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Expression of the breast and ovarian cancer susceptibility gene BRCA1 is down-regulated in sporadic breast and ovarian cancer cases. Therefore, the identification of genes involved in the regulation of BRCA1 expression might lead to new insights into the pathogenesis and treatment of these tumors. In the present study, an ''inverse genomics'' approach based on a randomized ribozyme gene library was applied to identify cellular genes regulating BRCA1 expression. A ribozyme gene library with randomized target recognition sequences was introduced into human ovarian cancer-derived cells stably expressing a selectable marker [enhanced green fluorescence protein (EGFP)] under the control of the BRCA1 promoter. Cells in which BRCA1 expression was upregulated by particular ribozymes were selected through their concomitant increase in EGFP expression. The cellular target gene of one ribozyme was identified to be the dominant negative transcriptional regulator Id4. Modulation of Id4 expression resulted in inversely regulated expression of BRCA1. In addition, increase in Id4 expression was associated with the ability of cells to exhibit anchorage-independent growth, demonstrating the biological relevance of this gene. Our data suggest that Id4 is a crucial gene regulating BRCA1 expression and might therefore be important for the BRCA1 regulatory pathway involved in the pathogenesis of sporadic breast and ovarian cancer.

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The expression of microRNAs is altered in various cancer types, leading to their definition as onco-and tumor-suppressor microRNAs. In our study, we investigated the role of miR-335 in the formation of sporadic human breast cancer and its involvement in the regulatory network of the breast cancer susceptibility gene BRCA1. To validate single components of the BRCA1 cascade, microRNA overexpression was performed in a cell culture model with subsequent protein analysis and luciferase reporter assays. Here, we were able to identify miR-335 as simultaneously regulating the known BRCA1 activators ERa, IGF1R, SP1 and the repressor ID4, including a feedback regulation of miR-335 expression by estrogens. Overexpression of miR-335 resulted in an upregulation of BRCA1 mRNA expression, suggesting a functional dominance of ID4 signaling. The relevance of the miR-335 regulation for human breast cancer was confirmed in primary sporadic breast cancer specimens with significantly decreased miR-335 levels (p < 0.05) in comparison to normal controls. Interestingly, the microRNA expression level correlated positively to the BRCA1 transcript level, supporting the hypothesis of a miR-335-mediated regulation of the tumor suppressor gene. Functionally, overexpression of miR-335 led to decreased cell viability and an increase in apoptosis, supporting its tumor-suppressive function. In summary, our data indicate that miR-335 affects different targets in the upstream BRCA1-regulatory cascade with impact on key cellular functions such as proliferation and apoptosis. Deregulation of the microRNA during breast cancer development and progression may thereby lead to an increased tumorigenic potential by inactivating crucial tumor-suppressive signals.In Western countries, breast cancer is the most frequent tumor found in women, with a low incidence in men. The tumor develops in a multistep process from ductal epithelial cells by triggers as yet poorly defined. Human breast cancers can be divided into two major subtypes, the inherited (5-10%) and the sporadic form, representing the greatest part. 1 The inherited subtype is predominantly characterized by germline mutations in one of the cancer susceptibility genes, known as BRCA1 and BRCA2, favoring a high predisposition to develop breast and ovarian cancer. 2 Although BRCA1 mutations have been found only rarely in the sporadic form, decreased expression levels of the gene were identified in these tumors. 3,4 As BRCA1 expression is embedded in a tightly controlled network, involving various activating and repressing factors, an altered transcriptional regulation may cause deregulation of the gene. Here, the expression of BRCA1 was determined to be hormone-dependent via direct activation through the estrogen (ERa), the aryl hydrocarbon (AhR) and the insulinlike growth factor 1 (IGF1R) receptors. [5][6][7] These are supported by hormone-independent factors such as the specific protein 1 (SP1) and inhibited via the inhibitor of DNA binding 4 (ID4). 8,9 These factors were identified as potent regula...

There is evidence that intraislet cellular communication and hormone delivery within the islets of Langerhans is controlled via capillary perfusion directed from the B cell core to the A/D cell mantle (intraislet portal system). To determine whether vascularization of freely transplanted islets repeats this "coreto-mantle" capillary perfusion, hamster islets were isolated by collagenase digestion and transplanted into a skinfold chamber of syngeneic animals (n = 12). 14 d after transplantation, the microvasculature of the islet grafts was analyzed by in vivo fluorescence microscopy. The capillary glomerulum-like network of the islet grafts (n = 109) was found supplied by individual arterioles, which regularly pierced the islet and broke into capillaries within the graft (96 /109 [88.1%1), resulting in capillary flow directed from the core to the islet's periphery. Only in 13 of 109 islets (11.9%) arterioles broke into capillaries at the outside margin ofthe islet and capillary flow was directed simultaneously to vessels located within the core, as well as the periphery of the graft. The islet's capillary network was drained by individual venules and intercapillary anastomoses between the newly formed islet capillaries and the preexisting capillaries of the host muscle tissue. Immunohistochemical staining revealed B cells located within the core, and A and D cells scattered in the periphery of the islets, indicating reestablishment of sequential B --A/D cellular perfusion of the grafts. Thus, freely transplanted islets develop an intra-islet portal system, similarly to that of pancreatic islets in situ. (J. Clin. Invest. 1994. 93:2280-2285 Key words: microcirculation * islets * transplantation * in vivo microscopy -hamster Introduction Hormone delivery within islets of Langerhans is controlled via the direction of islet cellular perfusion rather than paracrine interactions in the interstitial space ( 1). The intraislet portal system, which determines the order of microvascular perfusion, has been proposed to play the pivotal role in intraislet cellular communication, in particular according to the regulation of islet hormone secretion (2).Bonner-Weir and Orci (3) have been first to demonstrate that the islet's afferent arterioles do not break into capillaries at

Cyclin A1 is an alternative A-type cyclin that is essential for spermatogenesis, but it is also expressed in hematopoietic progenitor cells and in acute myeloid leukemia. Its functions during cell cycle progression of somatic cells are incompletely understood. Here, we have analysed the cell cycle functions of cyclin A1 in transformed and nontransformed cells. Murine embryonic fibroblasts derived from cyclin A1-deficient mice were significantly impaired in their proliferative capacity. In accordance, cyclin A1 À/À cells accumulated in G1 and G2/M phase while the percentage of S phase cells decreased. Also, lectin stimulated splenic lymphocytes from cyclin A1 À/À mice proliferated slower than their wild-type counterparts. Forced cyclin A1 overexpression in NIH3T3 cells and in U937 leukemic cells either by transient transfection or by retroviral infection enhanced S phase entry. Consequently, siRNA mediated silencing of cyclin A1 in highly cyclin A1 expressing ML1 leukemic cells significantly slowed S phase entry, decreased proliferation and inhibited colony formation. Taken together, these analyses demonstrate that cyclin A1 contributes to G1 to S cell cycle progression in somatic cells. Cyclin A1 overexpression enhances S phase entry consistent with an oncogenic function. Finally, cyclin A1 might be a therapeutic target since its silencing inhibited leukemia cell growth.

Ribozymes are small catalytic RNA molecules that can be engineered to enzymatically cleave RNA transcripts in a sequence-specific fashion and thereby inhibit expression and function of the corresponding gene product. With their simple structures and site-specific cleavage activity, they have been exploited as potential therapeutic agents in a variety of human disorders, including hepatitis C virus (HCV) infection. We have designed a hairpin ribozyme ( Ribozymes are small catalytic RNA molecules that hybridize to complementary sequences of a particular target mRNA and that can be engineered to enzymatically cleave and destroy RNA transcripts in a sequence-specific fashion, thereby preventing expression and function of the corresponding gene product. Ribozymes have been studied in connection with a variety of diseases and human disorders as potential therapeutic molecules (for recent reviews see references 2, 9, 15, 19, and 22). Hairpin ribozymes fold into a two-dimensional hairpin structure, consisting of a small catalytic region with four helical domains (see Fig. 1A). Based on Watson-Crick base pairing, the ribozyme binding arms (helices 1 and 2) hybridize to sequences flanking the cleavage site (GUC) within the target RNA, thereby determining specificity of the recognized target sequence.Recently, we introduced a novel "inverse genomics" procedure based on a retrovirus hairpin ribozyme library with randomized target recognition sequences for gene discovery in different experimental systems. These include (i) genes regulating the BRCA1 promoter (3), (ii) cellular genes mediating hepatitis C virus (HCV) internal ribosome entry site (IRES) activity (14), (iii) genes involved in anchorage-independent cell growth control (23), and (iv) genes involved in suppression of fibroblast transformation (16). Ribozymes that repeatedly conferred distinct cellular phenotypes were selected in these systems. Single ribozyme candidates were identified, and the binding sequence flanking the GUC site required for ribozyme cleavage was exploited to identify partial sequence information of the target gene responsible for the observed phenotype. For the HCV IRES project, a cellular selection scheme was developed using a reporter system based on herpes simplex virus (HSV) thymidine kinase (TK) as a negative selectable marker under translational control of the HCV IRES (14). This cellular selection system allowed the identification of ribozymes that actively inhibited HCV IRES-mediated translation of HSV TK and thereby conferred a ganciclovir (GCV)-resistance phenotype. By using the ribozyme binding sequences, potential cellular cofactors for HCV IRES were discovered (14). For these experiments, Rz3ЈX, a hairpin ribozyme engineered against the minus-strand HCV replication intermediate at position 40 within the terminal 98-nucleotide (nt) (3ЈX tail)

Summary. Glucocorticoids are broadly used for chemotherapy in childhood acute lymphoblastic leukaemia (ALL). The intracellular effects of glucocorticoids are mediated through the glucocorticoid receptor. The human glucocorticoid receptor gamma isoform (hGR-gamma) differs from the main isoform (hGR-alpha) by an additional amino acid within the DNA binding domain of the receptor protein. This may decrease hGR-alpha-mediated transcriptional activation. The importance of hGR-gamma expression in childhood ALL is unknown. To evaluate hGR-gamma mRNA expression levels, a real-time polymerase chain reaction (PCR)-based approach, allowing the selective amplification of hGR-gamma, was developed and optimized.We were able to demonstrate target selectivity of hGRgamma amplification using sequence-specific primers. Studying the structure of the 3¢ end of hGR-gamma, a combination of this isoform with other hGR isoforms could be demonstrated. Using analysis of hGR-gamma-specific amplification in comparison with the expression of hGRtotal (all isoforms) in leukaemic blasts from patients with either a good response to prednisone (PGR) or poor-prednisone response (PPR) in vivo, relative hGR-gamma expression was observed to be lower in cells from patients with PGR compared with PPR, in particular after 10 h of dexamethasone stimulation. These data were correlated with cell survival, demonstrating a more pronounced induction of apoptosis in cells from patients with PGR as compared with PPR.