Elevated expression of proprotein convertases alters breast cancer cell growth in response to estrogen and tamoxifen.

Two proprotein convertase cDNAs, PC1 and furin, were stably transfected into the human breast cancer cell line MCF-7. The PC1 or furin over-expressing cells possessed an altered morphology. When grown in vitro in a serum-free medium, the population doubling time of the convertase-transfected cells was twice that of wild-type (WT) cells. High concentrations of estradiol stimulated the growth of all three cell types to a similar extent; however, at low concentrations of estradiol, the convertase-transfected cells grew more slowly than WT cells. In athymic nude mice implanted with 5 mg estradiol pellets, the growth of tumors of convertase-transfected MCF-7 cells was stimulated to a degree similar to that of WT MCF-7 tumors. However, in mice implanted with lower-dose (1.5 mg) estradiol pellets, the tumors of PC1- or furin-transfected MCF-7 cells grew approximately five times slower than those of WT MCF-7 cells. In mice implanted with tamoxifen pellets, tumors of PC1- or furin-transfected MCF-7 cells regressed approximately five times slower than the WT tumors. This study shows that the over-expression of proprotein convertases confers a greater estrogen dependency and anti-estrogen resistance on human breast cancer cells.

Analysis of tissue- and hormone-specific regulation of the human prolactin-inducible protein/gross cystic disease fluid protein-15 gene in transgenic mice.

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.

A new member of the hormonally regulated rodent submaxillary gland glycoprotein gene family: cDNA cloning and tissue specific expression.

Polymerase chain reaction was used to amplify and identify two related rat submaxillary gland glycoprotein (rSMGGP and rSMGGP1) cDNAs. They were 489 bp and 594 bp long respectively. The shorter cDNA (rSMGGP) was identical to the previously published rat spot-I protein. The longer cDNA (rSMGGP1) had an additional (117 bp) unique nucleotide sequence in the 3' coding region, and the overall homology between the two cDNAs was 78%. rSMGGP also had a 68% homology to the mouse submaxillary gland glycoprotein (mSMGGP) cDNA. The predicted translated product of rSMGGP1 was 130 amino acids long, 39 amino acids longer than the rSMGGP. The region of greatest diversity between the putative peptides of the two rat cDNAs and the mouse cDNA was in the carboxy terminus. Northern blot analysis, using both rat cDNAs as probes, showed hybridization to an mRNA transcript (650 bases) in the submaxillary and lacrimal gland of the normal adult male and female rat. A larger transcript (approximately 700 bases) was induced under conditions of altered hormonal profiles: hypophysectomy, pregnancy/lactation, and castration. Dihydrotestosterone administration inhibited expression of the two transcripts in both the lacrimal and submaxillary glands of male and female rats. The labelled 117 bp DNA fragment unique to the rSMGGP1 cDNA hybridized only to the 700 base transcript in the rat lacrimal and submaxillary gland suggesting that differential exon usage produces the two variant mRNAs. The regulation of the SMGGP gene expression may provide yet another useful model for studying the mechanism of down-regulation of genes by androgen and the identification of tissue specific factors in the lacrimal and submaxillary gland.

Tissue-specific androgen-inhibited gene expression of a submaxillary gland protein, a rodent homolog of the human prolactin-inducible protein/GCDFP-15 gene.

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 prolactin-inducible protein (PIP/GCDFP-15) gene: cloning, structure and regulation.

The androgen and prolactin responsive prolactin-inducible protein (PIP)/gross cystic disease fluid protein (GCDFP-15) is expressed in benign and malignant human breast tumors and in such normal exocrine organs as sweat, salivary and lacrimal glands. In this paper we report the cloning and structure of the human gene, and describe potential mechanisms involved in its regulation by hormones. The entire PIP gene, 7 kb long, was found in a single recombinant phage clone. The gene has 4 exons ranging from 106 bp to 223 bp in length. Nuclear run-on experiments utilizing PIP genomic fragments to detect nascent PIP transcripts revealed that both androgen and prolactin increased transcription of the PIP gene. Neither hormone had any effect on the stability of PIP precursor RNA or mature mRNA. Therefore the PIP gene is an excellent model by which to study the molecular events associated with the actions of prolactin and androgen in the regulation of gene expression in mammalian cells.

Prolactin-inducible proteins in human breast cancer cells.

The mechanism of action of prolactin in target cells and the role of prolactin in human breast cancer are poorly understood phenomena. The present study examines the effect of human prolactin (hPRL) on the synthesis of unique proteins by a human breast cancer cell line, T-47D, in serum-free medium containing bovine serum albumin. [35S]Methionine-labeled proteins were analysed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Treatment of cells with hPRL (1-1000 ng/ml) and hydrocortisone (1 microgram/ml) for 36 h or longer resulted in the synthesis and secretion of three proteins having molecular weights of 11,000, 14,000, and 16,000. Neither hPRL nor hydrocortisone alone induced these proteins. Of several other peptide hormones tested, only human growth hormone, a hormone structurally and functionally similar to hPRL, could replace hPRL in causing protein induction. These three proteins were, therefore, referred to as prolactin-inducible proteins (PIP). Each of the three PIPs was purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and specific antibodies were generated to them in rabbits. By immunoprecipitation and immunoblotting (Western blot) of proteins secreted by T-47D cells, it was demonstrated that the three PIPs were immunologically identical to one another. In addition, the 16-kDa and 14-kDa proteins (PIP-16 and PIP-14), and not the 11-kDa protein (PIP-11), incorporated [3H]glycosamine. Furthermore, 2-deoxyglucose (2 mM) and tunicamycin (0.5 micrograms/ml), two compounds known to inhibit glycosylation, blocked the production of PIP-16 and PIP-14, with a concomitant increase in the accumulation of PIP-11. These results indicate PIP-16 and PIP-14 are glycosylated variants of PIP-11. Finally, in vitro translation of poly(A)+ messenger RNA followed by immunoprecipitation revealed a 12.5-kDa protein, possibly the precursor form of PIPs. In addition, T-47D cells treated with hPRL plus hydrocortisone contained 10-fold more mRNA for PIPs than control cells, suggesting that the hormones' action is at the level of gene expression. Our finding represents a first demonstration of prolactin regulation of gene expression in human target cells. The human breast cancer cells, T-47D, appear to be an excellent model to afford future studies on the molecular action of prolactin and on the possible role of prolactin in human breast cancer.

Clinical trials on chemical radiosensitization of malignant melanoma of the choroid.

The experimental investigations carried out in model systems and on plant, animal or human tissues have demonstrated that it is possible to increase significantly the radiosensitivity of pigmented cells, including hamster melanoma. On the basis of these data, a clinical pilot study was undertaken in order to establish whether a similar methodical approach will prove effective if applied to the radiotherapy of malignant melanoma of the choroid in patients treated with 60Co gamma-rays according to Stallard's technique. Cuprenil and Chelaton Polfa were used as radiosensitizers. The results of therapy differed distinctly in the two groups of patients (receiving or not receiving radiosensitizing drugs), the rate of tumour regression being much higher in those with a parallel pharmacological treatment.