3fd33020f7fcd72564f7 (MD5) Previous issue date: 2014 / Clostridium difficile toxins A and B (TcdA and TcdB) are homologous glycosyltransferases that inhibit a group of small GTPases within host cells, but several mechanisms underlying their pathogenic activity remain unclear. Here, we evaluated the effects of TcdA and TcdB on the Wnt/Beta-catenin pathway, the major driving force behind the proliferation of epithelial cells in colonic crypts. IEC-6 and RKO cells stimulated with Wnt3a-conditioned medium were incubated with 10, 50 and 100 ng/mL of TcdA or TcdB for 24h, resulting in a dose-dependent inhibition of the Wnt signaling, as demonstrated by a T-cell factor (TCF) reporter assay. This was further confirmed by immunofluorescence staining for nuclear localization of Beta-catenin and western blotting for Beta-catenin and c-Myc (encoded by a Wnt target gene). Moreover, our western blot analysis showed a decrease in the Beta-catenin protein levels, which was reversed by z-VAD-fmk, a pan-caspase inhibitor. Nonetheless, TcdA was still able to inhibit the Wnt/Beta-catenin pathway even in the presence of z-VAD-fmk, lithium chloride (a GSK3B inhibitor), or constitutively active Beta-catenin, as determined by a TCF reporter assay. Furthermore, pre-incubation of RKO cells with TcdA for 12h also attenuated Wnt3a-mediated activation of Wnt signaling, suggesting that inactivation of Rho GTPases plays a significant role in that inhibition. Taken together, these findings suggest that attenuation of the Wnt signaling by TcdA and TcdB is important for their anti-proliferative effects. / As toxinas A e B do Clostridium difficile (TcdA e TcdB) são glicosiltransferases homólogas que inibem um grupo de pequenas GTPases dentro da célula hospedeira, contudo, vários mecanismos envolvidos na atividade patogênica dessas toxinas permanecem desconhecidos. No presente estudo, avaliamos os efeitos das TcdA e TcdB na via de Wnt/Beta-catenina que representa a força motora principal responsável pela proliferação das células epiteliais nas criptas colônicas. Foram utilizadas células IEC-6 (células epiteliais de criptas de Rattus novergicus) e RKO (células de adenocarcinoma de cólon humano). Estas células foram estimuladas com meio condicionado contendo Wnt3a e incubadas com 10, 50 ou 100 ng/mL de TcdA ou 1, 5 ou 10 ng/mL de TcdB por 24h, resultando em uma inibição dose-dependente da via de sinalização canônica de Wnt, como demonstrado pelo ensaio de repórter de fator de célula T (TCF). Esse resultado foi corroborado pelos dados da imunofluorescência com marcação para a localização nuclear de Beta-catenina e por western blotting para Beta-catenina e c-MYC (gene-alvo da via de Wnt). Além disso, os dados do experimento de western blot evidenciaram uma diminuição dos níveis da proteína Beta-catenina, o qual foi prontamente revertido por z-VAD-fmk, um pan-inibidor de caspase. Entretanto, a TcdA ainda foi capaz de inibir a via de Wnt/Beta-catenina mesmo na presença do z-VAD-fmk, cloreto de lítio (um inibidor de GSK3Beta) ou plasmídeo de Beta-catenina constitutivamente ativo, como determinado pelo ensaio do TCF (TOPFlash/Luciferase). O estudo evidenciou ainda que a pré-incubação de células RKO com TcdA por 12h também atenuou a ativação da via de Wnt mediada por Wnt3a, o que sugere que a inativação de RhoGTPases, particularmente Rac1, possui um papel nessa inibição. Em conclusão, esses achados sugerem que a inibição da via canônica de Wnt pelas TcdA e TcdB representa um mecanismo importante da sua patogênese e explica seus efeitos anti-proliferativos.

Abstract Apoptosis is a biochemically regulated mechanism leading to the destruction of an individual cell. An inadequate apoptosis is partly responsible for uncontrolled survival of malignantly transformed cells and formation of cancer. The growth of a tumor depends on the proliferative capacity and destruction of tumor cells either through apoptosis or necrosis. In this work, the extent of apoptosis and the expression of apoptosis-regulating proteins were studied by 3'-end labeling of fragmented DNA (TUNEL) and immunohistochemistry in a set of 166 tissue samples consisting of 33 HCCs, 39 gallbladder carcinomas, 7 gallbladder dysplasias and 87 pancreatic carcinomas. In addition, p53 protein and P-glycoprotein expression was studied immunohistochemically. The extent of apoptosis was estimated by using apoptotic index, defined as a percentage of apoptotic cells in the entire tumor cell population. The present results show an average apoptotic index of 0.73% in HCC, 0.68% in gallbladder and 0.69% in pancreatic carcinoma. Bcl-2 positivity was found in only 3% of the HCCs, 10% of gallbladder and 13% of pancreatic carcinomas. Bax positivity was seen in all of the gallbladder and pancreatic carcinoma cases. Mcl-1 positivity was found in 87% of gallbladder and 86% of pancreatic tumors. The apoptotic index in bcl-2 positive cases was lower (0.35%) than in cases showing no immunoreactivity (0.64%) in pancreatic tumors (P = 0.013). Apoptotic index was higher in pancreatic tumors with strong bax immunoreactivity (0.70%) than in other cases (0.34%) (P = 0.002). Caspase 3, 6 and 8 expression was found in 92%, 92% and 73% of HCC, 95%, 77% and 77% of gallbladder carcinoma and 80%, 80% and 74% of pancreatic carcinoma cases, respectively. p53 positivity was found in 23% of hepatocellular, 57% of gallbladder and 41% of pancreatic carcinomas. P-glycoprotein was observed in 65% of the HCCs. Patients with Pgp positive tumors had a significantly shorter disease-free interval than those with Pgp negative tumors (P < 0.05). To evaluate the growth potential of HCC and pancreatic carcinoma, a growth index from the scores obtained for apoptosis, necrosis and cell proliferation was designed. Patients with a high degree of proliferation relative to the degree of necrosis and apoptosis (i.e. had a positive growth index) in HCC lesions had a significantly shorter survival (P = 0.004) and disease-free interval after operation (P = 0.019) than those with a tumor predominated by apoptosis and necrosis. Results were in line with HCC in pancreatic carcinoma, but the association did not reach statistical significance (P = 0.09). According to the results the extent of apoptosis was similar in HCCs, gallbladder and pancreatic carcinomas. These tumors also showed here a similar expression pattern of the bcl-2 family of proteins and caspases. None of the individual parameters associated significantly with apoptosis except for bcl-2 and bax in pancreatic carcinoma, neither was there any association between p53 and P-glycoprotein expression and apoptosis. Calculation of a growth index might be helpful in assessing the prognosis of patients with tumors with a scant stroma, such as HCC.

Le récepteur à neurotrophine TrkC a été identifié comme étant un récepteur à dépendance : en l'absence de son ligand NT-3, il déclenche l'apoptose. En effet, la survie des cellules qui expriment ces récepteurs dépend de la disponibilité en ligand, un mécanisme qui inhibe la prolifération incontrôlée et la migration des cellules tumorales. TrkC, en tant que récepteur à tyrosine kinase, est généralement considéré comme un proto-oncogène. Or nous montrons que l'expression TrkC est diminuée dans une grande fraction des cancers colorectaux humains, principalement par méthylation du promoteur de TrkC. En outre, ce mécanisme confère un avantage sélectif aux lignées cellulaires colorectales pour inhiber la mort des cellules tumorales. De plus, la réexpression de TrkC dans les lignées tumorales colorectales est associée à la mort des cellules tumorales et à l'inhibition in vitro des caractéristiques de transformation cellulaire, et in vivo de la croissance tumorale. Ensemble, ces données permettent de conclure que TrkC est un gène suppresseur de tumeur dans le cancer colorectal. Le mécanisme moléculaire par lequel TrkC déclenche l'apoptose implique le clivage de son domaine intracellulaire, ce qui libère un fragment pro-apoptotique (TrkC KF). Nous montrons que TrkC KF interagit avec Cobra1, un cofacteur de BRCA1, et que Cobra1 est nécessaire à l'apoptose induite par TrkC. Cobra1 conduit TrkC KF à la mitochondrie, où il favorise l'apoptose apoptosome-dépendante. Ainsi, nous proposons qu'en l'absence de NT-3, le clivage protéolytique de TrkC conduit à la libération d'un fragment tueur qui déclenche l'apoptose mitochondriale, via le recrutement de Cobra1 / The neurotrophin receptor TrkC was recently identified as a dependence receptor, and, as such, it triggers apoptosis in the absence of its ligand, NT-3. Indeed cells that express these receptors are thought to be dependent on ligand availability for their survival, a mechanism that inhibits uncontrolled tumor cell proliferation and migration. TrkC, as a classic tyrosine kinase receptor, is generally considered to be a proto-oncogene. We show that TrkC expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, we show that TrkC silencing by promoter methylation is a selective advantage for colorectal cell lines to limit tumor cell death. Furthermore, reestablished TrkC expression in colorectal cancer cell lines is associated with tumor cell death and inhibition of in vitro characteristics of cell transformation, as well as in vivo tumor growth. Together, these data support the conclusion that TrkC is a colorectal cancer tumor suppressor. TrkC triggers apoptosis in the absence of its ligand NT-3 : the molecular mechanism for apoptotic engagement involves the double cleavage of the receptor's intracellular domain, leading to the formation of a proapoptotic fragment (TrkC KF). We show that TrkC KF interfacts with Cobra1, a putative cofactor of BRCA1, and that Cobra1 is required for TrkC-induced apoptosis. Cobra1 shuttles TrkC KF to the mitochondria, where it promotes apoptosome-dependent apoptosis. Thus, we propose that, in the absence of NT-3, the proteolytic cleavage of TrkC leads to the release of a killer fragment that triggers mitochondria-dependent apoptosis via the recruitment of Cobra1