Dissertations / Theses on the topic 'E7 oncoprotein'

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-03T14:39:58Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese - Carolina Amaral (2).pdf: 3097271 bytes, checksum: a09eebe935773ec4b3446a95c4507f5c (MD5)
Made available in DSpace on 2017-04-03T14:39:58Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese - Carolina Amaral (2).pdf: 3097271 bytes, checksum: a09eebe935773ec4b3446a95c4507f5c (MD5) Previous issue date: 2015-12-15
FACEPE
Os Papilomavírus humano (HPV) infectam mucosas e epitélio contribuindo para o desenvolvimento de tumores benignos como também malignos. São amplamente conhecidos como causadores do câncer cervical, contudo, atualmente, vem apresentando evidências de associação com diversos outros tipos de canceres, como o câncer de pulmão. Sendo assim, o presente estudo avaliou a presença do DNA do HPV em tumores de pulmão de pacientes do Estado de Pernambuco bem como a expressão de suas oncoproteínas E6 e E7. Para isto, a detecção foi feita em amostras de tecidos de tumores frescos e parafinados de 63 pacientes. HPV estava presente em 52% das amostras, sendo detectados os tipos 16 e 18 com frequências de 81 e 19%, respectivamente. Quanto a presença do vírus nos diferentes tipos histológicos dos tumores, foi detectado HPV em 40% dos carcinomas escamosos, 33% dos adenocarcinomas, 18% dos carcinomas de células pequenas e 9% em carcinoma de células grandes. Através da técnica de imunohistoquimica detectou-se a presença das oncoproteinas virais E6 (anticorpo anti-HPV 16 e anti-HPV 18) e E7 (anticorpo anti-HPV 16 e anti-HPV 18) com frequências de 85 e 75%, respectivamente. Tal resultado confirma os resultados obtidos molecularmente quanto à presença do HPV e é sugestivo de que o vírus esteja em atividade nas células tumorais e provavelmente esteja desempenhando um papel na carcinogênese de pulmão. No entanto, mais estudos são necessários para se ter um maior esclarecimento sobre interação de E6 e E7 com proteínas celulares na tumorigenese pulmonar.
Small DNA viruses - Human Papillomavirus (HPV) - infect oral mucosa and the epthelium, which leads to the development of both benign and malign tumors. They are widely known as the principal causes of cervical cancer although currently there is evidence to show that they are associated with several other types of cancer, such as lung cancer. In the light of this, this study evaluated the presence of HPV in the tumors of lungs of patients from the State of Pernambuco, as well as the E6 and E7 oncoproteins expression. This involved carrying out the detection in tumor tissue samples that were fresh and paraffin-embedded and taken from 63 patients. HPV was found to be present in 52% of the samples, and types 16 and 18 were detected with frequencies of 81% and 19% respectively. With regard to the presence of the vírus in different histological types of tumors, HPV was detected in 40% of the squamous carcinomas, 33% of the adenocarcinomas, 18% of the small cell carcinomas and 9% in large cell carcinomas. The presence of the E6 (antibody anti-HPV 16 and anti-HPV 18) and E7 (antibody anti-HPV 16 and anti-HPV 18) oncoproteins was detected by means of the immunohistochemical technique and this confirmed the results obtained from a molecular analysis with regard to the presence of the virus and it is suggestive that the virus is active in tumor cells and is probably playing a role in lung carcinogenesis. However, further studies are required to have a clearer understanding of the interaction of E6 and E7 with the cell proteins in pulmonary tumorigenesis.

Thesis advisor: Junona Moroianu
Some non melanoma skin cancers (NMSC) have been associated with human papillomavirus (HPV) pathogenesis, like epidermodysplasia verruciformis (EV) and squamous cell carcinoma (SCC). EV is a genetically inherited skin disease that develops when the individuals are infected with cutaneous HPV types belonging to the β-genus, especially types 5 and 8. Transgenic mouse lineages expressing all early genes of cutaneous HPV8 develop papillomas, dysplasias and SCC after UV irradiation and this correlates with enhanced HPV8 oncogenes expression. We have previously discovered that the nuclear localization of mucosal HPV16 E7 and HPV11 E7 proteins is mediated by their zinc-binding domain via a Ran-dependent pathway and independent of nuclear import receptors and that a patch of hydrophobic residues within the zinc-binding domain of HPV16 E7 and HPV11 E7 proteins is responsible for their nuclear import via hydrophobic interactions with FG nucleoporins. Here we investigated the nucleocytoplasmic traffic of cutaneous HPV8 E7 protein using confocal microscopy to analyze the intracellular localization of EGFP-8E7, its subdomains and its mutants after transient transfections. We also investigated the nuclear import ability of GST-8E7, its subdomains and mutants using in vitro nuclear import assays in digitonin-permeabilized HeLa cells. In addition, we performed isolation assays to study the direct interaction between HPV8 E7 and two FG nucleoporins, Nup62 and Nup153 or the nuclear export receptor, CRM1. We found that the nuclear import of cutaneous HPV8 E7 is mediated by a nuclear localization signal (NLS) located within its zinc-binding domain. Furthermore, we determined that the hydrophobic residues within the 65LRLFV69 patch are responsible for the nuclear import and nuclear localization of HPV8 E7 via direct hydrophobic interactions with FG nucleoporins, Nup62 and Nup153, whereas the positively charged arginine 66 plays no significant role in the function of the NLS. In addition, we examined the nuclear export mechanism of cutaneous HPV8 E7 protein and showed that it has a leucine-rich nuclear export signal (NES) in its C-terminal domain that is recognized by the CRM1 nuclear export receptor. These studies are essential for understanding the nucleocytoplasmic traffic of cutaneous HPV8 E7 protein
Thesis (PhD) — Boston College, 2014
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Thesis advisor: Junona Moroianu
Human papillomaviruses (HPVs) have been estimated to be the most common sexually transmitted infection in the United States. In addition to condyloma accuminata, infection of the squamous basal epithelium by high-risk HPVs, notably type 16 (HPV16), has been shown to be the primary etiological agent in the majority of cervical carcinomas. The E7 major transforming protein of HPV16, along with E6, has been linked to tumorigenesis and malignancy. While the E7 protein itself possesses no enzymatic activity, its ability to bind a number of nuclear and cytoplasmic targets subverts a variety of cellular regulatory complexes and facilitates viral replication. Previous studies in the Moroianu Lab have shown the HPV16 E7 oncoprotein to translocate across the nuclear pore complex (NPC) in a facilitated manner dependent on a non-canonical, c-terminal, nuclear localization signal (cNLS) for import, and a consensus leucine-rich nuclear export sequence (NES) for export (28). While the leucine-rich NES has been characterized, a full examination of the cNLS has yet to be performed. Here we present evidence that the karyopherin independent nuclear import mediated by the cNLS of 16E7 is dependent on its c-terminal Zn binding domain. Furthermore, we demonstrate that nuclear import is mediated by the direct interaction of a small patch of hydrophobic residues, 65LRLCV69, with the FG domain of the central FG-nucleoporin Nup62. In addition, we examined a potential regulatory mechanism of 16E7 nucleocytoplasmic translocation. Previous work has shown that a serine conserved in the high-risk HPVs at position 71 is phosphorylated by an unknown kinase. Here we present evidence that while phosphorylation of S71 is not required for either 16E7 nuclear localization or nuclear export in HeLa cells, mimicking phosphorylation of the S71 residue results in a statistically significant shift in the distribution of localization phenotypes of the resultant cell population toward a larger percentage exhibiting more nuclear localization. These data suggest that nucleocytoplasmic transport of 16E7 is, at least in part, a regulated process
Thesis (PhD) — Boston College, 2013
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

High-risk human papillomaviruses (HPVs) are causative agents of most cervical cancers and a significant portion of other anogenital tract and oral carcinomas. The major oncogenic activities of HPV16 E6 and E7 oncoproteins are associated with the degradation of the p53 and retinoblastoma tumor suppressors, respectively. E6 also causes increased expression of the catalytic subunit of telomerase, hTERT. In addition, E6 and E7 contribute to carcinogenesis through induction of genomic instability. Accurate chromosome segregation during mitosis is essential for preservation of genomic stability and HPV16 E7 perturbs mitosis in several ways. HPV16 E7 induces the synthesis of supernumerary centrosomes and increases the incidence of multipolar mitoses, which can lead to chromosome missegregation. Moreover, HPV16 E7 expression causes a prometaphase delay, which usually reflects an activation of the mitotic spindle assembly checkpoint (SAC), yet some studies suggested that the SAC is abrogated in HPV16 E7-expressing cells.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-12T15:20:04Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Tese_Elyda Gonçalves_PPGG_2016.pdf: 2873444 bytes, checksum: a341f8fc45a442c022975db79d659268 (MD5)
Made available in DSpace on 2017-07-12T15:20:04Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Tese_Elyda Gonçalves_PPGG_2016.pdf: 2873444 bytes, checksum: a341f8fc45a442c022975db79d659268 (MD5) Previous issue date: 2016-03-11
CAPES
O câncer da mama é o tipo de câncer que mais acomete mulheres em todo o mundo. Diversos fatores estãoassociados ao desenvolvimento desta neoplasia, dentre elas as infecções virais. Entre os três vírus mais estudados como causa de carcinogênese mamária está oPapillomavirus humano (HPV). Assim, oobjetivo foi detectar e analisar o HPV emcarcinomasmamáriosde mulheres do Nordeste do Brasil. A detecção do DNA viral foi realizada PCR, as amostras positivasforam tipificadas por sequenciamento. A quantificação da carga viral e a determinação do status físico por qPCR, e a detecção as oncoproteínas de E6 e E7 de HPV por imunohistoquímica. O DNA de HPV foi detectado em 46,7% dos carcinomas de mama HPV-positivos. O HPV16foi omais prevalente, 92% dos casos. A carga viral do HPV apresentou uma média de 14,2 cópias em 104 células, noscarcinomas de mama. Além disso, em 57,2% dos carcinomas mamáriosHPV-positivas apresentaram o DNA viral integrado ao genoma do hospedeiro. Altas taxas de detecção das oncoproteínas E6(89,5%) e E7(90%) foram identificadas nos carcinomas de mama HPV-positivos. Já as proteínas supressoras de tumor, p53 e p16INK4A, apresentaram taxas menores 95,7% e 92,3% respectivamente. Os resultados deste estudo sugerem que o vírus esteja em atividade nas células tumorais e provavelmente desempenhem papel na carcinogênese mamária.
Breast cancer is the type of cancer that affects more women around the world. Several factors are associated with the development of cancer, among which viral infections. Of the three most-studied virus as a cause of mammary carcinogenesis is the Human papillomavirus (HPV). The objective was to detect and analyze HPV in breast carcinomas of women in northeastern Brazil. The detection of viral DNA was performed PCR positive samples were typed by sequencing. The quantification of viral load and to determine the physical status by qPCR, and detection of the oncoproteins E6 and HPV E7 by immunohistochemistry. HPV DNA was detected in 46.7% of HPV-positive breast carcinomas. HPV16 was the most prevalent, 92% of cases. The HPV viral load averaged 14.2 copies in 104 cells in breast carcinomas. Furthermore, 57.2% of HPV-positive breast carcinomas showed the integrated viral DNA into the host genome. High rates of detection of E6 (89.5%) and E7 (90%) were identified in HPV-positive breast tumors. Already the tumor suppressor protein p53 and p16INK4a, had lower rates 95.7% and 92.3% respectively. The results of this study suggests that the virus is active in tumor cells and probably play role in breast carcinogenesis.

Human papillomavirus (HPV) infections play a crucial role in human carcinogenesis. Greater than 96% of all cervical carcinomas are positive for high-risk HPV infections; especially types 16 and 18. High-risk HPV onco-proteins, E6 and E7, are consistently expressed in such cancers and function by inactivating p53 and pRb tumor suppressors, respectively. The presence of high-risk HPVs is also correlated with anogenital cancers. In this study, we examined the effect of high-risk HPV type 16 E6 and E7 oncoproteins in two normal human colorectal epithelial cell lines, NCE1 and NCE5. We report that the expression of E6/E7 proteins, alone, induced cellular transformation of both cell lines; consequently, NCE1-E6/E7 and NCE5-E6/E7 form colonies in soft agar with respect to their wild type cells. This is accompanied by cell cycle deregulation, as is demonstrated by the over-expression of cyclin dependant kinases (cdks) and their respective cyclins. Furthermore, we demonstrate that E6/E7 oncoprotein transduction induces migration of colorectal epithelial cells. More still, well analyzed Id gene expression, a family member of the helix-loop-helix (HLH) transcription factors involved in the regulation of cell invasion and metastasis of human cancer cells. In parallel, using tissue microarray analysis we found that the four members of the Id protein family are correlated with the presence of HPV type 16 and 18 in human colon cancer tissues. Our data suggests that high-risk HPV infections are sufficient to induce cellular transformation of normal human colorectal cells, in vitro. Furthermore, the correlation with the Id family of proteins may present a novel set of markers associated with HPV induced colorectal carcinogenesis. Our results may suggest a new approach to detect and prevent colorectal cancer.

Les virus du papillome humain (HPV) sont des virus à ADN double-brin encapsidés appartenant à la famille des Papillomaviridae ayant un tropisme distinct pour les épithéliums squameux de type muqueux ou cutanés. Jusqu'à présent, plus de 200 types de HPV ont été isolés et regroupés dans un arbre phylogénétique composé de 5 genres nommés alpha, beta, gamma, mu et nu. Parmi eux, les types HPV muqueux à haut risque appartenant au genre alpha ont été associés au cancer du col de l'utérus ainsi qu'à des sous-groupes de carcinomes ano-génitaux et de la tête et du cou. Ces virus sont responsables d'environ 5% de tous les cancers viro-induits. Les types bêta du HPV ont un tropisme pour la peau et pourraient être impliqués dans le développement du cancer de la peau non mélanique (NMSC), en association avec la lumière ultraviolette (UV). Ainsi, les modèles expérimentaux in vitro et in vivo ont démontré les propriétés de transformation des oncoprotéines E6 et E7 du type HPV bêta 38. De plus, des études sur le modèle de souris transgénique, où E6 et E7 du HPV38 sont exprimés au niveau de la couche basale non différenciée de l'épithélium sous le contrôle du promoteur du gène humain de la kératine (K14), ont montré une très forte susceptibilité de la peau à la carcinogenèse induite par les UV par rapport aux animaux de type sauvage. Tout aussi important que leur capacité à promouvoir la transformation cellulaire, les virus oncogènes ont développé différentes stratégies pour prendre le dessus sur le système immunitaire de l'hôte, favorisant ainsi l'établissement d'une infection persistante. Par conséquent, savoir si des virus oncogènes potentiels ont la capacité d'interférer avec la réponse immunitaire pourrait fournir des preuves supplémentaires de leur implication dans la cancérogenèse humaine. Ici, nous montrons que les oncoprotéines E6 et E7 de HPV38 suppriment l'expression de Tolllike 9 (TLR9), récepteur des ADN double-brins, en favorisant l'accumulation de ΔNp73α, un antagoniste de p53 et p73. Des expériences d'immunoprécipitation de la chromatine ont montré que ΔNp73α fait partie d'un complexe de régulation négative transcriptionnelle qui se lie à un élément de réponse NF-kappaB dans le promoteur TLR9. Fait intéressant, l'expression ectopique de TLR9 dans des cellules HPV38 E6E7 a entraîné une accumulation des inhibiteurs du cycle cellulaire p21WAF1/Cip1 et p27kip1, une réduction de l'activité kinase associée à CDK2 et l'inhibition de la prolifération cellulaire. Ensemble, ces données indiquent que TLR9 est impliqué dans d'autres événements, en plus de la réponse immunitaire innée. Par conséquent, nous avons constaté que le traitement des kératinocytes humains primaires (HPK) avec différents stress cellulaires, par exemple l'irradiation aux UV, la doxorubicine et le traitement H2O2, conduisent à une induction de la transcription de TLR9. Cet évènement induit par les UV est arbitré par le recrutement de plusieurs facteurs de transcription sur le promoteur TLR9, tels que p53, NF-kappaB p65 et c-Jun. L'expression de E6 et E7 de HPV38 affecte fortement le recrutement de ces facteurs de transcription sur le promoteur TLR9, avec comme conséquence l'affaiblissement de l'expression du gène TLR9. En résumé, nos données montrent que HPV38, de manière similaire à d'autres virus avec une activité oncogénique bien connue, peut inhiber 'expression de TLR9. Plus important encore, nous mettons en évidence une nouvelle fonction de TLR9 dans le contrôle de la réponse cellulaire aux stress et nous montrons que E6 et E7 de HPV38 sont capables d'interférer avec un tel mécanisme. Ces résultats confirment le rôle des types HPV bêta dans la carcinogenèse de la peau, en fournissant des informations supplémentaires sur leur contribution précise dans le processus multi-étapes de développement du cancer
The human papillomaviruses (HPV) consist of a group of capsid-enclosed double-stranded deoxyribonucleic acid (dsDNA) viruses from the Papillomaviridae family that display a distinct tropism for mucosal or cutaneous squamous epithelia. Until now, more than 200 types of HPV have been isolated and grouped into a phylogenetic tree composed of 5 genera (alpha, beta, gamma, mu and nu papillomaviruses). Among them, the mucosal high-risk HPV types that belong to the genus alpha have been associated with cervical cancer as well as a subset of anogenital and head and neck carcinomas. They are responsible for approximately 5% of all virus-induced cancers. Beta HPV types have a skin tropism and have been suggested to be involved, together with ultraviolet light (UV), in the development of non-melanoma skin cancer (NMSC). For instance, in vitro and in vivo experimental models highlight the transforming properties of beta HPV38 E6 and E7. Specifically, studies of transgenic mouse model, where HPV38 E6 and E7 are expressed in the undifferentiated basal layer of epithelia under the control of the Keratin 14 (K14) promoter, showed a very high susceptibility to UV-induced skin carcinogenesis in comparison to the wild-type animals. Equally important as their ability to promote cellular transformation, oncogenic viruses have different strategies to overtake the host immune system thus guaranteeing persistent infection. Therefore, understanding whether potential oncogenic viruses have the ability to interfere with the immune response could provide additional evidence relating to their involvement in human carcinogenesis. Here, we show that the E6 and E7 oncoproteins from HPV38 suppress the expression of the dsDNA innate immune sensor Toll-like receptor 9 (TLR9) by promoting the accumulation of ΔNp73α, an antagonist of p53 and p73. Chromatin immunoprecipitation experiments showed that ΔNp73α is part of a negative transcriptional regulatory complex that binds to a NF-κB responsive element within the TLR9 promoter. Interestingly, ectopic expression of TLR9 in HPV38 E6E7 cells resulted in an accumulation of the cell cycle inhibitors p21WAF/Cip1 and p27Kip1, reduction of CDK2-associated kinase activity and inhibition of cellular proliferation. Together these data indicate that TLR9 is involved in additional events, besides the innate immune response. Accordingly, we observed that the treatment of human primary keratinocytes (HPKs) with different cellular stresses, e.g. UV irradiation, doxorubicin and H2O2 treatment, results in TLR9 up-regulation. This UVinduced event is mediated by the recruitment of several transcription factors to the TLR9 promoter, such as p53, NF-kB p65 and c-Jun. The expression of HPV38 E6 and E7 strongly affect the recruitment of these transcription factors to the TLR9 promoter, with consequent impairment of TLR9 gene expression. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity, can down-regulate TLR9. Most importantly, we highlight a novel function of TLR9 in controlling the cellular response to stresses and we show that HPV38 E6 and E7 are able to interfere with such mechanism. These findings further support the role of beta HPV types in skin carcinogenesis, providing additional insight into their precise contribution to the multistep process of cancer development

Made available in DSpace on 2014-06-12T15:52:08Z (GMT). No. of bitstreams: 2 arquivo2820_1.pdf: 8386047 bytes, checksum: f165dd7cc5f2eedad7f74c08f159ecde (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011
Faculdade de Amparo à Ciência e Tecnologia do Estado de Pernambuco
O câncer cervical é o segundo tipo de neoplasia que mais acomete as mulheres no Brasil, com alta prevalência em Pernambuco. A associação desta patologia como o papilomavírus humano (HPV) já está bem estabelecida e atualmente, sabe-se que o HPV pode ser encontrado em vários outros locais de infecção, além da região genital. Com o intuito de apontar a distribuição corpórea do HPV pelo corpo humano foi realizada uma revisão da literatura buscando por sítios corpóreos em que o DNA viral já tinha sido identificado. Dentre os tipos virais de alto risco que mais acometem a população brasileira, sabe-se que o HPV16 aparece associado a mais de 50% dos achados. Baseado nisso, uma busca pela presença do DNA do HPV e pelos variantes virais do HPV16 foi realizada em mulheres de Pernambuco que apresentavam lesões genitais. Complementarmente, sabe-se que sistemas de expressão são amplamente utilizados para produção de diversas moléculas biológicas, sendo o bacteriano o mais rápido e fácil de ser utilizado. Visando melhor utilização do sistema de expressão em bactéria, desenvolvemos um método para detectar a produção de -galactosidase em cepas heterólogas de Escherichia coli. Esse sistemas bacterianos vem sendo utilizados para produção de diversas moléculas virais, como oncoproteínas virais. Baseado no levantamento bibliográfico realizado foi possível identificar DNA viral nos mais diferentes sítios corpóreos, inclusive com ausência de lesões clínicas, apontando para a possibilidade do HPV agir como um oportunista. Da população estudada, mais de 50% foram positivas para o HPV, com achados de múltiplas infecções com tipos virais distintos, onde o variante Europeu foi o mais frequente nos casos de HPV16 positivo. A linhagem Origami (DE3) de E.coli demonstrou-se eficiente no ensaio colorimétrico expressando o gene da -galactosidase com baixa produção de proteínas bacterianas. Baseado nisso, esse modelo bacteriano foi utilizado no processo de sub-clonagem do gene E7 do HPV16 permitindo após indução do promotor visualizar uma banda de 15 KDa na eletroforese de proteínas totais, banda provavelmente referente a oncoproteína viral. No entanto, faz-se necessário emprego de testes imunológicos com anticorpos específicos para confirmar sua produção e posterior purificação. A tipagem da população a respeito do variante do HPV mais predominante e produção da proteína E7 permitem o aumento nos conhecimentos dos diferentes mecanismos de interação do vírus com o hospedeiro e favorecem ao desenvolvimento de métodos diagnósticos e terapêuticos mais eficientes e específicos para as diferentes regiões do mundo

Made available in DSpace on 2014-06-12T15:51:44Z (GMT). No. of bitstreams: 2 arquivo2827_1.pdf: 3255075 bytes, checksum: 22005ee118cfc8ca27010124cd458070 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011
Conselho Nacional de Desenvolvimento Científico e Tecnológico
Leveduras vêm sendo utilizadas como sistemas de expressão para produção de proteínas de interesse biotecnológico. Dentre as diversas aplicações das proteínas heterólogas, as produzidas com finalidade terapêutica e para diagnóstico clínico vêm recebendo grande destaque, como por exemplo, a lectina frutalina e a oncoproteína E7 do Papilomavírus Humano (HPV). A frutalina, lectina extraída das sementes da fruta-pão, tem sido descrita como importante ferramenta no diagnóstico do câncer devido ao seu tropismo com células tumorais. Enquanto, a oncoproteína viral do HPV, E7, por ser encontrada em células tumorais relacionadas à infecção pelo HPV, torna-se um importante alvo para o desenvolvimento de vacinas profiláticas e terapêuticas contra esta infecção. Neste trabalho utilizamos as leveduras Pichia pastoris KM71H e Pichia pastoris GS115 como sistemas de expressão para a produção das proteínas frutalina e E7 do HPV16, respectivamente. A influência da fonte de carbono e do estresse iônico na produção da enzima β-galactosidase pela Kluyveromyces lactis DSM 3795 foi avaliada em diferentes condições de cultivo. Neste trabalho os níveis de frutalina recombinante (13,4 mg.L-1), obtidos pelo processo de batelada alimentada, foi 4 vezes maior que em testes com frascos aerados e a suplementação com elementos traços permitiu o aumento de 2.5 vezes na produção da proteína recombinante. Enquanto, o gene de E7 do HPV16 foi corretamente clonado e integrado ao genoma da P.pastoris GS115, sendo produzido 218,9mg.L-1 da proteína E7 com o peso molecular de 27KDa. A avaliação da influência da fonte de carbono e do estresse iônico na atividade da enzima β-galactosidase pela K. lactis DSM 3795 permitiu selecionar a lactose como a fonte de carbono ideal, pois favoreceu maior atividade específica da enzima (271,0 U.mg-1). Enquanto que, os cátions Na+, K+, Mg2+ potencializaram a atividade da enzima (410,4; 440,1; and 734.71 U.mg-1, respectivamente), o Ca2+ inibiu parcialmente a produção da β-galactosidase. Por fim, apesar da P. pastoris ter se mostrado um sistema eficiente para a produção de proteína heterólogas, ela ainda apresenta algumas limitações. Dessa maneira, o desenvolver estratégias que permitam a otimização de vetores para a produção dessas proteínas, representa um importante alvo no desenvolvimento de produtos para o diagnóstico, prevenção e tratamento do câncer

O câncer cervical é o segundo maior responsável por mortes atribuídas a câncer em mulheres e dados epidemiológicos tem demonstrado a associação entre a infecção do HPV e o desenvolvimento da neoplasia. Sabe-se que num dado momento da infecção pelo HPV, ocorre integração do genoma viral ao genoma da célula hospedeira e consequente hiperexpressão de dois oncogenes virais, E6 e E7, o que contribui fortemente para a transformação celular. O presente trabalho propõe o uso de vacinas terapêuticas expressando a oncoproteína E7 do HPV-16 geneticamente fusionada à porção amino terminal da flagelina FliCd de Salmonella enterica sv müenchen; e verificação de seu possível papel adjuvante. Vacinas de DNA foram construídas de modo a direcionar as proteínas hibridas ao espaço intracitoplasmático. A verificação da expressão in vitro foi feita utilizando transfecções de culturas celulares, seguida de imunofluorescência e imunodetecção. Em seguida, essas construções vacinais foram administradas em camundongos C57BL6. Ensaios de ELISPOT foram feitos para mensuração o nível de linfócitos T secretores de IFN-g. Os dados de imunofluorescência e imunodetecção demonstraram a correta expressão da proteína. Ensaios de proteção realizados com 5 x 106 células TC-1 administrada por via subcutânea mostraram 80% de proteção em camundongos que haviam recebido previamente 4 doses das vacinas de DNA por biobalística. Ensaios de ELISPOT mostraram em média 9 células do baço secretoras de IFN-g por 106 células do baço (INF-g+/106) responsivas ao peptídeo CD8 / E7 de forma específica. Nossos dados sugerem que a formulação vacinal possui um efeito terapêutico significativo frente ao desafio com as células tumorais TC-1. Em paralelo, foi construída a cepa de salmonela vacinal SL FlaE7 que não mostrou efeito protetor frente ao desafio com células TC-1.
The cervical cancer is the second major responsible for deaths attributed to cancer in women and epidemiologic data have been demonstrating the association between the infection of HPV and the development of this illness. It is known that in a certain moment of the infection with HPV, occurs the integration of the viral genome in the genome of the host cell and consequent over expression of two oncogenes, E6 and E7, it strongly contributes to the transformation of that cell. The present work proposes the use of DNA vaccines expressing the oncoprotein E7 of HPV-16 genetically fused to the portion A-terminal of the flagellin FliCd of Salmonella enterica sv müencheun and verification of your possible performance as adjuvant. The DNA vaccines were constructed in expression vector for eukaryotic to address the hybrid proteins to the intracitoplasmatic space. The verification of the expression in vitro was made using transfections of cellular cultures (COS-7) followed by immunofluorescence and western blot analyses. Soon after, those vaccines were injected in mice C57BL6 that were challenged then with 5^105 tumor cells TC-1 subcutaneous. ELISPOT assays were performed for measure the level of spleen cells secreting interferon g. The immunofluorescence and Western blot data are complementary demonstrating the correct expression of the protein. Protection assays showed 80% of protection in mice that had previously received 4 doses of the DNA vaccines in gene gun form. Assays of ELISPOT show 9 cells of the spleen secreting of IFN-g (average) for 106 cells of the spleen (INF-g /106). Our data suggest that the formulation of vaccines possesses a significant therapeutic effect front to the challenge with the tumor cells TC-1 accompanist splenocyte IFN-g secretory.

O câncer cervical é o segundo câncer mais comum entre mulheres no mundo. A maioria dos casos (83%) ocorre em países em desenvolvimento, onde são encontrados em estágios relativamente avançados e, conseqüentemente, a sobrevida média é de cerca de 49% após cinco anos. Portanto, uma vacina eficaz contra as infecções pelo HPV pode levar ao controle do câncer do colo do útero. Apesar de prevenir, a vacina profilática não é acessível em função do alto custo, além de não eliminar o vírus em mulheres já infectadas pelo HPV. Assim, propusemos o desenvolvimento de uma vacina terapêutica eficaz utilizando duas abordagens: VLPs (virus-like particles) quiméricas, que poderiam apresentar propriedades profiláticas e terapêuticas, obtidas da fusão das proteína L1 e E7; proteínas quiméricas obtidas a partir da fusão de epítopos das proteínas E6 e E7 do HPV16, com e sem ubiquitina. Após subclonagens, com a obtenção dos vetores pPICHOLI-L1ΔCE71-50 e pPICHOLI-L1ΔCE743-77, partiu-se para a indução da expressão das VLPs quiméricas em Pichia pastoris, das quais não foram detectadas expressão protéica. Realizaram-se inúmeras modificações no protocolo de indução. Mesmo após essas alterações não foi detectada nenhuma expressão das fusões L1ΔCE71-50 ou L1ΔCE743-77. Como alternativa de uma vacina terapêutica, nos propusemos a expressar em E. coli proteínas sintéticas originadas da fusão entre epítopos das proteínas E6 e E7 do HPV16, com ou sem Ubiquitina, visando aumentar a apresentação de peptídeos via MHC de classe I de modo a estimular a eliminação de células infectadas com HPV16, evitando e regredindo o desenvolvimento dessas células cancerosas. Com a proteína E6E7 solúvel e purificada, realizou-se um ensaio de imunização. Nesse experimento, 20% dos animais imunizados com a proteína E6E7 não apresentaram desenvolvimento de tumor após a inoculação de células TC1. Assim isso nos leva a crer que com o aumento da concentração de proteína e utilização de adjuvantes seria possível aumentar o número de animais resistentes ao desenvolvimento do tumor. Em um segundo experimento de imunização, comparamos as proteínas E6E7 e E6E7Ub, em duas concentrações, 15 e 40 µg, e também com ou sem o adjuvante whole cell pertussis (WCP). Independentemente da concentração e presença ou ausência de WCP, os grupos imunizados com E6E7Ub apresentaram proteção contra o tumor entre 80% e 100% dos camundongos, enquanto os grupos imunizados com E6E7 apresentaram proteção entre 0% e 25%. Esses resultados são promissores, ainda que preliminares, indicando um potencial de uso da proteína E6E7Ub como imunógeno para vacina terapêutica contra o câncer cervical induzido por HPV16
Cervical cancer is the second most common cancer among women worldwide. Most cases (83%) occur in developing countries, where they are found in relatively advanced stages and, consequently, the median survival is about 49% after five years. Therefore, an effective vaccine against HPV infections can lead to control of cancer of the cervix. Although preventable, the prophylactic HPV vaccine is not accessible to all due to their high cost and in addition the vaccine does not eliminate the HPV in infected women. We have therefore proposed the development of effective therapeutic vaccines using two approaches: chimeric VLPs (virus-like particles), endowed with prophylactic and therapeutic properties, obtained from the fusion protein L1 and E7; chimeric proteins derived from the fusion of epitopes of proteins E6 and E7 of HPV16 with and without ubiquitin. After subcloning, we obtained the vectors pPICHOLI-L1ΔCE71-50 and L1 pPICHOLI- L1ΔCE743-77. After transformation of yeast Pichia pastoris with these constructions, the cells were induced, but it was not possible to detect any recombinant protein expression. As an alternative, we proposed the expression of synthetic proteins in E. coli derived from the fusion between epitopes of E6 and E7 proteins of HPV16 with or without Ubiquitin, in order to enhance the presentation of peptides through MHC class I to stimulate the elimination of HPV16-infected cells, preventing and regressing the development of cancer cells. Soluble E6E7 protein was purified and, 20% of the animals immunized with this protein did not develop tumor after inoculation of TC1 cells. In a second immunization experiment we compared the proteins E6E7 and E6E7Ub, in two concentrations, 15 and 40µg, with or without the adjuvant whole cell pertussis (WCP). Regardless of concentration and presence or absence of WCP, all the groups immunized with E6E7Ub showed protection against tumor between 80% and 100%, while the groups immunized with E6E7 showed protection from 0% to 25%. These results are promising and although preliminary, indicate the potential of E6E7Ub protein as an immunogen, for a therapeutic vaccine against cervical cancer induced by HPV16

Production of vaccines and pharmaceutical proteins in plants is a promising nascent technology with a great potential to provide high-quality, safe and non-expensive production and delivery platform. In this work we studied the experimental vaccine against human papillomavirus based on modified plant pathogen - Potato virus X (PVX). The experimental vaccine is based on PVX virus particles decorated with genetically fused HPV-E7 oncoprotein. These chimeric virus particles should be able to activate strong and specific cellular immune response. However the modification of the PVX coat protein with such relatively large fused protein might influence its ability to form particles. In this work we have characterized some properties of such chimeric virus particles like solubility or ability infect host plant. (In Czech)

碩士
國立成功大學
生物化學研究所
95
Human papillomavirus, causative agent of cervical cancers, encodes the E6 and E7 oncogenes, whose simultaneous expression is pivotal for malignant transformation and maintenance. The E6 and E7 oncoproteins of high-risk HPV, particularly HPV 18 and 16, bind respectively to the p53 and Retinoblastoma (Rb) tumor suppressor proteins, which are involved in the regulation of cell growth. RNA interference (RNAi) is a sequence-specific RNA degradation process in the cytoplasm of eukaryotic cells that is induced by double-stranded RNA. Inhibition of HPV expression by means of RNAi has been reported in many papers. In this study, we have screened the effective siRNA/shRNA by siRNA validation system. By transfection of these shRNA to cervical cancer cell, we found that E6 and E7 shRNA induced accumulation of p53 by decreasing mRNA encoding E6 and E7 protein. We thought that HPV E6 and E7 siRNA as a candidate for gene-specific therapy for HPV-related cervical cancer. Since its discovery in 1969, EV71 has been recognized as a frequent cause of epidemics of hand-foot-mouse disease (HFMD) associated with several neurological sequelae in a small proportion of cases. In 1998, the largest EV71 outbreak in Taiwan, more than 90000 children with HFMD had been reported. Internal ribosomal entry site (IRES), structured RNA sequence, was thought that it is essential to recruit and activate translation machinery. We focus on prevention of EV71 infection by using small interfering RNA to inhibit IRES of EV71. we have screened effective siRNA against the expression of Taiwan/4643 EV71 IRES expression cassettes by screening system. We attempt to use siRNA we selected to knockdown expression of EV71 IRES. Then, the cell, EV71 infection, would be protected from death, and decreased the activation of EV71.

IGFBP-3 expressing rekombinant vaccinia viruses used for tumor therapy Insulin-like growth factor-binding protein-3 (IGFBP-3) is a major regulator of endocrine effects of IGF and is capable to suppress the growth of variety of cancer. Several studies have shown that IGFBP-3 can induce the apoptosis of cancer cells via IGF-dependent and IGF-independent mechanisms. In our study, we have constructed recombinant vaccinia viruses (VACV) expressing IGFBP-3 under the control of the early H5 and synthetic early/late (E/L) promoter to investigate the potential effect on cancer growth in our cervical cancer model. We have shown that the expression of IGFBP-3 alone had no effect on tumor growth. On the other hand, the co-expression of IGFBP-3 enhanced the anti-cancer effect of immunization with the fusion protein SigE7LAMP, which gave rise to the anti-cancer immunity directed against HPV16 induced tumors. We have shown that the double-recombinant P13-SigE7LAMP-H5-IGFBP-3 can enhance the protective immune responses against MK16/ABC induced tumors. Furthermore, we have show that both double-recombinant viruses P13-SigE7LAMP-H5- IGFBP-3 and P13-SigE7LAMP-E/L-IGFBP-3 can increase the anti-cancer effect of SigE7LAMP expression in the therapy of TC-1 induced tumors. Key words: IGFBP-3, IGF, VACV, HPV16, E7 oncoprotein,...

The latest model of tertiary structure of capsid protein of potato virus X (PVX CP) was used as a template to design new insertion sites suitable for the preparation of PVX-based antigen presentation system. Based on this model, seven insertion sites (A-G) located in putative surface loops were tested. As an antigen inserted into these sites was used 17 amino acids long epitope derived from human papillomavirus type 16 E7 oncoprotein (E7 epitope) fused with either 6xHis tag or StrepII tag in both possible orientations (6xHis-E7 and E7-6xHis, StrepII-E7 and E7-StrepII). Prior to plant expression, modified PVX CPs were expressed in Escherichia coli MC1061. The results showed that only PVX CP carrying StrepII-E7 or E7-StrepII in the insertion site A formed virus particles. The results from transient expression experiments with modified PVX CPs in Nicotiana benthamiana showed that only the insertion site A (located between 24th and 25th amino acid in the PVX CP) could tolerate all tested inserts. Importantly, viral particles were detected only in the presence of StrepII tag and their stability was affected by the insert orientation (StrepII-E7 vs. E7-StrepII) as only the viral particles presenting E7-StrepII could be purified. Besides the preparation of PVX-based antigen presentation system, an...

5 Abstract Even though prophylactic vaccine against human papillomavirus (HPV) is currently licensed, infections by the virus continue to be the major health problem mainly in developing countries. Considerable effort is being devoted to preparation of therapeutic vaccine and to decrease of the production costs of current vaccine. Viral proteins such as the E7 oncoprotein and the L2 capsid protein from HPV type 16 are promising targets for the development of the experimental anti-HPV vaccine. The aim of our work was optimization of expression of mutagenized E7 oncoprotein (E7ggg) fused to the C-terminus of Tobacco mosaic virus (TMV) coat protein (CP) or Potato virus X (PVX) CP in viral vectors derived from these plant viruses. The impact of linkers connecting CP and E7ggg fusion partners on expression and stability of fusion proteins was examined. The fusion proteins were first expressed in Escherichia coli (E. coli) MC1061 to assess the characteristics of the recombinant protein prior to their transient expression in both non-transgenic or transgenic Nicotiana benthamiana (N. benthamiana). We have obtained the high level expression in E. coli, but most of the expressed proteins based on TMV CP remained in insoluble inclusion bodies. To increase the ratio of soluble protein various molecular...

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
O carcinoma da orofaringe aumentou de incidência consideravelmente nas últimas décadas, ao contrário da tendência que se tem verificado para o cancro da cabeça e do pescoço. Isto deve-se a um novo subtipo de carcinoma da orofaringe associado ao Vírus do Papiloma Humano (HPV) que se apresenta com uma epidemiologia diferente. O doente com carcinoma da orofaringe HPV positivo é mais novo e sem os fatores de risco tipicamente associados ao cancro da cabeça e pescoço, como o tabaco ou álcool.O HPV é um vírus de DNA que infeta as células epiteliais humanas e tem efeito carcinogénico, caracterizado por ausência de mutação na P53 e desregulação da proteína do Retinoblastoma (Rb) assim como a sobre-expressão da proteína p16. Os diferentes mecanismos de carcinogénese aparentam ser o motivo pelo qual o carcinoma da orofaringe associado ao HPV apresenta melhor resposta ao tratamento e melhor sobrevivência global (OS).Devido às diferenças entre o carcinoma da orofaringe HPV positivo e negativo um novo sistema de estadiamento foi desenvolvido, o qual inclui imunohistoquímica (IHC) para a sobre-expressão da p16, um marcador alternativo para a deteção do HPV.Considerando os efeitos secundários do tratamento e a melhor sobrevivência do carcinoma da orofaringe HPV positivo, a morbilidade a curto e longo prazo tornou-se uma preocupação. Novos ensaios clínicos com enfoque na desintensificação do tratamento enquanto se mantem um bom resultado oncológico então a decorrer e virão trazer novas alterações à terapêutica do carcinoma da orofaringe associado ao HPV.
Oropharyngeal carcinoma has had a considerable increase in the last decades, contrary to the overall tendency of head and neck cancers. This is due to a new subtype of orofaryngeal cancer associated with human papillomavirus (HPV) which presents with different epidemiology. The patient of HPV positive oropharyngeal carcinoma is younger and without the typical risk factors of head and neck cancers, namely tobacco and alcohol consumption.HPV is a DNA virus that infects human epithelial cells and as the ability to induce carcinogenesis, characterized by the absence of a mutation on P53 and deregulation of the Retinoblastoma (Rb) protein as well as over-expression of p16 protein. The different mechanisms of carcinogenesis appear to be the reason why HPV associated oropharyngeal cancer presents with better response to treatment and better overall survival (OS). Because of all the differences between HPV positive and HPV negative oropharyngeal cancer a new staging system was required, which includes testing with immunohistochesmistry (IHC) for the over-expression of p16, a surrogate marker to HPV detection. Considering the side effects of treatment and the better survival of the HPV positive oropharyngeal cancer patients, the shot and long term morbidity became a concern. New clinical trials with a focus on treatment de-escalation while maintaining a good oncologic result are ongoing and will bring new changes to the therapy of oropharyngeal carcinoma associated with HPV.

Arrière-plan: les cellules tumorales circulantes (CTC) sont détectables dans de nombreux cancers et peuvent être utiles cliniquement pour le pronostic de la maladie, pour mesurer la récidive et pour prédire la sensibilité aux medicaments chimiothérapeutiques. Au cours des dernières années, l’études des CTC dans de nombreux cancers tels que le cancer du sein, du poumon, du côlon et de la prostate a grandement évolué. Alternativement, il y peu d'études à ce sujet concernant le cancer du col de l’utérus (CCU). Objectifs: Notre objectif est d’optimiser le processus d'enrichissement des CTC dans le CCU et la détection moléculaire des biomarqueurs E6 et E7. Matériel et Méthodes: Dans l’optique de mimer la présence de CTC dans le sang, nous avons dilué des cellules cancéreuses CaSki VPH16-positif provenant d’un CCU dans du sang humain prélevé sur des volontaires sains. Les CaSki ont été collectées suite à une centrifugation par densité avec le Ficoll, la lyse des globules rouges (RBC) et la lyse des RBC combinée avec un enrichissement positif et négatif à l’aide de marqueurs de surface cellulaire. Les CTC ont été détectées par la mesure d’expression des oncogènes E6 et E7 du virus du papillome humain (VPH), de la cytokératine 19 (CK19) et de la cycline p16INK4 en utilisant la technique quantitative en temps réel de Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR). Pour valider notre méthode de détection des CTC in vivo, nous avons recruté dix patientes atteintes d’un CCU VPH16 positif et six contrôles sains. Résultats: Dans le modèle de dilutions de cellules CaSki, la lyse des RBC seule ou combinée avec l'enrichissement négatif ou positif suggèrent des limites de détection de 1 CTC par mL de sang pour tous les biomarqueurs moléculaires utilisés. La sensibilité de détection est accrue lors de l'utilisation de l’enrichissement positif et négatif en réduisant le bruit de fond causé par les monocytes sanguins. Contrairement aux oncogènes E6 et E7, les marqueurs CK19 et p16INK4A ont été détectés chez des individus sains, les niveaux d'expression de base appropriés doivent donc être déterminés avec précision par rapport aux patientes CCU. Le gradient de densité par Ficoll a une limite de détection de seulement environ 1000 cellules par mL de sang. Enfin, les CTC ont été détectées dans 2/10 patientes en utilisant le marqueur CK19. Cependant, ces patientes étaient négatives pour les oncogènes E6/E7. Le marqueur p16INK4A était exprimé au même niveau dans tous les échantillons (CCU et normaux). Conclusion: Notre étude suggère que les oncogènes E6 et E7 du VPH16 sont les marqueurs biologiques les plus sensibles et spécifiques en qRT-PCR pour détecter les CTC dans le modèle de dilution de cellules de CCU dans le sang. Chez les patientes atteintes d’un CCU de stade précoce, seulement CK19 a révélé la présence potentielle de CTC, ce qui suggère que ces cellules sont rares à ce stade de la maladie. Mots clés: cancer du col de l’utérus, cellules tumorales circulantes, RT-qPCR, E6 et E7, CK19, p16INK4A, enrichissement immunomagnétique, détection moléculaire.
Background: Circulating tumor cells (CTCs) have been detected in many cancers and are used in multiple clinical applications including disease prognosis, tumor recurrence prediction and prediction of tumor sensitivity to chemotherapeutic drugs. Studies in most major solid cancer(s) (breast, lung, colon and prostate) are progressing rapidly, but there has been very little progress concerning uterine cervical cancer (UCC).Objective: our aim is to optimize enrichment processes and the molecular biomarker-based detection of human circulating tumor cells (CTCs) in uterine cervical cancer (UCC). Material & Methods: To mimic CTCs in patients, we designed an experimental spiking model where the CaSki HPV16-positive UCC cell line was serially diluted and spiked into human blood collected from healthy volunteers. CaSki CTCs were enriched using either Ficoll density centrifugation, red blood cell (RBC) lysis or RBC lysis combined with cell surface markers negative or positive enrichment. CTCs were detected using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to measure the gene expression of human papillomavirus (HPV) viral oncogenes (E6 and E7), cytokeratin 19 (CK19), or the cyclin dependent kinase inhibitor p16INK4A. Finally, ten HPV16- positive UCC patients and six healthy controls were recruited to validate CTCs detection in vivo. Result: In the spiking model, RBC lysis alone or combined with negative or positive enrichment suggests detection limits close to 1 CTC per mL of blood for all molecular biomarkers used. The sensitivity of detection increased when using positive and negative enrichment probably by reducing the peripheral blood mononuclear cell-derived RNA background. Unlike HPV oncogenes, CK19 and p16INK4A were detected in normal individuals, thus appropriate basal expression levels need to be accurately determined compared to cancer patients. Alternatively, Ficoll density gradient had a detection limit of only about 1000 cells per mL of blood. Finally CTCs were detected in 2/10 patients using CK19. None of the patients had E6/E7 transcripts and p16INK4A was expressed at similar level across all samples (cancer and healthy). Conclusion: qRT-PCR of HPV16 E6 and E7 is the most sensitive and specific biomarker used to detect CTCs in the spiking model. In early disease UCC patients, only CK19 revealed the presence of CTCs suggesting that these cells are rare at that stage of the disease. Keywords: uterine cervical cancer, circulating tumor cells, qRT-PCR, E6 and E7 oncoprotein, CK19, p16INK4A, immune-magnetic enrichment, molecular detection.

碩士
中國醫藥大學
生物科技學系碩士班
97
In 2007, the Department of Health information shows that cervical cancer is the sixth cause of female cancer death in Taiwan. It’s still the second most common malignancy and the second most common cause of cancer-related death in women worldwide from WHO information. Human papillomavirus has been closely linked with human cervical cancer. The objectives of this study are to 1). detect HPV E6 and E7 proteins expressed in cervical cancer cell lines using polyclonal and monoclonal antibody we generated specific to the HPV oncoproteins. 2). develop immunoassays for detection of HPV E6, E7 oncoproteins using pap smear samples (liquid based). 3). compare the results of HPV DNA test, immunoassays, and clinical diagnosis (histology or pap smear results). We collected cervical specimens from 204 women with cervix cell and 12 women with cervical cancer tissue. Our preliminary results show that HPV E6 and E7 oncoproteins are detected from cervical cancer cell lines such as HeLa (HPV18), CaSki (HPV16), but not in C33A (HPV negative) by western blot using the specific HPV antibody. ELISA data indicate direct detection of HPV E7 oncoprotein from various stages of cervical scrape samples. Compared ELISA results to PCR, high correlation was found in these two methods. Immunocytochemistry assay also demonstrates the overexpression of E6 and E7 oncoproteins present in the nuclear of abnormal cells from various stage of liquid based cervical scrape samples. Therefore, various formats, high throughput, user friendly immunoassays can be further developed.