Lisa Fiore

Pubertal changes in biochemical markers of growth.

Puberty is a crucial period of life during which dramatic hormonal changes induce notable modifications in linear growth, bone mass and body composition. These changes are associated with variations in some biochemical parameters such as markers of bone turnover and leptin, which may reflect changes in bone growth and fat mass, respectively. Children with growth hormone (GH) deficiency have reduced concentrations of bone markers, which increase during GH administration, while the levels of leptin decrease. There have been few studies analysing the behaviour of bone markers during puberty in GH-treated GH-deficient patients and no studies analysing the behaviour of leptin. Results from a longitudinal study showed that there was no change in serum osteocalcin, carboxy-terminal propeptide of type I procollagen, and cross-linked carboxy-terminal telopeptide of type I collagen levels during puberty in GH-treated GH-deficient children. Some studies have shown that changes in markers of bone turnover and leptin after short-term GH treatment may predict the growth response (at 6–12 months) to GH administration in GH-deficient children. At present, insufficient data are available for the clinical use of these parameters as markers of growth response during pubertal development and as predictors of long-term growth response to GH treatment in children with GH deficiency. Nevertheless, the use of more and possibly new markers might improve the accuracy of growth prediction models in the future.

Apoptosis and proliferation in thyroid carcinoma: Correlation with bcl-2 and p53 protein expression

The aim of this study was to determine the apoptotic cell death in 92 thyroid carcinomas of different histotypes (42 papillary, PTC; 12 poorly differentiated, PDC: 21 undifferentiated, UC; and 17 medullary, MC) by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL). Apoptotic index (Al, evaluated as a percentage of TUNEL-positive cells of neoplastic cells) was calculated in each tumour. The AI was very low in all subtypes of thyroid carcinoma, ranging from a median value of 0.2 in PTC to 1.4 in UC. The proliferative activity was determined by immunohistochemistry using monoclonal antibody, MIB-1. The percentage of proliferating cells was significantly different among the histotypes, increasing with tumour aggressiveness (from the mean value of 3.1 for PTC to 5.6 for PDC and 51.8 for UC). In addition, the ratio between proliferative activity and apoptosis was significantly higher in UC than in the other histotypes. The expression of bcl-2 and p53 protein (important in the modulation of cell death) was correlated (bcl-2, inverse correlation, r2 = 0.1, P = 0.04; p53, direct correlation, r2 = 0.11, P = 0.02) with apoptotic index in PTC.

Human Immunodeficiency Virus-1 Tat Induces Hyperproliferation and Dysregulation of Renal Glomerular Epithelial Cells

Human immunodeficiency virus-associated nephropathy (HIVAN) is etiologically related to the viral infection, but the mechanisms of virus-induced renal injury remain undetermined. Peculiar histopathological features of HIVAN are the enhanced proliferation and the loss of differentiation markers of glomerular epithelial cells (podocytes). We found that podocytes were not permissive to HIV-1 replication. In this study we investigated the effects of the HIV-1 regulatory protein Tat on primary cultures and on a continuous line of podocytes. Our results demonstrated that Tat induced hyperproliferation of these cells in a dose-dependent manner. This activity was primarily mediated by the basic domain of the viral protein. Proteoglycans were required for this phenomenon because Tat-induced increase of podocyte growth was significantly impaired by inhibition of proteoglycan synthesis with beta-D-xyloside. In podocyte cultures Tat promoted both the transcription and the release of basic fibroblast growth factor, which contributed to the enhanced cell proliferation. Moreover, Tat deregulated the podocyte phenotype causing down-regulation of maturity markers such as WT-1 and synaptopodin, alteration of cytoarchitecture, and impairment of permselectivity. Together, these results demonstrate that the interaction of extracellular Tat with podocytes can induce alterations that mimic the pathological changes of podocytes detected in HIVAN.

Down-regulation of the nm23.h1 gene inhibits cell proliferation

nm23 gene expression is strictly related to the state of cell growth. The level of its expression parallels the fraction of thymidine‐incorporating cells (S‐phase cells) in neoplastic mammary tissues and in the synchronously cycling fraction of MCF10A cells. nm23.h1 reaches a peak of expression in the S‐phase, and is present at very low level during the G0/G1 phase. Two strategies are used to demonstrate the direct involvement of the nm23.h1 gene in the process of cell proliferation. The first consists of transient inhibition of nm23.h1 expression by using anti‐sense oligonucleotide treatment; weak inhibitory effect on cell proliferation is observed. The second strategy involves the stable inhibition of nm23.h1 expression by transfection of MCF10A cells with a plasmid vector expressing the human nm23.h1 anti‐sense mRNA. The anti‐sense‐transfected cells show consistently slower proliferative activity than the control. Int. J. Cancer 73:297–302, 1997.

Establishment of a non-tumorigenic papillary thyroid cell line (FB-2) carrying the RET/PTC1 rearrangement.

A novel human thyroid papillary carcinoma cell line (FB‐2) has been established and characterized. FB‐2 cells harbor the RET/PTC1 chimeric oncogene in which the RET kinase domain is fused to the H4 gene. FB‐2 cells neither formed colonies in semisolid media nor induced tumors after heterotransplant into severe combined immunodeficient mice. However, HMGI(Y), HMGI‐C and c‐myc genes, which are associated to thyroid cell transformation, were abundantly expressed in FB‐2 cells but not in normal thyroid cells. FB‐2 cells only partially retained the differentiated thyroid phenotype. In fact, the PAX‐8 gene, which codes for a transcriptional factor required for thyroid cell differentiation, was expressed, while thyroglobulin, TSH‐receptor and thyroperoxidase genes were not. Moreover, FB‐2 cells produced high levels of interleukin (IL)‐6 and IL‐8.

High incidence of central precocious puberty in a bounded geographic area of northwest Tuscany: An estrogen disrupter epidemic?

The potential health consequences of human exposure to environmental estrogen disrupters are not known. Because many chemical compounds are environmentally persistent, toxic and estrogen-active, they can dysregulate the hypothalamic–pituitary–gonadal axis, potentially inducing reproductive disorders such as central precocious puberty (CPP). We performed a multi-center analysis of CPP distribution in northwest Tuscany (NWT), an area of 5990 km2 with 1 280 895 inhabitants. Study criteria consisted of recorded CPP diagnoses and prescriptions of gonadotropin-releasing hormone analogs from January 1, 1998 to December 1, 2003. Although similar CPP prevalences were found in four major cities of NWT (Livorno, Lucca, Massa and Pisa) (mean 30.4 per 100 000 children, standard deviation 18.6; p > 0.05), Viareggio area (< 300 km2) with 19 219 child inhabitants (0–14 years of age) had the highest CPP prevalence: more than 161 CPP cases per 100 000 children. Living in Viareggio area significantly increased the risk of CPP (relative risk (RR) 5.73, 95% confidence interval (CI) 3.5–9.3; rate/risk difference 0.133%, p < 0.05). Annual CPP incidence in the Viareggio area was relatively constant and significantly higher than in other NWT areas (RR 5.04, 95% CI 2.3–11.2; rate/risk difference 0.03%, p < 0.05). Indeed, 47% of total NWT cases were distributed in the countryside (300 km2) surrounding Viareggio. Specifically, three villages – Camaiore, Pietrasanta and Stazzema – in Viareggio presented the highest CPP frequency: 216.1, 393.5 and 274.0 CPP cases per 100,000 children, respectively (RR 9.59, 95% CI 1.71–16.6; rate/risk difference 0.26%, p < 0.05). Owing to the definite geographic distribution of CPP and because increasing distance (km) from Pietrasanta rarefied CPP frequency, we suggest environmental factors (e.g. estrogen disrupter pollution) as major CPP determinants in NWT.

Suppression of Fas Expression and Down-Regulation of Fas Ligand in Highly Aggressive Human Thyroid Carcinoma

The Fas-FasL system seems to mediate thyrocyte death in Hashimoto’s thyroiditis. In thyroid cancer, down-regulation of bcl-2 seems to alter apoptosis control. We compared the expression of immunoreactive Fas and FasL in normal thyroid with that of tumors ranging from benign to highly aggressive. Fas is essentially not expressed in normal thyrocytes, whereas FasL is expressed in approximately one-third of cases. Expression of both markers is significantly up-regulated in adenoma and in well-differentiated papillary and follicular carcinoma. In contrast, Fas is suppressed and FasL is strongly reduced in the most aggressive histological variants (poorly differentiated and undifferentiated carcinoma). Immunohistochemistry findings have been confirmed by analysis of Fas-FasL mRNA transcripts. In vitro studies showed that the Fas receptor of thyroid tumor cells was functional, because apoptosis was induced by an agonistic Fas antibody. Fas-expressing and Fas-resistant mammary cell lines were used as specificity controls. Together with our previous data inversely relating bcl-2 expression and thyroid tumor grade, the present findings further indicate that apoptotic pathways are altered in thyroid neoplasia. Thus, the Fas-FasL system may represent a marker of tumor aggressiveness.

Potentiation of the malignant phenotype of the undifferentiated ARO thyroid cell line by insertion of the bcl-2 gene.

We have reported that bcl‐2 is expressed in normal human thyroid epithelium and that its expression is down‐regulated in undifferentiated thyroid tumors. Production of IL‐6 was concomitantly down‐regulated in these forms. Based on these observations, we analyzed whether insertion of bcl‐2 would reverse the highly malignant phenotype of a thyroid cell line (ARO) derived from an undifferentiated carcinoma. This cell line fails to produce Bcl‐2 and IL‐6. By infection with a bcl‐2 retroviral vector, ARO cells expressing bcl‐2 (ARObcl‐2) were obtained. Compared with parental cells, expression of bcl‐2 was associated with enhancement of growth potential (DNA synthesis, in vitro proliferation rate, anchorage‐independent growth in semi‐solid media). Chemotaxis and invasive potential in Boyden chambers were also increased. bcl‐2‐expressing cells showed a reduced response to apoptotic stimuli (low‐serum conditions or anti‐neoplastic drugs). Large branched colonies were formed in Matrigel from ARObcl‐2 cells but not from parental cells. Finally, ARObcl‐2 cells showed a decreased latency of tumor appearance when injected into immunodeficient mice. Potentiation of the malignant phenotype of ARO cells by bcl‐2 was not ascribed to altered expression of (i) cytokine/growth factors (IL‐4, IL‐6, IL‐8, IL‐10, IL‐12, TGF‐α, TGF‐β), (ii) thyroid‐specific transcripts (TG, TPO, TSH‐R, PIGF, PAX‐8) or (iii) genes influencing tumor aggressiveness [VEGF, HMGI (Y), HMGI‐C]. Our data indicate that bcl‐2 potentiates the malignant phenotype of ARO cells not only by limiting the response to apoptotic stimuli but also by enhancing proliferation and tumor aggressiveness. Int. J. Cancer81:956–962, 1999.

Licoflavone C attenuates the genotoxicity of cancer drugs in human peripheral lymphocytes.

Flavonoids exhibit a wide spectrum of biological activities that can lead to beneficial effects for human health. The search for cytotoxic, genotoxic and/or antimutagenic natural compounds is therefore of great relevance, especially in cancer chemotherapy. In view of this, we screened the potential genotoxicity/antigenotoxicty of licoflavone C (LFLC) – a naturally occurring prenyl‐flavone extracted from Genista ephedroides – using the micronucleus (MN) assay on stimulated and cytochalasin B‐blocked human lymphocytes. LFLC did not increase the spontaneous MN level up to 600 µM final concentration where a strong toxicity was seen to occur. We therefore performed an antigenotoxicity assay against the two mutagenic anticancer drugs, mitomycin C (MMC) and daunorubicin (DAU), using two non‐toxic LFLC concentrations (0.1 µM and 1.0 µM). The MN frequencies induced by 0.025 µg/ml or 0.05 µg/ml DAU were significantly lowered by 45.4% or 46.6% and 41.8% or 44.8% at LFLC 0.1 and 1.0 µM, respectively. After treatment with 0.085 µg/ml or 0.17 µg/ml MMC, we detected a reduction in genotoxicity of 35.1% or 37.0% and of 38.0% or 35.8% at LFLC 0.1 and 1.0 µM, respectively. In conclusion, LFLC was proven to be protective toward the chromosome damage induced by DAU or MMC in cultured human peripheral lymphocytes. Copyright

Defective interleukin six expression and responsiveness in human mammary cells transformed by an adeno 5/SV40 hybrid virus.

Mammary epithelial cells (MECs) were isolated and cultured from mammary glands of healthy women undergoing reduction mammoplasty. Normal MECs were infected with the transforming hybrid virus adeno-5/SV40. Two transformed epithelial cell lines, M1 and M2, were obtained, characterised phenotypically and studied for the production of and the response to cytokines and growth regulators. In both cell lines, expression of the SV40 large T antigen was associated with loss of interleukin 6 (IL-6) production and responsiveness as well as with down-regulation of IL-8 and transforming growth factor (TGF)-alpha production. Both M1 and M2 cell lines were capable of forming colonies in semisolid media, but upon injection into severe combined immunodeficient (SCID) mice only M2 cells were tumorigenic. DNA synthesis in M1 cells was partially inhibited by serum or TNF-alpha and weakly stimulated by hydrocortisone (HC) and IL-8. In contrast, M2 cells were totally unresponsive to a variety of growth regulators. Both lines overexpressed the p53 protein at levels about 20-fold higher than those observed in primary MEC cultures, but no mutations of the p53 gene could be detected. The date confirm the view that the expression in human mammary cells of different oncogenes - including the SV40 T antigen - is frequently associated with alterations of cytokine production and responsiveness.