Giuseppe Bunone

Expression of Angiogenesis Stimulators and Inhibitors in Human Thyroid Tumors and Correlation with Clinical Pathological Features

Experimental evidence has shown, both in vitro and in animal models, that neoplastic growth and subsequent metastasis formation depend on the tumors ability to induce an angiogenic switch. This requires a change in the balance of angiogenic stimulators and inhibitors. To assess the potential role of angiogenesis factors in human thyroid tumor growth and spread, we analyzed their expression by semiquantitative RT-PCR and immunohistochemistry in normal thyroid tissues, benign lesions, and different thyroid carcinomas. Compared to normal tissues, in thyroid neoplasias we observed a consistent increase in vascular endothelial growth factor (VEGF), VEGF-C, and angiopoietin-2 and in their tyrosine kinase receptors KDR, Flt-4, and Tek. In particular, we report the overexpression of angiopoietin-2 and VEGF in thyroid tumor progression from a prevascular to a vascular phase. In fact, we found a strong association between tumor size and high levels of VEGF and angiopoietin-2. Furthermore, our results show an increased expression of VEGF-C in lymph node invasive thyroid tumors and, on the other hand, a decrease of thrombospondin-1, an angioinhibitory factor, in thyroid malignancies capable of hematic spread. These results suggest that, in human thyroid tumors, angiogenesis factors seem involved in neoplastic growth and aggressiveness. Moreover, our findings are in keeping with a recent hypothesis that in the presence of VEGF, angiopoietin-2 may collaborate at the front of invading vascular sprouts, serving as an initial angiogenic signal that accompanies tumor growth.

Induction of apoptosis by p75 neurotrophin receptor in human neuroblastoma cells.

The low-affinity nerve growth factor receptor p75NTR belongs to a membrane receptor superfamily whose members, in certain cell types, are able to transduce an apoptotic signal. To investigate the effect of p75NTR expression in neuroblastoma cells, we transfected the p75NTR cDNA into SK-N-BE cells, a neuroblastoma cell line that lacks expression of both p75NTR and TrkA. Cell clones expressing elevated levels of p75NTR showed a high degree of cell death by apoptosis, even in serum-supplemented medium. Moreover, the level of apoptosis correlated directly with the expression level of the receptor, indicating that p75NTR could activate the cell death program by itself. Clones expressing p75NTR showed a dramatic increase of cell death when switched into serum-free medium; these cultures rapidly extinguished. This apoptotic effect was greatly inhibited by NGF treatment. Our results support the hypothesis that p75NTR, when it is not bound by NGF, may play a role in neuronal selection during embryonic development and suggest that neuroblastomas may arise from immature neuroblasts that escape programmed cell death. Therefore, the loss of p75NTR expression in developing neural crest cells might be a primary event in the genesis of neuroblastoma.

Expression of β2m-Free HLA Class I Heavy Chains in Neuroblastoma Cell Lines

Flow cytometry with the specific monoclonal antibody (MoAb) L31 was used to analyse the expression of HLA class I heavy chains not bound with β2‐microglobulin (β2m) by neuroblastoma (NB) cell lines IMR‐32 and LA‐N‐1. The cells, which express barely detectable amounts of β2m‐free (L31‐positive molecules) and β2m‐complexed HLA class I antigens (W6.32‐ and BBM. I‐reactive molecules), expressed MHC class I molecules not bound to light chains upon differentiation with either retinoic acid or serum starvation. The expression was not accompanied by an increase of surface heterodimers. Conversely, recombinant interferon‐γ (rIFN‐γ) treatment led IMR‐32 and LA‐N‐1 cells to almost exclusively express β2m‐complexed HLA class I heavy chains. Surface β2m‐free MHC class I molecules displayed a molecular mass of ~45 kDa and did not bind exogenously added β2m. No changes in the synthesis of either HLA class I and β2m mRNAs or of L31 proteins were observed in differentiated NB cells, thus suggesting that the surface exposure of unusual HLA class I antigens is regulated post‐translationally. These findings indicate that, in addition to activated lymphocytes, the surface expression of β2m‐free class I heavy chains is a feature of other cell types, such as NB cells.

Assignment of the human heterogeneous nuclear ribonucleoprotein A1 gene (HNRPA1) to chromosome 12q13.1 by cDNA competitive in situ hybridization

Heterogeneous nuclear ribonucleoprotein (HNRP) core protein A1 is a major component of mammalian HNRP particles. The human HNRP A1 protein was shown to be encoded by a 4.6-kb gene, split into 10 exons, belonging to a multigene family of about 30 A1-specific sequences per haploid genome, many of which correspond to pseudogenes of the processed type. Here we report the mapping of the human HNRPA1 gene to band 12q13.1. Localization was performed by nonisotopic in situ hybridization using a phage genomic clone that contains the active HNRPA1 gene as well as 13.5-kb flanking sequences. To suppress hybridization to pseudogene sequences, unlabeled HNRPA1 cDNA was added in excess over the probe to the hybridization mixture.