Renato Mariani-Costantini

Immunohistochemical reactivity of a monoclonal antibody prepared against human breast carcinoma.

The reactivity profile of an IgM monoclonal antibody, MBR1, raised against the human breast cancer cell line MCF7, was studied in a variety of human tumours and non-neoplastic tissues by light microscopic immunohistochemistry. The range of reactivity included specific types of non-neoplastic epithelial cells and a number of epithelial tumours. Most mammary carcinomas reacted with MBR1, but adenocarcinomas and squamous carcinomas from different sites were also strongly positive. Different patterns of immunoreactivity were apparent in microscopically normal tissues, in tissues with inflammatory changes and in carcinomas. Heterogeneous staining, despite morphological similarities, was documented in neoplastic and non-neoplastic epithelial cells. The reactivity of MBR1 was different from that reported for other monoclonal antibodies, but revealed similarities to that of monoclonal antibodies and polyclonal sera against human milk fat globule membrane.

Instability at sequence repeats in melanocytic tumours.

To obtain information on the prevalence of microsatellite mutations in melanomas, we analysed the status of 14 repetitive loci characterized by structurally different non-coding and coding sequence repeats in a panel of 34 primary melanocytic tumours and in lymph node metastases matched to 13 cases. Instability at one or more of the non-coding dinucleotide repeats D2S123, D3S1611, D5S107 and D18S34 was detected in ten out of the 34 primary tumours (29%) and in ten of the 13 metastases (77%). There was no instability at the non-coding mononucleotide repeats BAT25, BAT26 and AP Δ3 or at the coding mononucleotide runs within the TGF βRII, IGFIIR, BAX, hMSH3 and hMSH6 genes. A five-repeats expansion of the coding E2F4 (CAG)n run was found in the only malignant melanoma of soft parts examined, which also showed instability at two dinucleotide loci, and in a superficial spreading melanoma, which was stable at the mononucleotide and dinucleotide repeats but was the only tumour that manifested instability at the SCA1 (CAG)n repeat. The absence of mutations at mononucleotide tracts indicates that, in the malignant melanomas tested, microsatellite instability was not associated with the microsatellite mutator phenotype characteristic of mismatch repair-deficient tumours. On the other hand, our results confirm that microsatellite instability at dinucleotide repeats increases with melanoma progression, and indicate that expansions of triplet repeats may occur in melanocytic tumours.

Immunotoxicity and Sensitizing Capacity of Metal Compounds Depend on Speciation

Immunotoxicity of metal compounds is an issue of great importance due to the recent industrial application of metals with unknown toxicity on the immune system and the discovery of metal intermediary compounds not sufficiently studied yet. In this report we show results of our study on the immunotoxicity of the following metals: the Platinum group elements (Platinum, Palladium, Rhodium), Titanium and Arsenic. We applied functional and non functional assays and investigated both innate and adaptive immune systems, in particular, cell proliferation, cytokine production by PBMCs and O−2 production by neutrophils. We obtained the following results: only some Ti compounds (Titanocene, Ti ascorbate and Ti oxalate) show immunotoxicity. Trivalent As compounds (Sodium arsenite and tetraphenyl arsonium chloride) are more immunotoxic than the other investigated As compounds. Genotoxicity of Pt group compounds is in the following order: Pt < Rh < Pd. Immunotoxicity of Pt group compounds is in the following order: Pd < Pt < Rh. Lymphocytes and macrophages show a different reaction of neutrophils to metal toxicity. We can conclude that these studies show that metal immunotoxicity depends on speciation. In general speciation provides additional and often essential information in evaluating metal toxicity. However, there are many difficulties in applying speciation in investigating toxico-kinetic aspects to many metals, mainly due to the lack of information about the existence and significance of species and to the lack of analytical methods for measuring species in biological samples.

In situ detection of c-myc mRNA in adenocarcinomas, adenomas, and mucosa of human colon.

We used a sensitive RNA:RNA in situ hybridization technique to study steady-state levels of c-myc proto-oncogene mRNA in primary human colon adenocarcinomas, villous adenomas, and normal mucosa samples. Frozen tissue sections, fixed in 4% buffered paraformaldehyde, were hybridized to 35S-labeled anti-sense transcripts of a c-myc clone and processed for autoradiography. The specificity of the hybridization was controlled by using 35S-labeled plasmid transcripts as a negative control, while RNA preservation in the tissue sample was assessed by using 35S-labeled anti-sense transcripts of a murine 28S rRNA clone. c-myc RNA was detectable in all the carcinomas (eight) and villous adenomas (four), but steady-state levels varied from high to low in different tumors with similar histology. Low levels of c-myc RNA were detected in epithelial stem cells of some of the normal mucosa samples (five). No genetic alterations of the c-myc locus were found by Southern analysis of DNAs extracted from the carcinomas.

Integrative genetic, epigenetic and pathological analysis of paraganglioma reveals complex dysregulation of NOTCH signaling

Head and neck paragangliomas, rare neoplasms of the paraganglia composed of nests of neurosecretory and glial cells embedded in vascular stroma, provide a remarkable example of organoid tumor architecture. To identify genes and pathways commonly deregulated in head and neck paraganglioma, we integrated high-density genome-wide copy number variation (CNV) analysis with microRNA and immunomorphological studies. Gene-centric CNV analysis of 24 cases identified a list of 104 genes most significantly targeted by tumor-associated alterations. The “NOTCH signaling pathway” was the most significantly enriched term in the list (Pxa0=xa00.002 after Bonferroni or Benjamini correction). Expression of the relevant NOTCH pathway proteins in sustentacular (glial), chief (neuroendocrine) and endothelial cells was confirmed by immunohistochemistry in 47 head and neck paraganglioma cases. There were no relationships between level and pattern of NOTCH1/JAG2 protein expression and germline mutation status in the SDH genes, implicated in paraganglioma predisposition, or the presence/absence of immunostaining for SDHB, a surrogate marker of SDH mutations. Interestingly, NOTCH upregulation was observed also in cases with no evidence of CNVs at NOTCH signaling genes, suggesting altered epigenetic modulation of this pathway. To address this issue we performed microarray-based microRNA expression analyses. Notably 5 microRNAs (miR-200a,b,c and miR-34b,c), including those most downregulated in the tumors, correlated to NOTCH signaling and directly targeted NOTCH1 in in vitro experiments using SH-SY5Y neuroblastoma cells. Furthermore, lentiviral transduction of miR-200s and miR-34s in patient-derived primary tympano-jugular paraganglioma cell cultures was associated with NOTCH1 downregulation and increased levels of markers of cell toxicity and cell death. Taken together, our results provide an integrated view of common molecular alterations associated with head and neck paraganglioma and reveal an essential role of NOTCH pathway deregulation in this tumor type.

Over-expression of the miR-483-3p overcomes the miR-145/TP53 pro-apoptotic loop in hepatocellular carcinoma

The miR-145-5p, which induces TP53-dependent apoptosis, is down-regulated in several tumors, including hepatocellular carcinomas (HCCs), but some HCCs show physiological expression of this miR. Here we demonstrate that in HCC cells carrying wild-type TP53 the steady activation of the miR-145 signaling selects clones resistant to apoptosis via up-regulation of the oncogenic miR-483-3p. Expression of the miR-145-5p and of the miR-483-3p correlated negatively in non-neoplastic liver (n=41; ρ=−0.342, P=0.028), but positively in HCCs (n=21; ρ=0.791, P<0.0001), which we hypothesized to be due to impaired glucose metabolism in HCCs versus normal liver. In fact, when liver cancer cells were grown in low glucose, miR-145-5p lowered miR-483-3p expression, allowing apoptosis, whereas when cells were grown in high glucose the levels of miR-483-3p increased, reducing the apoptotic rate. This indicates that depending on glucose availability the miR-145-5p has double effects on the miR-483-3p, either inhibitory or stimulatory. Moreover, resistance to apoptosis in clones overexpressing both miR-145-5p and miR-483-3p was abrogated by silencing the miR-483-3p. Our data highlight a novel mechanism of resistance to apoptosis in liver cancer cells harbouring wild type TP53 and suggest a potential role of miR-145-5p and miR-483-3p as druggable targets in a subset of HCCs.

Immunocytochemical identification of breast carcinoma cells in effusions using a monoclonal antibody.

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