To investigate whether polymorphic forms of the human dopamine D4 receptor have different functional characteristics, we have stably expressed cDNAs of the D4 .2, D4 .4, and D4 .7 isoforms in several cell lines . Chinese hamster ovary CHO-K1 cell lines expressing D4 receptor variants displayed pharmacological profiles that were in close agreement with previous data from transiently expressed D4 receptors in COS-7 cells . Dopamine stimulation of the D4 receptors resulted in a concentration-dependent inhibition of the forskolin-stimulated cyclic AMP (cAMP) levels . The potency of dopamine to inhibit cAMP formation was about twofold reduced for D4 .7 (EC so of -37 nM) compared with the D4 .2 and D4 .4 variants (EC SO of -16 nM) . Antagonists block the dopamine-mediated inhibition of cAMP formation with a rank order of potency of emonapride > haloperidol = clozapine > raclopride . There was no obvious correlation between the efficacy of inhibition of forskolin-stimulated cAMP levels and the D4 subtypes . Dopamine could completely reverse prostaglandin E 2 -stimulated cAMP levels for all three D4 receptor variants . Deletion of the repeat sequence does not affect functional activity of the receptor . The data presented indicate that the polymorphic repeat sequence causes only small changes in the ability of the D4 receptor to block cAMP production in CHO cells . Key Words : Dopamine receptors-D4-Cyclic AMP-Variants .

Purpose: This study examined the effectiveness of early and prolonged mu4D5 (the murine form of trastuzumab/ Herceptin) treatment in transgenic mice that overexpress human HER2 (huHER2), under the murine mammary tumor virus promoter, as a model of huHER2-overexpressing breast cancer.Experimental Design: Mice were randomly assigned to one of three treatment groups and received i.p. injections from 17 weeks of age until either 52 weeks of age or morbidity. Fourteen mice received 100 mg/kg mu4D5, 14 mice received 100 mg/kg antiherpes simplex virus glycoprotein D control antibody, and 11 mice received a diluent control.Results: High levels of huHER2 expression were detectable in mammary glands of young virgin founder mice. Mammary adenocarcinomas were frequently found in female founders and progeny at an average age of 28 weeks, with some progressing to metastatic disease. The incidence of mammary tumors was significantly reduced, and tumor growth inhibition was observed in mice receiving mu4D5 compared with control mice. In addition, Harderian gland neoplasms, highly associated with overexpression of hu-HER2 in this transgenic line, were entirely absent in the mu4D5 treatment group, indicating down-regulation of hu-HER2 in vivo activity.Conclusions: Early intervention with mu4D5 was of benefit in our transgenic mice at high risk for developing huHER2-overexpressing breast cancer. This study suggests a potential benefit of early treatment with Herceptin in HER2-positive primary breast cancer.

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Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events.

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