Laura del Senno

Chromosome 18q allelic loss and prognosis in stage II and III colon cancer

The prognostic significance of chromosome 18q allelic loss was evaluated in a series of 118 patients with curatively resected TNM stage II or stage III colon cancer. Chromosome 18q status was determined on frozen tumour samples, using microsatellite markers and the polymerase chain reaction (PCR). Mean follow‐up in surviving patients was 75.9 months. Chromosome 18q allelic loss was significantly related to tumour site, extramural venous invasion, flow cytometric nuclear DNA content and p53 protein expression. Patients whose tumour had no evidence of chromosome 18q allelic loss showed a better disease‐free and overall survival than patients whose tumour demonstrated 18q allelic loss. When patients were stratified by tumour stage, a significant survival advantage for patients whose tumour had no allelic loss on chromosome 18q was observed in stage II as well as in stage III disease. In particular, patients with stage II disease whose tumour had no chromosome 18q allelic loss demonstrated an excellent clinical outcome, with a 5‐year disease‐free survival rate of 96%. In contrast, the 5‐year disease‐free survival rate of patients with stage II disease and chromosome 18q allelic loss was only 54%. In multivariate analysis, status of chromosome 18q was the only significant independent prognostic factor for both disease‐free and overall survival. These results indicate that assessment of chromosome 18q status provides relevant prognostic information in colon cancer and might be employed in the selection of patients for adjuvant therapy. Int. J. Cancer (Pred. Oncol.) 79:390–395, 1998.© 1998 Wiley‐Liss, Inc.

Exposure to myelotoxic agents and myelodysplasia: case–control study and correlation with clinicobiological findings

To better define the role of exposure to myelotoxic agents in the genesis of myelodysplastic syndrome (MDS), we carried out (a) a case–control study for the determination of the relative risk (RR) of developing MDS, including 178 consecutive patients and 178 sex‐ and age‐matched controls; (b) a study of clinicobiological features in MDS arising after occupational exposure to myelotoxic agents and in MDS in ‘non‐exposed’ patients. The definition of the ‘exposure’ status was based on a predetermined questionnaire, with calculation of an ‘exposure’ index (hours/day × days/year × years). Cumulative exposure to pesticides or to organic solvents, for >2400 h, was recorded in 48 and 25 MDS patients, respectively, compared to 27 and four controls (P < 0.00001; RR 3.74; 95% confidence interval 2.02–5.37). Older age and an excess of refractory anaemia with ringed sideroblasts and refractory anaemia with excess of blasts was noted among ‘exposed’ MDS‐patients (group 1), compared to non‐exposed MDS‐patients (group 2). 68.3% patients in group 1 had clonal chromosome changes, compared with 43.2% patients in group 2. Complex karyotypes, −7/7q−, −5/5q−, +8, 7p and 17p aberrations were seen more frequently in group 1, whereas a normal karyotype, isolated 5q− or 20q− occurred more frequently in group 2. The association of exposure to myelotoxic agents with older age at presentation and with unfavourable chromosome changes accounted for the shorter survival observed in ‘exposed’ patients. These data show that occupational exposure to pesticides and organic solvents in our region resulted in an increased RR of developing MDS and that a distinct cytogenetic profile was associated with MDS in ‘exposed’ patients. These findings provide strong indirect evidence that these agents may play a role in the pathogenesis of MDS, preferentially targeting some of the chromosome regions which are frequently involved in therapy‐related myeloid neoplasias.

Human leukemia K-562 cells: induction of erythroid differentiation by 5-azacytidine.

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.

Deficiency of polycystin-2 reduces Ca2+ channel activity and cell proliferation in ADPKD lymphoblastoid cells

Polycystin‐2 (PC2), encoded by the PKD2 gene, mutated in 10‐15% of autosomal‐dominant polycystic kidney disease (ADPKD) patients, is a Ca2+‐permeable cation channel present in kidney epithelia and other tissues. As PC2 was found expressed in B‐lymphoblastoid cells (LCLs) and Ca2+ signaling pathways are important regulators of B cell function activities, we investigated whether PC2 plays some role in B‐LCLs. In LCLs, PC2 was found mainly in ER membranes but ~8 times less than in kidney HEK293 cells. The same reductions were found in PKD2 and PKD1 RNA; thus, PKD genes maintained, in LCLs, the same reciprocal proportion as they do in kidney cells. In LCLs obtained from subjects carrying PKD2 mutations (PKD2‐LCLs) and showing reduced PC2 levels, intracellular Ca2+ concentrations evoked by platelet‐activating factor (PAF), were significantly lower than in non‐PKD‐LCLs. This reduction was also found in PKD1‐LCLs but without PC2 reductions. Likewise, cell proliferation, which is controlled by Ca2+, was reduced in PKD2‐ and PKD1‐LCLs. Moreover, in LCLs with PKD2 nonsense mutations, aminoglycoside antibiotics reduced the PC2 defect by promoting readthrough of stop codons. Therefore, PC2 and PC1 are functionally expressed in LCLs, which provide a model, easily obtainable from ADPKD patients, to study PKD gene expression and function.

Richter's syndrome in a case of atypical chronic lymphocytic leukaemia with the t(11;14)(q13;q32): role for a p53 exon 7 gene mutation

Clinicobiological, histological, cytogenetic and molecular genetic studies were performed in a case of atypical B‐cell chronic lymphocytic leukaemia (B‐CLL) with the t(11;14)(q13;q32) evolving into Richters syndrome (RS) in order (a) to determine the clonal relationship between the cell of origin for B‐CLL and RS, and (b) to analyse genetic events underlying the disease progression in this patient.

Expression of polycystin-1 C-terminal fragment enhances the ATP-induced Ca2+ release in human kidney cells

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.

In vitro effects of estrogen on tgb and c-myc gene expression in normal and neoplastic human thyroids

The authors investigated the effects of 17 beta-estradiol (E) and thyroid-stimulating hormone (TSH) on tgb (coding for thyroglobulin), c-myc RNA levels, and [3H]thymidine (thy) incorporation in suspension cultures of normal, adenomatous and carcinomatous human thyroid follicles. The cultured follicles showed decreased tgb RNA and enhanced c-myc RNA levels. In the culture of normal and adenomatous samples E caused a significant increase of [3H]thy incorporation and tgb RNA levels, with no effect on c-myc RNA levels. No effect of E was observed in the carcinomatous thyroid culture. TSH induced a significant increase of [3H]thy incorporation and c-myc expression only in adenoma cultures and a significant increase of tgb RNA levels in both normal and adenomatous samples. TSH had no effect on the carcinoma. The results show that E, like TSH, stimulates in vitro the expression of the tgb gene in differentiated cells, without stimulating the expression of the c-myc proto-oncogene, suggesting a possible action of E on normal thyroid function and perhaps growth, even if not associated with increased c-myc expression.

Polycystin-1 regulates amphiregulin expression through CREB and AP1 signalling: implications in ADPKD cell proliferation.

In autosomal dominant polycystic kidney disease (ADPKD), renal cyst development and enlargement, as well as cell growth, are associated with alterations in several pathways, including cAMP and activator protein 1 (AP1) signalling. However, the precise mechanism by which these molecules stimulate cell proliferation is not yet fully understood. We now show by microarray analysis, luciferase assay, mutagenesis, and chromatin immunoprecipitation that CREB and AP1 contribute to increased expression of the amphiregulin gene, which codifies for an epidermal growth factor-like peptide, in ADPKD cystic cells, thereby promoting their cell growth. Increased amphiregulin (AR) expression was associated with abnormal cell proliferation in both PKD1-depleted and -mutated epithelial cells, as well as primary cystic cell lines isolated from ADPKD kidney tissues. Consistently, normal AR expression and proliferation were re-established in cystic cells by the expression of a mouse full-length PC1. Finally, we show that anti-AR antibodies and inhibitors of AP1 are able to reduce cell proliferation in cystic cells by reducing AR expression and EGFR activity. AR can therefore be considered as one of the key activators of the growth of human ADPKD cystic cells and thus a new potential therapeutic target.

Nonspecific Cation Current Associated with Native Polycystin-2 in HEK-293 Cells

Mutations in either PKD1 or PKD2 gene are associated with autosomal dominant polycystic kidney disease, the most common inherited kidney disorder. Polycystin-2 (PC2), the PKD2 gene product, and the related protein polycystin-L, function as Ca(2+)-permeable, nonselective cation channels in different expression systems. This work describes a nonspecific cation current (I(CC)) that is present in native HEK-293 cells and highly associated with a PC2-channel activity. The current is voltage dependent, activating for potentials that are positive to -50 mV and inactivating in a few milliseconds. It is sensitive to Cd(2+), Gd(3+), La(3+), SKF96365, and amiloride. After silencing of PC2 by RNA interfering, cells show a reduced current that is restored by transfection with normal but not truncated PC2. Consistently, I(CC) is abolished by perfusion with an anti-PC2 antibody. Furthermore, heterologous expression of the PC1 cytoplasmic tail significantly increases I(CC) peak amplitude compared with native cells. This is the first characterization of such a current in HEK-293 cells, a widely used expression system for ion channels. These cells, therefore, could be regarded as a suitable and readily accessible tool to study interactions between native PC2/PC1 complex and other membrane proteins, thus contributing to the understanding of autosomal dominant polycystic kidney disease pathogenesis.

Methylation analysis of the promoter F of estrogen receptor α gene: effects on the level of transcription on human osteoblastic cells

In this study, the methylation status of the distal promoter F of estrogen receptor alfa (ERalpha) gene in human osteoblastic cells was investigated. The activity of this promoter is responsible for the ERalpha gene transcription in bone tissue. The methylation status of promoter F was here evaluated, for the first time, by direct sequencing of bisulfite-treated genomic DNA, at 10 CpG specific sites localized in a region of about 800 bp. An heterogeneous methylation pattern was observed. The most notable difference was found at four particular CpGs, distant from the exon F transcription start site, showing a methylation status that correlates with the expression level, being ERalpha mRNA transcription reduced in a partially methylated cells but preserved in demethylated cells. The other CpG sites, localized around the transcription start site, were always demethylated except for MG-63 cells showing the lowest level of ERalpha expression. By quantitative RT-PCR analysis we demonstrated that ERalpha gene expression was higher in primary osteoblasts than in bone-derived cells (MG-63 and SaOS-2) and in all cases the ERalpha mRNA is represented by the isoform F. The same 10 CpG sites were investigated in non-osseous cell lines and were found fully methylated in ERalpha-negative breast cancer cells (MDA-MB-231) and completely demethylated in ERalpha-positive breast cancer cells (MCF7). The overall results suggest that methylation of the CpG sites inside ERalpha gene promoter F here analyzed may contribute to ERalpha transcriptional control, directly or indirectly, influencing the tissue specific expression of the gene.