Single molecule detection for in vitro diagnostics

In this paper we present a novel highly sensitive detection system for diagnostic applications. The system is designed to meet the needs of medical diagnostics for reliable measurements of pathogens and biomarkers in the low concentration regime. It consists of a confocal detection unit, micro-structured sampling cells, and a "Virtual lab" analysis software. The detection unit works with laser induced fluorescence and is designed to provide accurate and highly sensitive measurement at the single molecule level. Various sampling cells are micro-structured in glass, silicon or polymers to enable measurements under flow and nonflow conditions. Sampling volume is below one microliter. The "Virtual lab" software analyzes the light intensity online according to the patent pending "Accurate Stochastic Fluorescence Spectroscopy" (ASFS) developed by FluIT Biosystems GmbH. Tools for simulation and experiment optimization are included as well. Experimental results for various applications with relevance for in vitro diagnostics will be presented

Prostate apoptosis response gene-4 (par-4) abrogates the survival function of p185(BCR-ABL) in hematopoietic cells

Objective Prostate apoptosis response gene-4 (par-4) is deregulated in acute and chronic lymphatic leukemia. Given its pro-apoptotic role in neoplastic lymphocytes and evidence that par-4 antagonizes oncogenic Ras in solid tumors, we hypothesized that par-4 may act as a tumor suppressor impairing transformation induced by p185BCR-ABL. Materials and Methods The capacity of par-4 to interfere with factor independence induced by p185BCR-ABL and V12ras was evaluated by analysis of factor-independent growth of p185BCR-ABL/ par-4 and V12ras/par-4 tranduced cells. The expression of par-4 and p185BCR-ABL by the respective constructs was controlled by Western blot analysis. Activated Ras was detected by pull-down assay in the cell clones expressing p185BCR-ABL in the absence and presence of par-4. Results Expression of p185BCR-ABL causes factor independence, signifying a conversion toward a transformed phenotype in hematopoietic precursors. We demonstrate that par-4 completely abolishes factor independence induced by p185BCR-ABL and partially abrogates factor independence caused by activated V12ras. Evaluating the underlying molecular mechanisms, we show that par-4 hinders activation of oncogenic Ras and causes concomitant disruptions of p185BCR-ABL-mediated signaling. Conclusion We provide the first evidence that par-4 exhibits an antitransforming capacity by antagonizing p185BCR-ABL-induced factor-independent proliferation in hematopoietic cells

In the erythroleukemic cell line HEL Prostate-apoptosis-response-gene-4 (par-4) fails to down-regulate Bcl-2 and to promote apoptosis.

In a variety of malignant cells Prostate-apoptosis-response-gene-4 (Par-4) exhibits a pro-apoptotic influence sensitizing these cells to apoptosis-inducing agents by downregulating expression of Bcl-2. Considering the crucial role of Bcl-2 in the development of chemoresistance of acute myeloid leukemia (AML) cells, we here assessed the potential of Par-4 to down-regulate Bcl-2 and to induce apoptosis in the erythroleukemic cell line HEL. Testing a potential pro-apoptotic role of Par-4 upon incubation with various conventional chemotherapeutic drugs, novel agents such as the signal transduction inhibitor STI 571 and the histone deacetylase (HDAC)- inhibitor trichostatin A (TSA), as well as with the experimental substances Fas and TRAIL, we provide evidence that in the erythroleukemic cell line HEL expression of Par-4 is not sufficient to sensitize to any of these pro-apoptotic stimuli. We further demonstrate that--in contrast to previous reports in non-AML cells--Par-4 expression in HEL cells leads to an upregulation of Bcl-2. Moreover, Par-4-positive HEL cells exhibit a decreased level of the proapoptotic protein Bax as compared to Par-4- negative cells. In addition, Par-4 increases the expression of Daxx--whose downregulation is associated with augmented chemosensitivity--as well as expression of the procaspases-8, -9 and -10, whereas the levels of the procaspases-3 and -7 remain unaltered. In conclusion we here demonstrate that in the erythroleukemic cell line HEL--in contrast to other cell types Par-4 fails to promote apoptosis and outline the underlying molecular mechanisms

In lymphatic cells par-4 sensitizes to apoptosis by down-regulating bcl-2 and promoting disruption of mitochondrial membrane potential and caspase activation.

Inhibition of apoptosis is a hallmark of malignancies of the hematopoetic system. Previous studies in nonhematopoetic cells demonstrated that the prostate-apoptosis-response-gene-4 (Par-4) is up-regulated in cells undergoing programmed cell death and that Par-4 exerts its proapoptotic effect by down-regulating Bcl-2. After showing the aberrant expressional pattern of Par-4 in neoplastic lymphocytes as well as demonstrating inverse expressional patterns of Par-4 and Bcl-2 in malignant cells of patients suffering from acute lymphocytic leukemia, we assessed the functional consequences of Par-4 overexpression during apoptosis in Jurkat T lymphocytes. We show that in lymphatic cells Par-4 overexpression decreases the level of Bcl-2, whereas Bax, the proapoptotic counterpart of Bcl-2, retains unaltered levels. Moreover, Par-4 overexpression is accompanied by cleavage of poly(ADP-ribose) polymerase (PARP). Despite these effects, overexpression of Par-4 alone is not sufficient to induce apoptosis but markedly increases the rate of apoptosis on treatment with different chemotherapeutic agents. On chemotherapeutic treatment Par-4 overexpression enhances disruption of mitochondrial membrane potential, PARP-cleaving activity, as well as activation of caspase-3. The hypothesis of caspase-dependency of Par-4-promoted apoptosis is additionally supported by demonstrating complete abrogation of programmed cell death after pretreatment with a broad spectrum caspase-inhibitor. On inhibition of caspase-3 overexpression of Par-4 enables lymphatic cells to alternatively activate caspases-9, -6, and -7 by diminishing the influence of the inhibitors of apoptosis proteins (IAPs) cIAP1 and XIAP. Our study is the first to identify Par-4 as a proapoptotic protein in lymphatic cells, outlining a model of action evaluating the role of Bcl-2/Bax, as well as demonstrating the impact of Par-4 expression on PARP cleavage, disruption of mitochondrial membrane potential, caspase activation, and interactions with inhibitors of apoptosis proteins