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1.
Anticancer Res ; 40(1): 35-52, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31892551

RESUMO

BACKGROUND/AIM: Co-expression of c-Met and ALDH1A3 indicates a poor prognosis in stage III-IV breast cancers and contributes to cell proliferation and tumor formation by ALDH1-positive breast CSCs. PKCλ is overexpressed and contributes to a poor prognosis in several cancers. MATERIALS AND METHODS: A breast cancer genomics data set (METABRIC, n=2509) was downloaded and analyzed, as was the effect c-Met and PKCλ inhibitors on ALDH1high cell viability and tumor-sphere formation. RESULTS: c-Met expression correlates with expression of PKCλ in breast cancer. Stage III-IV breast cancer patients with c-Methigh PKCλhigh ALDH1A3high have a poorer prognosis than patients with c-Metlow PKCλlow ALDH1A3low Foretinib and auranofin suppressed cell viability and tumor-sphere formation by ALDH1high cells. These results suggest that c-Met and PKCλ are cooperatively involved in cancer progression and contribute to poor prognoses in breast cancer. CONCLUSION: c-Met and PKCλ are potentially useful prognostic markers and therapeutic targets in late-stage breast cancer.


Assuntos
Aldeído Oxirredutases/genética , Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Proteína Quinase C/genética , Proteínas Proto-Oncogênicas c-met/genética , Aldeído Oxirredutases/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Feminino , Humanos , Estimativa de Kaplan-Meier , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo
2.
World J Stem Cells ; 11(9): 705-721, 2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31616545

RESUMO

BACKGROUND: To solve the problem of liver transplantation donor insufficiency, an alternative cell transplantation therapy was investigated. We focused on amniotic epithelial cells (AECs) as a cell source because, unlike induced pluripotent stem cells, they are cost-effective and non-tumorigenic. The utilization of AECs in regenerative medicine, however, is in its infancy. A general profile for AECs has not been comprehensively analyzed. Moreover, no hepatic differentiation protocol for AECs has yet been established. To this end, we independently compiled human AEC libraries, purified amniotic stem cells (ASCs), and co-cultured them with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cell (HUVECs) in a 3D system which induces functional hepatic organoids. AIM: To characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cells. METHODS: AECs, MSCs, and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients. Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry, Alkaline phosphatase (AP) staining, and flow cytometry. An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay. AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics. The 2D and 3D culture were compared by relative gene expression using several differentiation protocols. ASCs, MSCs, and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging, periodic acid Schiff, and an indocyanine green (ICG) test. RESULTS: AECs have certain stemness markers such as EPCAM, SSEA4, and E-cadherin. One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers. Moreover, it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage. Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells. TJP1, associated with epidermal growth factor receptor, and MET, associated with hepatocyte growth factor receptor, were upregulated and may be important for hepatic differentiation. In conventional flat culture, the cells turned unviable and did not readily differentiate into hepatocytes. In 3D culture, however, hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol. Furthermore, the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity, hepatic-like glycogen storage, and ICG absorption/elimination. CONCLUSION: Human amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness. Under a 3D co-culture system, functional hepatic organoids were generated in a multicellular microenvironment.

3.
Int J Gynecol Pathol ; 38(4): 301-309, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30059452

RESUMO

We previously reported that aberrant expression of atypical protein kinase C λ/ι (aPKCλ/ι) in low-grade squamous intraepithelial uterine cervix lesions was associated with an increased risk of progression to higher grade. This study aimed to investigate aPKCλ/ι expression patterns in cervical squamous cell carcinoma (SCC) and its association with disease progression. We immunohistochemically assessed aPKCλ/ι expression in 168 SCC samples and 13 normal uterine cervix samples. In 69.0% of SCC cases, aPKCλ/ι was expressed more abundantly than in normal epithelium, but there was no significant association between aPKCλ/ι intensity and disease progression (P=0.087, Cochran-Mantel-Haenszel test). aPKCλ/ι in normal cervical epithelium was confined to the cytoplasm or intercellular junctions. In contrast, aPKCλ/ι was predominantly localized within the nucleus in 36.9% of SCC samples (P<0.001, χ test), and the prevalence was significantly increased relative to advanced tumor stage (P<0.001, Cochran-Mantel-Haenszel test). Moreover, patients with SCC with aPKCλ/ι nuclear localization had worse prognoses than those with cytoplasmic localization (P<0.001, log-rank test). aPKCλ/ι localization differed between the intraepithelial lesion and adjacent invasive cancer in 40% of cases, while the expression pattern was similar between primary and matched metastatic tumors. In conclusion, aPKCλ/ι nuclear localization in cervical cancer is associated with tumor progression and worse prognosis. This is the first report to show aberrant nuclear aPKCλ/ι localization in a subgroup of cervical cancer patients and its association with worse prognosis.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Neoplasias do Colo do Útero/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/patologia , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Prognóstico , Transporte Proteico , Neoplasias do Colo do Útero/patologia , Adulto Jovem
4.
Oncotarget ; 9(92): 36515-36529, 2018 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-30559934

RESUMO

Glyoxalase 1 (GLO1) is a ubiquitous enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis that induces apoptosis. In this study, we found that GLO1 gene expression correlates with neoplasm histologic grade (χ 2 test, p = 0.002) and is elevated in human basal-like breast cancer tissues. Approximately 90% of basal-like cancers were grade 3 tumors highly expressing both GLO1 and the cancer stem cell marker ALDH1A3. ALDH1high cells derived from the MDA-MB 157 and MDA-MB 468 human basal-like breast cancer cell lines showed elevated GLO1 activity. GLO1 inhibition using TLSC702 suppressed ALDH1high cell viability as well as the formation of tumor-spheres by ALDH1high cells. GLO1 knockdown using specific siRNAs also suppressed ALDH1high cell viability, and both TLSC702 and GLO1 siRNA induced apoptosis in ALDH1high cells. These results suggest GLO1 is essential for the survival of ALDH1-positive breast cancer stem cells. We therefore conclude that GLO1 is a potential therapeutic target for treatment of basal-like breast cancers.

5.
Anticancer Res ; 38(11): 6291-6297, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30396949

RESUMO

BACKGROUND/AIM: Atypical protein kinase C λ/ι (aPKCλ/ι) is a cell polarity-regulator localized in the tight junction and apical membrane in epithelial cells. Previous studies suggested that aPKCλ/ι overexpression and abnormal localization were involved in tumor progression in several cancers. We investigated the relationship between aPKCλ/ι and oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: The correlation between the aPKCλ/ι expression and the clinicopathological parameters in 76 OSCC cases was examined using immunohistochemical analyses. RESULTS: aPKCλ/ι overexpression was observed in 36.8% of cases. aPKCλ/ι expression was more intense in poorly differentiated OSCC and younger patients (<60 years of age). Although expression of aPKCλ/ι was not significantly associated with clinical parameters, the correlation was found between aPKCλ/ι localization and progression-free survival. CONCLUSION: This is the first study to assess the association of aPKCλ/ι expression in OSCC with clinical results. Expression and localization of aPKCλ/ι may be involved in the degree of malignancy in OSCC.


Assuntos
Carcinoma de Células Escamosas/patologia , Isoenzimas/metabolismo , Neoplasias Bucais/patologia , Proteína Quinase C/metabolismo , Regulação para Cima , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Estadiamento de Neoplasias , Prognóstico
6.
Biol Pharm Bull ; 41(4): 487-503, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29332929

RESUMO

Detection of anomalous cells such as cancer cells from normal blood cells has the potential to contribute greatly to cancer diagnosis and therapy. Conventional methods for the detection of cancer cells are usually tedious and cumbersome. Herein, we report on the use of a particle size analyzer for the convenient size-based differentiation of cancer cells from normal cells. Measurements made using a particle size analyzer revealed that size parameters for cancer cells are significantly greater (e.g., inner diameter and width) than the corresponding values for normal cells (white blood cells (WBC), lymphocytes and splenocytes), with no significant difference in shape parameters (e.g., circularity and convexity). The inner diameter of many cancer cell lines is greater than 10 µm, in contrast to normal cells. For the detection of WBC having similar size to that of cancer cells, we developed a PC software "Cancer Cell Finder" that differentiates them from cancer cells based on brightness stationary points on a cell surface. Furthermore, the aforementioned method was validated for cancer cell/clusters detection in spiked mouse blood samples (a B16 melanoma mouse xenograft model) and circulating tumor cell cluster-like particles in the cat and dog (diagnosed with cancer) blood samples. These results provide insights into the possible applicability of the use of a particle size analyzer in conjunction with PC software for the convenient detection of cancer cells in experimental and clinical samples for theranostics.


Assuntos
Neoplasias da Mama/patologia , Neoplasias Colorretais/patologia , Neoplasias Pulmonares/patologia , Melanoma Experimental/patologia , Neoplasias da Próstata/patologia , Animais , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Gatos , Linhagem Celular Tumoral , Forma Celular , Tamanho Celular , Neoplasias Colorretais/sangue , Neoplasias Colorretais/diagnóstico , Cães , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Leucócitos/citologia , Leucócitos/patologia , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/diagnóstico , Linfócitos/citologia , Linfócitos/patologia , Masculino , Melanoma Experimental/sangue , Melanoma Experimental/diagnóstico , Camundongos Endogâmicos C57BL , Neoplasias da Próstata/sangue , Neoplasias da Próstata/diagnóstico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Software , Baço/citologia , Baço/patologia , Propriedades de Superfície , Nanomedicina Teranóstica/instrumentação , Nanomedicina Teranóstica/métodos
7.
Genes Cancer ; 8(7-8): 628-639, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28966724

RESUMO

c-Met is a receptor-type tyrosine kinase, which is involved in a wide range of cellular responses such as proliferation, motility, migration and invasion. It has been reported to be overexpressed in various cancers. However, the role of c-Met in breast cancer stem cells (CSCs) still remains unclear. We herein, show that c-Met expression is significantly elevated in Basal-like type of breast cancer in comparison with other subtypes. High expression of c-Met strongly correlated with the expression of two CSC markers, ALDH1A3 and CD133 in breast cancers. In addition, breast cancers at tumor stage III-IV expressing both c-Methigh and ALDH1A3high had poor prognosis. Furthermore, treatment with c-Met inhibitors (Crizotinib, Foretinib, PHA-665752 and Tivantinib) in MDA-MB157 cells with high c-Met protein expression resulted in significant suppression in cell viability, contrary to MDA-MB468 cells with low c-Met protein expression. These c-Met inhibitors also suppressed cell viability and tumor-sphere formation of ALDH1high breast cancer cells with high c-Met expression. These results suggest that c-Met in ALDH1 positive CSCs seems to play an important role in breast cancer repopulation. Therefore, we conclude that c-Met is a potential therapeutic target in ALDH1 positive breast CSCs.

8.
Clin Breast Cancer ; 17(3): e135-e142, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27986439

RESUMO

INTRODUCTION: In general, it has been considered that estrogen receptor-positive (ER+) breast cancer has a good prognosis and is responsive to endocrine therapy. However, one third of patients with ER+ breast cancer exhibit endocrine therapy resistance, and many patients develop recurrence and die 5 to 10 years after diagnosis. In ER+ breast cancer, a major problem is to distinguish those patients most likely to develop recurrence or metastatic disease within 10 years after diagnosis from those with a sufficiently good prognosis. MATERIALS AND METHODS: We downloaded the messenger RNA expression data and the clinical information for 401 patients with ER+ breast cancer from the cBioPortal for Cancer Genomics. An information-theoretical approach was used to identify the prognostic factors for survival in patients with ER+ breast cancer and to classify those patients according to the prognostic factors. RESULTS: The information-theoretical approach contributed to the identification of KMT2C and SLC20A1 as prognostic biomarkers in ER+ breast cancer. We found that low KMT2C expression was associated with a poor outcome and high SLC20A1 expression was associated with a poor outcome. Both levels of KMT2C and SLC20A1 expression were significantly and strongly associated with the differentiation of survival. The 10-year survival rate for ER+ patients with low KMT2C and high SLC20A1 expression was 0%. In contrast, for ER+ patients with high KMT2C and low SLC20A1 expression, the 10-year survival rate was 86.78%. CONCLUSION: Our results strongly suggest that clinical examination of the expression of both KMT2C and SLC20A1 in ER+ breast cancer will be very useful for the determination of prognosis and therapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Bases de Dados Factuais , Proteína de Leucina Linfoide-Mieloide/metabolismo , Receptores Estrogênicos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Feminino , Humanos , Metástase Linfática , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Prognóstico , Taxa de Sobrevida
9.
Sci Rep ; 6: 19182, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26765121

RESUMO

Naïve CD8(+) T lymphocytes responding to microbial pathogens give rise to effector T cells that provide acute defense and memory T cells that provide long-lived immunity. Upon activation, CD8(+) T lymphocytes can undergo asymmetric division, unequally distributing factors to the nascent daughter cells that influence their eventual fate towards the effector or memory lineages. Individual loss of either atypical protein kinase C (aPKC) isoform, PKCζ or PKCλ/ι, partially impairs asymmetric divisions and increases CD8(+) T lymphocyte differentiation toward a long-lived effector fate at the expense of memory T cell formation. Here, we show that deletion of both aPKC isoforms resulted in a deficit in asymmetric divisions, increasing the proportion of daughter cells that inherit high amounts of effector fate-associated molecules, IL-2Rα, T-bet, IFNγR, and interferon regulatory factor 4 (IRF4). However, unlike CD8(+) T cells deficient in only one aPKC isoform, complete loss of aPKC unexpectedly increased CD8(+) T cell differentiation toward a short-lived, terminal effector fate, as evidenced by increased rates of apoptosis and decreased expression of Eomes and Bcl2 early during the immune response. Together, these results provide evidence for an important role for asymmetric division in CD8(+) T lymphocyte fate specification by regulating the balance between effector and memory precursors at the initiation of the adaptive immune response.


Assuntos
Divisão Celular Assimétrica , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/fisiologia , Diferenciação Celular , Proteína Quinase C/metabolismo , Imunidade Adaptativa , Animais , Técnicas de Inativação de Genes , Memória Imunológica , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/fisiologia
10.
Int J Gynecol Pathol ; 35(2): 106-17, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26535980

RESUMO

Atypical protein kinase C λ/ι (aPKCλ/ι) is a regulator of epithelial cellular polarity. It is also overexpressed in several cancers and functions in cell proliferation and invasion. Therefore, we hypothesized that aPKCλ/ι may be involved in development and progression of cervical intraepithelial neoplasia (CIN), the precancerous disease of cervical cancer induced by human papillomavirus. To do this, we investigated the relationship between aPKCλ/ι expression and CIN. aPKCλ/ι expression level and subcellular localization were assessed in 192 CIN biopsy samples and 13 normal epithelial samples using immunohistochemistry. aPKCλ/ι overexpression (normal epithelium, 7.7%; CIN1, 41.7%; CIN2/3, 76.4%) and aPKCλ/ι nuclear localization (normal epithelium, 0.0%; CIN1, 36.9%; CIN2/3, 78.7%) were higher in CIN samples than normal samples (P<0.05), suggesting that CIN grade is related to aPKCλ/ι overexpression and nuclear localization. Then, 140 CIN cases were retrospectively analyzed for 4-yr cumulative disease progression and regression rates using the Cox proportional hazards model. CIN1 cases with aPKCλ/ι overexpression or aPKCλ/ι nuclear localization had a higher progression rate than CIN1 cases with normal aPKCλ/ι expression levels or cytoplasmic localization (62.5% vs. 9.7% and 63.1% vs. 9.4%, respectively; P<0.001). Multivariate analysis indicated that human papillomavirus types 16 and 18, aPKCλ/ι overexpression (hazard ratio=4.26; 95% confidence interval, 1.50-12.1; P=0.007), and aPKCλ/ι nuclear localization (hazard ratio=3.59; 95% confidence interval, 1.24-10.4; P=0.019) were independent risk factors for CIN1 progression. In conclusion, aPKCλ/ι could be useful for the therapeutic management of patients with CIN, particularly those with non-human papillomavirus 16/18 types.


Assuntos
Biomarcadores Tumorais/análise , Neoplasia Intraepitelial Cervical/patologia , Proteína Quinase C/biossíntese , Neoplasias do Colo do Útero/patologia , Adulto , Neoplasia Intraepitelial Cervical/metabolismo , Neoplasia Intraepitelial Cervical/virologia , Progressão da Doença , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/epidemiologia , Prognóstico , Proteína Quinase C/análise , Estudos Retrospectivos , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia
11.
J Invest Dermatol ; 135(11): 2584-2592, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26076315

RESUMO

The atypical protein kinase C (aPKC)-partition-defective (PAR) complex regulates the formation of tight junctions and apico-basal epithelial polarity. To examine the role of this complex in the epidermis, we generated mutant mice harboring epidermal-specific deletion of aPKCλ (conditional knock-out (cKO)), a major component of the aPKC-PAR complex. The mutant mice exhibited abnormal hair follicle (HF) cycling, progressive losses of pelage hairs and vibrissae, and altered differentiation into the epidermis and sebaceous gland. We found that in the aPKCλ cKO mice HF stem cell (HFSC) quiescence was lost, as revealed by the decreased expression level of quiescence-inducing factors (Fgf18 and Bmp6) produced in Keratin 6-positive bulge stem cells. The loss of quiescence dysregulated the HFSC marker expression and led to the increase in Lrig1-positive cells, inducing hyperplasia of the interfollicular epidermis and sebaceous glands, and drove an increase in Lef1-positive matrix cells, causing a prolonged anagen-like phase. Persistent bulge stem cell activation led to a gradual depletion of CD34- and α6 integrin-positive HFSC reservoirs. These results suggest that aPKCλ regulates signaling pathways implicated in HFSC quiescence.


Assuntos
Folículo Piloso/citologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Células-Tronco/metabolismo , Animais , Animais Recém-Nascidos , Bromodesoxiuridina/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Células Epidérmicas , Epiderme/metabolismo , Deleção de Genes , Marcadores Genéticos , Folículo Piloso/patologia , Imuno-Histoquímica , Injeções Intraperitoneais , Camundongos , Camundongos Knockout , Isoformas de Proteínas/metabolismo , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Transdução de Sinais/genética
12.
J Cell Sci ; 128(13): 2244-58, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25977476

RESUMO

Epithelial apicobasal polarity has fundamental roles in epithelial physiology and morphogenesis. The PAR complex, comprising PAR-3, PAR-6 and atypical protein kinase C (aPKC), is involved in determining cell polarity in various biological contexts, including in epithelial cells. However, it is not fully understood how the PAR complex induces apicobasal polarity. In this study, we found that PAR-3 regulates the protein expression of Girdin (also known as GIV or CCDC88A), a guanine-nucleotide-exchange factor (GEF) for heterotrimeric Gαi subunits, at the transcriptional level by cooperating with the AP-2 transcription factor. In addition, we confirmed that PAR-3 physically interacts with Girdin, and show that Girdin, together with the Gαi3 (also known as GNAI3), controls tight junction formation, apical domain development and actin organization downstream of PAR-3. Taken together, our findings suggest that transcriptional upregulation of Girdin expression and Girdin-Gαi3 signaling play crucial roles in regulating epithelial apicobasal polarity through the PAR complex.


Assuntos
Proteínas de Transporte/metabolismo , Polaridade Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas dos Microfilamentos/genética , Transcrição Genética , Proteínas de Transporte Vesicular/genética , Animais , Sequência de Bases , Comunicação Celular , Cães , Regulação da Expressão Gênica , Células HeLa , Células Hep G2 , Humanos , Células Madin Darby de Rim Canino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso , Ligação Proteica , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais , Junções Íntimas/metabolismo , Fator de Transcrição AP-2/metabolismo
13.
J Immunol ; 194(5): 2249-59, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25617472

RESUMO

During an immune response against a microbial pathogen, activated naive T lymphocytes give rise to effector cells that provide acute host defense and memory cells that provide long-lived immunity. It has been shown that T lymphocytes can undergo asymmetric division, enabling the daughter cells to inherit unequal amounts of fate-determining proteins and thereby acquire distinct fates from their inception. In this study, we show that the absence of the atypical protein kinase C (PKC) isoforms, PKCζ and PKCλ/ι, disrupts asymmetric CD8(+) T lymphocyte division. These alterations were associated with aberrant acquisition of a pre-effector transcriptional program, detected by single-cell gene expression analyses, in lymphocytes that had undergone their first division in vivo and enhanced differentiation toward effector fates at the expense of memory fates. Together, these results demonstrate a role for atypical PKC in regulating asymmetric division and the specification of divergent CD8(+) T lymphocyte fates early during an immune response.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Divisão Celular/imunologia , Imunidade Inata , Isoenzimas/imunologia , Listeriose/imunologia , Proteína Quinase C/imunologia , Animais , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/microbiologia , Linfócitos T CD8-Positivos/patologia , Diferenciação Celular , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Células Dendríticas/patologia , Regulação da Expressão Gênica , Memória Imunológica , Isoenzimas/genética , Isoenzimas/metabolismo , Listeria monocytogenes/imunologia , Listeriose/enzimologia , Listeriose/microbiologia , Listeriose/patologia , Camundongos , Camundongos Knockout , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Transdução de Sinais , Análise de Célula Única , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/microbiologia , Subpopulações de Linfócitos T/patologia
14.
Oncol Lett ; 8(3): 977-984, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25120645

RESUMO

Colorectal flat-type tumors include laterally spreading tumors (LSTs) and flat depressed-type tumors. The former of which shows a predominant lateral spreading growth rather than an invasive growth. The present study examined the morphological characteristics of LSTs, in comparison with polypoid- or flat depressed-type tumors, along with the expression of atypical protein kinase C (aPKC) λ/ι, a pivotal cell polarity regulator, and the hallmarks of cell polarity, as well as with type IV collagen, ß-catenin and E-cadherin. In total, 37 flat-type (24 LSTs and 13 flat depressed-type tumors) and 20 polypoid-type colorectal tumors were examined. The LSTs were classified as 15 LST adenoma (LST-A) and nine LST cancer in adenoma (LST-CA). An immunohistochemical examination was performed on aPKC λ/ι, type IV collagen, ß-catenin and E-cadherin. The LST-A and -CA showed a superficial replacing growth pattern, with expression of ß-catenin and E-cadherin in the basolateral membrane and type IV collagen along the basement membrane. In addition, 86.6% of LST-A and 55.6% of LST-CA showed aPKC λ/ι expression of 1+ (weak to normal intensity staining in the cytoplasm compared with the normal epithelium). Furthermore, ~45% of the polypoid-type adenomas showed 2+ (moderate intensity staining in the cytoplasm and/or nucleus) and 66.7% of the polypoid-type cancer in adenoma were 3+ (strong intensity staining in the cytoplasm and nucleus). A statistically significant positive correlation was observed between the expression of aPKC λ/ι and ß-catenin (r=0.842; P<0.001), or type IV collagen (r=0.823; P<0.001). The LSTs showed a unique growth pattern, different from the expanding growth pattern presented by a polypoid tumor and invasive cancer. The growth characteristics of LST appear to be caused by adequate coexpression of ß-catenin, type IV collagen and aPKC λ/ι.

15.
J Proteome Res ; 13(11): 4686-94, 2014 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-25004236

RESUMO

We attempted to identify prognosis-related proteins expressed in early resection lung adenocarcinomas that had higher metastatic potential. Early resection of lung adenocarcinoma tissues were collected from patients who experienced recurrence within 5 years after surgery; these patients are defined here as the poor prognosis group. From these samples, we prepared frozen tissue sections and then isolated cancerous areas by laser capture microdissection to allow extraction of cancer tissue-derived soluble proteins. Shotgun LC-MS/MS analysis detected and identified a total of 875 proteins in these cancer tissues. Relative quantitative analysis revealed that 17 proteins were preferentially expressed in the poor prognosis group relative to the good prognosis group, which consisted of patients who did not exhibit recurrence. Among them, 14-3-3 beta/alpha and calnexin were reported to be potentially involved in tumor recurrence and the malignant properties of lung cancer. Here immunological analyses confirmed disease-associated expression of these proteins. In a cell-culture model using A549, targeted depletion of either 14-3-3 beta/alpha or calnexin reduced proliferation, invasion, and migration, suggesting that both proteins are involved in determining the malignant properties of lung cancer that contribute to poor prognosis.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteômica/métodos , Proteínas 14-3-3/metabolismo , Adenocarcinoma/cirurgia , Adenocarcinoma de Pulmão , Calnexina/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Cromatografia Líquida , Humanos , Microdissecção e Captura a Laser , Neoplasias Pulmonares/cirurgia , Invasividade Neoplásica/genética , Invasividade Neoplásica/fisiopatologia , Prognóstico , Recidiva , Espectrometria de Massas em Tandem
16.
Pancreatology ; 13(4): 360-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23890134

RESUMO

Pancreatic cancer is a lethal disease. Overall survival is typically 6 months from diagnosis. Determination of prognostic factors in pancreatic cancer that would allow identification of patients who could potentially benefit from aggressive treatment is important. However, until date, there are no established reliable prognostic factors for pancreatic cancer patients. Herein, we propose a beneficial biomarker which is significantly correlated with the prognosis in pancreatic cancer patients. Atypical protein kinase C λ/ι (aPKCλ/ι) is overexpressed and has been implicated in the progression of several cancers. We tested the expression levels of aPKCλ/ι in two types of pancreatic neoplasm, pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMNs), by immunohistochemistry. Examination of the aPKCλ/ι expression levels in surgically resected specimens of PDCA (n = 115) demonstrated that the expression levels of aPKCλ/ιin PDAC had prognostic implications, independent of the Tumor-Node-Metastasis classification and World Health Organization tumor grade. In the case of IPMNs (n = 46) also, the expression levels of aPKCλ/ιin IPMN were found to be of prognostic importance, independent of the World Health Organization histological grade or morphological type. Interestingly, high expression levels of aPKCλ/ι were significantly correlated with a worse histological grade (p = 0.010) and advanced stage of the tumor (p = 0.0050) in IPMN patients. These findings suggest that high expression levels of aPKCλ/ι could be involved in the malignant transformation of IPMNs. Based on these observations, we propose the expression level of aPKCλ/ι as a prognostic marker common to different types of pancreatic neoplasms.


Assuntos
Adenocarcinoma Mucinoso/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Isoenzimas/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteína Quinase C/metabolismo , Adenocarcinoma Mucinoso/patologia , Idoso , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/patologia , Prognóstico , Modelos de Riscos Proporcionais
17.
Biochem Biophys Res Commun ; 436(3): 519-24, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23764395

RESUMO

The Fc region of human IgG1 mediates effector function via binding to Fcγ receptors and complement activation. The H and L chains of IgG1 antibodies are joined by four interchain disulfide bonds. In this study, these bonds within the therapeutic IgG1 rituximab (RTX) were cleaved either by mild reduction followed by alkylation or by mild S-sulfonation; consequently, two modified RTXs - A-RTX (alkylated) and S-RTX (S-sulfonated) - were formed, and both were almost as potent as unmodified RTX when binding CD20 antigen. Unexpectedly, each modified RTX had a higher binding affinity for FcγRIIIA (CD16A) than did unmodified RTX. However, S-RTX and A-RTX were each less potent than RTX in an assay of antibody-dependent cellular cytotoxicity (ADCC). In this ADCC assay, each modified RTX showed decreased secretion of granzyme B, but no change in perforin secretion, from effector cells. These results provide significant information on the structures within IgG1 that are involved in binding FcγRIIIA, and they may be useful in the development of therapeutic antagonists for FcγRIIIA.


Assuntos
Anticorpos Monoclonais Murinos/química , Afinidade de Anticorpos , Dissulfetos/química , Proteólise , Receptores de IgG/química , Alquilação , Anticorpos Monoclonais Murinos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Técnicas de Cocultura , Granzimas/química , Humanos , Células Matadoras Naturais/química , Células Matadoras Naturais/imunologia , Perforina/química , Ligação Proteica , Receptores de IgG/imunologia , Rituximab
18.
Curr Biol ; 23(13): 1181-94, 2013 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-23791731

RESUMO

BACKGROUND: In preimplantation mouse embryos, the first cell fate specification to the trophectoderm or inner cell mass occurs by the early blastocyst stage. The cell fate is controlled by cell position-dependent Hippo signaling, although the mechanisms underlying position-dependent Hippo signaling are unknown. RESULTS: We show that a combination of cell polarity and cell-cell adhesion establishes position-dependent Hippo signaling, where the outer and inner cells are polar and nonpolar, respectively. The junction-associated proteins angiomotin (Amot) and angiomotin-like 2 (Amotl2) are essential for Hippo pathway activation and appropriate cell fate specification. In the nonpolar inner cells, Amot localizes to adherens junctions (AJs), and cell-cell adhesion activates the Hippo pathway. In the outer cells, the cell polarity sequesters Amot from basolateral AJs to apical domains, thereby suppressing Hippo signaling. The N-terminal domain of Amot is required for actin binding, Nf2/Merlin-mediated association with the E-cadherin complex, and interaction with Lats protein kinase. In AJs, S176 in the N-terminal domain of Amot is phosphorylated by Lats, which inhibits the actin-binding activity, thereby stabilizing the Amot-Lats interaction to activate the Hippo pathway. CONCLUSIONS: We propose that the phosphorylation of S176 in Amot is a critical step for activation of the Hippo pathway in AJs and that cell polarity disconnects the Hippo pathway from cell-cell adhesion by sequestering Amot from AJs. This mechanism converts positional information into differential Hippo signaling, thereby leading to differential cell fates.


Assuntos
Blastocisto/metabolismo , Polaridade Celular , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas dos Microfilamentos/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Junções Aderentes/metabolismo , Animais , Adesão Celular , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo
19.
PLoS One ; 8(12): e84036, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24391875

RESUMO

Cell polarity plays a critical role in neuronal differentiation during development of the central nervous system (CNS). Recent studies have established the significance of atypical protein kinase C (aPKC) and its interacting partners, which include PAR-3, PAR-6 and Lgl, in regulating cell polarization during neuronal differentiation. However, their roles in neuronal maintenance after CNS development remain unclear. Here we performed conditional deletion of aPKCλ, a major aPKC isoform in the brain, in differentiated neurons of mice by camk2a-cre or synapsinI-cre mediated gene targeting. We found significant reduction of aPKCλ and total aPKCs in the adult mouse brains. The aPKCλ deletion also reduced PAR-6ß, possibly by its destabilization, whereas expression of other related proteins such as PAR-3 and Lgl-1 was unaffected. Biochemical analyses suggested that a significant fraction of aPKCλ formed a protein complex with PAR-6ß and Lgl-1 in the brain lysates, which was disrupted by the aPKCλ deletion. Notably, the aPKCλ deletion mice did not show apparent cell loss/degeneration in the brain. In addition, neuronal orientation/distribution seemed to be unaffected. Thus, despite the polarity complex disruption, neuronal deletion of aPKCλ does not induce obvious cell loss or disorientation in mouse brains after cell differentiation.


Assuntos
Encéfalo/patologia , Diferenciação Celular , Polaridade Celular , Isoenzimas/fisiologia , Neurogênese/fisiologia , Neurônios/patologia , Proteína Quinase C/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Feminino , Glicoproteínas/genética , Glicoproteínas/metabolismo , Técnicas Imunoenzimáticas , Imunoprecipitação , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Cancer Sci ; 104(2): 259-65, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23163744

RESUMO

Overexpression of atypical protein kinase Cλ/ι (aPKCλ/ι), a regulator of cell polarity, is frequently associated with the poor prognoses of several cancers, including gastric cancer. Recent studies revealed a molecular link between aPKC and KIBRA, an upstream regulator of tumor suppressor Hippo pathway that regulates cell proliferation and apoptosis. Further, KIBRA directly inhibits the kinase activity of aPKC to regulate epithelial cell polarity. These observations suggest that the KIBRA-aPKC connection plays a role in cancer progression; however, clinical significance of the correlation between these factors remains unclear. Here we examined the correlation between KIBRA/aPKCλ/ι expression, as detected by immunohistochemistry, and clinicopathological outcomes in 164 gastric cancer patients using Fisher's exact test and Kaplan-Meier log-rank test. We found an intimate correlation between the expression level of KIBRA and aPKCλ/ι (P = 0.012). Furthermore, high expression of KIBRA is correlated with lymphatic (P = 0.046) and venous invasion (P = 0.039). The expression level of KIBRA by itself did not correlate with the prognosis; however, high expression of KIBRA in low aPKCλ/ι-expressing gastric cancer correlated with disease-specific (P = 0.037) and relapse-free survival (P = 0.041) by Kaplan-Meier with log-rank test and higher lymphatic invasion cases by Fisher's exact test (P = 0.042). Furthermore, overexpression of the aPKC-binding region of KIBRA disrupted tight junctions in epithelial cells. These results suggest that high expression of KIBRA in low aPKC-expressing cells causes massive loss of aPKC activity, leading to loss of polarity and invasiveness of gastric cancer cells.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Fosfoproteínas/biossíntese , Proteína Quinase C/biossíntese , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Comunicação Celular/genética , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Intervalo Livre de Doença , Cães , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estimativa de Kaplan-Meier , Metástase Linfática , Células Madin Darby de Rim Canino , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Prognóstico , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/genética , Junções Íntimas/genética , Junções Íntimas/metabolismo , Junções Íntimas/patologia
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