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Current systemic therapies for breast cancer are often limited by their non-specific mechanism of action, unwanted toxicities on normal tissues and short-term efficacy due to the emergence of drug resistance. Identification of the molecular alterations in key proteins involved in breast cancer cell proliferation and survival resulted in development of various signal transduction inhibitors as new treatment strategy. Preclinical data support the use of these agents in breast cancer, including estrogen receptor positive breast cancer patients in which signalling inhibitors might prevent or treat resistance to endocrine therapy. These compounds have generally shown an acceptable toxicity profile. However, little clinical activity of signalling inhibitors as monotherapy in breast cancer patients has been reported up to now. Furthermore, the preliminary results of clinical trials of combinations of signalling inhibitors and endocrine therapies published to day are rather disappointing. These negative findings are likely due to the occurrence of mechanisms of resistance to these drugs.

The vertebrate telencephalon is composed of many architectonically and functionally distinct areas and structures, with billions of neurons that are precisely connected. This complexity is fine-tuned during development by numerous genes. To identify genes involved in the regulation of telencephalic development, a specific subset of differentially expressed genes was characterized. Here, we describe a set of cDNAs encoded by genes preferentially expressed during development of the mouse telencephalon that was identified through a functional genomics approach. Of 832 distinct transcripts found, 223 (27%) are known genes. Of the remaining, 228 (27%) correspond to expressed sequence tags of unknown function, 58 (7%) are homologs or orthologs of known genes, and 323 (39%) correspond to novel rare transcripts, including 48 (14%) new putative noncoding RNAs. As an example of this latter group of novel precursor transcripts of micro-RNAs, telencephalic embryonic subtractive sequence (TESS) 24.E3 was functionally characterized, and one of its targets was identified: the zinc finger transcription factorZFP9. The TESS transcriptome has been annotated, mapped for chromosome loci, and arrayed for its gene expression profiles during neural development and differentiation (in Neuro2a and neural stem cells). Within this collection, 188 genes were also characterized on embryonic and postnatal tissue byin situhybridization, demonstrating that most are specifically expressed in the embryonic CNS. The full information has been organized into a searchable database linked to other genomic resources, allowing easy access to those who are interested in the dissection of the molecular basis of telencephalic development.

A rapid and sensitive radiometric assay for UDP-glucuronosyl-transferase (UGT) is described. UGT substrates are incubated in 96-well plates with microsomes in the presence of [14C]UDP-glucuronic acid, and 14C-labeled glucuronidation products are separated from the unreacted nucleotide sugar by solid-phase extraction using 96-well extraction plates. The assay was validated with 15 structurally diverse UGT substrates containing acidic, phenolic, and hydroxyl reacting groups. Glucuronidation velocities for these compounds were determined using human, rat, and dog liver microsomes, and reaction kinetics were studied with 1-naphthol and 4-methylumbelliferone. Results obtained with the new assay confirmed the previously reported rank order of glucuronidation velocity of several typical UGT substrates and the finding that the glucuronidation of most of these compounds is significantly faster in dog than in human liver microsomes. UGT specificity of five compounds was determined using recombinant human UGTs. The major UGT isoforms identified were UGT1A6, UGT1A7, and UGT1A9 for 4-methylumbelliferone; UGT1A6 and UGT1A8 for 1-naphthol; UGT2B7 for naloxone; UGT1A3 and UGT2B7 for ketoprofen; and UGT1A4 for trifluoperazine. Identical results were obtained with a conventional high-performance liquid chromatography method coupled to mass spectrometric detection. The new assay should prove valuable for rapidly benchmarking recombinant UGTs and microsomal preparations from different species and tissues, identifying high-turnover compounds during drug discovery, and reaction phenotyping studies. Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics.

1Pharmacogenomic Laboratory, INT Fondazione Pascale – CROM, Mercogliano (AV), Italy †Author for correspondence: Cell Biology & Biotherapy Unit, INT Fondazione Pascale, Naples, Italy Tel.: +39 081 590 3826 nicnorm@yahoo.com Kobunai et al. raised important questions concerning the molecular diagnostics of KRAS mutations in metastatic colorectal carcinoma (CRC) [1] that we are happy to address. In particular, they suggest that assays that are more sensitive than direct sequencing of the PCR product (PCR/sequencing) detect a higher rate of KRAS mutations even in samples with over 50% tumor cells. This phenomenon might in part be owing to the molecular heterogeneity of some CRC cells that contain clones of KRAS mutant and wild-type cells. They hypothesize, therefore, that using a limit of 30% tumor cells might lead patients to lose the benefit of treatment with an anti-EGFR agent. With regards to the points raised by Kobunai et al., we feel that it is important to underline the points discussed in the following sections. We confirmed that real-time PCR-based assays such as the DxS Therascreen kit (DxS Ltd, Manchester, UK) are more sensitive than PCR/sequencing [2]. However, our study, as well as a larger study by Tol et al. [3], found no statistically significant difference in the ability to detect KRAS mutations between DxS and PCR/sequencing in a huge cohort (a total of over 500 patients) of CRC samples with over 30% tumor cells. In a quality assessment of KRAS testing that was run in Germany, the error rate of PCR/sequencing and of more sensitive, real-time PCR-based techniques was comparable [4]. Similar data were obtained in an Italian, as yet unpublished quality assessment [Italian Association of Medical Oncology and the Italian Society of Surgical Pathology and

We identify a new enzymatic activity underlying metastasis in breast cancer and describe its susceptibility to therapeutic inhibition. We show that human prune (h-prune), a phosphoesterase DHH family appertaining protein, has a hitherto unrecognized cyclic nucleotide phosphodiesterase activity effectively suppressed by dipyridamole, a phosphodiesterase inhibitor. H-prune physically interacts with nm23-H1, a metastasis suppressor gene. The h-prune PDE activity, suppressed by dipyridamole and enhanced by the interaction with nm23-H1, stimulates cellular motility and metastasis processes. Out of 59 metastatic breast cancer cases analyzed, 22 (37%) were found to overexpress h-prune, evidence that this novel enzymatic activity is involved in promoting cancer metastasis.