The human PRL-inducible protein (PIP)/gross cystic disease fluid protein-15 is expressed in pathological conditions of the mammary gland and in several exocrine tissues, such as the lacrimal, salivary, and sweat glands. In human breast cancer cells, the expression of PIP/gross cystic disease fluid protein-15 is stimulated by androgen and PRL, and inhibited by estrogen. However, it is not known whether the expression of PIP in other tissues is under similar hormonal regulation. In the present study we employed reverse transcriptase-polymerase chain reaction followed by rapid amplification of complementary DNA (cDNA) ends to amplify the PIP cDNA homolog, the submaxillary gland protein (SMGP) in the mouse. The mouse PIP/SMGP cDNA encodes a putative secreted peptide of 144 amino acids with a 51% identity with human PIP. Using the mouse PIP/SMGP cDNA as a probe, we examined the tissue- and cell-specific expression of PIP/SMGP messenger RNA by in situ hybridization and Northern blot analysis of mouse and rat tissues. Hormonal regulation was also studied in the rat. PIP/SMGP messenger RNA expression was only detected in the lacrimal and submaxillary glands of the rodents. In the rat submaxillary gland, PIP/SMGP gene expression was confined to the acinar cells. In the male rat lacrimal gland, castration resulted in an increase in expression, and in both male and female rats, androgen replacement abolished PIP/SMGP gene expression. This pattern of regulation was not observed in the submaxillary gland and was actually reversed in human breast cancer cells. PRL had no effect on the regulation of PIP/SMGP in either salivary or lacrimal glands. Our study indicates that tissue-specific factors are important in determining the hormone responsiveness of the PIP/SMGP gene. Regulation of the PIP/SMGP gene in vivo may provide a useful model system to study the mechanism of down-regulation of expression by androgen in a tissue-specific manner.

The human prolactin-inducible protein/gross cystic disease fluid protein-15 (PIP/GCDFP-15) gene is expressed in more than 90% of human breast cancer biopsies but not in the normal mammary gland. However, it is expressed in several normal human apocrine glands such as the lacrimal and salivary glands. In human breast cancer cell lines, the gene is regulated by a number of hormones including androgen and prolactin. It is not known whether gene expression in normal tissues is under similar hormonal control. To understand the mechanisms by which hormone-and tissue-specific expression of the human PIP/GCDFP-15 gene are regulated in vivo, we generated transgenic mice using a 13·7 kb genomic DNA fragment containing the entire 7 kb human gene, together with 2·9 kilobases of 5 and 3·8 kilobases of 3 flanking sequences. The human PIP/GCDFP-15 transgene was found to be expressed in both the lacrimal and salivary glands but was not expressed in the mammary glands of transgenic mice. This tissue-specific pattern of the transgene expression in the mouse was very similar to that of the endogenous human PIP/GCDFP-15 gene, and to the endogenous mouse gene. In the mouse salivary glands, the transgene expression was highest in the parotid, considerably less in the submaxillary (submandibular) and absent in the sublingual glands. In the mouse lacrimal gland, as in the human breast cancer cell lines, the human PIP/GCDFP-15 transgene was also up-regulated by androgen. These studies demonstrate that the human gene with its 6·3 kb flanking sequences is able to confer gene expression in vivo in a tissue-specific and hormone-responsive manner.

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Metallothioneins (MTs) are low-molecular-weight proteins whose physiologic roles are the regulation of essential metals Cu and Zn, sequestration of heavy metals, and free radical scavenging. Induced production of MTs in a wide variety of organisms exposed to heavy metals has made them popular exposure indicators. While it has been postulated that the three different isoforms of MT play different physiologic roles, methods to discern induction separately have not been available. The development of real-time polymerase chain reaction (real-time PCR) primers and TaqMan probes to measure the two MT isoforms found in salmonid fish are described. Assuming a high degree of homology between the isoforms and within different groups of salmonids, the sequences for MT-I and MT-II from rainbow trout were used to develop primers and probes for lake trout using the Primer3 program. Two sections of each isoform that varied by only a few nucleotides were targeted. SYBR Green validated the primer specificity, and melt curve analysis further ensured that only one product was amplified. Analysis of archived samples from fish captured in unmanipulated reference lakes or from lakes experimentally treated with cadmium or ethynylestradiol (EE2) afforded an examination of seasonal and contaminant influences on MT-I and MT-II mRNA expression.