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2.
Leukemia ; 30(1): 229-37, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26174629

ABSTRACT

Aberrant post-transcriptional regulation by microRNAs (miRNAs) has been shown to be involved in the pathogenesis of acute myeloid leukemia (AML). In a previous study, we performed a large functional screen using a retroviral barcoded miRNA expression library. Here, we report that overexpression of miR-9/9* in myeloid 32D cell line (32D-miR-9/9*) had profound impact on granulocyte colony-stimulating factor-induced differentiation. Further in vitro studies showed that enforced expression of miR-9/9* blocked normal neutrophil development in 32D and in primary murine lineage-negative bone marrow cells. We examined the expression of miR-9/9* in a cohort of 647 primary human AMLs. In most cases, miR-9 and miR-9* were significantly upregulated and their expression levels varied according to AML subtype, with the highest expression in MLL-related leukemias harboring 11q23 abnormalities and the lowest expression in AML cases with t(8;21) and biallelic mutations in CEBPA. Gene expression profiling of AMLs with high expression of miR-9/9* and 32D-miR-9/9* identified ETS-related gene (Erg) as the only common potential target. Upregulation of ERG in 32D cells rescued miR-9/9*-induced block in neutrophil differentiation. Taken together, this study demonstrates that miR-9/9* are aberrantly expressed in most of AML cases and interfere with normal neutrophil differentiation by downregulation of ERG.


Subject(s)
Leukemia, Myeloid, Acute/genetics , MicroRNAs/physiology , Myeloid Progenitor Cells/metabolism , Neutrophils/cytology , Trans-Activators/genetics , Animals , Cell Differentiation , Gene Expression Regulation, Leukemic , Humans , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred C57BL , Transcriptional Regulator ERG
3.
Adv Otorhinolaryngol ; 62: 1-11, 2005.
Article in English | MEDLINE | ID: mdl-15608413

ABSTRACT

Last decade major advances have been made in the field of mouse model engineering. Newly developed conditional mouse models have overcome important drawbacks in conventional mouse models. Conditional mouse models are especially suited for the development of models of sporadic human carcinomas. These models can control gene (in)activation in a time and/or tissue-specific manner. Here, we review two important conditional mouse model systems, based on the Tet off/on and the Cre-Lox system. Furthermore possible applications of the Cre-Lox system in the development of a mouse model for HNSCC are being discussed. In the future, conditional mouse models for HNSCC can be used in the identification of new key genes in HNSCC tumorigenesis, and would furthermore serve as an indispensable tool for designing new treatment-modalities.


Subject(s)
Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Disease Models, Animal , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Animals , Biopsy, Needle , Female , Gene Expression Regulation, Neoplastic , Immunohistochemistry , Male , Mice , Mice, Transgenic , Netherlands , Sensitivity and Specificity
4.
Urol Res ; 30(4): 240-7, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12202942

ABSTRACT

Transitional epithelium of the urinary bladder can be damaged during, for example, catheterization, overstretching due to obstructed voiding, or partial resection. The subsequent repair process can be stimulated by specific proteins such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha). However, little is known about the role of EGF-like growth factors and their respective receptors in human urothelial repair. In this study, we examined the effects of EGF, TGFalpha, amphiregulin and heregulin-alpha (HRGalpha) on proliferation, wound closure, and the expression of their receptors c-erbB1-c-erbB4 in primary cultures of human urothelial cells in vitro. Under conditions representing intact urothelium, all EGF-like growth factors except HRGalpha induced proliferation. TGFalpha induced proliferation up to four times. Amphiregulin increased expression of c-erbB1. Treatment with either TGFalpha or amphiregulin resulted in higher c-erbB1 activation and c-erbB3 levels. None of the growth factors affected the constitutive expression of c-erbB2 and c-erbB4. In the repair model, both EGF and TGFalpha stimulated the wound closure most strongly. This was mainly achieved by increased cellular migration. Receptor expression was not affected by the addition of exogenous growth factor. The role of c-erbB2 in wound healing was further investigated with the use of antisense DNA. Wound closure could be delayed up to 50% by antisense c-erbB2 but not by mismatched or sense oligonucleotides. Excessive production (e.g. in bladder tumors) or application of EGF, TGFalpha or amphiregulin, but not HRGalpha may lead to either hyperplasia or a faster repair of damaged urothelium in vivo. These effects seem to be mediated not only via c-erbB1 but also via c-erbB2. Our results suggest that modified members of the EGF-EGFR family are potential targets for future therapies for bladder wound healing and malignancy.


Subject(s)
Epidermal Growth Factor/physiology , Receptor, ErbB-2/physiology , Regeneration/physiology , Ureter/physiology , Cells, Cultured , DNA, Antisense/pharmacology , Humans , Receptor, ErbB-2/genetics , Urothelium/physiology
6.
Virchows Arch ; 439(4): 552-9, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11710643

ABSTRACT

To investigate the importance of the microenvironment in bladder cancer invasion, a panel of six bladder carcinoma cell lines (SD, RT112, JON, 1207, T24, and J82) was tested in both in vitro and in vivo invasion assays. Furthermore, invasiveness was correlated with the expression of components of the E-cadherin-catenin complex. The E-cadherin-negative cell lines, T24 and J82, displayed a high in vitro invasive capacity, whereas the E-cadherin-positive cell lines, SD and JON, completely lacked in vitro invasive capacity. In contrast, in vivo invasion was noted for all cell lines, with the exception of cell line JON. Most notably, SD formed highly invasive tumors in vivo. The in vivo invasiveness of the E-cadherin-positive bladder carcinoma cell lines was associated with a heterogeneous expression of the E-cadherin-catenin complex. The discrepancy between in vitro and in vivo invasive behavior implies that, in vivo, the microenvironment plays an important role in the establishment of the invasive phenotype. In addition, it was found that orthotopic xenografting of 1207 and T24 bladder carcinoma cells resulted in site-specific tumor take and an enhanced tumor outgrowth and invasiveness, respectively, compared with heterotopic (i.e., subcutaneous) inoculation. We conclude that the site-specific growth and invasion of the bladder carcinoma cell lines in vivo and the observed assay specific invasion (in vitro vs in vivo) points to an effect of the local (bladder) microenvironment on tumor cell behavior.


Subject(s)
Carcinoma, Transitional Cell/pathology , Neoplasm Invasiveness/pathology , Trans-Activators , Urinary Bladder Neoplasms/pathology , Animals , Cadherins/biosynthesis , Carcinoma, Transitional Cell/metabolism , Chick Embryo , Cytoskeletal Proteins/biosynthesis , Female , Humans , Mice , Mice, SCID , Neoplasm Invasiveness/physiopathology , Neoplasm Transplantation/pathology , Transplantation, Heterologous , Tumor Cells, Cultured , Urinary Bladder Neoplasms/metabolism , alpha Catenin , beta Catenin
7.
Cancer Res ; 60(1): 177-83, 2000 Jan 01.
Article in English | MEDLINE | ID: mdl-10646871

ABSTRACT

High-grade transitional cell carcinomas (TCCs) of the urinary bladder are frequently associated with carcinoma in situ, which may replace large areas of the mucosa of the urinary tract. The invasive component of TCCs often reveals a loss of expression of the cell-cell adhesion molecule E-cadherin, but the role of E-cadherin in the development and expansion of intraepithelial neoplasia is unknown. To study the underlying mechanism of intraepithelial expansion (IEE), we have developed an IEE assay. Human TCC cell lines were investigated in this IEE assay for their capacity to replace the surrounding normal murine urothelial cells. In vitro IEE appeared to be prominent in three (SD, RT112, and 1207) of the four E-cadherin-positive cell lines. Although the two E-cadherin-negative cell lines (T24 and J82) were able to penetrate surrounding normal urothelium as single cells, they largely lacked the capacity of IEE. These results prompted us to investigate whether the cell-cell adhesion molecule E-cadherin is an important determinant for IEE. T24 cells that were transfected with full-length mouse E-cadherin cDNA displayed an enhanced IEE rate. Transfection did not influence their proliferative capacity, their pattern and level of integrin expression, or their ability to expand in the absence of surrounding urothelium. The data suggest that E-cadherin-mediated cohesiveness is an important factor in the IEE of bladder carcinoma cells. These observations argue for a dual, paradoxical role of E-cadherin in bladder tumorigenesis. On the one hand, E-cadherin promotes the expansion of intraepithelial neoplasia; on the other hand, its loss correlates with invasive behavior.


Subject(s)
Cadherins/physiology , Carcinoma in Situ/pathology , Carcinoma, Transitional Cell/pathology , Neoplasm Proteins/physiology , Urinary Bladder Neoplasms/pathology , Animals , Cadherins/genetics , Carcinoma in Situ/metabolism , Carcinoma, Transitional Cell/metabolism , Cell Adhesion , Chick Embryo , Female , Humans , Integrins/metabolism , Mice , Neoplasm Invasiveness , Neoplasm Proteins/genetics , Transfection , Tumor Cells, Cultured , Urinary Bladder Neoplasms/metabolism , Wound Healing
8.
Exp Cell Res ; 235(2): 395-402, 1997 Sep 15.
Article in English | MEDLINE | ID: mdl-9299164

ABSTRACT

A major problem in the management of bladder cancer is the high risk for recurrence of bladder tumors after transurethral resection. This has generally been attributed to the attachment and subsequent expansion of exfoliated tumor cells to the traumatized bladder wall. An in vitro cocultivation model was used to study the implantation and growth of human tumor cells in traumatized murine urothelium. Furthermore, we investigated in a time-course experiment whether stimulation of the regenerative activity of the normal urothelium by a growth factor could affect implantation and subsequent growth of bladder tumor cells. After inoculation on injured confluent cultures of murine urothelium, human T24 and SD bladder carcinoma cells preferentially attached to the denuded areas. SD cells expanded into the normal urothelium as a sharply demarcated tumor, while T24 cells infiltrated as single cells. Treatment of the primary urothelium with epidermal growth factor (EGF) stimulated the proliferation of the primary urothelium and reduced the implantation and growth of T24 considerably. EGF reduced the implantation of the SD tumor cells but could not prevent the further expansion at the expense of surrounding normal urothelium. Since EGF had no effect on migration or proliferation of SD or T24 cells, its modulation of expansive growth is most probably due to an increase in the regeneration of normal urothelium. This study suggests that recurrence of transitional cell carcinomas might in some instances be inhibited by stimulation of the regeneration of traumatized urothelium. The reported in vitro cocultivation model may be useful for studying additional factors involved in intraepithelial expansion of carcinoma cells.


Subject(s)
Carcinoma/pathology , Coculture Techniques/methods , Epidermal Growth Factor/pharmacology , Urinary Bladder Neoplasms/pathology , Urinary Bladder/pathology , Animals , Cell Division , Cell Movement , Female , Humans , Mice , Mice, Inbred C3H , Neoplasm Invasiveness , Regeneration , Tumor Cells, Cultured , Urinary Bladder/cytology , Urinary Bladder/physiology , Urothelium/cytology , Urothelium/pathology
9.
Lab Invest ; 75(2): 147-56, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8765315

ABSTRACT

Previous studies have indicated that growth factors such as epidermal growth factor, transforming growth factor alpha, and fibroblast growth factor 1 (FGF-1) have important regulatory functions in murine urothelial wound healing and tumorigenesis. Immunocytochemical analyses suggest that these factors are also involved in human urothelium. Yet, little is known about the functional effects of these growth factors on human urothelial cells. We established organoid-like primary cultures of normal human urothelium on porous membranes. Direct functional effects of growth factors were examined on confluent cultures reflecting intact urothelium. Immunocytochemistry was performed with a panel of specific antibodies against growth factors and their receptors on both cultures and the corresponding tissue sections. Lacking the appropriate antibodies, we performed reverse transcriptase PCR to detect FGF receptor mRNA in cultures and dissected tissue. The proliferation was stimulated by transforming growth factor alpha, FGF-1, and weakly by FGF-7, but not by FGF-2. TGF beta 1 inhibited proliferation. In contrast to mouse urothelium, none of the growth factors showed an effect on differentiation. The functional data correlate with the urothelial expression of epidermal growth factor receptors, TGF beta receptor types I and II, the (low) protein expression of FGF receptor 1, and the presence of FGF-7 receptor (FGF receptor 2 (IIIb)) mRNA. The organotypic nature of the cultures permits the study of growth factor interactions between urothelial cells. The data indicate that FGF-1, transforming growth factor alpha, and TGF beta 1 contribute differently to the maintenance of human urothelium.


Subject(s)
Fibroblast Growth Factors/physiology , Transforming Growth Factors/physiology , Ureter/cytology , Base Sequence , Cell Division/drug effects , Epithelial Cells , Epithelium/drug effects , Epithelium/ultrastructure , Fibroblast Growth Factors/biosynthesis , Fibroblast Growth Factors/metabolism , Humans , Models, Biological , Molecular Sequence Data , Organ Culture Techniques , Receptors, Fibroblast Growth Factor/biosynthesis , Receptors, Transforming Growth Factor beta/biosynthesis , Transforming Growth Factors/biosynthesis , Transforming Growth Factors/metabolism , Ureter/drug effects , Ureter/ultrastructure
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