Thèses sur le sujet « Oncogene Protein p21(ras) »

A incidência do melanoma cutâneo em pacientes adultos jovens tem aumentado consideravelmente nos últimos anos. Há, contudo, carência de conhecimentos clinicopatológicos e moleculares sobre os melanomas que ocorrem nessa faixa etária. O presente estudo teve por objetivo avaliar 132 casos de melanoma cutâneo primário em pacientes com idade entre 18 e 30 anos, com ênfase no estudo das características clínicas, histopatológicas e avaliação molecular das mutações nos genes BRAF, NRAS e KIT. Em relação aos achados clínicos e histopatológicos, houve predomínio de indivíduos do sexo feminino (61,4%), sendo o tronco o sítio anatômico mais comumente envolvido (44,3%) e o melanoma extensivo superficial o tipo histológico predominante (79,5%). A mutação V600E no gene BRAF (BRAFV600E) foi analisada em 93 casos, utilizando-se a técnica de RT-PCR. Essa mutação foi identificada em 38,7% (36/93) e, estatisticamente, associada à fase vertical de crescimento (p = 0,01), infiltrado inflamatório discreto (p = 0,02) e presença de mitose intradérmica (p = 0,004). Houve, ainda, forte indício de associação com a presença de ulceração (p = 0,05). Todas essas variáveis apresentaram associação com pior prognóstico do melanoma cutâneo. Observou-se predomínio da mutação BRAFV600E em regiões anatômicas relacionadas à exposição solar intermitente. Nenhum caso de melanoma com fenômeno de regressão apresentou mutação BRAFV600E (p < 0,05). Não houve associação significativa entre BRAFV600E e sexo, tipo histológico, nível de Clark, índice de Breslow, elastose solar, invasão angiolinfática e perineural, satelitose, nevo melanocítico coexistente e sobrevida. A pesquisa de mutações NRAS, pela técnica de RT-PCR, detectou frequência de 3,95% (3/76). As três mutações encontradas foram do tipo 61K e ocorreram em pacientes do sexo masculino e em região de cabeça e pescoço. As mutações BRAFV600E e NRAS, quando presentes, eram mutuamente exclusivas. A frequência de mutações KIT, analisadas por sequenciamento, foi de 11,1% (3/27). As três mutações identificadas estavam localizadas no éxon 9 (G510, G498S e 489I). Houve concomitância de casos com mutação KIT tanto com NRAS, como com BRAFV600E. Devido ao pequeno número de casos com mutação em KIT e NRAS, não foi possível estabelecer correlações clínicas e histopatológicas com esses genes. Este estudo é o primeiro a descrever as mutações G510D e G498S no gene KIT em melanomas cutâneos. No presente estudo, a mutação BRAFV600E, em melanomas cutâneos de adultos jovens, correlacionou-se com características anatomoclínicas de pior prognóstico em relação aos melanomas selvagens para BRAFV600E
The incidence of cutaneous melanoma in young adults has dramatically increased in recent years. However, there is scarce data about the clinicopathological and molecular characteristics on the melanomas occurring at this age group. The present study aimed to evaluate 132 patients aged between 18 and 30 years with primary cutaneous melanoma with emphasis on the study of clinical, histopathological characteristics and molecular evaluation of mutations in BRAF, NRAS and KIT genes. Regarding the clinical and histopathological findings, the following results were found: female predominance (61.4%), trunk was the most commonly anatomical site involved (44.3%) and superficial spreading melanoma, was the most common histological type (79.5 %). The V600E mutation in BRAF (BRAFV600E) gene was analyzed in 93 cases, using RT-PCR. It was present in 38.7% (36/93) and statistically related to the vertical growth phase (p = 0.01), mild inflammatory infiltration (p = 0.02) and the presence of intradermal mitosis (p = 0.004). There was, also, strongly evidence of an association with the presence of ulceration (p = 0.05). Worse prognosis was associated with these variables. There was a predominance of BRAFV600E mutation in anatomical regions related to intermittent sun exposure. No cases of melanoma with BRAFV600E mutation showed regression phenomenon (p < 0.05). There was no significant association between BRAFV600E and gender, histological type, Clark level, Breslow thickness, solar elastosis, angiolymphatic and perineural invasion, sattelitosis, coexisting melanocytic nevus and survival. The presence of a mutation in NRAS, by RT-PCR was seen in 3.95% (3/76) of the cases. All these three mutations were of type 61K, occurred in male patients and the head and neck region. BRAFV600E and NRAS mutations, when present, were mutually exclusive. The frequency of KIT mutations, analyzed by sequencing, was 11.1% (3/27). The three mutations identified in this gene were located in exon 9 (G510, G498S and 489I). Concomitant mutations were found between KIT and NRAS and BRAFV600E. Due to the small number of KIT and NRAS mutated cases, it was not possible to establish clinical and histopathological correlations and mutation status in these genes. This study was the first to describe the G510D and G498S mutations in KIT gene in cutaneous melanomas. In the present study, BRAFV600E mutation in cutaneous melanoma of young adults correlated with anatomic and clinical features of worse prognosis compared to wild type

A subfamília p21 RAS de pequenas GTPases, incluindo KRAS, HRAS e NRAS, participa de muitas redes de sinalização, incluindo proliferação celular, organização do citoesqueleto e apoptose, e é o alvo mais freqüente de mutações ativadoras em câncer. Mutações germinativas em KRAS e HRAS causam graves anormalidades desenvolvimentais levando às síndromes de Noonan, cárdio-facial-cutânea e Costello, porem mutações ativadoras germinativas em NRAS não foram descritas até hoje. A síndrome autoimune linfoproliferativa (ALPS) é o mais comum defeito genético de apoptose linfocitária, cursando com autoimunidade e acúmulo excessivo de linfócitos, particularmente do tipo T + CD4- CD8-. As mutações causadoras de ALPS descritas até hoje afetam a apoptose mediada por Fas, uma das vias extrínsecas de apoptose. Nós demonstramos aqui que os principais achados clínicos de ALPS, bem como uma predisposição para tumores hematológicos, podem ser causados por uma mutação heterozigota ativadora G13D no oncogene NRAS, sem causar prejuízo na apoptose mediada por Fas. O aumento na quantidade intracelular de NRAS ativo, ligado a GTP, induziu a um aumento da sinalização na via RAF/MEK/ERK, o que suprimiu a expressão da proteína pró-apoptótica BIM, e atenuou a apoptose intrínseca mitocondrial. Desta forma, uma mutação germinativa ativadora em NRAS causou um fenótipo clinico diferente do visto em pacientes com mutações em outros membros da família p21 RAS, cursando com um defeito imunológico seletivo, sem distúrbios generalizados do desenvolvimento
The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4-, CD8- ab T cells. Mutations in ALPS typically affect Fas-mediated apoptosis, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair Fas-mediated apoptosis. The increase in active, GTP-bound NRAS augmented RAF/MEK/ERK signaling which markedly decreased the pro-apoptotic protein BIM and attenuated intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, develops through progression of premalignant pancreatic intraepithelial neoplasias (PanINs). In mouse-models, KRAS-activation in acinar cells induced an acinar-to-ductal metaplasia (ADM), and mutation of the Kras oncogene is believed to initiate PanIN formation. ADM is also promoted by pancreatic injury, which cooperates with activated KRAS to stimulate PanIN and PDAC formation from metaplastic ducts. Our lab, and others, have shown that the downstream PI3K/AKT pathway is important for KRAS-mediated proliferation and survival in vitro and in vivo. Prior studies have demonstrated that full activation of AKT requires both PDK1- mediated phosphorylation of AKTT308 and mTOR complex 2 (mTORC2)-mediated phosphorylation of AKTS473. Given the importance of the PI3K/AKT signaling axis, I hypothesized that mTORC2 is required for KRAS-driven pancreatic tumorigenesis and investigated this relationship in mice by combining pancreasspecific expression of an activated KRASG12D molecule with deletion of the essential mTORC2 subunit RICTOR. In the context of activated KRAS, Rictor-null pancreata developed fewer PanIN lesions; these lesions lacked mTORC2 signaling and their proliferation and progression were impaired. Higher levels of nuclear cyclin dependent kinase inhibitors (CDKIs) were maintained in Rictor-null lesions, and nuclear BMI1, a known regulator of the CDKI Cdkn2a, inversely correlated with their expression.Rictor was not required for KRAS-driven ADM following acute pancreatitis, however the inverse correlation between CDKIs and BMI1 was maintained in this system. Treatment of PDX-Cre;KRASG12D/+;Trp53R172H/+ mice with an mTORC1/2 inhibitor delayed tumor formation, and prolonged the survival of mice with late stage PDAC. Knockdown of Rictor in established PDAC cell lines impaired proliferation and anchorage independent growth supporting a role for mTORC2 in fully transformed cells. These data suggest that mTORC2 cooperates with activated KRAS in the initiation and progression of PanIN lesions and is required for the transformation and maintenance of PDAC. My work illustrates phenotypic differences between pancreatic loss of Rictor and PDK1 in the context of KRAS, broadens our understanding of this signaling node and suggests that mTORC2 may potentially be a viable target for PDAC therapies.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, develops through progression of premalignant pancreatic intraepithelial neoplasias (PanINs). In mouse-models, KRAS-activation in acinar cells induced an acinar-to-ductal metaplasia (ADM), and mutation of the Kras oncogene is believed to initiate PanIN formation. ADM is also promoted by pancreatic injury, which cooperates with activated KRAS to stimulate PanIN and PDAC formation from metaplastic ducts. Our lab, and others, have shown that the downstream PI3K/AKT pathway is important for KRAS-mediated proliferation and survival in vitro and in vivo. Prior studies have demonstrated that full activation of AKT requires both PDK1- mediated phosphorylation of AKTT308 and mTOR complex 2 (mTORC2)-mediated phosphorylation of AKTS473. Given the importance of the PI3K/AKT signaling axis, I hypothesized that mTORC2 is required for KRAS-driven pancreatic tumorigenesis and investigated this relationship in mice by combining pancreasspecific expression of an activated KRASG12D molecule with deletion of the essential mTORC2 subunit RICTOR. In the context of activated KRAS, Rictor-null pancreata developed fewer PanIN lesions; these lesions lacked mTORC2 signaling and their proliferation and progression were impaired. Higher levels of nuclear cyclin dependent kinase inhibitors (CDKIs) were maintained in Rictor-null lesions, and nuclear BMI1, a known regulator of the CDKI Cdkn2a, inversely correlated with their expression.Rictor was not required for KRAS-driven ADM following acute pancreatitis, however the inverse correlation between CDKIs and BMI1 was maintained in this system. Treatment of PDX-Cre;KRASG12D/+;Trp53R172H/+ mice with an mTORC1/2 inhibitor delayed tumor formation, and prolonged the survival of mice with late stage PDAC. Knockdown of Rictor in established PDAC cell lines impaired proliferation and anchorage independent growth supporting a role for mTORC2 in fully transformed cells. These data suggest that mTORC2 cooperates with activated KRAS in the initiation and progression of PanIN lesions and is required for the transformation and maintenance of PDAC. My work illustrates phenotypic differences between pancreatic loss of Rictor and PDK1 in the context of KRAS, broadens our understanding of this signaling node and suggests that mTORC2 may potentially be a viable target for PDAC therapies.

Introdução: A doença renal crônica continua sendo um desafio no campo da pesquisa médica. Atualmente um interesse crescente tem surgido no intuito de avaliar o potencial de células tronco em retardar o avanço de doenças crônicas progressivas. Material e Métodos: Para determinar o efeito dessas células em um modelo de progressão de doença renal crônica foram usadas células linhagem negativa (Lin ) separadas magneticamente e injetadas em ratos submetidos à injúria renal. Ratos singênicos Fischer 344 foram submetidos à nefrectomia 5/6 (Nx) e divididos em 3 grupos: Nx (não tratados); NxSC1 (submetidos à infusão de 2 106 células Lin no 15º dia de pós-operatório); e NxSC3 (submetidos à infusão de 2 106 células Lin no 15º, 30º e 45º dias de pós-operatório). No 60º dia de pós-operatório clearance de inulina, imunohistoquímica e immunoblotting foram realizados. Resultados: Os animais submetidos à nefrectomia apresentaram redução do clearance de inulina (0,33 ± 0,02 ml/min/100g peso corpóreo), proteinúria (12 ± 0,5 mg/24hs) , anemia e hipertensão (145 ± 7,7 mmHg) compatíveis com doença renal crônica. A infusão de células Lin- resultou em atenuação da proteinúria (p<0,05) com relação aos animais não tratados a despeito de não ter havido diferença nos níveis de pressão arterial e aldosterona plasmática. Esses achados foram similares entre os grupos tratados com uma ou com três infusões de células. Adicionalmente a infusão de células resultou em redução do índice de glomeruloesclerose e da área intersticial relativa (p<0,05), menor infiltração do tecido renal por macrófagos e linfócitos e menor proliferação celular. A expressão tecidual do p21 e de VEGF já foi associada à aceleração da progressão da lesão renal crônica. No nosso modelo ambas as proteínas tiveram sua expressão reduzida. A redução da expressão tecidual de eNOS tem sido implicada na progressão da doença renal. Em nosso modelo houve aumento dessa expressão após infusão das células Conclusões: A infusão de células Linatenuou todos os marcadores de injúria renal em um modelo de doença precoce possivelmente através de um mecanismo imunomodulador.
Progressive renal failure continues to be a challenge. The use of bone marrowderived stem cells (SCs) represents a means of meeting that challenge. We used lineage-negative (Lin-) SCs to test the hypothesis that Lin- cell infusion decreases renal injury. Syngeneic Fischer 344 rats were submitted to 5/6 nephrectomy and divided into 3 groups: Nx (untreated); NxSC1 (receiving 2 × 106 Lin- cells on postnephrectomy day 15); and NxSC3 (receiving 2 × 106 Lin- cells on postnephrectomy days 15, 30 and 45). Controls were unoperated/untreated. On postnephrectomy day 60, clearance studies, immunohistochemistry and immunoblotting were performed. Lin- cell infusion effectively reduced postnephrectomy proteinuria, glomerulosclerosis, anemia, renal infiltration of immune cells and monocyte chemoattractant protein-1 protein expression, as well as decreasing the interstitial area. Immunostaining for proliferating cell nuclear antigen showed that, in comparison with controls, Nx rats presented greater cell proliferation, whereas NxSC1 rats and NxSC3 rats presented less cell proliferation than did Nx rats. Protein expression of p21 and VEGF increased after nephrectomy and decreased after Lin- cell infusion. Protein expression of eNOS reduced after nephrectomy and increased after cell infusion. These data suggest that SC treatment ameliorates progressive end-stage renal disease.

O bócio multinodular (BMN) é definido como aumento da glândula tireóide devido a proliferação de tireócitos e caracteriza-se pela heterogeneidade no crescimento e função das células foliculares. É uma patologia comum, com aumento da prevalência em áreas com deficiência de iodo, sendo este o principal fator etiológico do BMN. O BMN é considerado uma neoplasia benigna da tireóide. A patogênese desta disfunção ainda não foi inteiramente elucidada. Nesta revisão serão abordados os mecanismos envolvidos na patogênese e os principais aspectos etiológicos e clínicos do BMN.
Multinodular goiter (MNG) is an enlargement of the thyroid gland and is characterized by heterogeneity in growth and function of thyroid follicular cells. It is a common pathology, with higher prevalence in iodine deficiency areas. Iodine deficiency is the main etiologic factor for MNG. MNG have been considered a true thyroid neoplasm. The pathogenesis of multinodular goiter is not yet clarified. The purpose of this review is to summarize the current knowledge of MNG with respect to the pathology, etiologic and clinical characteristics.

The RAS oncogenes KRAS, NRAS and HRAS are mutated in one third of human cancers where they exhibit different mutation patterns. A potential factor contributing to this mutation bias is the variation of RAS expression levels. Here, I investigate some of the determinants of RAS protein abundance. First, I examine whether codon bias among RAS genes and within other cancer gene families plays a role in cell context-specific expression. I further describe a tRNA expression program that favors oncogene translation in proliferating cells. Second, I investigate why oncogenic RAS mutants exhibit a higher protein abundance than the RAS wild type. In this context, I study the underlying mechanisms leading to this variation and more specifically how protein-protein interactions between RAS and its downstream binding partners change the protein turnover of RAS and therefore, its protein abundance. Overall, this thesis provides insight into the possible relevance of RAS protein synthesis and protein degradation as determinants of RAS mutation patterns in human cancers.
Els oncogens KRAS, NRAS i HRAS estan mutats en un terç dels càncers en humans on hi exhibeixen patrons de mutació diferents. Un possible factor que contribueix a aquest biaix de mutació és la variació dels nivells d'expressió de RAS. En aquesta tesi investigo els elements determinants de l'abundància de la proteïna RAS. Primer, examino si el biaix de codó entre els gens RAS i entre gens d'altres famílies implicades en càncer contribueix a les diferències d'expressió, en funció del context cel·lular. Així mateix, descric un programa d'expressió de tRNA que facilita la traducció d'oncogens en cèl·lules proliferatives. En segon lloc, investigo per què mutants oncogènics de RAS tenen una abundància de proteïna més elevada que la RAS salvatge. Així mateix, estudio els mecanismes subjacents responsables d'aquesta variació i més concretament el paper de les interaccions de RAS amb altres proteïnes en la regulació de la seva abundància. Així doncs, aquesta tesi estudia la possible rellevància dels mecanismes de síntesi i degradació de la proteïna RAS en els patrons de mutació en càncer.

Il a déjà été montré que l'activation du récepteur AT[indice inférieur 2] (récepteur de type 2) de l'angiotensine II (Ang II) induit la différenciation morphologique de la lignée cellulaire neuronale NG108-15. Il a été trouvé que cette différenciation morphologique est accompagnée d'une augmentation des niveaux de tubuline polymérisée ainsi que des niveaux de MAP 2 associé aux microtubules (Laflamme et al., 1996). Parmi les cibles intracellulaires impliquées dans les effets du récepteur AT[indice inférieur 2], on a noté une forte diminution de l'activité de p21[indice supérieur ras] suite à un traitement à l'Ang II (Gendron et al., 1999). Au cours des présents travaux, nous avons donc étudié le rôle de cette diminution de la forme active de p21[indice supérieur ras] dans l'induction de la différenciation neuronale. Nous avons créé une lignée cellulaire à transfection stable exprimant de façon inductible un mutant dominant négatif de p21[indice supérieur ras]. Dans ces cellules, l'inhibition de p21[indice supérieur ras] induit l'élongation des neurites se traduisant par une augmentation de la polymérisation de la tubuline. Nous avons de plus noté que l'Ang II, via l'activation du récepteur AT[indice inférieur 2], stimule, de façon lente mais soutenue, l'activité des MAPKs ERK1 et ERK2, un effet qui s'observe même chez les cellules transfectées avec p21[indice supérieur ras] inactif (p21N17Ras). De ces observations, nous pouvons conclure que le récepteur AT[indice inférieur 2] de l'Ang II induit des effets opposés sur p21[indice supérieur ras] et p42/p44 MAPK. Nos résultats montrent aussi que l'inhibition de p21[indice supérieur ras] est suffisante à l'induction de l'élongation neuritique mais que cette différenciation morphologique implique une activation obligatoire des MAPKs p42/p44 puisque l'effet de l'Ang II ou de l'inhibition de p21[indice supérieur ras] sur l'élongation des neurites est bloqué par le PD 98059, un inhibiteur spécifique de MEK1 (IC50 de 10 [mu]M).

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

No
Zinc oxide (ZnO) nanoparticles are the mostly used engineered metal oxide nanoparticles in consumer products. This has increased the likelihood of human exposure to this engineered nanoparticle (ENPs) through different routes. At present, the majority of the studies concerning ZnO ENPs toxicity have been conducted using in vitro and in vivo systems. In this study, for the first time we assessed the effect of ZnO ENPs on the major cellular pathways in the lymphocytes of healthy individuals as well as in susceptible patients suffering from lung cancer, chronic obstructive pulmonary disease (COPD) and asthma. Using the differential expression analysis, we observed a significant (P < 0.05) dose-dependent (10, 20 and 40 microg/ml for 6h) increase in the expression of tumour suppressor protein p53 (40, 60 and 110%); Ras p21 (30, 52 and 80%); c-Jun N-terminal kinases; JNKs) (28, 47 and 78%) in lung cancer patient samples treated with ZnO ENPs compared to healthy controls. A similar trend was also seen in COPD patient samples where a significant (P < 0.05) dose-dependent increase in the expression of tumour suppressor protein p53 (26, 45 and 84%), Ras p21 (21, 40 and 77%), JNKs (17, 32 and 69%) was observed after 6h of ZnO ENPs treatment at the aforesaid concentrations. However, the increase in the expression profile of tested protein was not significant in the asthma patients as compared to controls. Our results reiterate the concern about the safety of ZnO ENPs in consumer products and suggest the need for a complete risk assessment of any new ENPs before its use.

碩士
國立臺灣大學
牙醫學研究所
82
In this study, we used immunohistochemical method to assess the expression of p21 ras, p53 and proliferating cell nuclear an- tigen (PCNA) in 50 cases of oral submucous fibrosis (ＯＳ Ｆ), 10 cases of normal oral mucosa, 10 cases of leukoplakia, 5 cases of epithelial dysplasia, and 5 cases of verrucous carcinoma. By statistic analysis, we further correlated the expression of p21 ras, p53 and PCNA in ＯＳＦ with betel nut chewing, cigarette smoking, alcohol drinking as well as the depth of fibrosis and the degree of inflammation in subepithelial connective tissue. By immunohistochemistry, the positive staining of p21 ras was most often found in the cytoplasm and nuclei of epithelial cells of ＯＳＦ oral mucosa. The pattern of p21 ras positive staining of ＯＳＦwas similar to that of normal oral mucosa. The positive staining of p53 was most frequently discovered in the nuclei of basal and suprabasal epithelial cells of ＯＳＦ oral mucosa. The pattern of p53 positive staining of ＯＳＦ was similar to those of leukoplakia and epithelial dysplasia. The positive staining of PCNA was most commonly noted in the nuclei of basal, suprabasal and lower spinous epithelial cells. The pattern of PCNA positive staining of ＯＳＦ was very close to those of leukoplakia, epith- elial dysplasia, and verrucous carcinoma. The expression of p53 in the whole layer of epithelial cells of ＯＳＦ oral mucosa was positively and significantly correlated to the duration of chew- ing betel nuts (P<0.05). There was a significant negative corre- lation between the expression of PCNA in the whole layer of epi- thelial cells of ＯＳＦ oral mucosa and the total number of ciga- rettes smoked by the ＯＳＦ patients (P<0.05). We concluded thatＯＳＦ was undoubtful an oral precancerous lesion, because the p53 and PCNA positive staining patterns of ＯＳＦ were similar to those of oral leukoplakia and epithelial dysplasia.

碩士
國立臺灣大學
牙醫科學研究所
84
We used the immunohistochemical method to study the expressions of p53 protein, PCNA and p21 ras in 30 cases of mucoepidermoid carcinoma (MEC), 37 cases of adenoid cystic carcinoma(ACC), 5 cases of normal submandibular gland and 7 cases of minor salivary gland adjacent to mucocele. The p53 positive staining rates were 66.7% and 70.3% for 30 MECs and 37 ACCs, respectively. Overexpression of p53 protein in MECs was significantly associated with advanced clinical staging (P<0.01) and perineural invasion(P<0.01), but not with histologic grading of malignancy.Expression of p53 protein in MECs of major salivary gland (MjMECs) was also significantly and positively correlated to clinical staging of cancer. The PCNA positive rates were both 100% for 30 MECs and 37 ACCs, and the mean PCNA indices were 59.8 .plmin.19.9% and 56.8 .plmin.20.7% for MECs and ACCs,respectively .High PCNA expression was found in MECs with deep invasion (P< 0.01) as well as in ACCs with perineural invasion (P<0.05) and cellular anaplasia (P<0.05). However, there was a significant negative correlation found between PCNA indices and MjMECs with lymph node metastasis(P<0.05) or with high grade malignancy (P< 0.05). Women tended to have higher PCNA expression in MECs in minor salivary gland (MiMECs) than men (P<0.01). The p21 ras positive staining rates were 76.7% for 30 MECs and 94.6% for 37 ACCs. High p21 ras expression in MECs was observed in cancers of low clinical staging (P<0.01), low grade malignancy (P<0.01), no deep invasion (P<0.001) and no lymph node metastasis (P<0.01). In MjMECs, low p21 ras expression was significantly associated with tumors with deep invasion (P<0.01) and high grade malignancy (P< 0.01). In MiMECs, high p21 ras expression tended to be more frequently seen in women than in men (P<0.01) and in tumors without deep invasion than tumors with deep invasion (P<0.01).

Noncovalent factors, such as shape complementarity and electrostatic driving forces, almost exclusively cause the affinity and specificity for which two or more biological macromolecules organize into a functioning complex. The human oncoprotein p21Ras (Ras) and a structurally identical but functionally distant analog, Rap1A (Rap), exhibit high selectivity and specificity when binding to downstream effector proteins that cannot be explained through structural analysis alone. Both Ras and Rap bind to Ral guanine nucleotide dissociation stimulator (RalGDS) with affinities that differ tenfold instigating diverse cellular functions; it is hypothesized that this specificity of RalGDS to discriminate between GTPases is largely electrostatic in nature. To investigate this hypothesis, electrostatic fields at the binding interface between mutants of RalGDS bound to Rap or Ras are measured using vibrational Stark effect (VSE) spectroscopy, in which spectral shifts of a probe oscillator’s energy is related directly to that probe’s local electrostatic environment and measured by Fourier transform infrared spectroscopy (FTIR). After calibration, the probe is inserted into a known position in RalGDS where it becomes a highly local, sensitive, and directional reporter of fluctuations of the protein’s electrostatic field caused by structural or chemical perturbations of the protein. The thiocyanate (SCN) vibrational spectroscopic probe was systematically incorporated throughout the binding interface of RalGDS. Changes in the absorption energy of the thiocyanate probe upon binding were directly related to the change of the strength of the local electrostatic field in the immediate vicinity of the probe, thereby creating a comprehensive library of the binding interactions between Ras-RalGDS and Rap-RalGDS. The measured SCN absorption energy on the monomeric protein was compared with solvent-accessible surface area (SASA) calculations with the results highlighting the complex structural and electrostatic nature of protein-water interface. Additional SASA studies of the nine RalGDS mutants that bind to Ras or Rap verified that experimentally measured thiocyanate absorption energies are negatively correlated with exposure to water at the protein-water interface. By changing the solvent composition, we confirmed that the cyanocysteine residues that are more exposed to solvent experienced a large difference in absorption energy. These studies reinforce the hypothesis that differences in the electrostatic environment at the binding interfaces of Ras and Rap are responsible for discriminating binding partners.
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