Biosynthesis of human myeloperoxidase.

Members of Chordata peroxidase subfamily [1] expressed in mammals, including myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), and thyroid peroxidase (TPO), express conserved motifs around the heme prosthetic group essential for their activity, a calcium-binding site, and at least two covalent bonds linking the heme group to the protein backbone. Although most studies of the biosynthesis of these peroxidases have focused on MPO, many of the features described occur during biosynthesis of other members of the protein subfamily. Whereas MPO biosynthesis includes events typical for proteins generated in the secretory pathway, the importance and consequences of heme insertion are events uniquely associated with peroxidases. This Review summarizes decades of work elucidating specific steps in the biosynthetic pathway of human MPO. Discussion includes cotranslational glycosylation and subsequent modifications of the N-linked carbohydrate sidechains, contributions by molecular chaperones in the endoplasmic reticulum, cleavage of the propeptide from proMPO, and proteolytic processing of protomers and dimerization to yield mature MPO. Parallels between the biosynthesis of MPO and TPO as well as the impact of inherited mutations in the MPO gene on normal biosynthesis will be summarized. Lastly, specific gaps in our knowledge revealed by this review of our current understanding will be highlighted.

Loss of miR-10a activates lpo and collaborates with activated Wnt signaling in inducing intestinal neoplasia in female mice.

miRNAs are small regulatory RNAs that, due to their considerable potential to target a wide range of mRNAs, are implicated in essentially all biological process, including cancer. miR-10a is particularly interesting considering its conserved location in the Hox cluster of developmental regulators. A role for this microRNA has been described in developmental regulation as well as for various cancers. However, previous miR-10a studies are exclusively based on transient knockdowns of this miRNA and to extensively study miR-10a loss we have generated a miR-10a knock out mouse. Here we show that, in the Apc(min) mouse model of intestinal neoplasia, female miR-10a deficient mice develop significantly more adenomas than miR-10(+/+) and male controls. We further found that Lpo is extensively upregulated in the intestinal epithelium of mice deprived of miR-10a. Using in vitro assays, we demonstrate that the primary miR-10a target KLF4 can upregulate transcription of Lpo, whereas siRNA knockdown of KLF4 reduces LPO levels in HCT-116 cells. Furthermore, Klf4 is upregulated in the intestines of miR-10a knockout mice. Lpo has previously been shown to have the capacity to oxidize estrogens into potent depurinating mutagens, creating an instable genomic environment that can cause initiation of cancer. Therefore, we postulate that Lpo upregulation in the intestinal epithelium of miR-10a deficient mice together with the predominant abundance of estrogens in female animals mainly accounts for the sex-related cancer phenotype we observed. This suggests that miR-10a could be used as a potent diagnostic marker for discovering groups of women that are at high risk of developing colorectal carcinoma, which today is one of the leading causes of cancer-related deaths.

Regulation of sodium/iodide symporter and lactoperoxidase expression in four human breast cancer cell lines.

BACKGROUND: Agents capable of increasing radioiodine concentration by stimulating the sodium/iodide symporter (NIS) expression have been extensively investigated for the treatment of certain well-differentiated breast cancers. AIM: In this study, we analyzed the regulation of the NIS and lactoperoxidase (LPO) gene expression in 4 different human breast cancer cell lines, representative of different histotypes of breast cancer. METHODS: MCF-7, T-47D, MDA-MB231, and HCC-1937 (the latter carrying the BRCA-1 mutation) were exposed to different stimulators and the levels of NIS and LPO mRNA measured by a quantitative RT-PCR. RESULTS: All-trans-Retinoic Acid (RA), Dexamethasone (DEX), Trichostatin A (TSA), and Sodium Butyrate (NaB) induced the expression of NIS mRNA in MCF-7 and T-47D cell lines, whereas HCC-1937 and MBA-MB231 were slightly responsive only to the histone-deacetylase inhibitors TSA and NaB. Minor stimulatory effects were detected on LPO mRNA in MCF-7 and T-47D treated with TSA and NaB or RA only in MCF-7, while no effect was detectable in the other two cell lines. CONCLUSIONS: These data indicate that retinoic acid, alone or in combination with DEX, as well as HDAC-inhibitors are very promising agents for a radioiodine- based therapy in a large spectrum of breast cancers, including neoplasms from both basal and ductal cells, especially for the well-differentiated estrogen-dependent tumors. Other molecules or other drug combinations should be tested to extend the same strategy to the less differentiated and more aggressive tumor cells, including those carrying the BRCA mutation.

Uptake and gene expression with antitumoral doses of iodine in thyroid and mammary gland: evidence that chronic administration has no harmful effects.

Several studies have demonstrated that moderately high concentrations of molecular iodine (I(2)) diminish the symptoms of mammary fibrosis in women, reduce the occurrence of mammary cancer induced chemically in rats (50-70%), and have a clear antiproliferative and apoptotic effect in the human tumoral mammary cell line MCF-7. Nevertheless, the importance of these effects has been underestimated, in part because of the notion that exposure to excess iodine represents a potential risk to thyroid physiology. In the present work we demonstrate that uptake and metabolism of iodine differ in an organ-specific manner and also depend on the chemical form of the iodine ingested (potassium iodide vs. I(2)). Further, we show that a moderately high I(2) supplement (0.05%) causes some of the characteristics of the "acute Wolff-Chaikoff effect"; namely, it lowers expression of the sodium/iodide symporter, pendrin, thyroperoxidase (TPO), and deiodinase type 1 in thyroid gland without diminishing circulating levels of thyroid hormone. Finally, we confirm that I(2) metabolism is independent of TPO, and we demonstrate that, at the doses used here, which are potentially useful to treat mammary tumors, chronic I(2) supplement is not accompanied by any harmful secondary effects on the thyroid or general physiology. Thus, we suggest that I(2) could be considered for use in clinical trials of breast cancer therapies.

Expression and characterization of bovine lactoperoxidase by recombinant baculovirus.

Lactoperoxidase (LPO) is a heme-containing oxidation-reduction enzyme present in milk. In this study, the gene encoding bovine lactoperoxidase (bLPO) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLPO was isolated. A bLPO-related recombinant baculovirus-expressed protein of 78 kDa was detected using anti-bLPO antibodies. After digestion with N-glycosidase F, the molecular weight of the recombinant bLPO (rbLPO) decreased. In addition, rbLPO reacted with lectin, indicating that the protein was glycosylated. The rbLPO activity and heme content in the culture supernatants increased upon addition of delta-aminolevulinic acid, which is a heme precursor. Differences in the delta-aminolevulinic acid-dependent circular dichroism spectrum and rbLPO pepsin hydrolysis were observed. These results suggest that the secondary structure and structural stability of rbLPO depends on the heme environment. Our data suggest that this bLPO expression system is useful for studying structure, catalytic mechanisms, and biological function.

Similarities and differences in the ultrastructure of two hormone-dependent and one independent human breast carcinoma grown in athymic nude mice: comparison with the rat DMBA-induced tumor and normal secretory mammocytes.

The human breast cancer cell lines MCF-7 (ER positive), ZR 75-1 (ER positive) and MDA-MB 231 (ER negative) form solid tumors within one week following inoculation into athymic nude mice. Tumor formation by MCF-7 and ZR 75-1 cells was dependent upon estrogen, whereas MDA-MB 231 cells formed tumors in ovariectomized mice with or without supplemental estrogen. Ultrastructural comparison of tumors formed by the three human breast carcinoma lines in athymic nude mice indicated that lactoperoxidase activity, milk protein and fat globule formation were virtually absent from all three tumors. The estrogen-dependent tumors (MCF-7, ZR 75-1), however, had more desmosomes, intermediate-sized microfilaments and collagen than the estrogen-independent tumor (MDA-MB 231). When the ultrastructure of the three human tumors was compared to the hormone-dependent, DMBA-induced rat mammary carcinoma and to the normal lactating rat mammocytes, the following observations were evident: a) the estrogen-dependent human tumors closely resembled the normal rat tissue in the distribution of desmosomes and collagen, b) the rat mammary carcinoma differed from both the estrogen-dependent and -independent human tumors, in having milk protein, milk fat globules and intense lactoperoxidase activity. The results indicate that these hormone-dependent and -independent human mammary tumors maintained in athymic nude mice differ markedly in their ultrastructure from the lactating rat mammocytes and the rat DMBA-induced mammary carcinoma.

Bacteriostatic activity in cow's milk from udders infected with Streptococcus agalactiae and Staphylococcus aureus.

Milk samples from infected udders contained more lactoperoxidase and more thiocyanate than before infection. Irritation of 10 quarters of 5 cows caused the increase in the bacteriostatic activity of milk. Bacteriostatic activity of milk from the udders infected with staphylococci dropped after several weeks of chronic mastitis.