HPV16 Oncoproteins Induce MMPs/RECK-TIMP-2 Imbalance in Primary Keratinocytes: Possible Implications in Cervical Carcinogenesis

Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome

Downregulation of the RECK-tumor and metastasis suppressor gene in glioma invasiveness

Invasive behavior is the pathological hallmark of malignant gliomas, being responsible for the failure of surgery, radiation, and chemotherapy. Matrix metalloproteinases (MMPs) are essential for proper ECM remodeling and invasion. The tumor and metastasis suppressor RECK protein regulates at least three members of the MMPs family: MMP-2, MMP-9, and MT1-MMP. In order to mimic the in vivo invasion process, A172 and T98G, respectively, non-invasive and invasive human glioblastoma cell lines, were cultured onto uncoated (control) or type I collagen gel-coated surface, and maintained for up to 7 days to allow establishment of the invasive process. We show that the collagen substrate causes decreased growth rates and morphological alterations correlated with the invasive phenotype. Electronic transmission microscopy of T98G cells revealed membrane invaginations resembling podosomes, which are typically found in cells in the process of crossing tissue boundaries, since they constitute sites of ECM degradation. Real time PCR revealed higher RECK mRNA expression in A172 cells, when compared to T98G cells and, also, in samples obtained from cultures where the invasive process was fully established. Interestingly, the collagen substrate increases RECK expression in A172 cells and the same tendency is displayed by T98G cells. MMPs-2 and -9 displayed higher levels of expression and activity in T98G cells, and their activities are also upregulated by collagen. Therefore, we suggest that: (1) RECK down regulation is critical for the invasiveness process displayed by T98G cells; (2) type 1 collagen could be employed to modulate RECK expression in glioblastoma cell lines. Since a positive correlation between RECK expression and patients survival has been noted in several types of tumors, our results may contribute to elucidate the complex mechanisms of malignant gliomas invasiveness

RECK expression in cervicitis, CIN I, CIN II/III and invasive cervical carcinomas.

<p>n = 66;</p>*<p>Weak = no staining or faint staining;</p>**<p>Strong = moderate or strong staining.</p

HPV16 oncoproteins down-regulate RECK and TIMP-2 in HFKs.

<p><i>A,</i> Monolayer cultures of primary HFKs were transduced with pLXSN-based retroviral vectors expressing HPV16 E6wt and/or E7wt. RECK levels were determined by Western blot. Beta-actin was used as loading control. FF287 cell line was used as a positive control. <i>B,</i> Immunofluorescence detection of RECK (<i>green</i>) in HFKs expressing HPV oncoproteins. RECK is clearly detected in control and HPV16 E6wt expressing HFKs, while E7wt and E6E7-expressing cells exhibit reduced protein levels (<i>arrows</i>). Nuclei were counterstained with DAPI (<i>blue</i>). Original magnification 1000×. <i>C,</i> TIMP-2 was determined by ELISA (Biotrak). <i>P<0.05</i>.</p

RECK is down-regulated in CIN II/III and invasive carcinoma samples.

<p>Representative immunoreactivity of RECK in (A) Cervicitis, (B) cervical intraepithelial neoplasia II (CIN II), (C) cervical intraepithelial neoplasia III (CIN III) and (D) cervical invasive carcinoma. Note the strong RECK membrane staining pattern in cervicitis (black arrow), a diminished cytoplasmastic staining in CIN II/III and a very weak RECK staining in invasive carcinoma. Inset in (D): Sample incubated in the absence of primary antibody.</p

Expression of HPV-16 E6/E7 down-regulates RECK in organotypic cultures.

<p><i>A,</i> Representative immunoreactivity of RECK in control and HPV16 E6E7 expressing rafts cultures. Note the strong RECK membrane staining pattern (black arrow) in control compared to the faint staining in HPV16 E6E7 expressing rafts cultures. <i>B,</i> Quantitative real-time PCR (qRT-PCR) of <i>RECK</i> expression in organotypic cultures. The relative expression levels were normalized to tubulin. Bars represent the means of triplicate experiments; <i>bars</i>, ±SE.</p

Pró-letramento: experiências pedagógicas em matemática e linguagem

O livro trata das relações da Unesp com o programa “Pró-Letramento – Mobilização pela Qualidade da Educação”, realizado pelo Ministério da Educação e Cultura (MEC) em parceria com universidades que integram a Rede Nacional de Formação Continuada. Trata-se de um programa voltado para professores com vista à melhoria da qualidade do ensino e da aprendizagem da leitura e da escrita em Matemática e Linguagem nos anos iniciais do ensino fundamental. O papel das universidades é o de principalmente servirem como centros de pesquisa e de criação de atividades, sendo o corpo de pesquisadores da Unesp composto por especialistas nas áreas de Matemática, Linguagem e Educação em geral, que atuam inclusive com o apoio da educação a distância. Desde a criação do Pró-Letramento, em 2006, a Unesp está envolvida, por exemplo, com a formatação de cursos e com a elaboração do material a ser utilizado, baseado sempre na problematização dos conteúdos e das práticas cotidianas dos professores. Esse material traz ainda orientações de atividades a serem desenvolvidas entre os professores e aplicadas em sala de aula

Make EU trade with Brazil sustainable

Brazil, home to one of the planet's last great forests, is currently in trade negotiations with its second largest trading partner, the European Union (EU). We urge the EU to seize this critical opportunity to ensure that Brazil protects human rights and the environment