Alberto Gulino

Echocardiographic evidence for a direct effect of GH/IGF-I hypersecretion on cardiac mass and function in young acromegalics

The interpretation of echocardiographic abnormalities in acromegalic patients is complicated by non‐specific age‐related diseases, many of which are commoner in acromegaly. We have therefore investigated the cause‐effect relationship between GH/IGF‐I hypersecretion and precocious cardiovascular abnormalities in a series of young acromegalic patients.

Activation of MMP-2 by human GCT23 giant cell tumour cells induced by osteopontin, bone sialoprotein and GRGDSP peptides is RGD and cell shape change dependent

We show that osteopontin (OPN), bone sialoprotein (BSP) and GRGDSP peptides, in solution, induce activation of metalloproteinase‐2 (MMP‐2) secreted by human GCT23 giant cell tumour cells. Activation of MMP‐2 is RGD sequence dependent, possibly involves anti‐αVβ3 integrins, is preceded by a change from spread to rounded cell morphology and is mimicked by the actin depolymerising agent cytochalasin B. Cells that had spread on OPN, BSP and GRGDSP substrata failed to activate MMP‐2, but subsequent addition of soluble GRGDSP induced rounding and MMP‐2 activation. Activation induced by GRGDSP and cytochalasin B was cell mediated, inhibited by EDTA, tissue inhibitor of metalloproteinase‐2 (TIMP‐2) and carboxyl terminal MMP‐2 consistent with a role for membrane type (MT)‐MMP but did not involve urokinase, plasmin or thrombin activity. Activation induced by GRGDSP and cytochalasin B, but not cell rounding, was inhibited by herbimycin A, cycloheximide and actinomycin D, suggesting a role for tyrosine kinases, protein and RNA synthesis, but was not associated with changes in mRNA for MT‐MMP‐1, MMP‐1, MMP‐2, TIMP‐1 or TIMP‐2. GRGDSP and cytochalasin B enhanced levels of membrane‐associated pro‐ and active form MMP‐1 and MMP‐2 but not MT‐MMP‐1, stimulated cell surface MMP‐1 staining and induced that of MT‐MMP‐1, MMP‐2 and TIMP‐2. This was consistent with the possible relocation of constitutive MT‐MMP‐1 to the cell surface as a prerequisite for subsequent cell surface MMP‐2/TIMP‐2/MT‐MMP‐1 complex formation and to the potential induction of conditions favourable for reciprocal cell surface MMP‐1/MMP‐2 activation. Our data provide a novel insight into interactions between RGD containing bone matrices, GCT cells and MMPs of potential relevance to GCT pathology. Int. J. Cancer 77:82–93, 1998.© 1998 Wiley‐Liss, Inc.

In vivo modulation of the distribution of thymocyte subsets : effects of estrogen on the expression of different T cell receptor Vβ gene families in CD4-, CD8- thymocytes

Estrogen treatment of mice has been shown to deplete CD4+, CD8+ double-positive (DP) thymocytes and to alter the relative proportion of CD4+ and CD8+ single-positive (SP) thymocytes. In this work, we have studied the effect of the steroid hormone 17 beta-estradiol (E2) on the different subsets of CD4-/CD8- double-negative (DN) thymocytes by analyzing the expression of CD5, CD3-epsilon and of several V beta gene family products of the T cell antigen receptor (TCR). After in vivo administration of E2 a significant decrease in the number and proportion of dull CD5+, CD3-, beta-TCR- DN thymocytes was observed. In contrast E2 treatment significantly increased the proportion of bright CD5+, CD3+, beta-TCR+ DN cells. The E2-induced increase in DN/TCR+ cells was observed for subsets expressing V beta 6, V beta 8, and V beta 11, but not V beta 3 gene products of the TCR. Thus, estrogen administration results in a selective inbalance of the DN thymocyte subsets by depleting an immature, dull CD5+, CD3-, TCR beta- DN subset, while enriching a mature, bright CD5+, CD3+, TCR beta+ DN subset of cells. In addition to TCR beta+ DN thymocytes, an increased proportion of CD4+ and CD8+ SP thymocytes expressing V beta 8, V beta 6, and V beta 11, but not V beta 3, TCR proteins was also observed after E2 administration. An involvement of intrathymic cytokine production in mediating the hormone action is suggested by the ability of estrogen to increase the levels of IL-1 alpha mRNA of intact thymus. Our data suggest that estrogen exerts its effects on a broad range of immature cells, including dull CD5+, CD3-, beta-TCR- DN and DP thymocytes.

The growth arrest and downregulation of c-myc transcription induced by ceramide are related events dependent on p21 induction, Rb underphosphorylation and E2F sequestering.

Ceramide is an intracellular lipid mediator generated through the sphingomyelin cycle in response to several extracellular signals. Ceramide has been shown to induce growth inhibition, c-myc downmodulation and apoptosis. In this paper we examined the mechanism by which ceramide induces growth suppression and the role of the G1-CDK/pRb/E2F pathway in this process. The addition of exogenous, cell-permeable C2-ceramide to the Hs 27 human diploid fibroblast cell line resulted in a dose-dependent induction of the p21WAF1/CIP1/Sdi1 kinase inhibitor with reduction of cyclin-D1 associated kinase activity. Furthermore, significant dephosphorylation of pRb was observed, with increased association of pRb and the E2F transcription factor into a transcriptionally inactive complex. Ceramide was also capable of inhibiting the transcriptional activity of a CAT reporter vector driven by E2F binding sites containing c-myc promoter transfected into Hs 27 cells. The requirement of the pRb protein for ceramide-induced c-myc downregulation was supported by the failure of ceramide to inhibit promoter activity in HeLa cells, in which pRb function is abrogated by the presence of the E7 Papilloma virus oncoprotein, and in pRb-deleted SAOS2 AT cells. Ceramide-induced downregulation of the c-myc promoter was restored in SAOS2 #1 cells in which a functional Rb gene was reintroduced. Our studies demonstrate that pRb dephosphorylation, induced by ceramide, is at least partly necessary for c-myc downregulation, and therefore the CDK-Rb-E2F pathway appears to be a target for the ceramide-induced modulation of cell cycle regulated gene transcription.

Estrogen receptors: new perspectives in breast cancer management

The imbalance between proliferative and differentiative estrogenic effect, caused by quantitative and qualitative alteration of the estrogen receptor (ER) expression, may play a determinant role in mammary neoplastic transformation. Our studies demonstrate that ER levels are significantly higher in human mammary neoplastic tissues when compared to perineoplastic tissues and that increased ER expression is associated with ER gene hypomethylation. During progressive multifactorial carcinogenesis, ER overexpression may represent an early step in neoplastic transformation. In fact, high levels of ER represent good markers of differentiation and can predict the likelihood of benefiting from anti-estrogen therapy. Nevertheless, about 35% of ER-positive breast cancers are resistant to endocrine therapy and 10% of ER-negative tumors behave as hormone-sensitive tumors. Recent studies on ER mRNA variants, which naturally occur in human breast tumors, demonstrated mutations, deletions and alternative splicings, yielding deletions of exons 3, 4, 5 and 7. ER variants exhibited altered functions or changed the responsiveness to hormonal therapy. Analysis of these variants could be a useful parameter to better predict tumor responsiveness to anti-estrogen therapy. Recently, a regain of hormonal responsiveness by ER-negative breast cancer cells has been reported following ER gene transfection. However, estradiol treatment inhibits rather than stimulates cell growth as well as the metastatic and invasive potential of the ER gene transduced cells. Transfer of the ER gene may be considered as a new therapeutic approach in the management of hormone-independent breast cancer.

Prevalence of hypertension in acromegalic patients: clinical measurement versus 24-hour ambulatory blood pressure monitoring.

Hypertension is thought to play an important role in the pathogenesis of acromegalic cardiomyopathy. So far, hypertension has been defined by clinical measurement, with considerable variations reported concerning its prevalence in acromegalics.

Interleukin-6 and CAAT/enhancer binding protein β-deficient mice act as tools to dissect the IL-6 signalling pathway and IL-6 regulation

Interleukin-6 (IL-6) is a pleiotropic cytokine playing important roles in immunity, hemopoiesis and inflammation. IL-6 signalling is known to involve the activation of two independent transcription factors: Stat3 (through phosphorylation by Jak kinases) and C/EBP beta (through activation of the ras pathway). In addition, C/EBP beta is believed to act as a transcriptional activator of the IL-6 gene itself. Making use of IL-6-deficient mice, we have recently demonstrated that IL-6 is essential for the induction of acute phase mRNAs in the liver upon localized tissue damage, but not upon systemically induced inflammation. Here we show that the defective mRNA induction is paralleled by a defective activation of Stat3, thus establishing a direct relationship between IL-6 function, Stat3 activation and acute phase genes induction. On the other hand, making use of C/EBP beta-deficient mice, we show that the induction of IL-6 by a variety of stimuli does not require C/EBP beta activity. In contrast to the predicted activating role of C/EBP beta, IL-6 levels are increased in the C/EBP beta-deficient mice, suggesting that C/EBP beta may act as a down-modulator of the IL-6 gene. Through the generation of C/EBP beta, IL-6 double mutant mice we show that IL-6 hyperproduction is responsible for the development of the Castlemans like lymphoproliferative disease described in the C/EBP beta-deficient mice, since the disorder is completely blocked by inactivating the IL-6 gene.

Advanced carcinoma of the pancreas: Phase II study of combined chemotherapy, β-interferon, and retinoids

Because of the poor response of pancreatic cancer to conventional therapy, the authors performed a phase II pilot study to evaluate whether beta-interferon and retinoids, added to active chemotherapeutic agents, could increase response rate and survival in a group of patients who had metastatic disease. Twenty-three chemotherapy-naive patients were treated as follows: epirubicin, 60 mg/m2, and mitomycin C, 10 mg/m2, intravenously on day 1; folinic acid, 200 mg/m2, and 5-fluorouracil (5-FU), 370 mg/m2, intravenously for 5 consecutive days. beta-Interferon, 1 x 10(6) IU/m2, subcutaneously three times a week, and retinol palmitate, 50,000 IU orally twice a day, were given between chemotherapy cycles. Patients having responses and disease stabilization were maintained with the same dose of beta-interferon and retinol palmitate. Treatment was given every 4 weeks for four courses or until onset of progression. A median of three courses of chemotherapy was delivered to each patient. All patients were evaluable. Eight patients responded (35%) and 8 (35%) had stable disease. Median time to progression and survival for all patients were, respectively, 6.1 months and 11 months. Toxicity was severe: 60% of patients had hematologic toxicity, 40% had gastrointestinal toxicity, 13% had cardiac toxicity, and 1 patient had a hemolitic-uremic syndrome. The combination of chemotherapy, beta-interferon, and retinoids shows activity in metastatic pancreatic carcinoma. Toxicity was high but patients who had responses and disease stabilization had prolonged symptom palliation.

Heavy-metal modulation of the human intercellular adhesion molecule (ICAM-1) gene expression

The intercellular adhesion molecule 1 (ICAM-1) can be induced on many different cell types by a set of various modulators (IL1 beta, TNF, LPS, IFN-gamma), which are released during the inflammatory process. We have investigated the possibility that other factors, related to the stress and biophysical perturbations of the inflammatory response, may also modulate ICAM-1. Here, we report that heavy metals, in particular zinc, can enhance the expression of the ICAM-1 gene on cells actively involved at different levels during inflammation. Kinetic studies of ICAM-1 gene expression shows a maximum level of induction 4 h after treatment with metals, followed by a rapid decrease to basal levels within 12 h. The effect on enhanced gene expression is mostly due to a rapid increase of the transcriptional rate as shown by nuclear run-on experiments. In B lymphoblastoid cells, but not in fibroblasts, the increase in RNA expression seems significantly greater that the subsequent increase in protein expression, suggesting that a further point of post-transcriptional regulation of ICAM-1 occurs and may be linked to the cellular specificity. may be linked to the cellular specificity.

RETINOIC ACID-ENHANCED INVASION THROUGH RECONSTITUTED BASEMENT MEMBRANE BY HUMAN SK-N-SH NEUROBLASTOMA CELLS INVOLVES MEMBRANE-ASSOCIATED TISSUE-TYPE PLASMINOGEN ACTIVATOR

Al‐trans retinoic acid (RA) enhanced human, S‐type, SK‐N‐SH neuroblastoma cell invasion of reconstituted basement membrane in vitro but did not induce terminal differentiation of this cell line. In contrast to basal invasion, which was urokinase (uPA)‐ and plasmin‐dependent, RA‐enhanced invasion was dependent on tissue‐type plasminogen activator (t‐PA) and plasmin activity. Neither basal nor RA‐enhanced invasion involved TIMP‐2 inhibitable metalloproteinases. Enhanced invasion was associated with the induction of t‐PA expression, increased expression of the putative t‐PA receptor amphoterin, increased association of t‐PA with cell membranes and increased net membrane‐associated PA activity. Enhanced invasion was not associated with significant changes in the expression of uPA or its membrane receptor UPAR; plasminogen activator inhibitors PAI‐1 and PAI‐2; metalloproteinases MMP‐1, MMP‐2, MMP‐3, MMP‐9 and membrane type MMP1; or tissue inhibitors of metalloproteinases TIMP‐1 and TIMP‐2. RA stimulated the association of t‐PA with the external cell membrane surface, which could be inhibited by heparin sulphate but not by mannose sugars or chelators of divalent cations, consistent with a role for amphoterin. Our data indicate that RA can promote the malignant behavior of S‐type neuroblastoma cells refractory to RA‐mediated terminal differentiation by enhancing their basement membrane invasive capacity. We suggest that this results from the action of a novel, RA‐regulated mechanism involving stimulation of t‐PA expression and its association with the cell membrane leading to increased PA‐dependent matrix degradation. Int. J. Cancer 73:740–748, 1997.