The process of oncogenic transformation in vitro has been examined in an attempt to define the molecular mechanisms of carcinogenesis. Transformation in Ki-MSV-infected rat adrenal cortex cells appears to be a multistep process (Auersperg et al., 1981), as does the process of transformation in other nonestablished cells (Land et at., 1983b). The Ki-MSV-infected adrenal cortex cells initially express a partially transformed phenotype and after further passaging progress to a highly transformed phenotype (Calderwood and Auersperg, 1984). Examination of Ki-MSV-infected adrenal cortex cells indicated that progression to a highly transformed phenotype could occur in the absence of significant changes in the level of the expression of the viral ras oncogene. These results indicated that an oncogenically activated ras gene could be expressed in these nonestablished cells in the absence of transformation. Since ras and myc cooperate to transform primary fibroblasts the effect of the co-introduction of myc on Ki-MSV-induced transformation of adrenal cortex cells was examined. It could be demonstrated that myc and ras cooperate to transform the adrenal cortex cells more efficiently than either oncogene alone, but that the infected cultures initially only express some of the phenotypes associated with transformation. The appearance of a fully transformed phenotype, as monitored by growth in soft agar, was not expressed until several passages after infection. An analysis of the Ki-MSV/MMCV-infected cultures indicated that some of the phenotypes associated with activated oncogenes in immortalized cell lines appeared to be suppressed in the coinfected adrenal cortex cells. Transformation by ras and myc appears to require a further cellular change resulting in a loss of the suppression of oncogene action. The emergence of transformed cultures from the Ki-MSV-infected rat adrenal cortex cells was correlated with the reduced expression of a novel ras-related protein of 27000 Mr. Transformation induced by src and myc was also examined. These two oncogenes appeared to cooperate in a two step pathway of transformation that was not susceptible to cellular suppression. The transformed phenotype did not appear to be entirely free of external influence as the growth rate of the transformed cells could be modified by culture conditions. The ability of myc to cooperate with src and ras in the transformation of the early passage adrenal cortex cells provides further support for mutistep carcinogenesis. The effect of oncogenes on steroidogenesis was examined in the Y-1 adrenocortical tumour cell line. The effect of the virally borne oncogenes on growth and morphology of the Y-1 cells was relatively subtle. The oncogenes appear to stimulate the production of fluorogenic steroids, each in a distinct fashion. A model of transformation can be derived in which the roles of the oncogenes and their interaction with the cell can be evaluated. The differences in the pathways of transformation for the two combinations of oncogenes illustrates the potential complexity of the transformation process and provides an in vitro model system for further study. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis (doctoral)--Rijksuniversiteit te Utrecht.

Epstein-Barr virus (EBV) induced infectious mononucleosis (IM) is characterized by the activation and expansion of T lymphocytes and the induction of cytotoxic responses able to mediate the lysis of EBV-uninfected, allogeneic MHC mismatched and EBV-infected autologous target cells. Freshly isolated peripheral blood mononuclear cells (PBMC) were used to examine the nature of these cellular immune responses. Activated lymphocytes, as identified by HLA-DR expression, associated with EBV induced IM were shown to be a heterogeneous population containing significantly elevated cytotoxic/suppressor (CD8+) T cells, helper/inducer (CD4+) T cells and natural killer (NK, CD16+) cells. CD8+ T cells were the primary activated population, representing 24% of the total lymphocyte population and 60-70% of the CD8+ T cell population. The activated CD4+ T cells and natural killer (NK) cells accounted for 7% and 4% of the total lymphocyte population, respectively. Analysis of serum soluble interleukin 2 receptors (IL-2R) and CD8 molecules demonstrated significantly (p<0.001) elevated levels in the sera of IM patients compared with normal controls. These elevated levels of serum IL-2R am CD8 molecules correlated, (r=0. 67 and r=0.82, respectively) with increased percentages of CD8/HLA-DR positive T cells (i.e., activated CD8 T cells). Increased levels of soluble cell surface molecules peaked during the acute phase and normalized as the patients progressed toward convalescence. Individual patients demonstrated strong correlations between the percentage of CD8/HLA-DR positive cells and soluble CD8 levels. The functional significance of the serum IL-2R and CD8 molecules is presently unknown. However, the strong correlative data between serum CD8, and to a lesser extent IL-2R, and CD8 T cell activation suggests that serum CD8 levels may provide a sensitive measure of CD8 T cell activation in systemic infections. The ability of freshly isolated acute IM PBMC to lyse allogeneic, EBV-infected lymphoblastoid cell lines (LCL), demonstrated the ability of acute IM effector cells to lyse MHC mismatched target cells. Effector cells from acute IM patients lysed allogeneic DM-LCL and AF-LCL target cells by 34% (n=7) and 23% (n=6), respectively, compared with 4% (n=5) and 0% (n=5), respectively, for normal controls. MAb-dependent complement depletion of CD3+ or CD8+ cells with anti-CD3 and anti-CD8 mAb decreased the non-MHC restricted cytolysis of LCL by 96% and 89%, respectively. In contrast, complement depletion with NK-cell specific mAbs Leu 11b and NKH-1, resulted in only a slight decrease (<35%) in the lysis of these LCL (46%). Depletion with anti-HLA-DR also significantly (p<0.001) decreased the lysis of LCL. Depletions with anti-CD4 demonstrated no decrease in LCL-lysis. MAbs OKT3 and OKT8 inhibited the non-MHC restricted cytolysis by 87% and 82%, respectively. We interpret these results as evidence that, 1) lysis of allogeneic cells is mediated primarily by CD3+, CD8+, HLA-DR+, cytotoxic T lymphocytes (CTL); and 2) these acute IM cytotoxic T cells utilize the T cell receptor and the CD8 antigen as an accessory molecule. An active role for target cell MHC class I molecules in the recognition and subsequent lysis of target cells is supported by a number of observations: 1) the MHC class I reactive mAbs W6/32 and BBM.1 significantly (p<0.05) inhibited the lysis of 63463-LCL by 65% and 57%, respectively; 2) acute IM effector T cells did not lyse the MHC class I negative Daudi cell line; 3) allogeneic MHC class I matched LCL mediated strong competitive inhibition (72% at 10:1 competitor to target cell ratio) vs 29% competitive inhibition for an allogeneic MHC class I mismatched LCL; and 4) NK-cell depleted effector cells from one patient mediated preferential lysis of the K562 cell line expressing MHC class I. HLA-A2 molecules. We interpret these results as evidence that target cell MHC class I molecules (or associated determinants) are the target antigen(s) for the allogeneic MHC cytotoxic response. The role of EBV in this acute allogeneic response was examined using target cell lines devoid of EBV genome. Acute IM CTL mediated lysis of the allogeneic HSB-2 T cell line (45%), and allogeneic HTLV-I transformed T cell lines (16%). The lysis of the HSB-2 T cell line was inhibited by anti-OKT3 (58% inhibition), W6/32 (53%) and BBM.1 (42%). Similarily, lysis of HTLV-I T cell lines was inhibited by W6/32 (69% inhibition), BBM.1 (69%) and OKT3 (38%). These data demonstrate that EBV antigenic expression is not required for allogeneic recognition and subsequent lysis of these allogeneic target cells. Effector cells from acute IM patients (n=5) were able to lyse their autologous EBV-infected LCL (mean lysis=21%), but were unable to lyse the EBV-uninfected autologous HTLV-I T cell line. These same effectors, however, were able to mediate lysis of both allogeneic B cell lines (21% lysis) and allogeneic T cell lines (8% lysis). These data are consistent with the observations by Strang et al. (1987a), who recently cloned virus specific/MHC-restricted CTL cloned from acute IM PBMC. These virus specific/MHC-restricted T cells presumably mediate the lysis of the autologous EBV-transformed B cell lines but not the autologous EBV-uninfected T cell lines. Whether the CTL which lyse the autologous EBV-transformed LCL are also responsible for the observed allogeneic reactivity was examined with cold target competition using autologous and allogeneic LCL. Lysis of autologous LCL was inhibited only by autologous competitor cells (64% inhibition compared with 24% for allogeneic LCL). Likewise, lysis of the allogeneic LCL was inhibited only by the allogeneic competitor cells (85% inhibition compared with 30% for autologous LCL). These data demonstrated no competition between allogeneic and autologous LCL and therefore support the concept that lysis of autologous LCL and allogeneic target cells is mediated by distinct effector populations. These data help us to understand the unusual immune response observed during acute IM. The strong allogeneic cytotoxic response is thought to represent polyclonal CD8 T cell activities induced by EBV-infected and transformed B cells which circulate in vivo. In addition, a population of CD8 CTL exist which mediate the lysis of autologous EBV-transformed B cells. These CTL likely represent virus-specific/MHC-restricted CTL and presumably play a major role in the control of EBV infections. The role, if any, of the markedly expanded alloreactive CTL population in the elimination of EBV infected and transformed B cells remains to be clarified.

The objective of this study was to identify possible roles of the class II antigen-associated, electrophoretically invariant chain (Ii) in class II antigen functions through analysis of the kinetics of synthesis, processing, and turnover of Ii in various cellular systems of Ii hyperexpression. Using [35S]methionine metabolic labeling of microsomal membrane proteins and analysis in one-dimensional SDS polyacrylamide gradient gels and two-dimensional electrophoretic gels, enhanced expression of Ii was demonstrated in leukemic cells of a subset of patients with hairy cell leukemia (HCL), in normal peripheral blood cells freshly transformed with EBV, and in Burkitt's lymphoma cell lines treated with n-butyrate. As part of an initial effort to identify leukemic cell subset-defining, membrane-associated molecules which might then be tested as targets for, or regulators of, the anti-leukemic cell immune response, subsets of HCL patients were identified on the basis of leukemic cell enhanced expression of 35,000 and 15,000 dalton proteins (p35 and p15). A similar enhanced expression of the p35 molecule was demonstrated in peripheral blood or cord blood lymphocytes freshly transformed with Epstein-Barr virus (EBV), as compared to long-term cultured EBV cell lines. Further structural characterization of the HCL p35 by one-dimensional SDS-PAGE and two-dimensional gel electrophoresis of HCL total microsomal membrane proteins, anti-class II antigen immunoprecipitates, and anti-Ii immunoprecipitates showed that the HCL p35 molecule was the human class II antigen-associated Ii chain. In order to determine the functional significance of altered Ii expression on class II antigen function, the relative kinetics of Ii synthesis, processing, and turnover was examined in an in vitro model system of regulated Ii synthesis. Treatment of the Burkitt's lymphoma cell line, Jijoye, and its class II antigen-negative, mutant, daughter cell line, P3HR-1, with 4 mM n-butyrate for 48 hr, enhanced the rate of synthesis of the Ii chain. One-dimensional SDS-PAGE and two-dimensional gel, electrophoretic analysis of anti-Ii and anti-class II antigen immunoprecipitates isolated from pulse-/pulse-chase-/or continuously labeled, control and butyrate-treated P3HR-1 and Jijoye cells demonstrated enhanced levels of synthesis of the dominant, most basic form of Ii in the butyrate-treated samples (to a greater degree in Jijoye cells). The butyrate enhancement of Ii synthesis occurred in the presence or absence of detectable class II alpha and beta chains, as did the relative turnover, although the terminal processing of Ii to an electrophoretically slower and more acidic form was dependent upon its association with class II antigens. The level of the dominant, most basic form of Ii expressed in the hairy leukemic cells equaled that in butyrate-treated Jijoye cells. However, hairy leukemic cell, class II antigen-associated Ii was not terminally processed. The results of these experiments were consistent with the following. In lymphoblastoid cells, two pathways for Ii turnover might exist. One is through association with class II antigen complexes and progressive carbohydrate-chain terminal processing, and a second is not associated with class II antigens and such processing. Class II antigens may direct the processing of Ii towards some function (rather than vice versa). Butyrate treatment rather uniquely enhances the synthesis of Ii in some lymphoid cells in the presence or absence of class II antigens. Hairy leukemic cells demonstrate an inability to terminally process Ii which might be relevant to the function of Ii and/or class II antigens on those cells.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 124-147).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

A infecção persistente por HPV-16 está fortemente associada ao risco de desenvolvimento de neoplasias do colo do útero, vagina, vulva, pênis, canal anal e orofaringe. O estudo detalhado da variabilidade nucleotídica intra-típica de HPV-16 resultou em importantes achados no que concerne à filogenia e evolução viral, e à história natural das infecções. Variantes Asiático-Americanas (AA) e E-350G de HPV-16 foram associadas com maior risco de persistência da infecção viral e desenvolvimento de câncer de colo de útero quando comparadas à variante Européia protótipo (E-P ou E-350T), embora esta ainda apresente alto risco quando comparada aos outros tipos virais. Mais recentemente, diferenças funcionais entre as proteínas E6/E7 das distintas variantes moleculares de HPV- 16 estão sendo descritas, a fim de explicar as diferenças nas associações epidemiológicas observadas. Dados do nosso grupo apontaram para a transcrição aumentada do gene MEK2 especificamente em queratinócitos humanos primários (PHKs) transduzidos com E6/E7 da variante E-350G. Pelo exposto, objetivou-se: (1) Analisar os níveis de ativação de proteínas efetoras das vias de transdução de sinal mediadas por MAPK e PI3K/AKT em queratinócitos imortalizados por E6/E7 de três variantes moleculares de HPV-16 (AA, E-P, E-350G); (2) Analisar os efeitos das proteínas E6/E7 dessas variantes sob as vias de MAPK quanto à indução de fatores de transcrição; (3) Analisar o potencial transformante de PHKs imortalizados pelas diferentes variantes, e em cooperação com a proteína celular c-MYC; (4) Analisar o potencial de migração e invasão em PHKs imortalizados pelas diferentes variantes de HPV-16, e em cooperação com a proteína celular c-MYC. Neste estudo observou-se que a variante AA de HPV-16 induziu a maior ativação das vias de sinalização estudadas (MAPK, e PI3K/AKT). Ademais, PHKs imortalizados por esta variante apresentaram maior capacidade de migração, de invasão através de uma matriz de colágeno, além de maior potencial transformante. Adicionalmente, as células imortalizadas pela variante AA apresentaram maior expressão da proteína mesenquimal vimentina e diminuição dos níveis da proteína epitelial E-caderina, sugerindo ativação parcial de Transição Epitélio Mesênquima (EMT) nestes queratinócitos. Ademais, quando o oncogene c-MYC foi co-transduzido nas diferentes linhagens infectadas por E6/E7 de HPV-16, foi observado que em PHKs imortalizados pela variante AA também houve maior ativação da via de MAPK-ERK, maior migração, e um potencial transformante semelhante, em relação às células co-transduzidas pela variante E-350G e c-MYC. Em conjunto, estes dados sugerem que a variante AA de HPV-16 possui vantagem seletiva sob as outras variantes em promover transformação celular, migração e invasão, e isto poderia explicar, ao menos em parte, a maior prevalência desta variante no câncer cervical. Os resultados gerados neste estudo são de extrema relevância para avaliar o impacto da variabilidade intra-típica de HPV-16 sobre o potencial oncogênico observado em estudos epidemiológicos / Persistent infection with HPV-16 is strongly associated with risk of developing neoplasia in the uterine cervix, vagina, vulva, penis, anal canal and oropharynx. The detailed study of HPV-16 intra-typical nucleotide variability resulted in important findings regarding phylogeny and viral evolution, and the natural history of infections. Asian-American (AA) and E-350G variants of HPV-16 were associated with increased risk of persistent viral infection and development of cervical cancer compared to the European prototype (E-P or E-350T), although this variant still presents higher risk when compared to other viral types. More recently, functional differences between the E6/E7 proteins of distinct molecular variants of HPV-16 are being described, in order to explain the differences in the epidemiological associations observed. Data from our group pointed to increased transcription of the MEK2 gene specifically in primary human keratinocytes (PHKs) transducing E6/E7 of the E-350G variant. Consequently, the aims of this study were: 1) To examine the activation levels of effector proteins of the signal transduction pathways mediated by MAPK and PI3K/AKT in PHKs immortalized by E6/E7 of three different molecular variants of HPV-16 (AA, E-P, E-350G); (2) To analyze the effects of E6/E7 of different molecular variants of HPV-16 upon MAPK pathways concerning the induction of transcription factors; (3) To analyze the transforming potential of PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC; (4) To analyze the potential of migration and invasion in PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC. In this study we observed that the AA variant of HPV-16 induced higher activation of both signaling pathways studied (MAPK, and PI3K/AKT). Furthermore, this variant presented increased migration capacity, higher invasion through a collagen matrix, and greater transforming potential. Moreover, cells immortalized by the AA variant showed higher expression of the mesenchymal protein vimentin and a decrease of the epithelial protein E-cadherin, suggesting partial activation of Epithelial Mesenchymal Transition (EMT). In addition, when the c-MYC oncogene was co-transduced in the different cells lines infected with HPV-16 E6/E7, we observed that in PHKs immortalized by the AA variant there was also an enhanced activation of the MAPK-ERK pathway, a higher ability to migrate, and similar transformation potential in comparison with cells co-transduced with the E-350G variant and c-MYC. Taken together, this data suggest that the AA molecular variant of the HPV-16 has a selective advantage over the other variants to promote cell transformation, migration and invasion, and this could partly explain the higher prevalence of this variant in cervical cancer. The results generated in this study are very important to assess the impact of intra-typical variability of HPV-16 on the oncogenic potential observed in epidemiological studies

A infecção persistente por HPV-16 está fortemente associada ao risco de desenvolvimento de neoplasias do colo do útero, vagina, vulva, pênis, canal anal e orofaringe. O estudo detalhado da variabilidade nucleotídica intra-típica de HPV-16 resultou em importantes achados no que concerne à filogenia e evolução viral, e à história natural das infecções. Variantes Asiático-Americanas (AA) e E-350G de HPV-16 foram associadas com maior risco de persistência da infecção viral e desenvolvimento de câncer de colo de útero quando comparadas à variante Européia protótipo (E-P ou E-350T), embora esta ainda apresente alto risco quando comparada aos outros tipos virais. Mais recentemente, diferenças funcionais entre as proteínas E6/E7 das distintas variantes moleculares de HPV- 16 estão sendo descritas, a fim de explicar as diferenças nas associações epidemiológicas observadas. Dados do nosso grupo apontaram para a transcrição aumentada do gene MEK2 especificamente em queratinócitos humanos primários (PHKs) transduzidos com E6/E7 da variante E-350G. Pelo exposto, objetivou-se: (1) Analisar os níveis de ativação de proteínas efetoras das vias de transdução de sinal mediadas por MAPK e PI3K/AKT em queratinócitos imortalizados por E6/E7 de três variantes moleculares de HPV-16 (AA, E-P, E-350G); (2) Analisar os efeitos das proteínas E6/E7 dessas variantes sob as vias de MAPK quanto à indução de fatores de transcrição; (3) Analisar o potencial transformante de PHKs imortalizados pelas diferentes variantes, e em cooperação com a proteína celular c-MYC; (4) Analisar o potencial de migração e invasão em PHKs imortalizados pelas diferentes variantes de HPV-16, e em cooperação com a proteína celular c-MYC. Neste estudo observou-se que a variante AA de HPV-16 induziu a maior ativação das vias de sinalização estudadas (MAPK, e PI3K/AKT). Ademais, PHKs imortalizados por esta variante apresentaram maior capacidade de migração, de invasão através de uma matriz de colágeno, além de maior potencial transformante. Adicionalmente, as células imortalizadas pela variante AA apresentaram maior expressão da proteína mesenquimal vimentina e diminuição dos níveis da proteína epitelial E-caderina, sugerindo ativação parcial de Transição Epitélio Mesênquima (EMT) nestes queratinócitos. Ademais, quando o oncogene c-MYC foi co-transduzido nas diferentes linhagens infectadas por E6/E7 de HPV-16, foi observado que em PHKs imortalizados pela variante AA também houve maior ativação da via de MAPK-ERK, maior migração, e um potencial transformante semelhante, em relação às células co-transduzidas pela variante E-350G e c-MYC. Em conjunto, estes dados sugerem que a variante AA de HPV-16 possui vantagem seletiva sob as outras variantes em promover transformação celular, migração e invasão, e isto poderia explicar, ao menos em parte, a maior prevalência desta variante no câncer cervical. Os resultados gerados neste estudo são de extrema relevância para avaliar o impacto da variabilidade intra-típica de HPV-16 sobre o potencial oncogênico observado em estudos epidemiológicos / Persistent infection with HPV-16 is strongly associated with risk of developing neoplasia in the uterine cervix, vagina, vulva, penis, anal canal and oropharynx. The detailed study of HPV-16 intra-typical nucleotide variability resulted in important findings regarding phylogeny and viral evolution, and the natural history of infections. Asian-American (AA) and E-350G variants of HPV-16 were associated with increased risk of persistent viral infection and development of cervical cancer compared to the European prototype (E-P or E-350T), although this variant still presents higher risk when compared to other viral types. More recently, functional differences between the E6/E7 proteins of distinct molecular variants of HPV-16 are being described, in order to explain the differences in the epidemiological associations observed. Data from our group pointed to increased transcription of the MEK2 gene specifically in primary human keratinocytes (PHKs) transducing E6/E7 of the E-350G variant. Consequently, the aims of this study were: 1) To examine the activation levels of effector proteins of the signal transduction pathways mediated by MAPK and PI3K/AKT in PHKs immortalized by E6/E7 of three different molecular variants of HPV-16 (AA, E-P, E-350G); (2) To analyze the effects of E6/E7 of different molecular variants of HPV-16 upon MAPK pathways concerning the induction of transcription factors; (3) To analyze the transforming potential of PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC; (4) To analyze the potential of migration and invasion in PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC. In this study we observed that the AA variant of HPV-16 induced higher activation of both signaling pathways studied (MAPK, and PI3K/AKT). Furthermore, this variant presented increased migration capacity, higher invasion through a collagen matrix, and greater transforming potential. Moreover, cells immortalized by the AA variant showed higher expression of the mesenchymal protein vimentin and a decrease of the epithelial protein E-cadherin, suggesting partial activation of Epithelial Mesenchymal Transition (EMT). In addition, when the c-MYC oncogene was co-transduced in the different cells lines infected with HPV-16 E6/E7, we observed that in PHKs immortalized by the AA variant there was also an enhanced activation of the MAPK-ERK pathway, a higher ability to migrate, and similar transformation potential in comparison with cells co-transduced with the E-350G variant and c-MYC. Taken together, this data suggest that the AA molecular variant of the HPV-16 has a selective advantage over the other variants to promote cell transformation, migration and invasion, and this could partly explain the higher prevalence of this variant in cervical cancer. The results generated in this study are very important to assess the impact of intra-typical variability of HPV-16 on the oncogenic potential observed in epidemiological studies