Achille Cittadini

Anomalous dystroglycan in carcinoma cell lines

Dystroglycan is a receptor responsible for crucial interactions between extracellular matrix and cytoplasmic space. We provide the first evidence that dystroglycan is truncated. In HC11 normal murine and the 184B5 non‐tumorigenic mammary human cell lines, the expected β‐dystroglycan 43 kDa band was found but human breast T47D, BT549, MCF7, colon HT29, HCT116, SW620, prostate DU145 and cervical HeLa cancer cells expressed an anomalous ≈31 kDa β‐dystroglycan band. α‐Dystroglycan was udetectable in most of the cell lines in which β‐dystroglycan was found as a ≈31 kDa species. An anomalous ≈31 kDa β‐dystroglycan band was also observed in N‐methyl‐N‐nitrosurea‐induced primary rat mammary tumours. Reverse transcriptase polymerase chain reaction experiments confirmed the absence of alternative splicing events and/or expression of eventual dystroglycan isoforms. Using protein extraction procedures at low‐ and high‐ionic strength, we demonstrated that both the 43 kDa and ≈31 kDa β‐dystroglycan bands harbour their transmembrane segment.

Targeted inhibition of the epidermal growth factor receptor‐tyrosine kinase by ZD1839 (‘Iressa’) induces cell‐cycle arrest and inhibits proliferation in prostate cancer cells

The epidermal growth factor (EGF) plays a role in the development of prostate cancer, which becomes essential after androgen resistance has emerged. The EGF receptor (EGFR) is therefore a potential target for anticancer therapy. We evaluated the effects of ZD1839 (‘Iressa’), an orally active EGFR—tyrosine kinase inhibitor, on prostate cancer cell lines. The effects of ZD1839 were evaluated on the anchorage dependent and independent growth of androgen‐responsive (LNCaP) and androgen‐independent (DU145 and PC3) cells by a cell proliferation assay, cell counting, and soft agar analysis. Flow cytometric analysis and Western blotting were used to assess the effects on the cell‐cycle and on protein expression levels, respectively. ZD1839 caused a dose‐ and time‐dependent growth inhibition in all three cell lines. A dose‐dependent supra‐additive increase in growth inhibition was observed when ZD1839 was combined with the antiandrogen flutamide or ionizing radiation (IR). The antiproliferative effect of ZD1839 was mainly cytostatic and associated with a block in the G0/G1 phase of the cell‐cycle, evident after about 12 h of treatment. In the DU145 cells this block was associated with an increase in expression of the CDK inhibitor p27Kip1, both in the cytoplasmic and nuclear fractions. The increase in p27Kip1 was not evident in the LNCaP and PC3 cells. No changes were observed in the expression of cyclin D1 protein. These results demonstrate the antiproliferative effects of ZD1839 on the growth of prostate cancer cells and suggest that inhibition of EGFR‐associated signal transduction pathway might represent a promising novel therapeutic strategy for the treatment of prostate cancer. J. Cell. Physiol. 201: 97–105, 2004.

DHA induces apoptosis by altering the expression and cellular location of GRP78 in colon cancer cell lines.

n-3 polyunsaturated fatty acids exert growth-inhibitory and pro-apoptotic effects in colon cancer cells. We hypothesized that the anti-apoptotic glucose related protein of 78kDa (GRP78), originally described as a component of the unfolded protein response in endoplasmic reticulum (ER), could be a molecular target for docosahexaenoic acid (DHA) in these cells. GRP78 total and surface overexpression was previously associated with a poor prognosis in several cancers, whereas its down-regulation with decreased cancer growth in animal models. DHA treatment induced apoptosis in three colon cancer cell lines (HT-29, HCT116 and SW480), and inhibited their total and surface GRP78 expression. The cell ability to undergo DHA-induced apoptosis was inversely related to their level of GRP78 expression. The transfection of the low GRP78-expressing SW480 cells with GRP78-GFP cDNA significantly induced cell growth and inhibited the DHA-driven apoptosis, thus supporting the essential role of GRP78 in DHA pro-apoptotic effect. We suggest that pERK1/2 could be the first upstream target for DHA, and demonstrate that, downstream of GRP78, DHA may exert its proapoptotic role by augmenting the expression of the ER resident factors ERdj5 and inhibiting the phosphorylation of PKR-like ER kinase (PERK), known to be both physically associated with GRP78, and by activating caspase-4. Overall, the regulation of cellular GRP78 expression and location is suggested as a possible route through which DHA can exert pro-apoptotic and antitumoral effects in colon cancer cells.

Magnesium depletion causes growth inhibition, reduced expression of cyclin D1, and increased expression of P27KIP1 in normal but not in transformed mammary epithelial cells

In this study, we have evaluated the effects of extracellular magnesium restriction on the growth and cell cycle parameters of normal (HC11) and transformed (MCF‐7) breast epithelial cell lines. Cells were incubated in medium with different concentrations of Mg2+ (from 0.5 to 0 mM) and the growth rates were determined by [3H]‐thymidine incorporation and cell counting. The growth of the HC11 cells was drastically inhibited by Mg2+ depletion whereas the MCF‐7 cells were only slightly inhibited (about 50% and 15%, respectively, after incubation in 0.05 mM Mg for 48 h). Cell cycle analyses showed a decrease in the percentage of cells in the S phase when both cell lines were incubated at low Mg2+ concentration. However, while the percentage of cells in both the G0/G1 and G2/M phases was increased in the HC11 cells, only the percentage of cells in the G2/M phase was increased in the MCF‐7 cell line. Extracellular magnesium depletion was associated with increased expression of the cyclin‐dependent kinase inhibitor p27Kip1 and decreased expression of cyclin D1 in the HC11 but not in the MCF‐7 cells. We also demonstrated that Mg2+ depletion does not inhibit kinase activities in the normal HC11 cells and that Mg2+‐restricted HC11 cells are still responsive to the epidermal growth factor (EGF)‐ and insulin‐mediated stimulation of cell growth. These data suggest that normal but not transformed mammary epithelial cells are inhibited by extracellular Mg2+ restriction and that this effect might be mediated by changes in the levels of expression of both cyclin D1 and p27Kip1. J. Cell. Physiol. 180:245–254, 1999.

Superficial papillary urothelial carcinomas in young and elderly patients: a comparative study

To compare the clinicopathological and immunohistochemical findings of superficial papillary transitional cell carcinomas in ‘young’ and ‘elderly’ patients, as the natural history and prognosis of bladder tumours in young patients remains a matter of debate.

Magnesium and tumors: Ally or foe?

Magnesium plays a crucial role in many cell functions such as energy metabolism, protein and DNA syntheses, and cytoskeleton activation. Proliferating cells have long been known to contain more magnesium than quiescent cells, and experimental conditions that decreased magnesium availability affected cell proliferation rate. There is little information about how tumor growth influenced systemic availability of magnesium in a patient, nor is it clear whether treatment-associated changes of magnesaemia influenced tumor growth and dissemination. Hypomagnesaemia is observed during multi-agent therapies with cisplatin or the anti-EGFR antibody, cetuximab. The latter was shown to cause hypomagnesaemia by impeding EGF-dependent activation of TRPM6, the main cation channel responsible for Mg transcellular absorption in the intestine and kidney. Limited observations also suggest that hypomagnesaemia could favorably influence tumor response to cetuximab. All such findings brought magnesium into the arena of clinical oncology, but potential caveats should be kept in mind before considering practical implications. We briefly review that magnesium causes pleiotropic, often diverging effects on tumor growth, vascularization, and metastatization, such that both favorable and unfavorable effects can be identified. Inflammatory responses to hypomagnesaemia should also be considered. Translating biology into clinical facts will therefore require a deeper understanding of such a complexity.

Increased expression of CD133 and reduced dystroglycan expression are strong predictors of poor outcome in colon cancer patients

BackgroundExpression levels of CD133, a cancer stem cell marker, and of the α-subunit of the dystroglycan (α-DG) complex, have been previously reported to be altered in colorectal cancers.MethodsExpression levels of CD133 and α-DG were assessed by immunohistochemistry in a series of colon cancers and their prognostic significance was evaluated.ResultsScattered cells positive for CD133 were rarely detected at the bases of the crypts in normal colonic mucosa while in cancer cells the median percentage of positive cells was 5% (range 0–80). A significant correlation was observed with pT parameter and tumor stage but not with tumor grade and N status. Recurrence and death from disease were significantly more frequent in CD133-high expressing tumors and Kaplan-Meier curves showed a significant separation between high vs low expressor groups for both disease-free (pu2009=u20090.002) and overall (pu2009=u20090.008) survival.Expression of α-DG was reduced in a significant fraction of tumors but low α-DG staining did not correlate with any of the classical clinical-pathological parameters. Recurrence and death from the disease were significantly more frequent in α-DG-low expressing tumors and Kaplan-Meier curves showed a significant separation between high vs low expressor tumors for both disease-free (pu2009=u20090.02) and overall (pu2009=u20090.02) survival. Increased expression of CD133, but not loss of α-DG, confirmed to be an independent prognostic parameters at a multivariate analysis associated with an increased risk of recurrence (RRu2009=u20092.4; pu2009=u20090.002) and death (RRu2009=u20092.3; pu2009=u20090.003).ConclusionsLoss of α-DG and increased CD133 expression are frequent events in human colon cancer and evaluation of CD133 expression could help to identify high-risk colon cancer patients.

Magnesium restriction induces granulocytic differentiation and expression of P27Kip1 in human leukemic HL‐60 cells

When cultured in Mg restricted medium, human leukemic HL‐60 cells develop morphological and functional granulocytic differentiation. In 0.03 mM Mg, cells display the distinctive features of differentiation, without appreciable inhibition of proliferation. In 0.01 mM Mg, cells show terminal differentiation, accompanied by clear inhibition of proliferation. Such cells accumulate in the G0/G1 phase and subsequently die via apoptosis, similar to HL‐60 cells that have been induced to differentiate by DMSO. These phenotypic changes are associated with a marked increase in the expression level of the cyclin dependent kinase inhibitor p27Kip1. Cyclin E expression is also slightly increased in Mg restricted cells, whereas no changes are observed in the expression level of cyclin D1. We also show that during differentiation cell total Mg decreases, whereas [Mg2+]i increases in both Mg‐depleted and DMSO‐treated cells. These data suggest that the maturation process is paralleled by a redistribution of intracellular Mg, leading to a shift from the bound to the free form. These changes could modulate the kinetics of Mg‐dependent enzyme(s) that are involved in the control of the differentiation pathway. We propose that this model may represent an useful tool for the study of the mechanisms of cell differentiation and related events, such as aging and death. J. Cell. Biochem. 70:313–322, 1998.

An ultrastructural study of calcium induced degenerative changes in dissociated heart cells

Abstract Dissociated myocytes were prepared by enzymatic perfusion of adult rabbit hearts, and fixed following exposure to various concentrations of Ca2+. Only slight ultrastructural changes were noted in myocytes which were maintained in relaxation [(Ca2+) m ] or in cyclic contractile activity [(Ca2+) ≅ 0.4 μ m ]. In these cells, slight swelling permitted clear demonstration of interesting ultrastructural details such as tight connections between Z bands and sarcolemma, attachment of filaments to these structures, and relationships of sarcoplasmic reticulum and T tubules. When the Ca2+ concentration was raised above 1.0 μ m , sarcomeres exhibited extreme and irreversible shortening, followed by myofilament disorganization, disappearance of Z lines, aggregation of most thick and some thin filaments forming a central mass, and outward displacement of organelles. Under these conditions numerous filaments were attached randomly to the sarcolemma and to intracellular membranes, demonstrating their inherent capacity to bind to membranous structures. The ultrastructural alterations observed in our experiments are similar to those described for the “calcium paradox”, which is likely to be a common pathogenic mechanism of myocardial cell damage.

The tumor suppressor HINT1 regulates MITF and β-catenin transcriptional activity in melanoma cells.

Histidine triad nucleotide-binding protein 1 (HINT1) is a haploinsufficient tumor suppressor gene that inhibits the Wnt/β-catenin pathway in colon cancer cells and Microphthalmia-associated transcription factor (MITF) activity in human mast cells. MITF and β-catenin play a central role in melanocyte and melanoma cell survival, and this study aimed to investigate the effects of HINT1 on the MITF and β-catenin pathways in malignant melanoma cells. We found that HINT1 inhibits MITF and β-catenin transcriptional activity, and both proteins can be co-immunoprecipitated with an anti-HINT1-specific antibody in melanoma cell lines. Stable, constitutive overexpression of the HINT1 protein in human melanoma cells significantly impaired cell proliferation in vitro and tumorigenesis in vivo. These effects were associated with a decreased expression of cyclin D1 and BCL2, well known MITF and β-catenin transcription targets, respectively. We also demonstrated that BCL2 and cyclin D1 can partially rescue the HINT1-driven phenotype. Moreover, we found in ChIP assays that HINT1 binds the chromatin at MITF and β-catenin sites in BCL2 and cyclin D1 promoters, respectively, and that mSIN3a and HDAC1, well known transcriptional repressors, can be co-immunoprecipitated with an anti-HINT1-specific antibody. These findings support the tumor suppressor activity of HINT1 gene in melanoma cells by promoting the formation of non-functional complexes with oncogenic transcription factors like MITF and β-catenin.