RESUMO
Appropriate 'in vivo' models are crucial for studying breast cancer biology and evaluating the efficacy of therapeutic agents. Thus we engineered a novel transgenic mouse line expressing the human Ki-Ras bearing an activating mutation (Ki-Ras(G12V)) selectively in the mammary epithelium after lactation. These mice develop invasive ductal adenocarcinomas with 100% incidence within 3-9 months after Ki-Ras(G12V) induction. Immunophenotyping revealed that the mammary tumors express luminal markers, are positive for estrogen and progesterone receptors, negative for HER2 and have a low proliferation index. Moreover, cell lines derived from such tumors are estrogen-responsive and, when transplanted into nude mice, form tumors that respond to the antiestrogen ICI 182780. In conclusion, the mammary tumors of these transgenic mice and the derived cell lines exhibit key features of the major form of human breast cancer, that is, luminal A subtype and thus have a high potential for breast cancer research and treatment.
Assuntos
Adenocarcinoma/genética , Epitélio/metabolismo , Receptor alfa de Estrogênio/genética , Genes ras/genética , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/genética , Adenocarcinoma/metabolismo , Animais , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Fulvestranto , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Mutação de Sentido Incorreto , Células Tumorais CultivadasRESUMO
Erratum to: Breast Cancer Res Treat (2012), 134:569581, DOI 10.1007/s10549-012-2090-9. Uunfortunately, authors could not find the original film from which the figure was drawn. Therefore, as suggested by the Editor, they have repeated the relative experiment, and ask to publish this new figure as a correction. The authors apologize for any inconvenience that it may cause.
RESUMO
Sexual hormones, estrogens and androgens, determine biological response in a tissue- and gender-specific manner and have a pivotal role in endocrine-mediated tumorigenesis. In situ estrogen production by aromatase is a critical determinant for breast cancer growth and progression. On the contrary, clinical and in vitro studies indicate that androgens have a protective role in mammary carcinogenesis. Here, we demonstrated, in hormone-dependent breast cancer cells, the existence of a functional interplay between the androgen receptor (AR), the orphan nuclear receptor DAX-1 and the aromatase enzyme involved in the inhibition of the estrogen-dependent breast cancer cell proliferation exerted by androgen signaling. Indeed, our results revealed, in MCF-7 cells, that ligand-activated AR induces the expression of the orphan nuclear receptor DAX-1 by direct binding to a newly identified androgen-response-element within the DAX-1 proximal promoter. In turn, androgen-induced DAX-1 is recruited, in association with the corepressor N-CoR, within the SF-1/LRH-1 containing region of the aromatase promoter, thereby repressing aromatase expression and activity. In elucidating a novel mechanism by which androgens, through DAX-1, inhibit aromatase expression in breast cancer cell lines, these findings reinforce the theory of androgen- opposing estrogen-action, opening new avenues for therapeutic intervention in estrogen-dependent breast tumors.
Assuntos
Aromatase/metabolismo , Proliferação de Células , Receptor Nuclear Órfão DAX-1/genética , Estrogênios/fisiologia , Androgênios/farmacologia , Apoptose , Aromatase/genética , Sequência de Bases , Neoplasias da Mama , Receptor Nuclear Órfão DAX-1/metabolismo , Repressão Enzimática , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Nandrolona/análogos & derivados , Nandrolona/farmacologia , Neoplasias Hormônio-Dependentes , Regiões Promotoras Genéticas , Receptores Androgênicos/metabolismo , Elementos de RespostaRESUMO
ERα function is crucial for the development of normal mammary gland as well as in the process of progression of breast cancer cells. Signals that target receptor levels contribute to regulate estrogens effects in the cells. An intricate cross-regulation has been documented between ERα and TGF-ß down-stream molecules: SMAD2, SMAD3, and SMAD4, that can bind ERα and regulate their signaling. Thus, identification of natural anticancer drugs able to influence the latter molecule might provide alternative choices for breast cancer treatment. Taking into account our previous published data we wanted to study the effect of 5-Methoxypsoralen (bergapten) on ERα and on TGF-ß pathway. We reported that bergapten, a coumarin containing compound, effectively depletes ERα in MCF-7 breast cancer sensitive cells and in tamoxifen-resistant clone. The decrease of ERα protein after bergapten treatment results from the ubiquitine-proteasome pathway as demonstrated by the use of MG-132. IP experiments with ER antibody, demonstrated that the protein has physical interaction with SMAD4 and poly-ubiquitine and the amount of ubiquitinated receptor, linked to SMAD4, is greater under bergapten. The crucial role played by SMAD4, in this process, emerges from the observation that in breast cancer cells, silencing of SMAD4, resulted in increased expression of endogenous ERα in both control and bergapten-treated cells, compared to wild- type cells. The same results were confirmed in siRNA TGF-ß RII cells. The results suggest a novel negative regulation of ERα by TGF-ß/SMAD4 in breast cancer cells and indicate that the SMAD4 protein is involved in the degradation of ERα induced by bergapten. We propose that bergapten may efficiently act as a natural antitumoral agent, able to deplete ERα from breast cancer tamoxifen-sensitive and resistant cells, thereby retraining the effect of membrane signals targeting ERα and in such way its mitogenic potentiality.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Metoxaleno/análogos & derivados , Proteína Smad4/metabolismo , Ubiquitinação , 5-Metoxipsoraleno , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Estrogênios/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Metoxaleno/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologiaRESUMO
Human estrogen receptors alpha and beta are crucially involved in the regulation of mammary growth and development. Normal breast tissues display a relative higher expression of ER beta than ER alpha, which drastically changes during breast tumorogenesis. Thus, it is reasonable to suggest that a dysregulation of the two estrogen receptor subtypes may induce breast cancer development. However, the molecular mechanisms underlying the potential opposing roles played by the two estrogen receptors on tumor cell growth remain to be elucidated. In the present study, we have demonstrated that ER beta overexpression in breast cancer cells decreases cell proliferation and down-regulates ER alpha mRNA and protein content, along with a concomitant repression of estrogen-regulated genes. Transient transfection experiments, using a vector containing the human ER alpha promoter region, showed that elevated levels of ER beta down-regulated basal ER alpha promoter activity. Furthermore, site-directed mutagenesis and deletion analysis revealed that the proximal GC-rich motifs at -223 and -214 are critical for the ER beta-induced ER alpha down-regulation in breast cancer cells. This occurred through ER beta-Sp1 protein-protein interactions within the ER alpha promoter region and the recruitment of a corepressor complex containing the nuclear receptor corepressor NCoR, accompanied by hypoacetylation of histone H4 and displacement of RNA-polymerase II. Silencing of NCoR gene expression by RNA interference reversed the down-regulatory effects of ER beta on ER alpha gene expression and cell proliferation. Our results provide evidence for a novel mechanism by which overexpression of ER beta through NCoR is able to down regulate ER alpha gene expression, thus blocking ER alpha's driving role on breast cancer cell growth.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , Elementos de Resposta , Fator de Transcrição Sp1/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fator de Crescimento Insulin-Like I/fisiologia , Correpressor 1 de Receptor Nuclear/genética , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , RNA Polimerase II/metabolismoRESUMO
AIM: To validate a silhouette-based scale, the Body Image Dimensional Assessment (BIDA), an instrument for the screening of body dissatisfaction in large samples. MATERIALS AND METHODS: Five-hundred ninety-two both gender non-clinical participants and 57 patients with eating disorders (ED) were administered the BIDA and the Body Dissatisfaction subscale of the Eating Disorder Inventory 2 (BD-EDI2). The BIDA consists of only 4 items to answer with reference to a series of four silhouettes not age- nor gender-related using a numeric scale that allows the quantification of the degree of Body Dissatisfaction, Sexual Body Dissatisfaction, Comparative Body Dissatisfaction and the calculation of the final Body Dissatisfaction Index (BDI). RESULTS AND CONCLUSIONS: The study has shown that the BIDA has good reliability and validity as well as high predictive capability at a threshold BDI≥30 (sensitivity = 83.3% and specificity = 92.1%). By virtue of the rapid timing of administration, the BIDA can be a useful screening instrument of body dissatisfaction in non clinical populations to detect people at risk for ED and a follow-up instrument in clinical setting.
Assuntos
Transtornos Dismórficos Corporais/diagnóstico , Transtornos da Alimentação e da Ingestão de Alimentos/diagnóstico , Adolescente , Adulto , Imagem Corporal , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Psicometria/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Inquéritos e QuestionáriosRESUMO
Tamoxifen (Tam) treatment is a first-line endocrine therapy for estrogen receptor-α-positive breast cancer patients. Unfortunately, resistance frequently occurs and is often related with overexpression of the membrane tyrosine kinase receptor HER2. This is the rationale behind combined treatments with endocrine therapy and novel inhibitors that reduce HER2 expression and signaling and thus inhibit Tam-resistant breast cancer cell growth. In this study, we show that activation of farnesoid X receptor (FXR), by the primary bile acid chenodeoxycholic acid (CDCA) or the synthetic agonist GW4064, inhibited growth of Tam-resistant breast cancer cells (termed MCF-7 TR1), which was used as an in vitro model of acquired Tam resistance. Our results demonstrate that CDCA treatment significantly reduced both anchorage-dependent and anchorage-independent epidermal growth factor (EGF)-induced growth in MCF-7 TR1 cells. Furthermore, results from western blot analysis and real-time reverse transcription-PCR revealed that CDCA treatment reduced HER2 expression and inhibited EGF-mediated HER2 and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation in these Tam-resistant breast cancer cells. Transient transfection experiments, using a vector containing the human HER2 promoter region, showed that CDCA treatment downregulated basal HER2 promoter activity. This occurred through an inhibition of nuclear factor-κB transcription factor binding to its specific responsive element located in the HER2 promoter region as revealed by mutagenesis studies, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Collectively, these data suggest that FXR ligand-dependent activity, blocking HER2/MAPK signaling, may overcome anti-estrogen resistance in human breast cancer cells and could represent a new therapeutic tool to treat breast cancer patients that develop resistance.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptor ErbB-2 , Receptores Citoplasmáticos e Nucleares/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ácido Quenodesoxicólico/administração & dosagem , Ácido Quenodesoxicólico/metabolismo , Ácido Quenodesoxicólico/farmacologia , Imunoprecipitação da Cromatina , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Ensaio de Desvio de Mobilidade Eletroforética , Fator de Crescimento Epidérmico/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Isoxazóis/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêuticoRESUMO
In the present study, the molecular mechanism underlying the up-regulatory effect of estradiol (E2) on mouse insulin receptor substrate-1 (IRS-1) promoter was investigated in CHO cells on which the same promoter had first been functionally characterized. The mouse IRS-1 promoter bears four consensus half Estrogen Responsive Elements (ERE) sequences and thirteen AP-1- and ten Sp1-binding elements. We performed molecular dissection of this promoter gene providing 3' different deleted constructs, containing the same AP-1 rich region with a progressively increased number of ERE half sites located downstream. None of these constructs was responsive to E2, while a downstream region (nt -1420 to -160) rich in GC elements was induced by E2. However, the latter region lost its intrinsic E2 responsiveness when the whole IRS-1 promoter was mutated for deletion in all four ERE half sites. Deletion analysis of the ERE half sites demonstrated that only ERE located at the position -1500 to -1495, close to the GC-rich region, was able to maintain the induced activatory effect of E2 on the IRS-1 gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the region containing the half ERE/Sp1 (nt -1500 to -1477) as the one conferring E2 responsiveness to the whole promoter. This effect occurs through the functional interaction between E2/ERalpha and Sp1.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Animais , Sequência de Bases , Western Blotting , Células CHO , Linhagem Celular Tumoral , Cricetinae , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Proteínas Substratos do Receptor de Insulina , Camundongos , Mutagênese Sítio-Dirigida , Ligação ProteicaRESUMO
We have recently shown that a human CD4+ T cell line (CEM-SS) acquires the permissiveness to M-tropic strains and primary isolates of HIV-1 after transplantation into SCID mice. This permissiveness was associated with the acquisition of a memory (CD45RO+) phenotype as well as of a functional CCR5 coreceptor. In this study, we have used this model for invest-igating in vivo the relationships between HIV-1 infection, apoptosis and T cell differentiation. When an in vivo HIV-1 infection was performed, the CEM cell tumors grew to a lower extent than the uninfected controls. CEM cells explanted from uninfected SCID mice (ex vivo CEM) underwent a significant level of spontaneous apoptosis and proved to be CD45RO+, Fas+ and Fas-L+, while Bcl-2 expression was significantly reduced as compared to the parental cells. Acute HIV-1 infection markedly increased apoptosis of uninfected ex vivo CEM cells, through a Fas/Fas-L-mediated autocrine suicide/fratricide, while parental cells did not undergo apoptosis following viral infection. The susceptibility to apoptosis of ex vivo CEM cells infected with the NSI strain of HIV-1, was progressively lost during culture, in parallel with the loss of Fas-L and marked changes in the Bcl-2 cellular distribution. On the whole, these results are strongly reminiscent of a series of events possibly occurring during HIV-1 infection. After an initial depletion of bystander CD4+ memory T cells during acute infection, latently or chronically infected CD4+ T lymphocytes are progressively selected and are protected against spontaneous apoptosis through the development of an efficient survival program. Studies with human cells passaged into SCID mice may offer new opportunities for an in vivo investigation of the mechanisms involved in HIV-1 infection and CD4+ T cell depletion.
Assuntos
Apoptose , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/patologia , HIV-1/fisiologia , Receptor fas/metabolismo , Animais , Linfócitos T CD4-Positivos/transplante , Linhagem Celular , Humanos , Camundongos , Camundongos SCID , Neoplasias Experimentais/fisiopatologia , FenótipoRESUMO
Although several studies are available on the in vitro inhibitory activities of type I interferon (IFN) on HIV-1 replication, the role of these cytokines in the pathogenesis of AIDS is still matter of conjecture. Both beneficial and adverse effects have been envisaged and considered as a possible rationale for the development of either IFN or anti-IFN therapies in HIV-1-infected patients. In the present study, we have evaluated the efficacy of human type I IFN on HIV-1 infection and virus-induced depletion of human CD4 T cells in two models established in SCID mice. In SCID mice transplanted with human U937 cells (U937-SCID mouse model), continuous treatment with type I consensus IFN (CIFN) resulted in a total suppression of HIV-1 infection. This inhibitory effect was superior to that obtained after AZT treatments. Results from an ensemble of experiments in SCID mice transplanted with either control or genetically modified human U937 cells transduced with a Tat-inducible IFN-alpha gene (LTR-IFN-A2 U937) indicated that low levels of IFN-alpha, produced locally as a result of virus infection, were extremely effective in inhibiting acute HIV infection and virus replication. Of interest, LTR-IFN-A2 U937 cells conferred a strong anti-HIV-1 protection to coinjected bystander U937 cells. Notably, experiments with SCID mice reconstituted with human PBL (hu-PBL-SCID mouse model) showed that treatment with CIFN inhibited HIV-1 replication more effectively than AZT treatment. Remarkably, treatment with CIFN resulted in a clear-cut protection from the virus-induced depletion of human CD4 T cells, which was also associated with the generation of an antibody response toward HIV-1 antigens in 50% of the virus-injected xenografts. These results suggest that type I IFN efficiently preserves human CD4(+) cells from virus-induced damage in hu-PBL-SCID mice, not only by inducing an antiviral state in target cells but also by stimulating anti-HIV-1 human immune responses in vivo.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Interferon Tipo I/farmacologia , Animais , Linfócitos T CD4-Positivos/imunologia , Transplante de Células , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Depleção Linfocítica , Camundongos , Camundongos SCID , Transplante de Neoplasias , Transplante Heterólogo , Células U937 , Replicação Viral/efeitos dos fármacosRESUMO
In a previous study, we had found that the extent of T-cell dysfunctions induced by a T-tropic strain of human immunodeficiency virus type 1 (HIV-1) in SCID mice reconstituted with human peripheral blood lymphocytes (hu-PBLs) (hu-PBL-SCID mice) was related to the in vivo state of activation of the human lymphocytes. In this article, we compared the effect of infection of hu-PBL-SCID mice with either T-tropic (X4) or M-tropic (R5) strains of HIV-1 by performing virus inoculation at either 2 h or 2 weeks after the hu-PBL transfer, when the human T cells exhibited a marked activation state or a predominant memory phenotype, respectively. A comparable level of infection was found when hu-PBL-SCID mice were challenged with either the SF162 R5 or the IIIB X4 strain of HIV at 2 h postreconstitution, while at 2 weeks, the R5 virus infection resulted in a higher level of HIV replication than the X4 virus. The R5 strain induced a marked human CD4(+) T-cell depletion along with a drop in levels of human immunoglobulin M in serum and release of soluble factors at both infection times, while the X4 virus induced severe immune dysfunctions only at 2 h. Of interest, injection of hu-PBLs into SCID mice resulted in a marked up-regulation of CCR5 on human CD4(+) T cells. The percentage of CXCR4(+) cells did not change after transplantation, even though a significant decrease in antigen expression was observed. Comparative experiments with two molecular clones of HIV-1 (X4 SF2 and R5 SF162) and two envelope recombinant viruses generated from these viruses showed that R5 viruses (SF162 and the chimeric env-SF162-SF2) caused an extensive depletion of human CD4(+) T cells in SCID mice at both 2 h and 2 weeks after reconstitution, while the X4 viruses (SF2 and the chimeric env-SF2-SF162) induced CD4 T-cell depletion only when infection was performed at the 2-h reconstitution time. These results emphasize the importance of the state of activation/differentiation of human CD4(+) T cells and gp120-coreceptor interactions at the time of primary infection in determining HIV-1 pathogenicity in the hu-PBL-SCID mouse model.
Assuntos
Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/patogenicidade , Animais , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Modelos Animais de Doenças , HIV-1/isolamento & purificação , Humanos , Imunoglobulina M/imunologia , Depleção Linfocítica , Camundongos , Camundongos SCID , Receptores CCR5/biossíntese , Receptores CXCR4/biossíntese , Proteínas do Envelope Viral/fisiologiaRESUMO
In this article, we show that passage in SCID mice rendered a human CD4(+) T-cell line (CEM cells) highly susceptible to infection by macrophage-tropic (M-tropic) strains and primary clinical isolates of human immunodeficiency virus type 1 (HIV-1). This in vivo-acquired permissiveness of CEM cells was associated with the induction of a CD45RO+ phenotype as well as of some beta-chemokine receptors. Regulated upon activation, normal T-cell expressed and secreted chemokine entirely inhibited the ability of M-tropic HIV-1 strains to infect these cells. These findings may lead to new approaches in investigating in vivo the capacity of different HIV strains to exploit chemokine receptors in relation to the dynamics of the activation and/or differentiation state of human CD4(+) T cells.
Assuntos
Síndrome da Imunodeficiência Adquirida/etiologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Macrófagos/virologia , Receptores CCR5/fisiologia , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/virologia , Animais , Sequência de Bases , Linfócitos T CD4-Positivos/patologia , Diferenciação Celular , Linhagem Celular , Primers do DNA/genética , Feminino , Humanos , Antígenos Comuns de Leucócito/fisiologia , Ativação Linfocitária , Camundongos , Camundongos SCID , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Receptores CCR5/genética , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/fisiologia , Regulação para Cima , Cultura de VírusRESUMO
The cellular response to HIV infection was determined by analysing the expression of cellular proteins in uninfected and HIV-1 infected U937 cells using two-dimensional protein electrophoresis. HIV infected U937 cells constitute a useful model for the study of the chronic productive infection of cells of the monocyte/macrophage lineage by the human immunodeficiency virus. Our data suggest that the expression of 70 proteins is modified following HIV infection: the expression of approximately half of these proteins was found to be increased, while that of the other half was repressed. We estimate that the expression of around fifteen of these proteins was markedly changed following HIV infection. These results suggest that HIV infection results in the modified expression of approximately 0.5% of total cellular proteins. To our knowledge, this study represents the first global quantitative analysis of the cellular response to HIV infection in a model of chronic infection of cells of the monocyte-macrophage lineage.
Assuntos
HIV/fisiologia , Macrófagos/virologia , Monócitos/virologia , Biossíntese de Proteínas , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Humanos , Macrófagos/metabolismo , Monócitos/metabolismoRESUMO
In this study we attempted to develop a new xenochimeric model for HIV infection in SCID mice, characterized by an easy engraftment of target cells, high levels of viremia and long-lasting HIV-1 infection. SCID mice were injected subcutaneously with uninfected human U937 cells and cell-free HIV-1 (IIIB strain) or HIV-1-infected human peripheral blood lymphocytes (PBL). Mice were evaluated for tumor growth, viral infection at the tumor level (DNA-polymerase chain reaction (PCR), RNA-PCR) and immunostaining for the p55/p18 HIV protein) and p24 antigenemia or serum HIV-1 RNA copies. Pretreatment of mice with antibodies to either mouse-IFN alpha/beta or granulocytes resulted in a tumor take and levels of p24 antigenemia higher than in control mice. In mice treated with these antibody preparations, there was a long-lasting HIV infection with the presence of high levels of circulating infectious virus (serum p24 values up to 4000 pg/ml and serum RNA copies up to 5 x 10(7)/ml over 3 months, with the majority of the cells expressing HIV-antigens at the tumor site). Intraperitoneal treatment of SCID mice with AZT (480 mg/kg per day) resulted in a complete inhibition of both p24 and RNA HIV-1 copies in the serum, together with a marked reduction in the number of infected cells and the levels of virus expression at the tumor site. We conclude that some specific features of this model (i.e. easy establishment, high reproducibility, well defined kinetics of virus infection, massive and long persistent viremia) underline the special advantages of its use for testing new antiviral therapies.
Assuntos
Infecções por HIV/terapia , HIV-1/crescimento & desenvolvimento , Modelos Biológicos , Animais , Fármacos Anti-HIV/uso terapêutico , Feminino , Infecções por HIV/virologia , HIV-1/metabolismo , Humanos , Camundongos , Camundongos SCID , Transplante Heterólogo , Células Tumorais Cultivadas , Zidovudina/uso terapêuticoRESUMO
Constitutive expression of IFN-alpha5 and IFN-beta was detected in different lymphoid cells including peripheral blood mononuclear cells from normal individuals following amplification of IFN mRNA by reverse transcriptase-polymerase chain reaction and direct sequencing of the amplified product. The activated form of the interferon-induced transcription factor complex ISGF3 was also detected in nuclear extracts from uninduced cells. Culture supernatants from uninduced U937 cells were also found to activate an ISRE cloned upstream of the luciferase reporter gene, indicating the presence of endogenous IFN activity equivalent to approximately 0.3 to 0.5 IU/mL. This endogenous IFN was also shown to play a role in maintaining the basal level of expression of the major histocompatibility class I genes in lymphoid cells. These results suggest that IFN-alpha5 and IFN-beta are produced at low levels in normal tissues and play an important role in the regulation of cell function and in the maintenance of homeostasis.
Assuntos
Interferon-alfa/biossíntese , Interferon beta/biossíntese , Leucócitos/imunologia , Monócitos/imunologia , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Sequência Consenso , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Genes MHC Classe I , Humanos , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Luciferases/biossíntese , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
In this study, we describe the kinetics of host immune reactions occurring in mice with severe combined immunodeficiency (SCID) at different times after the intraperitoneal injection of human peripheral blood mononuclear leukocytes (huPBL). At 24 hr, a massive neutrophil recruitment and an induced expression of a wide spectrum of murine cytokine mRNA (i.e., interleukin [IL]-1 beta, IL-4, IL-6, IL-10, IL-12, tumor necrosis factor [TNF]-alpha and interferon [IFN]-gamma) occurred in the huPBL-SCID mouse peritoneal cavity. By using ELISAs specific for mouse cytokines, large amounts of IL-1-alpha, TNF-alpha, IL-6, and IFN-gamma were detected in the peritoneal washings of huPBL-SCID mice 1 day after intraperitoneal injection. IL-6 and IFN-gamma production persisted for up to 2 weeks after PBL transplantation. Medullary and extramedullary expansion of the SCID mouse hematopoietic cells also occurred in the chimeras as early as 1 week after injection, together with a marked thymic differentiation (murine CD4+/CD8+ cells) at 10-12 weeks after transplantation. On the whole, these results indicate that, after huPBL injection, SCID mice mount a complex multistage immune response. These host reactions should be taken into consideration for any accurate interpretation of results obtained using the huPBL-SCID model. The control of responses (by means of specific antibodies to murine cytokines and to granulocytes or through the use of anti-inflammatory drugs) may be helpful in improving the engraftment of huPBL in SCID mice and in furthering our knowledge of the T and B cell-independent natural immune reactions.
Assuntos
Citocinas/fisiologia , Hematopoese , Leucócitos/imunologia , Camundongos SCID/imunologia , Cavidade Peritoneal/patologia , Timo/citologia , Animais , Diferenciação Celular , Expressão Gênica , Humanos , Inflamação/patologia , Injeções Intraperitoneais , Camundongos , Neutrófilos/imunologia , RNA Mensageiro/genéticaRESUMO
Low levels of beta interferon (IFN) mRNA are transcribed in freshly explanted murine peritoneal macrophages. Nuclear runoff transcription assays show that this "constitutive" IFN-beta-mRNA transcription does not increase in macrophages treated either with lipopolysaccharide or with IFN-gamma, which induce a marked accumulation of this mRNA and greatly increase IFN secretion. Therefore, these agents promote accumulation of IFN-beta mRNA by posttranscriptional mechanisms. The IFN-alpha 2 gene is also constitutively transcribed by macrophages, but the corresponding mRNA does not accumulate in lipopolysaccharide-treated cells.
Assuntos
Interferon Tipo I/genética , Macrófagos/imunologia , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Animais , Células Cultivadas , Regulação da Expressão Gênica , Interferon gama/farmacologia , Cinética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
The levels of expression of histocompatibility antigens on the cell membrane and their gene expression in non-metastatic and in highly metastatic Friend leukemia cells (FLC) were measured and the levels of expression of these antigens were correlated with the different in vivo behaviour of the tumor cells. Highly metastatic in vivo passaged FLC (either interferon-sensitive 745 or interferon alpha/beta-resistant 3Cl-8 cells) expressed higher levels of class I H-2K and H-2D antigens on their cell membrane with respect to the non-metastatic in vitro passaged counterparts. The increased expression of H-2 class I antigens was associated with an increased transcription of H-2K and H-2D genes. As both in vitro and in vivo passaged FLC have been shown to be resistant in vitro to the natural killer (NK) cell activity, we tried to correlate the levels of expression of histocompatibility antigens with the in vivo clearance of [125I]UDR-labeled FLC. However, no correlation was found between the levels of expression of H-2 antigens and the in vivo clearance of tumor cells. In fact, in vivo passaged FLC (tested either after 1 or after 15 in vitro passages) expressed virtually identical levels of H-2 antigens; however, the freshly explanted in vivo passaged FLC exhibited markedly lower levels of clearance from the lung, spleen and liver (when injected i.v. in DBA/2 mice) with respect to the corresponding FLC cultivated for several passages in vitro. Pretreatment of in vitro passaged 745 FLC with either interferon alpha/beta or interferon gamma resulted in the acquisition of some metastatic potential of FLC to the liver when interferon-treated FLC were subsequently injected i.v. in DBA/2 mice; such in vitro treatments resulted in a 2-3-fold increase in the expression of H-2K antigens versus the control untreated FLC. We suggest that such increases could represent some advantages for the homing properties of tumor cells and/or for the tumor progression, by mechanisms different from the resistance to the NK cells.