RECK controls breast cancer metastasis by modulating a convergent, STAT3-dependent neoangiogenic switch.

Metastasis is the primary cause of cancer-related death in oncology patients. A comprehensive understanding of the molecular mechanisms that cancer cells usurp to promote metastatic dissemination is critical for the development and implementation of novel diagnostic and treatment strategies. Here we show that the membrane protein RECK (Reversion-inducing cysteine-rich protein with kazal motifs) controls breast cancer metastasis by modulating a novel, non-canonical and convergent signal transducer and activator of transcription factor 3 (STAT3)-dependent angiogenic program. Neoangiogenesis and STAT3 hyperactivation are known to be fundamentally important for metastasis, but the root molecular initiators of these phenotypes are poorly understood. Our study identifies loss of RECK as a critical and previously unknown trigger for these hallmarks of metastasis. Using multiple xenograft mouse models, we comprehensively show that RECK inhibits metastasis, concomitant with a suppression of neoangiogenesis at secondary sites, while leaving primary tumor growth unaffected. Further, with functional genomics and biochemical dissection we demonstrate that RECK controls this angiogenic rheostat through a novel complex with cell surface receptors to regulate STAT3 activation, cytokine signaling, and the induction of both vascular endothelial growth factor and urokinase plasminogen activator. In accordance with these findings, inhibition of STAT3 can rescue this phenotype both in vitro and in vivo. Taken together, our study uncovers, for the first time, that RECK is a novel regulator of multiple well-established and robust mediators of metastasis; thus, RECK is a keystone protein that may be exploited in a clinical setting to target metastatic disease from multiple angles.

Domain specific overexpression of TIMP-2 and TIMP-3 reveals MMP-independent functions of TIMPs during Xenopus laevis development.

Extracellular matrix remodelling mediates many processes including cell migration and differentiation and is regulated through the enzymatic action of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). TIMPs are secreted proteins, consisting of structurally and functionally distinct N- and C-terminal domains. TIMP N-terminal domains inhibit MMP activity, whereas their C-terminal domains may have cell signalling activity. The in vivo role of TIMP N- and C-terminal domains in regulating developmental events has not previously been demonstrated. Here we investigated the roles of TIMP-2 and TIMP-3 N- and C-terminal domains in Xenopus laevis embryos. We show that overexpression of TIMP-2 N- and C-terminal domains results in severe developmental defects and death, as well as unique changes in MMP-2 and -9 expression, indicating that the individual domains may regulate MMPs through distinct mechanisms. In contrast, we show that only the N-terminal, but not the C-terminal domain of TIMP-3, results in developmental defects.

Mechanical simulation of muscle loading on the proximal femur: analysis of cemented femoral component migration with and without muscle loading.

OBJECTIVE: This study examines the effect of including muscle forces in fatigue tests of cemented total hip arthroplasty reconstructions. DESIGN: An experimental device capable of applying the joint reaction force, the abductor force, the vastus lateralis force, and the tensor fasciae latae force to the implanted femur is described. BACKGROUND: Current in vitro fatigue tests of cemented total hip arthroplasty reconstructions do not apply physiological muscle loads. Experimental and numerical studies report significant differences in stresses obtained in the cement mantle depending on the loads applied. The differing stresses may alter the outcome of an in vitro test. METHODS: Ten femoral components were reproducibly implanted into proximal composite femurs. Five of these femoral components were tested using a loadprofile which included muscle loading, five were tested without muscle loading. The migration of each femoral component was monitored continuously during dynamic fatigue tests. RESULTS: Clinically comparable migration amounts were found for both sets of femoral components, with the femoral components tested with muscle loading experiencing lower mean migration, lower mean inducible displacement, and less experimental scatter. CONCLUSIONS: The inclusion of muscle forces seems to stabilise the femoral component during the test. In vitro fatigue tests of cemented total hip arthroplasty reconstructions should include muscle loading to provide increased confidence in the results obtained. RELEVANCE: This study examines how the migration of cemented femoral hip prostheses is influenced by muscle forces. Hip prostheses are one of the few medical devices for which pre-clinical testing protocols have emerged, and this study ascertains whether or not the inclusion of muscle forces is necessary for pre-clinical tests. The conclusion that muscle loading should be included, and that it is important for the development of a new generation of standardised tests to provide enhanced patient protection against functionally poor prostheses.

Abduction paralysis with hypotropia: sign of weakness of a contralateral superior oblique.

background The clinical diagnosis of extraocular motor paralysis that is caused by severe cranial trauma can often be complicated. The resulting clinical picture can make the identification of all the components of potentially treatable oculomotor problems difficult. methods We examined five cases of complete abducens nerve paralysis with marked downshoot in attempted abduction seen after severe cranial trauma. results With the patients looking in the field of gaze of the paralysis, a marked infraductive movement of the paralytic eye occurred while the other eye maintained fixation. Other clinical findings confirmed this to be a secondary deviation due to a paresis of the contralateral superior oblique. conclusion Patients with a paralysis of the lateral rectus following a severe cranial trauma who demonstrate a marked downshoot of the involved eye should be suspected of having a paresis of the contralateral superior oblique. This diagnosis has helped us effectively to treat this vertical incomitance by a simple weakening procedure of the contralateral inferior oblique.

The use of binocular visual acuity in the assessment of intermittent exotropia.

BACKGROUND: It has been suggested that a decrease in distance stereoacuity in patients with intermittent exotropia is a good indicator of diminishing control. However, there has been no adequate explanation for this reported reduction in distance stereoacuity in these patients. We postulate that the decrease in stereoacuity is related to blurred visual acuity created by an increasing demand on accommodation, which these patients use in an attempt to control the exodeviation. This can best be assessed by measuring binocular visual acuity (BVA). Analysis of BVA could provide a useful clinical tool to evaluate control measures used by patients with intermittent exotropia. METHODS: A prospective study of patients with intermittent exotropia, ranging in age from 6 to 60 years, was performed. Only those patients with the presence of either basic or divergence excess (simulated or true) type exodeviation were included in the study. The data analysis included the age of these patients, age at onset of the deviation, monocular and binocular visual acuity, oculomotor and fusional status, and near and distance stereoacuity. RESULTS: Data from 36 patients show that the measurements of BVA correlated well with a corresponding loss of distance stereoacuity but not with the size of the deviation. CONCLUSION: The decrease of stereoacuity reported in patients with exotropia can be explained by increased accommodation and decreased distance BVA. This measurement can be a simple method of quantifying the fusional control of patients with intermittent exotropia.

Structure and chromosomal location of mouse and human CD52 genes.

Human CD52 (CAMPATH-1 antigen) is an abundant surface molecule on lymphocytes and a favoured target for lymphoma therapy and immunosuppression. It comprises a small glycosylphosphatidylinositol (GPI) anchored peptide to which a large carbohydrate moiety is attached. Structurally similar proteins include the proposed mouse homologue, B7 antigen (B7-Ag; not to be confused with the CD28 ligand), and human and mouse CD24. Sequence similarities between CD52 and B7-Ag precursors are concentrated over the signal peptides and the sequences cleaved during GPI attachment. While the short mature peptides are not apparently homologous, the N-linked glycosylation site is retained in both. We describe similarities in exon-intron organisation, syntenic chromosome positions (human CD52, 1p36; mouse B7-Ag, chromosome 4, between Dsil and D4Nds16) and sequence homology in the promoter regions which strongly suggests that B7-Ag is the mouse homologue of CD52. The structure of these genes is also similar to that of mouse CD24, suggesting a common ancestor. Promoter activities and transcription start sites were also analysed. These results suggest that human CD52 and mouse B7-Ag gene expressions are controlled by TATA-less promoters.

Acute pentylenetetrazol injection reduces rat GABAA receptor mRNA levels and GABA stimulation of benzodiazepine binding with No effect on benzodiazepine binding site density.

The effects of a single convulsive dose of pentylenetetrazol (PTZ, 45 mg/kg i.p.) on rat brain gamma-aminobutyric acid type A (GABAA) receptors were studied. Selected GABAA receptor subunit mRNAs were measured by Northern blot analysis (with beta-actin mRNA as a standard). Four hours after PTZ, the GABAA receptor gamma2-mRNA was decreased in hippocampus, cerebral cortex, and cerebellum; alpha1-mRNA was decreased in cerebellum; and beta2 subunit mRNA was decreased in cortex and cerebellum. The alpha5 subunit mRNA level was not altered. Those mRNAs that had been reduced were increased in some brain regions at the 24-h time point, and these changes reverted to control levels by 48 h. PTZ effect on GABAA receptors was also studied by autoradiographic binding assay with the benzodiazepine agonist [3H]flunitrazepam (FNP), the GABAA agonist [3H]muscimol, and the benzodiazepine antagonist [3H]flumazenil. There was an overall decrease in [3H]FNP binding 12 but not 24 h after PTZ treatment. In contrast, [3H]muscimol binding was minimally affected, and [3H]flumazenil binding was unchanged after PTZ treatment. Additional binding studies were performed with well-washed cerebral cortical homogenates to minimize the amount of endogenous GABA. There was no PTZ effect on specific [3H]FNP binding. However, there was a significant reduction in the stimulation of [3H]FNP binding by GABA. The results showed that an acute injection of PTZ caused transient changes in GABAA receptor mRNA levels without altering receptor number but affected the coupling mechanism between the GABA and benzodiazepine sites of the GABAA receptor.

Elimination of the immunogenicity of therapeutic antibodies.

The immunogenicity of therapeutic Abs limits their long-term use. The processes of complementarity-determining region grafting, resurfacing, and hyperchimerization diminish mAb immunogenicity by reducing the number of foreign residues. However, this does not prevent anti-idiotypic and anti-allotypic responses following repeated administration of cell-binding Abs. Classical studies have demonstrated that monomeric human IgG is profoundly tolerogenic in a number of species. If cell-binding Abs could be converted into monomeric non-cell-binding tolerogens, then it should be possible to pretolerize patients to the therapeutic cell-binding form. We demonstrate that non-cell-binding minimal mutants of the anti-CD52 Ab CAMPATH-1H lose immunogenicity and can tolerize to the "wild-type" Ab in CD52-expressing transgenic mice. This finding could have utility in the long-term administration of therapeutic proteins to humans.

High level transcription of the complement regulatory protein CD59 requires an enhancer located in intron 1.

CD59 is a complement regulatory protein and may also act as a signal-transducing molecule. CD59 transgenic mice have been generated using a CD59 minigene (CD59 minigene-1). Although this minigene contained a 4.6-kilobase pair 5'-flanking region from the human CD59 gene as a promoter, the expression levels of the CD59 mRNA were substantially lower than those observed in humans, suggesting that CD59 gene expression might also require other transcriptional regulatory elements such as an enhancer. To investigate the transcriptional regulation of the CD59 gene, we used three cell lines that express CD59 at different levels. We have identified DNase I-hypersensitive sites in intron 1 in HeLa cells, which express CD59 at high levels, but not in Jurkat (intermediate level) or Raji cells (low level). Furthermore, cell line-specific enhancer activity was detected in a fragment containing these DNase I-hypersensitive sites. The CD59 enhancer was mapped to between -1155 and -888 upstream of the 5'-end of exon 2. To investigate the enhancer activity in vivo, a new CD59 minigene was constructed by the addition of the enhancer fragment into CD59 minigene-1. High expressor CD59 transgenic mice were generated using the new minigene.

Gene structure of human CD59 and demonstration that discrete mRNAs are generated by alternative polyadenylation.

We have isolated the CD59 gene from human genomic libraries. The gene is distributed over more than 27 x 10(3) base-pairs and consists of one 5'-untranslated exon and three coding exons. The gene structure is similar to that of mouse Ly-6 with the exception of the larger size of CD59 introns. Northern blot analysis using six different probes located in the 3'-region of the gene shows that more than four different CD59 mRNA molecules are generated by alternative polyadenylation. Three of these polyadenylation sites were predicted from previously published cDNA sequences. We have isolated a fourth from Jurkat poly(A)+ RNA by the procedure of rapid amplification of cDNA ends. Alternative polyadenylation may be due to the RNA secondary structure around the typical polyadenylation signal, AAUAAA.

Transfection of human CD59 complementary DNA into rat cells confers resistance to human complement.

We have examined the role of the human CD59 antigen in inhibiting complement-mediated lysis by transfer and expression of a CD59 cDNA in rat cells. A cDNA encoding CD59 was subcloned into the expression vector pSFSVneo and stably transfected into the rat T cell line NB2-6TG. Indirect immunofluorescence staining using the anti-CD59 monoclonal antibody YTH53.1 demonstrated the presence of human CD59 antigen on transfected cells and its attachment to the cell surface by a rat glycolipid anchor. Transfected cells were found to contain a single 3.3-kb species of CD59 mRNA by Northern blot hybridization. Immunoblotting revealed that this encoded a protein band of the same size as that observed in human erythrocytes. To determine the biological effect of expression of human CD59 in rat cells, an assay was devised which measured the relative lysis of transfected cells compared to untransfected cells in the presence of human complement and a lytic monoclonal antibody. It was observed that CD59-transfected rat cells are less susceptible to lysis by human complement and that this effect was blocked by a F(ab')2 fragment of YTH53.1. These experiments provide a direct demonstration that CD59 can function as an homologous complement restriction factor for nucleated cells.

Phosphatidylethanolamine synthesis in ethanolamine-responsive and -nonresponsive cells in culture.

Mammalian cells can be classified into two types based upon whether or not they show growth response to ethanolamine (Etn) in culture. The content of phosphatidylethanolamine (PE) in phospholipid and incorporation of radioactive Etn into the cells were examined in the Etn-responsive and -nonresponsive cells in order to elucidate the mechanisms of growth stimulation by Etn. In all Etn-responsive cells tested, 5 microM Etn significantly altered the composition of cellular phospholipid compared to that grown without Etn, while Etn-nonresponsive cells had a similar phospholipid composition whether the growth medium contained Etn or not. Using two rat mammary carcinoma cell lines, 64-24 (responsive type) and 22-1 (nonresponsive type), further studies were carried out. In 64-24 cells there was a proportional increase in PE content as the dosage of Etn in the medium was increased. The increase in PE content leveled off at 10 microM. Further, the increase in PE content was correlated with increased rate of growth. In contrast, PE content or growth rate did not change at all in 22-1 cells. In 64-24 cells radioactive Etn (0.1-50 microM) was incorporated four- to five-fold more efficiently into phospholipid, and the aqueous pool of precursors of PE was ten times less as compared to 22-1 cells, indicating that Etn-responsive cells utilize Etn supplied in the medium to synthesize PE far more efficiently than Etn-nonresponsive cells. De novo synthesis of PE must not be sufficient to support optimum growth in Etn-responsive cells.