Für die Diagnose und das Monitoring des Sézary Syndrome haben sich zwei hauptsächliche Herangehensweisen etabliert: die Immunophänotypisierung und PCR basierte T-Zell-Rezeptor (TCR)-Gen-Rearrangement Analyse. Die verminderte Expression von T-Zell-Antigenen auf im Blut zirkulierenden Lymphozyten ist beim Sézary Syndrom beschrieben, insbesondere der Antigene CD7 und CD26. Jedoch ist die Verteilung des genetisch definierten Tumorzellklons auf die CD7 und CD26 positiven bzw. negativen Subpopulationen bisher nicht untersucht worden. Aus diesem Grund etablierten wir eine qualitative sowie eine quantitative klonspezifische PCR und bestimmten die Verteilung der klonalen Zellen auf diese Subpopulationen. Diese Daten wurden anschließend in Beziehung zur Klinik und zur Prognose der Patienten gesetzt. Für jeden unserer fünf Sézary Syndrom Patienten wurde die Sequenz des klonalen TCR-gamma und TCR-beta Rearrangementes ermittelt. Es erfolgte eine durchflußzytometrische Analyse der PBMC hinsichtlich der CD7 und CD26 Expression. Anschließend wurde für jeden Fall eine klonspezifische qualitative und quantitative (real-time) PCR etabliert. Mit Hilfe dieser PCR untersuchten wir das Vorhandensein und die Frequenz klonaler Zellen in den sortierten Subpopulationen. Die FACS-Analyse ergab bei unseren Patienten im Vergleich zu den Kontrollen eine verminderte CD7 und CD26 Expression der Lymphozyten. In der qualitativen PCR konnten klonale Zellen sowohl in der CD7+ und CD7- Population gezeigt werden, gleiches gilt für die CD26 Populationen. Die quantitative PCR zeigte, dass der dominante T-Zell-Klon sowohl CD7+ als auch CD7- sein kann, wohingegen CD26- Tumorzellen bei weitem überwiegen. Hinsichtlich Klinik und Prognose deutete sich an, das ein CD7+ Tumorzellklon eine bessere Prognose für den Patienten bedeuten kann. Zusammenfassung: Die Tumorzellen des Sézary Syndrom zeigen einen CD3+CD7+CD26- sowie CD3+CD7-CD26- Phänotyp. Aus diesem Grund kann bei dieser Erkrankung die Tumorzelllast im Blut über die Bestimmung der CD3+CD26- Zellen erfolgen. Die Signifikanz eines CD7+ Tumorzellkolons in Bezug zur Prognose muß an einem größeren Patientenkollektiv überprüft werden. / Introduction: For diagnosis and monitoring of the Sézary syndrome (SS) two crucial approaches are applied: immunophenotyping and PCR based T-cell receptor (TCR)-gene rearrangement analysis. The absence of T-cell antigens, in particular CD7 and CD26, on peripheral circulating tumor cells has been described in SS patients. However, the prevalence of the genetically identified malignant T-cell clone in CD7 and CD26 positive and negative subsets in SS has never been investigated so far. Thus we have performed qualitative and quantitative PCR based turmor cell determination in the corresponding FACS-separated T-cell fractions. These data were then correlated to clinical outcome and prognosis. Methods: For each of five SS patients with a total of 15 peripheral blood samples the sequences of clonal TCR-gamma and/or TCR-beta rearrangements were established. Then clone-specific qualitative und quantitative (real-time) PCR have been developed for each case. Using these PCR, we estimated presence and frequency of tumor cells in FACS-sorted CD3+CD7+/- as well as CD3+CD26+/- T-cell subsets. Results: The qualitative PCR showed that the malignant T-cells can be both: CD7 positive or negative, the same is true for CD26. The quantitative PCR demonstrated that CD7+ as well as CD7- tumor cells can occur simultaneously whereas CD26- tumor cells predominate by far. The individual tumor cell phenotypes did not fluctuate substantially in the disesase course, but considerable interindividual variations were seen. Furthermore we found in our patients, that CD7+ tumor cells may be a factor for a more favourable prognosis. Conclusions: The tumor cells in SS have mostly a CD3+CD7+CD26- as well as CD3+CD7- CD26- phenotyp. Thus, the tumor burden in this disease can be assessed by an estimation of the CD26- T-cells. The significance of a CD7+ t-cell clone for a better prognosis has to be re-evaluated in a larger patient-collective.

In Canada, colorectal cancer is the second leading cause of cancer death for both men and women. There are many different factors that contribute to the progression and spread of the disease. However, increasing evidence now suggests that the tumor microenvironment plays a paramount role in these processes. CD26 is a multifunctional, cell-surface glycoprotein that has intrinsic enzyme activity, binds adenosine deaminase and interacts with the extracellular matrix. Through its various functions it serves to constrain cancer progression. For example, it is known to cleave CXCL12, the ligand for CXCR4. The CXCL12:CXCR4 axis is normally involved in cancer metastasis by promoting cancer cell migration, invasion and proliferation. Down-regulation of CD26 is observed in certain cancers - this has been shown in vitro to occur in response to certain soluble mediators. The first part of this study looked at the effects of glucose and its metabolic product lactate on CD26 expression in colorectal carcinoma cells. Our study showed that CD26 expression is lower in cancer cells that are grown in low-glucose, high-lactate conditions, which replicates the situation within a tumor. The second part of this study examined the effect of adenosine, a purine nucleoside, on colorectal carcinoma cells and supportive stromal cells - cancer-associated HS675.T fibroblasts (CAFs) and Met-5a mesothelial cells. Adenosine increased the proliferation of CAFs and increased CXCL12 mRNA in both stromal cell lines. It also increased MMP-13 mRNA in stromal cells as well as colorectal cancer cells, suggesting that adenosine may promote progression and metastasis through various mechanisms. The last section focused on the ability of cellular products and 3-dimensional tissue topology to coordinate and affect the behaviour of the different cell populations. Here we show that secretory products from colorectal cancer cells promote CAF proliferation but inhibit mesothelial cell proliferation, and are also able to modulate MMP-13 expression. Finally, certain responses are enhanced in multicellular spheroids. In conclusion, the tumor microenvironment represents a major consideration in the treatment of solid tumors. Our data suggest that various soluble mediators, such as adenosine, may have therapeutic implications in cancer treatment and might represent novel targets for future research.

Liu, Limei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-156). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

CD26/DPPIV is a dipeptidyl peptidase that cleaves and destroys a variety of substrates such as the chemokine SDF-1α, a chemokine expressed along bone marrow endothelium, which is essential for the recruitment of hematopoietic stem cells (HSCs) via binding with its receptor CXCR4 to the bone marrow. Thus, CD26 is thought to interfere with the second step, chemokine/chemokine receptor interactions, of the cellular migration paradigm. To further study the role of CD26 in the migration of HSCs, we screened several human leukemic cell lines to find a model cell line that expresses active CD26 and discovered that the pro-monocytic cell line, U937 was optimal for this purpose. U937 cells were used to optimize a variety of assays including an CD26 activity assay and transwell migration assay with and without the use of a CD26 inhibitor, Diprotin A. Then, we isolated short-term and long-term HSCs from the bone marrow of C57BL/6N mice using a combination of surface markers and a fluorescence-activated cell sorter. The expression levels of Step 2’s homing molecules were measured by FACS in both fractions of HSCs. Interestingly, we detected differences in the expression of CD26 between these two populations that may help explain the inability of long-term HSCs to migrate to the bone marrow. Thus, through the use of a CD26 inhibitor the long-term HSCS migration to the bone marrow could be enhanced, leading to a prolonged and efficient stem cell engraftment activity. Such studies are could help develop protocols to improve stem cell engraftment for patients suffering from hematological diseases such as leukemia.

Indiana University-Purdue University Indianapolis (IUPUI) / Hematopoietic cell transplantation (HCT) is an important modality used to treat patients with hematologic diseases and malignancies. A better understanding of the biological processes controlling hematopoietic cell functions such as migration/homing, proliferation and self-renewal is required for improving HCT therapies. This study focused on the role of two biologically relevant proteins, dipeptidylpeptidase IV (DPPIV/CD26) and Ras homologue enriched in brain 2 (Rheb2), in modulating hematopoietic cell engraftment. The first goal of this study was to determine the role of the protein DPPIV/CD26 in modulating the engraftment of human umbilical cord blood (hUCB) CD34+ stem/progenitor cells using a NOD/SCID mouse xenograft model, and based upon previous work demonstrating a role for this enzyme in Stromal-Derived Factor-1/CXCL12 mediated migration and homing. Related to this first goal, pretreatment with an inhibitor of DPPIV/CD26 peptidase activity increased engraftment of hUCB CD34+ cells in vivo in recipient Non Obese Diabetic/Severe Combined Immunodeficiency (NOD/SCID) mice while not disturbing their differentiation potential following transplantation. These results support using DPPIV/CD26 inhibition as a strategy for enhancing the efficacy of cord blood transplantation. The second goal was to determine, by overexpression, the role of the Rheb2 in affecting the balance between proliferation and in vivo repopulating activity of mouse hematopoietic cells. Rheb2 is known to activate the mammalian target of rapamycin (mTOR) pathway, a pathway important in hematopoiesis. Rheb2 overexpression increased the proliferation and mTOR signaling of two hematopoietic cell lines, 32D and BaF3, in response to delayed IL-3 addition. In primary mouse hematopoietic cells, Rheb2 overexpression enhanced the proliferation and expansion of hematopoietic progenitor cells (HPCs) and phenotypic hematopoietic stem cells (HSCs) in vitro. In addition, HPC survival was enhanced by Rheb2 overexpression. Using in vivo competitive repopulation assays, Rheb2 overexpression transiently expanded immature HPC/HSC populations shortly after transplantation, but reduced the engraftment of total transduced cells. These findings support previous work showing that signaling proteins able to enhance the proliferative status of hematopoietic stem cells often cause exhaustion of self-renewal and repopulating ability. These studies of hematopoietic engraftment modulated by both of these molecules provide information which may be important to future work on HCT.

O câncer cervical é uma neoplasia muito prevalente na população feminina e está associado à infecção pelo papilomavírus humano (HPV). As oncoproteínas E6 e E7 de HPV de alto risco são as principais responsáveis pelas alterações celulares que levam ao desenvolvimento deste tipo tumoral. A dipeptidil peptidase IV (DPPIV/CD26) é uma enzima que exerce importantes funções relacionadas à progressão tumoral. Diversos estudos demonstram alterações na expressão e atividade desta proteína em diferentes tipos de câncer. Tendo em vista a relação entre a DPPIV/CD26 e o câncer, e que ainda não existem estudos relacionando esta proteína ao câncer cervical, neste estudo inicialmente investigamos a expressão e atividade da DPPIV/CD26 em linhagens celulares de carcinoma cervical humano (SiHa, HeLa e C33A) e em queratinócitos imortalizados (HaCaT). Nossos resultados demonstram uma baixa expressão da DPPIV/CD26 nas linhagens celulares estudadas, sendo praticamente indetectável na linhagem HeLa. Foi verificada a atividade enzimática dipeptidilpeptidásica tanto ligada à membrana quanto solúvel em todas as linhagens. Na presença do inibidor de DPPIV/CD26 (fosfato de sitagliptina) observamos que a linhagem SiHa apresentou um aumento na migração celular, e assim sugerimos que ao menos em parte a migração nesta linhagem é regulada pela atividade enzimática da DPPIV/CD26. A fim de investigar a relação da expressão da DPPIV/CD26 com as oncoproteínas E6 e E7 do HPV, avaliamos sua expressão em queratinócitos normais e transduzidos com estas oncoproteínas. Verificamos que queratinócitos expressando E6 de HPV de alto risco apresentam uma redução na expressão da DPPIV/CD26, e esta regulação parece ser dependente da degradação da p53. Considerando que as linhagens celulares estudadas apresentam baixa expressão e atividade da DPPIV/CD26, para melhor compreender a importância da expressão desta proteína, nós induzimos a superexpressão da DPPIV/CD26 em linhagem de câncer cervical (HeLa) para posterior avaliação dos efeitos em diferentes mecanismos tumorais. Os resultados demonstram uma redução no crescimento de células expressando DPPIV/CD26, sendo este efeito independente da atividade enzimática. Além disso, foi demonstrado que a indução da expressão de DPPIV/CD26 não afeta os mecanismos de migração e adesão celular na linhagem HeLa. Sendo assim, acreditamos que o esclarecimento do papel da DPPIV/CD26 no contexto do câncer cervical possibilita que novas abordagens diagnósticas e terapêuticas sejam implementadas no futuro. / Cervical cancer is a very prevalent neoplasm in female population and is associated with human papillomavirus (HPV) infection. The high risk HPV E6 and E7 oncoproteins are responsible for cellular alterations that lead to the development of this tumor type. The dipeptidyl peptidase IV (DPPIV/CD26) is an enzyme that exerts important functions related to tumor progression. Several studies have shown changes in the expression and activity of this protein in different types of cancer. Considering the relationship between DPPIV/CD26 and cancer, and that there are still no studies relating this protein to cervical cancer, in the present study we first investigated the DPPIV/CD26 expression and activity in human cervical carcinoma cell lines (SiHa, HeLa and C33A) and in immortalized keratinocytes (HaCaT). Our results demonstrate a low DPPIV/CD26 expression in the studied cell lines, being almost undetectable in HeLa cell line. The dipeptidylpeptidasic enzymatic activity was verified both membrane bound and in the soluble form in all cell lines. In the presence of the DPPIV/CD26 inhibitor (sitagliptin phosphate) we observed that SiHa cell line showed an increase in cell migration, thus we suggest that at least in part cell migration in this cell line is regulated by DPPIV/CD26 enzymatic activity. In order to investigate the relationship between DPPIV/CD26 expression and HPV E6 and E7 oncoproteins, we evaluated the expression of this protein in normal keratinocytes or transduced with these oncoproteins. We have found that keratinocytes expressing high-risk HPV E6 present a reduction in DPPIV/CD26 expression, and this regulation appears to be dependent on p53 degradation. Considering that the cell lines studied have low DPPIV/CD26 expression and activity, in order to better understand the importance of the expression of this protein, we induced the DPPIV/CD26 overexpression in a cervical cancer cell line (HeLa) for further evaluation of the effects on different tumor mechanisms. The results demonstrate a reduction in cell growth of DPPIV/CD26 expressing cells, being this effect independent of the enzymatic activity. In addition, it has been demonstrated that the induction of DPPIV/CD26 expression does not affect the cell migration and adhesion mechanisms in the HeLa cell line. Thus, we believe that the elucidation of the DPPIV/CD26 role in the context of cervical cancer enables new diagnostic and therapeutic approaches to be implemented in the future.

O câncer cervical é uma neoplasia muito prevalente na população feminina e está associado à infecção pelo papilomavírus humano (HPV). As oncoproteínas E6 e E7 de HPV de alto risco são as principais responsáveis pelas alterações celulares que levam ao desenvolvimento deste tipo tumoral. A dipeptidil peptidase IV (DPPIV/CD26) é uma enzima que exerce importantes funções relacionadas à progressão tumoral. Diversos estudos demonstram alterações na expressão e atividade desta proteína em diferentes tipos de câncer. Tendo em vista a relação entre a DPPIV/CD26 e o câncer, e que ainda não existem estudos relacionando esta proteína ao câncer cervical, neste estudo inicialmente investigamos a expressão e atividade da DPPIV/CD26 em linhagens celulares de carcinoma cervical humano (SiHa, HeLa e C33A) e em queratinócitos imortalizados (HaCaT). Nossos resultados demonstram uma baixa expressão da DPPIV/CD26 nas linhagens celulares estudadas, sendo praticamente indetectável na linhagem HeLa. Foi verificada a atividade enzimática dipeptidilpeptidásica tanto ligada à membrana quanto solúvel em todas as linhagens. Na presença do inibidor de DPPIV/CD26 (fosfato de sitagliptina) observamos que a linhagem SiHa apresentou um aumento na migração celular, e assim sugerimos que ao menos em parte a migração nesta linhagem é regulada pela atividade enzimática da DPPIV/CD26. A fim de investigar a relação da expressão da DPPIV/CD26 com as oncoproteínas E6 e E7 do HPV, avaliamos sua expressão em queratinócitos normais e transduzidos com estas oncoproteínas. Verificamos que queratinócitos expressando E6 de HPV de alto risco apresentam uma redução na expressão da DPPIV/CD26, e esta regulação parece ser dependente da degradação da p53. Considerando que as linhagens celulares estudadas apresentam baixa expressão e atividade da DPPIV/CD26, para melhor compreender a importância da expressão desta proteína, nós induzimos a superexpressão da DPPIV/CD26 em linhagem de câncer cervical (HeLa) para posterior avaliação dos efeitos em diferentes mecanismos tumorais. Os resultados demonstram uma redução no crescimento de células expressando DPPIV/CD26, sendo este efeito independente da atividade enzimática. Além disso, foi demonstrado que a indução da expressão de DPPIV/CD26 não afeta os mecanismos de migração e adesão celular na linhagem HeLa. Sendo assim, acreditamos que o esclarecimento do papel da DPPIV/CD26 no contexto do câncer cervical possibilita que novas abordagens diagnósticas e terapêuticas sejam implementadas no futuro. / Cervical cancer is a very prevalent neoplasm in female population and is associated with human papillomavirus (HPV) infection. The high risk HPV E6 and E7 oncoproteins are responsible for cellular alterations that lead to the development of this tumor type. The dipeptidyl peptidase IV (DPPIV/CD26) is an enzyme that exerts important functions related to tumor progression. Several studies have shown changes in the expression and activity of this protein in different types of cancer. Considering the relationship between DPPIV/CD26 and cancer, and that there are still no studies relating this protein to cervical cancer, in the present study we first investigated the DPPIV/CD26 expression and activity in human cervical carcinoma cell lines (SiHa, HeLa and C33A) and in immortalized keratinocytes (HaCaT). Our results demonstrate a low DPPIV/CD26 expression in the studied cell lines, being almost undetectable in HeLa cell line. The dipeptidylpeptidasic enzymatic activity was verified both membrane bound and in the soluble form in all cell lines. In the presence of the DPPIV/CD26 inhibitor (sitagliptin phosphate) we observed that SiHa cell line showed an increase in cell migration, thus we suggest that at least in part cell migration in this cell line is regulated by DPPIV/CD26 enzymatic activity. In order to investigate the relationship between DPPIV/CD26 expression and HPV E6 and E7 oncoproteins, we evaluated the expression of this protein in normal keratinocytes or transduced with these oncoproteins. We have found that keratinocytes expressing high-risk HPV E6 present a reduction in DPPIV/CD26 expression, and this regulation appears to be dependent on p53 degradation. Considering that the cell lines studied have low DPPIV/CD26 expression and activity, in order to better understand the importance of the expression of this protein, we induced the DPPIV/CD26 overexpression in a cervical cancer cell line (HeLa) for further evaluation of the effects on different tumor mechanisms. The results demonstrate a reduction in cell growth of DPPIV/CD26 expressing cells, being this effect independent of the enzymatic activity. In addition, it has been demonstrated that the induction of DPPIV/CD26 expression does not affect the cell migration and adhesion mechanisms in the HeLa cell line. Thus, we believe that the elucidation of the DPPIV/CD26 role in the context of cervical cancer enables new diagnostic and therapeutic approaches to be implemented in the future.

O câncer cervical é uma das neoplasias mais prevalentes, sendo a segunda mais frequente em mulheres no Brasil. A exoprotease dipeptidil-peptidase IV (DPPIV), também conhecida como CD26, é uma enzima encontrada em uma diversidade de células, e sua atividade enzimática e interação com outras proteínas parecem ser fundamentais para o controle da transformação maligna e progressão tumoral. Esta enzima é encontrada ancorada na membrana celular e também como uma isoforma solúvel (DPPIV/sCD26), ativa em fluidos biológicos. Pesquisas demonstram que alterações em sua expressão e atividade têm sido observadas em diversos tumores. Tendo em vista a relação entre a DPPIV/CD26 e o câncer, neste estudo investigamos a atividade e expressão da DPPIV/CD26 em linhagens celulares de câncer cervical (SiHa, HeLa and C33A) e de queratinócitos imortalizados (HaCaT). A atividade enzimática também foi monitorada na presença do inibidor específico da DPPIV/CD26, o fosfato de sitagliptina. Avaliamos também a relação desta enzima com os mecanismos de migração e adesão celular. Nossos resultados demonstram que todas as linhagens estudadas apresentam atividade enzimática DPPIV/CD26 ligada à membrana e solúvel, sendo superior nas linhagens SiHa e HaCaT. Confirmamos que esta atividade é atribuída à DPPIV/CD26 pela inibição da sua atividade enzimática na presença de fosfato de sitagliptina. Uma maior expressão do gene da DPPIV/CD26 foi observada nas linhagens HaCaT e SiHa, uma baixa expressão na C33A, sendo praticamente indetectável na HeLa. Estes dados corroboram os resultados obtidos para a atividade enzimática. Foi observada uma maior capacidade migratória na linhagem HeLa, quando comparada a SiHa, porém na presença de fosfato de sitagliptina, a linhagem SiHa apresentou um aumento na migração. Além disso, na presença do inibidor, tanto a linhagem HeLa quanto a SiHa exibiram uma redução na adesão celular. Este estudo demonstra a presença da enzima DPPIV/CD26 em células de câncer cervical, revelando diferentes níveis de expressão, e sua relação com a migração e adesão celular. / Cervical cancer is one of the most prevalent neoplasias, being the second most frequent in women in Brazil. The exoprotease dipeptidyl peptidase IV (DPPIV), also known as CD26, is an enzyme found in a variety of cells, and its enzymatic activity and interaction with other proteins appear to be essential for the control of malignant transformation and tumor progression. This enzyme is found anchored to the cell membrane and also as a soluble isoform (DPPIV/sCD26), active in biological fluids. Studies have shown changes in their expression and activity in several tumor types. Given the relationship between DPPIV/CD26 and cancer, in the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and immortalized keratinocytes (HaCaT). Enzymatic activity was also monitored in the presence of the specific DPPIV/CD26 inhibitor, sitagliptin phosphate. We also evaluated the relationship of this enzyme with cell migration and adhesion. Our results show that all cell lines studied exhibit DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form, being higher in SiHa and HaCaT. We confirm that this activity is attributed to DPPIV/CD26 by its inhibition in the presence of sitagliptin phosphate. We observed a higher expression of DPPIV/CD26 in HaCaT and SiHa cell lines, a low expression in C33A and in HeLa cells this expression was almost undetectable. These data corroborate the results obtained for enzymatic activity. We observed a higher migratory capacity of HeLa, when compared to SiHa, but in the presence of sitagliptin phosphate, SiHa showed an increase in migration. Furthermore, in the presence of the inhibitor, SiHa and HeLa cells exhibited a reduction in cell adhesion. This study demonstrates the presence of DPPIV/CD26 in cervical cancer cells, revealing a differential expression and its relationship with cell migration and adhesion.

O câncer cervical é uma neoplasia muito prevalente na população feminina e está associado à infecção pelo papilomavírus humano (HPV). As oncoproteínas E6 e E7 de HPV de alto risco são as principais responsáveis pelas alterações celulares que levam ao desenvolvimento deste tipo tumoral. A dipeptidil peptidase IV (DPPIV/CD26) é uma enzima que exerce importantes funções relacionadas à progressão tumoral. Diversos estudos demonstram alterações na expressão e atividade desta proteína em diferentes tipos de câncer. Tendo em vista a relação entre a DPPIV/CD26 e o câncer, e que ainda não existem estudos relacionando esta proteína ao câncer cervical, neste estudo inicialmente investigamos a expressão e atividade da DPPIV/CD26 em linhagens celulares de carcinoma cervical humano (SiHa, HeLa e C33A) e em queratinócitos imortalizados (HaCaT). Nossos resultados demonstram uma baixa expressão da DPPIV/CD26 nas linhagens celulares estudadas, sendo praticamente indetectável na linhagem HeLa. Foi verificada a atividade enzimática dipeptidilpeptidásica tanto ligada à membrana quanto solúvel em todas as linhagens. Na presença do inibidor de DPPIV/CD26 (fosfato de sitagliptina) observamos que a linhagem SiHa apresentou um aumento na migração celular, e assim sugerimos que ao menos em parte a migração nesta linhagem é regulada pela atividade enzimática da DPPIV/CD26. A fim de investigar a relação da expressão da DPPIV/CD26 com as oncoproteínas E6 e E7 do HPV, avaliamos sua expressão em queratinócitos normais e transduzidos com estas oncoproteínas. Verificamos que queratinócitos expressando E6 de HPV de alto risco apresentam uma redução na expressão da DPPIV/CD26, e esta regulação parece ser dependente da degradação da p53. Considerando que as linhagens celulares estudadas apresentam baixa expressão e atividade da DPPIV/CD26, para melhor compreender a importância da expressão desta proteína, nós induzimos a superexpressão da DPPIV/CD26 em linhagem de câncer cervical (HeLa) para posterior avaliação dos efeitos em diferentes mecanismos tumorais. Os resultados demonstram uma redução no crescimento de células expressando DPPIV/CD26, sendo este efeito independente da atividade enzimática. Além disso, foi demonstrado que a indução da expressão de DPPIV/CD26 não afeta os mecanismos de migração e adesão celular na linhagem HeLa. Sendo assim, acreditamos que o esclarecimento do papel da DPPIV/CD26 no contexto do câncer cervical possibilita que novas abordagens diagnósticas e terapêuticas sejam implementadas no futuro. / Cervical cancer is a very prevalent neoplasm in female population and is associated with human papillomavirus (HPV) infection. The high risk HPV E6 and E7 oncoproteins are responsible for cellular alterations that lead to the development of this tumor type. The dipeptidyl peptidase IV (DPPIV/CD26) is an enzyme that exerts important functions related to tumor progression. Several studies have shown changes in the expression and activity of this protein in different types of cancer. Considering the relationship between DPPIV/CD26 and cancer, and that there are still no studies relating this protein to cervical cancer, in the present study we first investigated the DPPIV/CD26 expression and activity in human cervical carcinoma cell lines (SiHa, HeLa and C33A) and in immortalized keratinocytes (HaCaT). Our results demonstrate a low DPPIV/CD26 expression in the studied cell lines, being almost undetectable in HeLa cell line. The dipeptidylpeptidasic enzymatic activity was verified both membrane bound and in the soluble form in all cell lines. In the presence of the DPPIV/CD26 inhibitor (sitagliptin phosphate) we observed that SiHa cell line showed an increase in cell migration, thus we suggest that at least in part cell migration in this cell line is regulated by DPPIV/CD26 enzymatic activity. In order to investigate the relationship between DPPIV/CD26 expression and HPV E6 and E7 oncoproteins, we evaluated the expression of this protein in normal keratinocytes or transduced with these oncoproteins. We have found that keratinocytes expressing high-risk HPV E6 present a reduction in DPPIV/CD26 expression, and this regulation appears to be dependent on p53 degradation. Considering that the cell lines studied have low DPPIV/CD26 expression and activity, in order to better understand the importance of the expression of this protein, we induced the DPPIV/CD26 overexpression in a cervical cancer cell line (HeLa) for further evaluation of the effects on different tumor mechanisms. The results demonstrate a reduction in cell growth of DPPIV/CD26 expressing cells, being this effect independent of the enzymatic activity. In addition, it has been demonstrated that the induction of DPPIV/CD26 expression does not affect the cell migration and adhesion mechanisms in the HeLa cell line. Thus, we believe that the elucidation of the DPPIV/CD26 role in the context of cervical cancer enables new diagnostic and therapeutic approaches to be implemented in the future.

O câncer cervical é uma das neoplasias mais prevalentes, sendo a segunda mais frequente em mulheres no Brasil. A exoprotease dipeptidil-peptidase IV (DPPIV), também conhecida como CD26, é uma enzima encontrada em uma diversidade de células, e sua atividade enzimática e interação com outras proteínas parecem ser fundamentais para o controle da transformação maligna e progressão tumoral. Esta enzima é encontrada ancorada na membrana celular e também como uma isoforma solúvel (DPPIV/sCD26), ativa em fluidos biológicos. Pesquisas demonstram que alterações em sua expressão e atividade têm sido observadas em diversos tumores. Tendo em vista a relação entre a DPPIV/CD26 e o câncer, neste estudo investigamos a atividade e expressão da DPPIV/CD26 em linhagens celulares de câncer cervical (SiHa, HeLa and C33A) e de queratinócitos imortalizados (HaCaT). A atividade enzimática também foi monitorada na presença do inibidor específico da DPPIV/CD26, o fosfato de sitagliptina. Avaliamos também a relação desta enzima com os mecanismos de migração e adesão celular. Nossos resultados demonstram que todas as linhagens estudadas apresentam atividade enzimática DPPIV/CD26 ligada à membrana e solúvel, sendo superior nas linhagens SiHa e HaCaT. Confirmamos que esta atividade é atribuída à DPPIV/CD26 pela inibição da sua atividade enzimática na presença de fosfato de sitagliptina. Uma maior expressão do gene da DPPIV/CD26 foi observada nas linhagens HaCaT e SiHa, uma baixa expressão na C33A, sendo praticamente indetectável na HeLa. Estes dados corroboram os resultados obtidos para a atividade enzimática. Foi observada uma maior capacidade migratória na linhagem HeLa, quando comparada a SiHa, porém na presença de fosfato de sitagliptina, a linhagem SiHa apresentou um aumento na migração. Além disso, na presença do inibidor, tanto a linhagem HeLa quanto a SiHa exibiram uma redução na adesão celular. Este estudo demonstra a presença da enzima DPPIV/CD26 em células de câncer cervical, revelando diferentes níveis de expressão, e sua relação com a migração e adesão celular. / Cervical cancer is one of the most prevalent neoplasias, being the second most frequent in women in Brazil. The exoprotease dipeptidyl peptidase IV (DPPIV), also known as CD26, is an enzyme found in a variety of cells, and its enzymatic activity and interaction with other proteins appear to be essential for the control of malignant transformation and tumor progression. This enzyme is found anchored to the cell membrane and also as a soluble isoform (DPPIV/sCD26), active in biological fluids. Studies have shown changes in their expression and activity in several tumor types. Given the relationship between DPPIV/CD26 and cancer, in the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and immortalized keratinocytes (HaCaT). Enzymatic activity was also monitored in the presence of the specific DPPIV/CD26 inhibitor, sitagliptin phosphate. We also evaluated the relationship of this enzyme with cell migration and adhesion. Our results show that all cell lines studied exhibit DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form, being higher in SiHa and HaCaT. We confirm that this activity is attributed to DPPIV/CD26 by its inhibition in the presence of sitagliptin phosphate. We observed a higher expression of DPPIV/CD26 in HaCaT and SiHa cell lines, a low expression in C33A and in HeLa cells this expression was almost undetectable. These data corroborate the results obtained for enzymatic activity. We observed a higher migratory capacity of HeLa, when compared to SiHa, but in the presence of sitagliptin phosphate, SiHa showed an increase in migration. Furthermore, in the presence of the inhibitor, SiHa and HeLa cells exhibited a reduction in cell adhesion. This study demonstrates the presence of DPPIV/CD26 in cervical cancer cells, revealing a differential expression and its relationship with cell migration and adhesion.