Systemic administration of anti-urokinase plasminogen activator receptor monoclonal antibodies induces hepatic fibrin deposition in tissue-type plasminogen activator deficient mice.

BACKGROUND: Degradation of extracellular matrix proteins, such as fibrin, is pivotal to tumor invasion. Inhibition of the interaction between urokinase plasminogen activator (u-PA) and its receptor (u-PAR), and hence pro-u-PA activation, is an attractive approach to anti-invasive cancer therapy. A number of inhibitors exist for the human system, but because of species specificity none of these are efficient in mice. We have recently generated an inhibitory monoclonal antibody (mAb) against mouse u-PAR (mR1) by immunization of u-PAR-deficient mice. OBJECTIVES: To evaluate the effect of mR1 in vivo in a physiological setting sensitive to deregulated fibrinolysis, we have administered mR1 systemically and quantitated the effect on liver fibrin accumulation. METHODS: Wild-type and tissue-type plasminogen activator (t-PA) deficient mice were administered with mR1, or control antibody, during 6 weeks. Thereafter, the livers were retrieved and the amount of liver fibrin measured by unbiased morphometrical analysis of immunofluorescence signal. RESULTS: Systemic administration of mR1 caused significantly increased fibrin signal in anti-u-PAR treated t-PA-deficient mice compared to mock-treated, which mimics the phenotype of u-PAR;t-PA double-deficient mice. Fibrin and fibronectin accumulated within the sinusoidal space and was infiltrated by inflammatory cells. Analysis of small and rare hepatic fibrin plaques observed in t-PA-deficient mice showed infiltrating macrophages that, contrary to surrounding Kuppfer cells, expressed u-PAR. CONCLUSION: We show that u-PAR-expressing macrophages are involved in cell-mediated fibrinolysis of liver fibrin deposits, and that the antimouse-u-PAR mAb is effective in vivo and thus suited for studies of the effect of targeting the u-PA/u-PAR interaction in mouse cancer models.

Patched 2, located in 1p32-34, is not mutated in high stage neuroblastoma tumors.

Neuroblastoma is a childhood malignancy originating from cells of the sympathetic nervous system, exhibiting a marked diversity in outcome, with spontaneous regression at one end of the spectrum and severe disease and death at the other end. Features associated with frequent recurrence, a poor prognosis, and high tumor stage are loss of heterozygosity in the distal region of chromosome 1p and amplification of the N-myc gene. Patched 2 is a novel homologue to the tumor suppressor gene Patched 1, and has been mapped to 1p32-34, a part of chromosome 1 frequently deleted in high stage neuroblastoma tumors. RT-PCR analysis of 9 neuroblastoma cell lines showed expression of both Patched 1 and 2. We analyzed 14, mainly high stage, neuroblastoma tumors for mutations in the Patched 2 gene with denaturing HPLC using the Wave DNA fragment analysis system. In four tumor samples variations were detected within the coding sequence, and two of them gave rise to amino-acid substitutions. These variations were, however, also detected in normal DNA from the respective patients. We conclude that Patched 2 is expressed, but not frequently mutated, in high stage neuroblastomas and is therefore not likely to be involved in the genesis of this tumor.

HASH-1 and E2-2 are expressed in human neuroblastoma cells and form a functional complex.

The basic helix-loop-helix (bHLH) transcription factor mammalian achaete-scute homolog-1 (MASH-1 in mouse and HASH-1 in human) is essential for proper development of olfactory and most peripheral autonomic neurons, and for the formation of distinct neuronal circuits within the central nervous system. We have previously shown that HASH-1 is expressed in neuroblastoma tumors and cell lines, and in this study we have used the yeast two-hybrid system to isolate HASH-1 interacting proteins from a human neuroblastoma cDNA library. Two of the isolated clones contained cDNA from the E2-2 gene (also known as ITF2/SEF2-1). We show that E2-2 interacts with HASH-1 in both yeast and mammalian cells. The HASH-1/E2-2 complex binds an E-box (CACCTG) in vitro, and transactivates an E-box containing reporter construct in vivo. Furthermore, E2-2 seems to be one of the major HASH-1 interacting proteins in extracts from neuroblastoma cells. In conclusion, E2-2 forms a functional complex with HASH-1, and might therefore be involved in the development of specific parts of the central and peripheral nervous systems.