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Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary monogenic disorder characterized by development and enlargement of kidney cysts that lead to loss of renal function. It is caused by mutations in two genes (PKD1 and PKD2) encoding for polycystin-1 and polycystin-2 proteins which regulate different signals including cAMP, mTOR and EGFR pathways. Abnormal activation of these signals following PC1 or PC2 loss of function causes an increased cell proliferation which is a typical hallmark of this disease. Despite the promising findings obtained in animal models with targeted inhibitors able to reduce cystic cell growth, currently, no specific approved therapy for ADPKD is available. Therefore, the research of new more effective molecules could be crucial for the treatment of this severe pathology. In this regard, we have studied the effect of berberine, an isoquinoline quaternary alkaloid, on cell proliferation and apoptosis in human and mouse ADPKD cystic cell lines. Berberine treatment slows cell proliferation of ADPKD cystic cells in a dose-dependent manner and at high doses (100 μg/mL) it induces cell death in cystic cells as well as in normal kidney tubule cells. However, at 10 μg/mL, berberine reduces cell growth in ADPKD cystic cells only enhancing G0/G1 phase of cell cycle and inhibiting ERK and p70-S6 kinases. Our results indicate that berberine shows a selected antiproliferative activity in cellular models for ADPKD, suggesting that this molecule and similar natural compounds could open new opportunities for the therapy of ADPKD patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-kappaB signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293(CTT)), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-kappaB nuclear levels and NF-kappaB-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-kappaB promoter activation was mediated by PKCalpha because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293(CTT) cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-kappaB inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKCalpha-mediated NF-kappaB signalling and cell survival.

Polycystin-1 (PC1) is a membrane protein expressed in tubular epithelia of developing kidneys and in other ductal structures. Recent studies indicate this protein to be putatively important in regulating intracellular Ca(2+) levels in various cell types, but little evidence exists for kidney epithelial cells. Here we examined the role of the PC1 cytoplasmic tail on the activity of store operated Ca(2+) channels in human kidney epithelial HEK-293 cell line. Cells were transiently transfected with chimeric proteins containing 1-226 or 26-226 aa of the PC1 cytoplasmic tail fused to the transmembrane domain of the human Trk-A receptor: TrkPC1 wild-type and control Trk truncated peptides were expressed at comparable levels and localized at the plasma membrane. Ca(2+) measurements were performed in cells co-transfected with PC1 chimeras and the cytoplasmic Ca(2+)-sensitive photoprotein aequorin, upon activation of the phosphoinositide pathway by ATP, that, via purinoceptors, is coupled to the release of Ca(2+) from intracellular stores. The expression of TrkPC1 peptide, but not of its truncated form, enhanced the ATP-evoked cytosolic Ca(2+) concentrations. When Ca(2+) assays were performed in HeLa cells characterized by Ca(2+) stores greater than those of HEK-293 cells, the histamine-evoked cytosolic Ca(2+) increase was enhanced by TrkPC1 expression, even in absence of external Ca(2+). These observations indicate that the C-terminal tail of PC1 in kidney and other epithelial cells upregulates a Ca(2+) channel activity also involved in the release of intracellular stores.

Osteoblasts have been shown to express both isoforms of estrogen receptor (ER alpha and ER beta). As a tool for the study of endogenous regulation of these genes the decoy strategy was employed. Human MG-63 osteoblast-like cells were transfected with a DNA decoy molecule containing a putative negative cis-element (DNA-102) located in the C distal promoter of ER alpha gene. Using real-time quantitative RT-PCR, we found that the DNA-102, but not scrambled DNA, produced a 36-fold increase in the level of total ER alpha mRNA and a 12-fold increase in the level of mRNA for the F isoform that is transcribed from the upstream F promoter, which is predominantly used in osteoblasts. This effect appears to be controlled by estrogen since 17-beta-estradiol downregulated the mRNA increase. Notably, the same decoy was able to induce a 6-fold increase in ER beta mRNA transcription, indicating the coregulation of the ER alpha and ER beta expression. An increase in OPN but not in BMP4 expression was also observed. In addition, in decoy-treated cells, the cell growth decreased together with an increase in alkaline phosphatase activity. These findings indicated that DNA-102 decoy was able to induce a more differentiated osteoblastic phenotype. The augmentation of ER alpha and ER beta expression by the decoy approach may offer a further possibility for patient response to estrogenic therapy in the treatment of diseases related to estrogen deficiency.

We have previously found that the androgen receptor gene is expressed both in normal and adenomatous human adrenal cortex and in the NCI-H295 human adrenocortical cancer cell line. Furthermore, we have observed that dihydrotestosterone (DHT) at physiological concentrations (10(-11) M) inhibits human adrenocortical cell growth in vitro and slightly decreases c-myc RNA levels in NCI-H295 cells. As c-myc is probably not the main mechanism mediating DHT-induced inhibition of cell growth, other genes controlling cell proliferation may be involved. Transforming Growth Factor beta (TGF beta) is a regulatory peptide that acts by both autocrine and paracrine mechanisms to control proliferation and differentiation, and there is previous evidence that TGF beta may exert an antimitotic effect on human fetal adrenal cells in vitro. This study examines a possible role for TGF beta 1 in mediating the DHT-induced reduction of human adrenocortical cell growth. TGF beta 1 and its receptor (TGF beta RII) are expressed in DHT-treated and nontreated NCI-H295 cells; on Northern blot analysis 24-h treatment with DHT (10(-11) M) produced a small increase in TGF beta RII RNA, and quantitative RT-PCR showed a 1.5-fold increase in TGF beta 1 RNA levels. These findings suggest that TGF beta 1 and its receptor may be involved in DHT-induced inhibition of human adrenocortical cell growth.

In order to clarify the effect of retinoids on thyroid cell growth and function, the presence of retinoic acid receptors (RARs) and the action of retinoic acid (RA) on DNA synthesis and on thyroid peroxidase (TPO) and thyroglobulin (TGB) mRNA expression were investigated in primary cultures of human thyroid follicular cells. A time and dose-dependent reduction in 3H-thymidine (3H-thy) incorporation was found in cells exposed for 48 h to all-trans-RA up to 1 microM. A cytotoxic effect was found only with the higher dose of 50 microM. The RA-induced decrease of 3H-thy incorporation was reflected by parallel change in DNA content of cell monolayers. The inhibitory effect of 1 microM RA on 3H-thy incorporation ranged from 28.5 +/- 4.6% in normal cells to 42 +/- 3.2% in adenomatous cells. In addition, 1 microM RA significantly reduced basal and TSH-induced TPO mRNA levels in normal, goitrous and adenomatous cells, but did not alter TGB mRNA levels. Furthermore, in these cells the study of RAR alpha and beta mRNA showed the presence of two major RAR alpha mRNA transcripts of approximately 3.5 and 2.8 Kb in size, whereas RAR beta mRNA was undetectable. Overall, our data indicate that RAR alpha gene is expressed in human thyrocytes and that RA may be involved in the regulation of the human thyroid by reducing proliferation and function of follicular cells.

An upstream region of the human estrogen receptor gene (ER) spanning from -2776 to -38 in relation to a previously described exon 1 was sequenced and analyzed for the possible presence of open reading frames (ORF) and regulatory motifs. We identified a long 459 bp ORF beginning at -2301. Most of the transcriptional regulatory motifs present within the ER upstream sequence are distributed either upstream or downstream of this long ORF which displays a TATA box, a CACC motif and a canonical cap site in the 5' flanking region.

In this article we show that the cytidine analog 5-azacytidine is able to induce differentiation of the human leukemia K-562 cell line. Erythroid induction is associated with (a) an increase of the overall globin synthesis and globin mRNA accumulation, (b) a relative increase of fetal with respect to embryonic globins, and (c) a decrease of the proliferative capacity of hemoglobin-containing cells. In addition, we have analysed the DNA methylation pattern at the cleavage sites of MspI and HpaII restriction enzymes, which are known to cleave differently CCGG DNA sequences when 5-methylcytosine is present. These experiments indicate that in K-562 cells treated with 5-azacytidine, DNA becomes hypomethylated, suggesting that genetic programmes leading to an erythroid phenotype may be activated by a reduction of DNA methylation.

A possible role of the c-myc oncogene in the neoplastic transformation of the human thyroid has been investigated. The structure, the methylation status, and the copy number of this oncogene have been analyzed in normal and in tumor thyroid DNAs by Southern blotting technique and dot blot hybridization. Among six carcinomas, four presented abnormal c-myc DNA structure: Three cases showed a mutation in the 5' flanking region of the gene, originating a new EcoRI site; the other case showed a deletion of 5 kb involving the first exon of the gene. This deletion was also observed in the white blood cells of the same individual. In addition, in three carcinomas a double dose of the oncogene has been demonstrated. In all the carcinomas examined, undermethylation of the c-myc oncogene has been observed. These findings suggest that c-myc oncogene alterations might be involved in the malignant transformation of the human thyroids and can be considered as tumor markers.

The correlation between expression and extent of DNA methylation of c-myc and c-abl oncogenes has been investigated in the human leukemic K-562 cell line before and after 5-azacytidine-mediated erythroid induction. RNA accumulation was analyzed by cytoplasmic dot hybridization and DNA methylation by using HpaII and MspI endonucleases, which differently cleave the CCGG sequence depending on cytosine methylation. Both the oncogenes are expressed in uninduced cells; however, whereas the c-myc expression does not change following 5-azacytidine treatment, the c-abl expression sharply decreases. The HpaII pattern shows that the c-myc DNA region is undermethylated and that the c-abl gene is hypermethylated both before and after the erythroid induction. Nevertheless, in both the genes 5-azacytidine produces variations in the MspI pattern compatible with mCmCGG to CmCGG demethylations.

Estrogen Receptor (ER) RNA has been studied in ER positive and ER negative cells, grown in alpha medium and fetal calf serum (FCS), with and without estrogen addition. ER mRNA was detected only in ER positive cells. When cells were grown without estrogen addition, a progressive and transient increase of ER RNA levels was observed. When cells were cultured with estradiol, the ER RNA increase took place early and ER RNA levels remained 2 times higher than in untreated cells. Therefore, a down-regulation of ER RNA is not apparent in neoplastic breast cells. We hypothesize that the increase of ER expression is caused by stabilization of ER transcripts, due to the estrogen induced ER cytoplasmic depletion.

A cDNA for coagulation factor XII has been used to investigate the presence of gene lesions and restriction fragment length polymorphisms in two brothers with Hageman trait and their family. A TaqI polymorphic fragment has been found in the two propositi and in 11 members of the paternal lineage. This polymorphism, absent in the normal population, is correlated with the reduction of factor XII activity and enables the identification of heterozygous factor XII deficiency. Factor XII gene deletion as the cause of Hageman trait in this family has been excluded. A restriction map has been constructed, and the TaqI polymorphic site has been localized within the 5' portion of the gene. The mutation in the polymorphic site is probably the cause of the factor XII deficiency. Data suggest the presence of one factor XII gene per haploid genome.

We have studied the expression of the c-myc and c-abl oncogenes in two human leukemic K562 cell lines which do express hemoglobin genes retaining a differential rate of cell proliferation. Our data indicate that in hemin-induced K562(S) cells the expression of c-abl oncogene decreases and appears to be related to a decrease in the proliferation capacity rather than to the activation of differentiated functions. The K562(hC) cell line, which produces large amounts of Hb Gower 1 retaining an efficient rate of cell proliferation, expresses indeed the c-abl oncogene at high level.