Carmela Beger

Expression and structural analysis of glucocorticoid receptor isoform gamma in human leukaemia cells using an isoform-specific real-time polymerase chain reaction approach

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 hGR‐gamma 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 hGR‐total (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.

MicroRNA miR-335 is crucial for the BRCA1 regulatory cascade in breast cancer development

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 ERα, 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.

Involvement of proteasome alpha-subunit PSMA7 in hepatitis C virus internal ribosome entry site-mediated translation.

ABSTRACT 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 (Rz3′X) targeting the HCV minus-strand replication intermediate at position 40 within the 3′X tail. Surprisingly, Rz3′X was found to induce ganciclovir (GCV)-resistant colonies in a bicistronic cellular reporter system with HCV internal ribosome entry site (IRES)-dependent translation of herpes simplex virus thymidine kinase (TK). Rz3′X-transduced GCV-resistant HeLa reporter cells showed substantially reduced IRES-mediated HCV core protein translation compared with control vector-transduced cells. Since these reporter systems do not contain the HCV 3′X tail sequences, the results indicate that Rz3′X probably exerted an inhibitory effect on HCV IRES activity fortuitously through another gene target. A novel technique of ribozyme cleavage-based target gene identification (cleavage-specific amplification of cDNA ends) (M. Krüger, C. Beger, P. J. Welch, J. R. Barber, and F. Wong-Staal, Nucleic Acids Res. 29:e94, 2001) revealed that human 20S proteasome α-subunit PSMA7 mRNA was a target RNA recognized and cleaved by Rz3′X. We then showed that additional ribozymes directed against PSMA7 RNA inhibited HCV IRES activity in two assay systems: GCV resistance in the HeLa IRES TK reporter cell system and a transient transfection assay performed with a bicistronicRenilla-HCV IRES-firefly luciferase reporter in Huh7 cells. In contrast, ribozymes were inactive against IRES of encephalomyocarditis virus and human rhinovirus. Additionally, proteasome inhibitor MG132 exerted a dose-dependent inhibitory effect on HCV IRES-mediated translation but not on cap-dependent translation. These data suggest a principal role for PSMA7 in regulating HCV IRES activity, a function essential for HCV replication.

Cyclin A1, the alternative A-type cyclin, contributes to G1/S cell cycle progression in somatic cells

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−/− cells accumulated in G1 and G2/M phase while the percentage of S phase cells decreased. Also, lectin stimulated splenic lymphocytes from cyclin A1−/− 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.

Constitutional mismatch repair deficiency and childhood leukemia/lymphoma – report on a novel biallelic MSH6 mutation

Biallelic mutations of mismatch repair genes cause constitutional mismatch repair deficiency associated with an increased risk for childhood leukemia/lymphoma. We report on a case with constitutional mismatch repair deficiency caused by a novel MSH6 mutation leading to a T-cell lymphoma and colonic adenocarcinoma at six and 13 years of age, respectively. A review of the literature on hematologic malignancies in constitutional mismatch repair deficiency showed that in almost half of the 47 known constitutional mismatch repair deficiency families, at least one individual is affected by a hematologic malignancy, predominantly T-cell lymphomas. However, diagnosing constitutional mismatch repair deficiency may be difficult when the first child is affected by leukemia/lymphoma, but identification of the causative germline mutation is of vital importance: (i) to identify relatives at risk and exclude an increased risk in non-mutation carriers; (ii) to prevent hematopoietic stem cell transplantation from sibling donors also carrying a biallelic germline mutation; and (iii) to implement effective surveillance programs for mutation carriers, that may reduce constitutional mismatch repair deficiency-associated mortality.

Down-Regulation of BRCA1 in Chronic Pancreatitis and Sporadic Pancreatic Adenocarcinoma

Purpose: BRCA1 and BRCA2 are considered to be breast cancer susceptibility genes that may also contribute to pancreatic cancer development because family studies revealed mutation carriers to have an increased risk of developing pancreatic cancer. However, as demonstrated for breast and ovarian cancer, inactivation of BRCA in sporadic diseases is based on alteration in gene expression or functional alteration. Experimental Design: To study a potential correlation of BRCA1 and BRCA2 to chronic pancreatitis and development of sporadic pancreatic adenocarcinoma, we have analyzed the expression of these genes by quantitative PCR and performed immunohistochemical analyses in normal pancreatic tissues, chronic pancreatitis, and pancreatic cancer specimens. Results: BRCA1 expression was down-regulated in chronic alcoholic pancreatitis, in particular on the RNA level. Furthermore, our data indicate suppressed BRCA1 expression in pancreatic cancer on both the RNA and protein levels. Quantitative analysis of BRCA1 protein expression demonstrated regular staining in 50% of tumor specimens tested and reduced staining in 50% of tumor specimens tested. Correlation with the clinical outcome revealed a significantly better 1-year overall survival for patients with BRCA1-regular as compared with BRCA1-reduced or BRCA1-absent tumors. In contrast, no substantial differences in BRCA2 expression were found in chronic pancreatitis and pancreatic cancer samples. Conclusions: Our data demonstrate alteration of BRCA1 expression in chronic pancreatitis and sporadic pancreatic adenocarcinoma. We, for the first time, provide evidence for a role of BRCA1 in pancreatic carcinogenesis of noninherited tumors and for clinical outcome.

Use of ribozymes to inhibit gene expression.

Publisher Summary Ribozymes are small RNA molecules with endoribonuclease activity that hybridize to complementary sequences of particular target mRNA transcripts through Watson-Crick base pairing and exert catalytic cleavage activity. The development of ribozymes to specifically target a gene involved in a disease state usually includes two main steps: (1) identification of active ribozymes by in vitro cleavage assays and (2) ribozyme-mediated inhibition of gene expression in vivo . As the laboratory is working mainly with hairpin ribozymes against human immunodeficiency virus (HIV), this chapter uses hairpin ribozymes as an example to illustrate these processes. The chapter demonstrates how hairpin ribozymes have been developed to a clinical application against HIV. Ribozyme technology for expression knock out can potentially be applied to different diseases, including infectious diseases, cancers, and cardiovascular disorders.

DNA damage response involves modulation of Ku70 and Rb functions by cyclin A1 in leukemia cells

Cyclin A1 plays a critical role in hematopoietic malignancies, notably, acute myeloid leukemia. The molecular mechanisms of cyclin A1 action are incompletely understood. Here, we show that cyclin A1 functions are mediated by the retinoblastoma and the Ku70 pathway. High levels of cyclin A1 and the associated CDK2 kinase activity were associated with increasing levels of phosphorylated retinoblastoma in vivo. UV irradiation induced a switch of the CDK2 towards cyclin A1, with accordance to changes in CDK2 kinase activity. The C‐terminus of cyclin A1 directly interacted with Ku70, and DNA binding activity of Ku70 was modulated by cyclin A1/CDK2 and phosphatase treatment. Cyclin A1‐deficiency induced by shRNA increased apoptosis that is induced by DNA damage and death receptor ligands. Taken together, these analyses demonstrate that cyclin A1 exerts antiapoptotic functions by interacting with retinoblastoma and Ku proteins in leukemia cells.

Kinetics of the in vivo expression of glucocorticoid receptor splice variants during prednisone treatment in childhood acute lymphoblastic leukaemia.

The in vivo glucocorticoid response in childhood acute lymphoblastic leukaemia (ALL) correlates with the response to multi‐agent chemotherapy. However, it is still unclear, whether the expression levels of glucocorticoid receptor (GR) splice variants facilitate the escape from glucocorticoid‐induced apoptosis and hence contribute to glucocorticoid resistance.

Homozygous alpha-thalassaemia: Prenatal diagnosis, risks, options and counselling

Sir, We agree with Dr Dickerhoff [1] that a-thalassaemia should be excluded in women from Southeast Asia showing a low MCV. As is the case for a great variety of diseases, it is optimal to reach diagnosis as early as possible in order to discuss the risks, options and consequences of the disease with the couple. First, this enables careful monitoring of the pregnancy and the initiation of potential therapeutic procedures in order to reduce fetal and maternal risks. Second, in case of an abortion or termination of pregnancy, knowledge of the disease allows optimal planning to minimize the risk of complications. Furthermore, we are in consensus with Dr Dickerhoff’s third point discussing prenatal diagnosis of thalassaemic syndromes, in order to inform parents adequately about the options and risks. These points have been mentioned in our article [2]. We would like to point out that information about prenatal diagnosis and possible consequences should be performed as part of a genetic counselling session, taking into account guidelines for non-directiveness [3]. Parents should be informed non-directively, giving them detailed information about risks during pregnancy, possibility and risks of prenatal testing, problems of the disease if diagnosis confirms homozygous a-thalassaemia, and possible consequences, in order to enable them make their own decisions based on informed consent.