Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 43
Filtrar
1.
Int J Cancer ; 135(5): 1028-37, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24474556

RESUMO

Ovarian cancer is the deadliest gynecological malignancy in Western countries. Early detection, however, is hampered by the fact that the origin of ovarian cancer remains unclear. Knowing that in a high percentage of endometrioid ovarian cancers Wnt/ß-catenin signaling is activated, and in view of the hypothesis that ovarian cancer may originate from the distal oviduct, we studied mice in which Wnt/ß-catenin signaling was activated in Müllerian duct-derived tissues. Conditional adenomatous polyposis coli (Apc) knockout mice were used to study the activation of Wnt/ß-catenin signaling in Müllerian duct-derived organs. These Pgr(Cre/+);Apc(ex15lox/lox) mice (n = 44) were sacrificed at 10, 20, 40 and 80 weeks and uterus, oviduct, ovaries and surrounding fat tissues were assessed using immunohistochemistry. Using nuclear ß-catenin staining, Wnt/ß-catenin signaling activation was confirmed in the entire epithelium of the adult Müllerian duct (fimbriae, oviduct and endometrium), but was absent in ovarian surface epithelium cells (OSEs). Besides endometrial hyperplasia, in 87.2% of mice intraepithelial lesions of the distal oviduct were found, whereas OSEs remained unaffected. In addition, 62.5% of mice developed tumors in the distal and fimbrial part of the oviduct. In the ovaries, mainly at young age, in 16.3% of mice, simple epithelial cysts were noted, which developed further into endometrioid ovarian tumors, resembling human endometrioid ovarian cancer (27.9% of mice). Next to this, locoregional growth in the utero-ovarian ligament was also shown. Here, for the first time, mutations (activation of Wnt/ß-catenin) in the distal oviduct result in precursor lesions that develop into ovarian tumors, resembling human endometrioid ovarian cancer.


Assuntos
Carcinoma Endometrioide/patologia , Modelos Animais de Doenças , Tubas Uterinas/patologia , Camundongos , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Via de Sinalização Wnt/genética , Polipose Adenomatosa do Colo/genética , Animais , Carcinoma Endometrioide/genética , Carcinoma Epitelial do Ovário , Hiperplasia Endometrial/patologia , Endométrio/patologia , Células Epiteliais/patologia , Feminino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
2.
J Pathol ; 230(1): 48-58, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23288720

RESUMO

Endometrioid endometrial cancer arises through a gradual series of histological changes, each accompanied by specific alterations in gene expression and activity. Activation of the Wnt-ß-catenin pathway and loss of PTEN activity are frequently observed in endometrial cancers. However, the specific roles played by alterations in these pathways in the initiation and progression of endometrial cancer are currently unclear. Here, we investigated the effects of loss of Pten and Apc gene function in the mouse endometrium by employing tissue-specific and inducible mutant alleles, followed by immunohistochemical (IHC) and loss of heterozygosity (LOH) analysis of their corresponding cancerous lesions. Loss of the Apc function in the endometrium leads to cytoplasmic and nuclear ß-catenin accumulation in association with uterine hyperplasia and squamous cell metaplasia, but without malignant transformation. Loss of Pten function also resulted in squamous metaplasia but, in contrast to loss of Apc function, it initiates endometrial cancer. On the other hand, loss of Apc function in the endometrium accelerates Pten-driven endometrial tumourigenesis. Analysis of compound heterozygous mice confirmed that somatic loss of the wild-type Pten allele represents the rate-limiting initiation step in endometrial cancer. Simultaneous loss of Pten and Apc resulted in endometrial cancer characterized by earlier onset and a more aggressive malignant behaviour. These observations are indicative of the synergistic action between the Wnt-ß-catenin and Pten signalling pathways in endometrial cancer onset and progression.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias do Endométrio/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Carcinoma de Células Escamosas/patologia , Progressão da Doença , Neoplasias do Endométrio/patologia , Feminino , Deleção de Genes , Inativação Gênica , Genes APC/fisiologia , Perda de Heterozigosidade/genética , Masculino , Camundongos , Camundongos Knockout , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Útero/anormalidades , Útero/metabolismo
3.
Int J Cancer ; 130(12): 2874-85, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21815142

RESUMO

Human papillomavirus (HPV) infections may result in benign hyperplasia, caused by low-risk HPV types, or (pre)malignant lesions caused by high-risk HPV types. The molecular basis of this difference in malignant potential is not completely understood. Here, we performed gene profiling of different HPV infected vulvar tissues (condylomata acuminata (n = 5), usual type vulvar intraepithelial neoplasia (uVIN) (n = 9)) and control samples (n = 14) using Affymetrix Human U133A plus 2 GeneChips. Data were analyzed using OmniViz®, Partek® and Ingenuity® Software. Results were validated by real-time RT-PCR and immunostaining. Although similarities were observed between gene expression profiles of low- and high-risk HPV infected tissues (e.g., absence of estrogen receptor in condylomata and uVIN), high-risk HPV infected tissues showed more proliferation and displayed more DNA damage than tissues infected with low-risk HPV. These observations were confirmed by differential regulation of cell cycle checkpoints and by increased expression of DNA damage-biomarkers p53 and γH2AX. Furthermore, FANCA, FANCD2, BRCA1 and RAD51, key players in the DNA damage response, were significantly upregulated (p < 0.05). In addition, we compared our results with publicly available gene expression profiles of various other HPV-induced cancers (vulva, cervix and head-and-neck). This showed p16(INK4a) was the most significant marker to detect a high-risk HPV infection, but no other markers could be found. In conclusion, this study provides insight into the molecular basis of low- and high-risk HPV infections and indicates two main pathways (cell cycle and DNA damage response) that are much stronger affected by high-risk HPV as compared to low-risk HPV.


Assuntos
Alphapapillomavirus , Pontos de Checagem do Ciclo Celular , Dano ao DNA , Reparo do DNA , Infecções por Papillomavirus/genética , Vulva/patologia , Doenças da Vulva/genética , Proteína BRCA1/biossíntese , Biomarcadores Tumorais , Condiloma Acuminado/genética , Condiloma Acuminado/metabolismo , Condiloma Acuminado/patologia , Condiloma Acuminado/virologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , DNA Viral/análise , DNA Viral/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/biossíntese , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/biossíntese , Feminino , Perfilação da Expressão Gênica , Histonas/biossíntese , Humanos , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Rad51 Recombinase/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Vulva/virologia , Doenças da Vulva/patologia , Doenças da Vulva/virologia
4.
Int J Cancer ; 128(10): 2463-9, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-20658529

RESUMO

Imiquimod has been shown to be an effective treatment for usual type vulvar intraepithelial neoplasia (uVIN). Since local inflammation and burning are common side effects, patients often use nonsteroidal anti-inflammatory drugs (NSAIDs). Our study investigated whether NSAID-use, which has been documented to inhibit the cell-mediated immune response, interferes with the outcome of imiquimod treatment. Monocyte-derived dendritic cells (moDCs) and Langerhans cells (moLCs) were cultured in the presence of NSAIDs. The expression of relevant surface markers (CD80, CD86, CD40, HLA-DR, CCR6 and CCR7), stimulatory function, and cytokine production were evaluated. Furthermore, we analyzed in uVIN patients whether frequent NSAID-use had an effect on the clinical response and on immunocompetent cell counts before and after imiquimod treatment. Although an effect was observed on the expression of moDC and moLC maturation markers, NSAIDs did not affect the ability of moDCs and moLCs to stimulate allogeneic T-cell proliferation, or the production of cytokines in an allogeneic T-cell stimulation assay. In agreement with this, in uVIN patients treated with imiquimod, no interference of frequent NSAID-use with clinical outcome was observed. However, we did notice that high CD1a(+) and CD207(+) cell counts in frequent NSAID-users before treatment seemed to predict a favourable response to imiquimod treatment. Our data indicate that NSAID-use does not seem to interfere with moDC and moLC function and does not interfere with immunomodulatory properties of imiquimod in uVIN patients. Therefore, NSAIDs can safely be used to reduce imiquimod side effects in uVIN patients during treatment.


Assuntos
Aminoquinolinas/uso terapêutico , Anti-Inflamatórios não Esteroides/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma in Situ/tratamento farmacológico , Neoplasias Vulvares/tratamento farmacológico , Biópsia , Carcinoma in Situ/imunologia , Separação Celular , Interações Medicamentosas , Feminino , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Imiquimode , Imuno-Histoquímica , Neoplasias Vulvares/imunologia
5.
Gynecol Oncol ; 121(1): 157-62, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21239049

RESUMO

OBJECTIVE: Recently we reported on the efficacy of imiquimod for treating vulvar intraepithelial neoplasia (VIN) in a placebo-controlled, double-blinded randomized clinical trial (RCT). Four weeks after treatment, a complete response was observed in 35% of patients and a partial response in 46%. All complete responders remained disease-free at 12 months follow-up. In the current investigations, we assessed long-term follow-up at least 5 years after the initial RCT. METHODS: Twenty-four of 26 imiquimod-treated patients who had participated in the initial RCT were seen for follow-up. Primary endpoint was durability of clinical response to imiquimod assessed by naked eye vulvar examination and histology. Long-term clinical response was correlated to lesion size before start of the initial RCT. Secondary endpoints were mental health, global quality of life, body image and sexual function in relation with long-term clinical response. RESULTS: Median follow-up period was 7.2 years (range 5.6-8.3 years). VIN recurred in one of nine complete responders. Of the initial partial responders, two became disease-free after additional imiquimod treatment. In the other partial responders, VIN recurred at least once after the initial RCT. In long-term complete responders, lesion size at study entry was smaller and these patients had a significantly better global quality of life at follow-up than patients with residual disease and/or recurrence after imiquimod treatment. CONCLUSIONS: In case of a complete response, imiquimod is effective in the long-term. Furthermore, patients with a long-term complete response had a significantly better global quality of life than patients who recurred after imiquimod treatment.


Assuntos
Aminoquinolinas/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma in Situ/tratamento farmacológico , Neoplasias Vulvares/tratamento farmacológico , Adulto , Imagem Corporal , Carcinoma in Situ/patologia , Carcinoma in Situ/psicologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/psicologia , Progressão da Doença , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Imiquimode , Pessoa de Meia-Idade , Invasividade Neoplásica , Qualidade de Vida , Sexualidade , Neoplasias Vulvares/patologia , Neoplasias Vulvares/psicologia
6.
Int J Cancer ; 127(12): 2831-40, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21351262

RESUMO

Recently, we reported on the efficacy of imiquimod for treatment of usual type vulvar intraepithelial neoplasia (uVIN). A histologic regression of uVIN to normal tissue was observed in 58% of patients. As success of treatment is related to clearance of high-risk human papilloma virus (HPV), the aim of our study was to assess differences in immune cell counts and in the expression of p16(INK4a) in VIN tissue before and after imiquimod treatment, in relation to HPV clearance and clinical response. Vulvar tissue samples taken prior to imiquimod treatment and 4 weeks after treatment were tested for the presence of HPV. Previously determined immune cell counts (CD1a, CD207, CD208, CD123/CD11c, CD94, CD4, CD8 and CD25/HLA-DR) in epidermis and dermis of 25 VIN patients and 19 healthy controls were completed with the counts for CD14 and CD68. The expression of p16(INK4a) was investigated by immunohistochemistry in 15 patients. Before imiquimod treatment, both HPV cleared and HPV noncleared patients showed mainly in the dermis significantly upregulated immune cell counts compared to healthy controls. However, in patients that cleared HPV and showed histologic regression already 4 weeks after imiquimod treatment, immune cell counts and p16(INK4a) expression were normalized. In conclusion, our data indicate that imiquimod-induced clearance of HPV results in normalization of counts for certain immune cells and is strongly correlated with histologic regression of the disease.


Assuntos
Aminoquinolinas/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma in Situ/imunologia , Contagem de Linfócitos , Papillomaviridae/efeitos dos fármacos , Infecções por Papillomavirus/imunologia , Neoplasias Vulvares/imunologia , Adulto , Biomarcadores Tumorais/metabolismo , Carcinoma in Situ/tratamento farmacológico , Carcinoma in Situ/virologia , Estudos de Casos e Controles , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , DNA Viral/genética , Feminino , Humanos , Imiquimode , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Infecções por Papillomavirus/tratamento farmacológico , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase , Prognóstico , Neoplasias Vulvares/tratamento farmacológico , Neoplasias Vulvares/virologia , Adulto Jovem
7.
Acta Obstet Gynecol Scand ; 89(6): 741-8, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20504079

RESUMO

No standard screening programs exist to detect vulvar carcinoma or its precursor lesions, and therefore gynecologists, dermatologists and other healthcare providers in this field should be aware of the clinical features, behavior and management of the different existing premalignant vulvar lesions, squamous vulvar intraepithelial neoplasia (VIN), vulvar Paget's disease and melanoma in situ. In 2004, a new classification for squamous VIN was introduced by the International Society for the Study of Vulvar Disease, subdividing squamous VIN into the HPV-related usual type, and into differentiated type, which is associated with lichen sclerosus. This review describes the relevant aspects of squamous VIN, vulvar Paget's disease and melanoma in situ, its epidemiological characteristics, diagnosis, management and malignant potential.


Assuntos
Carcinoma in Situ/diagnóstico , Melanoma/diagnóstico , Doença de Paget Extramamária/diagnóstico , Lesões Pré-Cancerosas/diagnóstico , Neoplasias Cutâneas/diagnóstico , Neoplasias Vulvares/diagnóstico , Carcinoma in Situ/epidemiologia , Carcinoma in Situ/terapia , Epitélio/patologia , Feminino , Humanos , Melanoma/epidemiologia , Melanoma/terapia , Doença de Paget Extramamária/epidemiologia , Doença de Paget Extramamária/terapia , Lesões Pré-Cancerosas/epidemiologia , Lesões Pré-Cancerosas/terapia , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/terapia , Neoplasias Vulvares/epidemiologia , Neoplasias Vulvares/terapia
8.
Endocrinology ; 149(4): 1942-50, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18096667

RESUMO

In many type I endometrial cancers, the PTEN gene is inactivated, which ultimately leads to constitutively active Akt and the inhibition of Forkhead box O1 (FOXO1), a member of the FOXO subfamily of Forkhead/winged helix family of transcription factors. The expression, regulation, and function of FOXO1 in endometrial cancer were investigated in this study. Immunohistochemical analysis of 49 endometrial tumor tissues revealed a decrease of FOXO1 expression in 95.9% of the cases compared with the expression in normal endometrium. In four different endometrial cancer cell lines (ECC1, Hec1B, Ishikawa, and RL95), FOXO1 mRNA was expressed at similar levels; however, protein levels were low or undetectable in Ecc1, Ishikawa, and RL95 cells. Using small interfering RNA technology, we demonstrated that the low levels of FOXO1 protein were due to the involvement of Skp2, an oncogenic subunit of the Skp1/Cul1/F-box protein ubiquitin complex, given that silencing Skp2 increased FOXO1 protein expression in Ishikawa cells. Inhibition of Akt in Ishikawa cells also increased nuclear FOXO1 protein levels. Additionally, progestins increased FOXO1 protein levels, specifically through progesterone receptor B (PRB) as determined by using stably transfected PRA-specific and PRB-specific Ishikawa cell lines. Finally, overexpression of triple mutant (Tm) FOXO1 in the PR-specific Ishikawa cell lines caused cell cycle arrest and significantly decreased proliferation in the presence and absence of the progestin, R5020. Furthermore, TmFOXO1 overexpression induced apoptosis in PRB-specific cells in the presence and absence of ligand. Taken together, these data provide insight into the phosphoinositide-3-kinase/Akt/FOXO pathway for the determination of progestin responsiveness and the development of alternate therapies for endometrial cancer.


Assuntos
Neoplasias do Endométrio/metabolismo , Fatores de Transcrição Forkhead/fisiologia , Receptores de Progesterona/fisiologia , Linhagem Celular , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/patologia , Endométrio/química , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/análise , Humanos , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptores de Progesterona/análise , Proteínas Quinases Associadas a Fase S/fisiologia
9.
Int J Cancer ; 123(3): 616-22, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18498128

RESUMO

Usual type VIN is a premalignant disorder caused by persistent HPV infection. High prevalence of VIN in immuno-suppressed women suggests that a good innate and adaptive immune response is important for defense against HPV. Here, we explored expression levels of chemokines and related these to the presence or absence of immuno-competent cells (dendritic and T-cells) in affected (HPV-positive VIN) and non-affected (HPV-negative) vulvar tissues from the same patients. Combining microarray data with quantitative real-time RT-PCR, it was observed that several important chemokines were differentially expressed between VIN and control samples (up-regulation of IL8, CXCL10, CCL20 and CCL22 and down-regulation of CXCL12, CCL21 and CCL14). Furthermore, an increased number of mature dendritic cells (CD208+) seemed to be bottled up in the dermis, and although a T-cell response (increased CD4+ and CD8+ cells) was observed in VIN, a much larger response is required to clear the infection. In summary, it seems that most mature dendritic cells do not receive the proper chemokine signal for migration and will stay in the dermis, not able to present viral antigen to naive T-cells in the lymph node. Consequently the adaptive immune response diminishes, resulting in a persistent HPV infection with increased risk for neoplasia.


Assuntos
Alphapapillomavirus , Quimiocinas/metabolismo , Células Dendríticas/imunologia , Hospedeiro Imunocomprometido , Infecções por Papillomavirus/complicações , Linfócitos T/imunologia , Infecções Tumorais por Vírus/complicações , Displasia do Colo do Útero/imunologia , Neoplasias do Colo do Útero/imunologia , Adulto , Quimiocinas/genética , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Análise em Microsséries , Pessoa de Meia-Idade , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/virologia , Regulação para Cima , Neoplasias do Colo do Útero/virologia , Displasia do Colo do Útero/virologia
10.
Hum Reprod ; 23(2): 298-305, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18077316

RESUMO

BACKGROUND: Combined hormone treatments in post-menopausal women have different clinical responses on uterus and vagina; therefore, we investigated differences in steroid signalling between various hormone therapies in these tissues. METHODS: A total of 30 post-menopausal women scheduled for hysterectomy were distributed into four subgroups: control-group (n = 9), Tibolone-group (n = 8); estradiol (E(2))-group (n = 7); E(2) + medroxyprogesterone acetate (MPA)-group (n = 6). Medication was administered orally every day for 21 days prior to removal of uterus and upper part of the vagina. Tissue RNA was isolated, and gene expression profiles were generated using GeneChip technology and analysed by cluster analysis and significance analysis of microarrays. Apoptosis and cell proliferation assays, as well as immunohistochemistry for hormone receptors were performed. RESULTS: 21-days of treatment with E(2), E(2) + MPA or tibolone imposes clear differential gene expression profiles on endometrium and myometrium. Treatment with E(2) only results in the most pronounced effect on gene expression (up to 1493 genes differentially expressed), proliferation and apoptosis. Tibolone, potentially metabolized to both estrogenic and progestagenic metabolites, shows some resemblance to E(2) signalling in the endometrium and, in contrast, shows significant resemblance to E(2) + MPA signalling in the myometrium. In the vagina the situation is entirely different; all three hormonal treatments result in regulation of a small number (4-73) of genes, in comparison to signalling in endometrium and myometrium. CONCLUSION: Endometrium and myometrium differentially respond to the hormone therapies and use completely different sets of genes to regulate similar biological processes, while in this experiment the upper part of the vagina is hardly hormone responsive.


Assuntos
Endométrio/metabolismo , Terapia de Reposição de Estrogênios , Miométrio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vagina/metabolismo , Análise por Conglomerados , Quimioterapia Combinada , Estradiol/uso terapêutico , Feminino , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Acetato de Medroxiprogesterona/uso terapêutico , Norpregnenos/uso terapêutico
11.
J Steroid Biochem Mol Biol ; 109(3-5): 219-23, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18434135

RESUMO

Tamoxifen is used as adjuvant treatment for postmenopausal breast cancer patients. The mechanism of action of tamoxifen in breast cancer patients is that tamoxifen inhibits growth of cancer cells by competitive antagonism for estrogens at the estrogen receptor (ER). In the endometrium, tamoxifen has an effect that varies with the ambient concentration of estrogen: in premenopausal women (high estrogen levels), tamoxifen displays an estrogen-antagonistic effect, while in postmenopausal women (low estrogen levels), tamoxifen displays an estrogen-agonistic mode of action. Here, using microarray technology we have compared estrogen signaling with tamoxifen signaling in the human endometrium. It was observed that on the one hand tamoxifen-treatment results in modulation of expression of specific genes (370 genes) and on the other hand tamoxifen-treatment results in modulation of a set of genes which are also regulated by estrogen treatment (142 genes). Upon focusing on regulation of proliferation, we found that tamoxifen-induced endometrial proliferation is largely accomplished by using the same set of genes as are regulated by estradiol. So, as far as regulation of proliferation goes, tamoxifen seems to act as estrogen agonist. Furthermore, tamoxifen-specific gene regulation may explain why tamoxifen-induced endometrial tumors behave more aggressively than sporadic endometrial tumors.


Assuntos
Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Estrogênios/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologia , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Pólipos/patologia
12.
Birth Defects Res A Clin Mol Teratol ; 82(9): 627-35, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18655124

RESUMO

BACKGROUND: A cleft of the lip with or without the palate (CLP) is a frequent congenital malformation with a heterogeneous etiology, for which folic acid supplementation has a protective effect. To gain more insight into the molecular pathways affected by natural folate, we examined gene expression profiles of cultured B-lymphoblasts from CLP patients before and after the addition of 5-methyltetrahydrofolate (5-mTHF) to the cultures. METHODS: Immortalized B-lymphoblasts from five children with CLP were cultured in folate-deficient medium for 5 days. 5-mTHF was added to a concentration of 30 nM. Gene expression patterns were then evaluated before and after supplementation using Human Genome U133 Plus 2.0 arrays. Data analysis was performed with Omniviz and the GEPAS analysis suite. Differential genes were categorized into biological pathways with Ingenuity Pathway systems. Differential expression was validated by quantitative RT-PCR. RESULTS: Using supervised clustering, with a false discovery rate <1%, we identified 144 and 409 significantly up-regulated and down-regulated probesets, respectively, after 5-mTHF addition. The regulated genes were involved in a variety of biological pathways, including one carbon pool and cell cycle regulation, biosynthesis of amino acids and DNA/RNA nucleotides, protein processing, apoptosis, and DNA repair. CONCLUSIONS: The large variety of the identified folate responsive pathways fits with the modifying role of folate via the methylation pathway. From the present data we may conclude that folate deficiency deranges normal cell development, which might contribute to the development of CLP. The role of these folate responsive genes in CLP development is intriguing and needs further investigation.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Ácido Fólico/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genoma Humano/fisiologia , Transdução de Sinais/genética , Linhagem Celular Transformada , Criança , Fenda Labial/metabolismo , Fissura Palatina/metabolismo , Regulação para Baixo/genética , Feminino , Humanos , Masculino , Projetos Piloto , Regulação para Cima/genética
13.
Mol Endocrinol ; 20(9): 2122-40, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16645042

RESUMO

Modulators of cofactor recruitment by nuclear receptors are expected to play an important role in the coordination of hormone-induced transactivation processes. To identify such factors interacting with the N-terminal domain (NTD) of the progesterone receptor (PR), we used this domain as bait in the yeast Sos-Ras two-hybrid system. cDNAs encoding the C-terminal MYST (MOZ-Ybf2/Sas3-Sas2-Tip60 acetyltransferases) domain of HBO1 [histone acetyltransferase binding to the origin recognition complex (ORC) 1 subunit], a member of the MYST acetylase family, were thus selected from a human testis cDNA library. In transiently transfected CV1 cells, the wild-type HBO1 [611 amino acids (aa)] enhanced transcription mediated by steroid receptors, notably PR, mineralocorticoid receptor, and glucocorticoid receptor, and strongly induced PR and estrogen receptor coactivation by steroid receptor coactivator 1a (SRC-1a). As assessed by two-hybrid and glutathione-S-transferase pull-down assays, the HBO1 MYST acetylase domain (aa 340-611) interacts mainly with the NTD, and also contacts the DNA-binding domain and the hinge domains of hormone-bound PR. The HBO1 N-terminal region (aa 1-340) associates additionally with PR ligand-binding domain (LBD). HBO1 was found also to interact through its NTD with SRC-1a in the absence of steroid receptor. The latter coassociation enhanced specifically activation function 2 activation function encompassed in the LBD. Conversely, the MYST acetylase domain specifically enhanced SRC-1 coupling with PR NTD, through a hormone-dependent mechanism. In human embryonic kidney 293 cells expressing human PRA or PRB, HBO1 raised selectively an SRC-1-dependent response of PRB but failed to regulate PRA activity. We show that HBO1 acts through modification of an LBD-controlled structure present in the N terminus of PRB leading to the modulation of SRC-1 functional coupling with activation function 3-mediated transcription. Importantly, real-time RT-PCR analysis also revealed that HBO1 enhanced SRC-1 coactivation of PR-dependent transcription of human endogenous genes such as alpha-6 integrin and 11beta-hydroxydehydrogenase 2 but not that of amphiregulin. Immunofluorescence and confocal microscopy of human embryonic kidney-PRB cells demonstrated that the hormone induces the colocalization of HBO1 with PR-SRC-1 complex into nuclear speckles characteristic of PR-mediated chromatin remodeling. Our results suggest that HBO1 might play an important physiological role in human PR signaling.


Assuntos
Histona Acetiltransferases/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Receptores de Progesterona/metabolismo , Transcrição Gênica/genética , Ativação Transcricional/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Hormônios/metabolismo , Humanos , Ligantes , Coativador 1 de Receptor Nuclear , Complexo de Reconhecimento de Origem/genética , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de Progesterona/genética , Receptores de Esteroides/metabolismo , Transdução de Sinais , Fatores de Transcrição
14.
Oncogene ; 23(33): 5607-15, 2004 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-15184881

RESUMO

The protein REPS2 is implicated in growth factor receptor-mediated endocytosis and signalling, and its expression is downregulated in androgen-independent prostate cancer cells. Herein, the NF-kappaB subunit p65 is identified as a human REPS2 protein partner, interacting with the EH domain of REPS2. Using crystal structure data from literature and experimental data from yeast and mammalian two-hybrid analysis, the results indicate that the NPF-motif in p65 acts as binding site for the EH domain in REPS2. However, in cultured prostate cancer cells, the REPS2-p65 interaction is triggered upon stimulation with phorbol ester (PMA). This indicates that PMA-sensitive signalling pathways can affect the interaction between REPS2 and p65. During prostate cancer progression from androgen-dependent to androgen-independent growth, downregulation of REPS2 is accompanied by upregulation of NF-kappaB activity. This might involve loss of REPS2-p65 interaction, which would lead to increased NF-kappaB activity. Androgen-deprivation causes apoptosis of prostate cancer cells, and activated NF-kappaB is a known inhibitor of apoptosis. Hence, decreased expression of REPS2 might be a key factor, causing prostate cancer cells to become resistant to induction of apoptosis by androgen deprivation.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , NF-kappa B/metabolismo , Neoplasias da Próstata/enzimologia , Proteínas/fisiologia , Animais , Células COS , Proteínas de Ligação ao Cálcio , Chlorocebus aethiops , Humanos , Masculino , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição RelA/metabolismo , Triptofano/fisiologia
15.
Oncogene ; 22(19): 2920-5, 2003 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-12771942

RESUMO

During progression of prostate cancer, cellular changes occur, leading to a transition from androgen-dependent to androgen-independent growth. One aspect of this transition is a switch from androgens to growth factors, like epidermal growth factor (EGF), as primary regulators of proliferation. We examined the involvement of REPS2/POB1 in this process. REPS2/POB1 is an EH domain-containing protein, reported to be involved in signalling via RalBP1 and to play a role in endocytosis of EGF receptors. Furthermore, the protein is relatively highly expressed in androgen-dependent as compared to androgen-independent human prostate cancer cell lines and xenografts. Next to the known REPS2/POB1 protein, an open reading frame encoding REPS2/POB1, with 139 additional amino-acid residues at the NH(2)-terminus, was cloned and found to be expressed in prostate cancer cells. Overexpression, by transient transfection, of both forms of REPS2/POB1 in prostate cancer cell lines, induced apoptosis within 48 h. At shorter time intervals after transfection, signalling towards a TPA response element luciferase reporter was found to be inhibited. From these experiments, it is concluded that REPS2/POB1, through its influence on the Ral signalling pathway, is involved in growth factor signalling. Decreased expression of REPS2/POB1 during progression of prostate cancer may therefore result in loss of control of growth factor signalling and consequently in loss of control of cell proliferation.


Assuntos
Proteínas de Transporte/metabolismo , Regulação para Baixo , Peptídeos e Proteínas de Sinalização Intracelular , Fosfoproteínas/metabolismo , Neoplasias da Próstata/metabolismo , Apoptose/genética , Sequência de Bases , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/genética , Biblioteca Gênica , Substâncias de Crescimento/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Fosfoproteínas/genética , Neoplasias da Próstata/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Células Tumorais Cultivadas
16.
Endocr Relat Cancer ; 12(1): 135-48, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15788645

RESUMO

Prostate cancer development often includes a shift from androgen-dependent to androgen-independent growth. It is hypothesized that, during this transition, growth factors like the epidermal growth factor (EGF) gain importance as activators of tumour cell proliferation. To study this, androgen- and EGF-regulation of growth and gene-expression was analysed in the androgen-dependent human prostate cancer cell line LNCaP-FGC (FGC) and its androgen-independent derivative line LNCaP-LNO (LNO). It was observed that androgen-dependent FGC cells require exposure to either androgens or EGF to proliferate. This is in contrast to androgen-independent LNO cells that showed significant proliferation in medium depleted of androgens and growth factors. Gene expression data were obtained for the androgen-dependent FGC and androgen-independent LNO cells cultured in the presence or absence of androgens (synthetic R1881) or EGF for different time periods. Expression profiling showed that many cell cycle genes, including a number of androgen- and EGF-regulated genes, are constitutively activated in androgen-independent LNO cells. Furthermore, the overlap between changes in gene expression activated by androgen and EGF receptor signalling pathways was found to be very high (75%). These results partly explain why androgen-independent LNO cells can proliferate in the absence of androgenic stimulation. However, possibly other, so far unknown, signal transduction pathways that induce and maintain proliferation, have also been activated.


Assuntos
Androgênios/farmacologia , Biomarcadores Tumorais/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias da Próstata/metabolismo , Transdução de Sinais , Perfilação da Expressão Gênica , Humanos , Masculino , Neoplasias Hormônio-Dependentes/genética , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/genética , Células Tumorais Cultivadas
17.
J Soc Gynecol Investig ; 12(1): 58-64, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15629674

RESUMO

OBJECTIVES: Estrogen-stimulated proliferation of the normal and malignant human endometrium is balanced by the differentiating properties of progesterone. This study evaluated the role of insulin-like growth factor (IGF) signaling in steroid-induced modulation of endometrial cancer cell proliferation. METHODS: We used the human endometrial, estrogen-responsive ECC-1 and progesterone-responsive PRAB-36 cell lines. Proliferation studies with IGFs in combination with either estrogen or progesterone were conducted. Furthermore, the mRNA and protein expression of insulin-like growth factor-binding proteins (IGFBPs) was evaluated. RESULTS: Using the ECC-1 cell line, we observed that estrogen-induced proliferation is modulated via the IGF-receptor signaling pathway, and that IGF-1-induced stimulation of proliferation does not influence estrogen receptor signaling. Furthermore, expression of the main modulators of IGF action, the IGFBPs, was found to be regulated by estrogen and progesterone in both cell lines. IGFBP-4 was up-regulated by estrogen in the ECC-1 cell line, and IGFBP-3 and IGFBP-6 were down-regulated by progesterone in the PRAB-36 cell line. CONCLUSION: Estrogen-induced stimulation of proliferation of ECC-1 endometrial cancer cells is partly achieved via IGF signaling. Furthermore, the IGFBPs are regulated by estrogens as well as progestagens and could potentially play a role in the modulation of endometrial cancer cell proliferation.


Assuntos
Carcinoma/patologia , Proliferação de Células , Neoplasias do Endométrio/patologia , Estrogênios/farmacologia , Somatomedinas/farmacologia , Feminino , Humanos , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Transdução de Sinais , Células Tumorais Cultivadas , Regulação para Cima
18.
Clin Cancer Res ; 9(11): 4190-9, 2003 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-14519645

RESUMO

PURPOSE: In endometrial cancer, loss of progesterone receptors (PR) is associated with more advanced disease. This study aimed to investigate the mechanism of action of progesterone and the loss of its receptors (PRA and PRB) in development of endometrial cancer. EXPERIMENTAL DESIGN: A 9600-cDNA microarray analysis was performed to study regulation of gene expression in the human endometrial cancer subcell line Ishikawa PRAB-36 by the progestagen medroxy progesterone acetate (MPA). Five MPA-regulated genes were selected for additional investigation. Expression of these genes was studied by Northern blot and by immunohistochemistry in Ishikawa subcell lines expressing different PR isoforms. Additionally, endometrial cancer tissue samples were immunohistochemically stained to study the in vivo protein expression of the selected genes. RESULTS: In the PRAB-36 cell line, MPA was found to regulate the expression of a number of invasion- and metastasis-related genes. On additional investigation of five of these genes (CD44, CSPG/Versican, Tenascin-C, Fibronectin-1, and Integrin-beta 1), it was observed that expression and progesterone regulation of expression of these genes varied in subcell lines expressing different PR isoforms. Furthermore, in advanced endometrial cancer, it was shown that loss of expression of both PR and E-cadherin was associated with increased expression CD44 and CSPG/Versican. CONCLUSION: The present study shows that progestagens exert a modulatory effect on the expression of genes involved in tumor cell invasion. As a consequence, loss of PR expression in human endometrial cancer may lead to development of a more invasive phenotype of the respective tumor.


Assuntos
Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Regulação Neoplásica da Expressão Gênica/genética , Receptores de Progesterona/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Proteoglicanas de Sulfatos de Condroitina/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/genética , Lectinas Tipo C , Acetato de Medroxiprogesterona/farmacologia , Invasividade Neoplásica/genética , Proteínas de Neoplasias/genética , Tenascina/genética , Versicanas
19.
Biochem J ; 383(Pt 2): 267-76, 2004 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-15239671

RESUMO

Phosphorylation of the human AR (androgen receptor) is directly correlated with the appearance of at least three AR isoforms on an SDS/polyacrylamide gel. However, it is still not clear to what extent phosphorylation is involved in the occurrence of isoforms, which sites are phosphorylated and what are the functions of these phosphosites. The human AR was expressed in COS-1 cells and AR phosphorylation was studied further by mutational analyses and by using reversed-phase HPLC and MS. The reversed-phase HPLC elution pattern of the three isoforms revealed that Ser-650 was phosphorylated constitutively. After de novo synthesis, only Ser-650 was phosphorylated in the smallest isoform of 110 kDa and both Ser-650 and Ser-94 were phosphorylated in the second isoform of 112 kDa. The hormone-induced 114 kDa isoform shows an overall increase in phosphorylation of all the isolated peptides. The activities of the Ser-Ala substitution mutant S650A (Ser-650-->Ala) was found to be identical with wild-type AR activation in four different cell lines and three different functional analyses, e.g. transactivation, N- and C-terminal-domain interaction and co-activation by transcriptional intermediary factor 2. This was also found for mutants S94A and S515A with respect to transactivation. However, the S515A mutation, which should eliminate phosphorylation of the potential mitogen-activated protein kinase site, Ser-515, resulted in an unphosphorylated form of the peptide containing Ser-650. This suggests that Ser-515 can modulate phosphorylation at another site. The present study shows that the AR isoform pattern from AR de novo synthesis is directly linked to differential phosphorylation of a distinct set of sites. After mutagenesis of these sites, no major change in functional activity of the AR was observed.


Assuntos
Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Animais , Células COS , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Metribolona/farmacologia , Peso Molecular , Mutagênese Sítio-Dirigida , Mutação , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Androgênicos/genética , Transcrição Gênica
20.
Cancer Lett ; 356(2 Pt B): 434-42, 2015 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-25304370

RESUMO

Endometrial cancer is the leading gynecologic cancer in women in the United States with 52,630 women predicted to be diagnosed with the disease in 2014. The objective of this study was to determine if progesterone (P4) receptor membrane component 1 (PGRMC1) influenced endometrial cancer cell viability in response to chemotherapy in vitro and in vivo. A lentiviral-based shRNA knockdown approach was used to generate stable PGRMC1-intact and PGRMC1-deplete Ishikawa endometrial cancer cell lines that also lacked expression of the classical progesterone receptor (PGR). Progesterone treatment inhibited mitosis of PGRMC1-intact, but not PGRMC1-deplete cells, suggesting that PGRMC1 mediates the anti-mitotic actions of P4. To test the hypothesis that PGRMC1 attenuates chemotherapy-induced apoptosis, PGRMC1-intact and PGRMC1-deplete cells were treated in vitro with vehicle, P4 (1 µM), doxorubicin (Dox, 2 µg/ml), or P4 + Dox for 48 h. Doxorubicin treatment of PGRMC1-intact cells resulted in a significant increase in cell death; however, co-treatment with P4 significantly attenuated Dox-induced cell death. This response to P4 was lost in PGRMC1-deplete cells. To extend these observations in vivo, a xenograft model was employed where PGRMC1-intact and PGRMC1-deplete endometrial tumors were generated following subcutaneous and intraperitoneal inoculation of immunocompromised NOD/SCID and nude mice, respectively. Tumors derived from PGRMC1-deplete cells grew slower than tumors from PGRMC1-intact cells. Mice harboring endometrial tumors were then given three treatments of vehicle (1:1 cremophor EL: ethanol + 0.9% saline) or chemotherapy [Paclitaxel (15 mg/kg, i.p.) followed after an interval of 30 minutes by CARBOplatin (50 mg/kg)] at five day intervals. In response to chemotherapy, tumor volume decreased approximately four-fold more in PGRMC1-deplete tumors when compared with PGRMC1-intact control tumors, suggesting that PGRMC1 promotes tumor cell viability during chemotherapeutic stress. In sum, these in vitro and in vivo findings demonstrate that PGRMC1 plays a prominent role in the growth and chemoresistance of human endometrial tumors.


Assuntos
Apoptose , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/prevenção & controle , Proteínas de Membrana/metabolismo , Receptores de Progesterona/metabolismo , Animais , Western Blotting , Neoplasias do Endométrio/patologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitose , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA