We investigated the expression pattern of the breast cancer associated gene LIV‐1 on mRNA and protein level in 111 human breast cancer patients by in situ hybridization as well as immunohistochemistry and focused on the unknown potential of LIV‐1 expression levels as a prognostic marker. To our knowledge, this is the first study on endogenous LIV‐1 protein expression. Results of our study indicate that LIV‐1 mRNA and protein expression levels are only weakly correlated, suggesting posttranscriptional regulatory mechanisms. Furthermore, LIV‐1 mRNA quantity in combination with a positive ER status seem to represent a better marker than the progesterone receptor status according to the prognostic significance for relapse free survival (RFS). A negative correlation of LIV‐1 protein levels with tumor size, grade and stage reflects an association of LIV‐1 protein expression with less aggressive tumors. High LIV‐1 protein expression seems to be associated with a longer relapse free and overall survival in breast cancer patients with invasive ductal carcinoma. This association, however, seems to be dependent from other prognostic markers. Our data suggest that LIV‐1 is a promising candidate for a novel marker for breast cancer patients with better outcome. Furthermore, our study presents a revised cDNA sequence of LIV‐1 and demonstrates the localization of endogenous LIV‐1 in the endoplasmic reticulum. (Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html). © 2005 Wiley‐Liss, Inc.

SummaryThe czc determinant, which mediates resistance to Co 2þ , Zn 2þ and Cd 2þ in Alcaligenes eutrophus CH34 by cation efflux, is regulated by a two-component regulatory system composed of the sensor histidine kinase CzcS and the response activator CzcR (in addition to other components previously described). Regulatory genes are arranged in an upstream regulatory region (URR) and a downstream regulatory region (DRR). Transcription of czcCBA and of the URR was regulated by heavy-metal cations. DNA sequencing of the region downstream of czcD revealed the presence of the czcR and czcS genes which together with czcD form the DRR. Regulation of the DRR was studied with a czcD::lacZ translational fusion and a czcS::lux transcriptional fusion. Expression of both genes is also regulated by heavy metals. The genes of the URR yielded three mRNAs of approx. 1200, 500 and 200 nucleotides, respectively. The genes czcCBA for the cation/proton antiporter CzcCBA were transcribed by one operon as a transcript of 6200 nucleotides.

The androgen dependency of the genes coding for the cysteine-rich secretory proteins (CRISP) was analysed in their main sites of expression. Male mice were treated with the gonadotropin-releasing hormone antagonist Ac-DNapAla-DClPhAla-DPyrAla-Ser-Tyr-DCtl-Leu-Lys(Mor)-Pro-DAla-NH 2 [DNapAla, D-2-naphthyl-Ala ; DClPhAla, D-4-chlorphenyl-Ala ; DPyrAla, D-pyridyn-3-yl-Ala ; DCtl, D-citrulline ; Lys(Mor), L-2-amino-6-(morpholin-4-yl)-hexanoic acid], and CRISP RNA levels were assessed by northern blot and competitive reverse transcriptase-mediated (RT)-PCR. In the salivary gland, CRISP-1 and to a lesser extent CRISP-3 expression was markedly reduced, in spite of an up-regulation of androgen receptor transcript levels. A down-regulation of CRISP-1 expression was also observed in the epididymis. Conversely, the levels of the testicular CRISP-2 transcripts were hardly affected at all. Female mice were ovariectomised and treated with testosterone propionate, and their salivary gland RNAs analysed. CRISP-1 and CRISP-3 RNA levels were significantly increased, and these effects were prevented by a concomitant treatment with the antiandrogen flutamide. Androgen receptor transcript levels were not affected by androgen administration but increased following antiandrogen treatment. CRISP expression during postnatal development was monitored by northern blot analysis. CRISP-1 and CRISP-2 transcripts were detected as early as 22 days after birth in the epididymis and testis, respectively, whereas CRISP-3 mRNA was visible only from day 30 in the salivary gland. A sharp increase of all CRISP levels was noted on day 40, coincident with the onset of sexual maturity. Altogether these results indicate that despite their high similarity, the CRISP genes are differentially regulated by androgens.Keywords : androgen; cysteine-rich secretory protein; epididymis; salivary gland; testis.The cysteine-rich secretory protein (CRISP) family was orig-ily of plants, which are induced upon infection by pathogens as inally described in the mouse [1, 2] and comprises evolutionarily part of the hypersensitive defense reaction [16, 17]. conserved polypeptides with a potential involvement in innate CRISP-1 is the best characterized member of the family and immunity. The CRISP genes are located on mouse chromosome its gene is mainly expressed in the corpus and cauda regions of 17 and human chromosome 6, in close vicinity to the major the epididymis, and in the male submandibular gland [1, 2, 9]. histocompatibility complex region [3Ϫ6]. They are expressed in Specific binding of its rat counterpart named acidic epididymal the specific granules of human neutrophils [7], in murine B cell glycoprotein and DE to spermatozoa heads has been reported, precursors [8], by glands with an exocrine function and by mu-leading to speculation about a role in sperm maturation [18Ϫ cosal epithelial surfaces [1, 2, 9, 10], which suggests a role in 20], but this could not be confirmed in mouse or human [9, 10, non-specific defense reactions, similar to that of defensins [11]...

The mRNA for cysteine-rich secretory protein-3 (CRISP-3) was originally identified in the mouse salivary gland as an androgen-dependent transcript, and is closely related to CRISP-1 and CRISP-2 which are abundantly expressed in the epididymis and testis respectively. Overlapping phage clones encompassing the entire length of the CRISP-3 gene were isolated from a lambda EMBL3 genomic library and analysed. DNA sequencing revealed that the gene consisted of eight exons ranging between 55 and 740 bp in size, and seven introns. All exon-intron junctions conformed to the GT/AG rule established for eukaryotic genes. The length of the introns was determined by PCR and was found to vary between 1.0 and 3.7 kb, indicating that the gene spans over 20 kb of the mouse genome. Primer extension allowed the mapping of the major transcription initiation site to an adenine located at the appropriate position downstream of a bona fide TATA box, in a region corresponding well to the eukaryotic consensus sequence. Over 800 bp of CRISP-3 promoter region were determined and two regions almost exactly matching the androgen-responsive element consensus RGWACANNNTGTWCY detected. In addition, sequences described in the Drosophila melanogaster Sgs-3 gene as being involved in its salivary gland-specific expression as well as two putative OTF- and GATA-binding elements were also found.

In mice, cysteine-rich secretory protein-1 (CRISP-1) is mainly found in the epididymis and also, to a lesser extent, in the salivary gland of males, where androgens control its expression. We have now isolated and characterized overlapping phage clones covering the entire length of the CRISP-1 gene. DNA sequencing revealed that the gene is organized into eight exons, ranging between 55 and 748 bp in size, and seven introns. All exon-intron junctions conformed to the GT/AG rule established for eukaryotic genes. The intron length, as determined by PCR, varied between 1.05 and 4.0 kb so that the CRISP-1 gene spans over 20 kb of the mouse genome. The transcription-initiation site was determined by primer extension and localized at the expected distance downstream of a consensus TATA box. Approximately 3.7 kb of the CRISP-1 promoter region were isolated and sequenced, and several stretches fitting the androgen-responsive element consensus were found. Those that most resembled the consensus were analysed by electrophoretic mobility-shift assay and found to form specific complexes with the liganded androgen receptor in vitro, but with different affinities. Putative binding elements for the transcription factors Oct, GATA, PEA3, CF1. AP-1 and AP-3 were also found in the promoter region.