Stefano Martinotti

Characterization of a Novel Human Serine Protease That Has Extensive Homology to Bacterial Heat Shock Endoprotease HtrA and Is Regulated by Kidney Ischemia

We report the isolation and characterization of a cDNA encoding the novel mammalian serine protease Omi. Omi protein consists of 458 amino acids and has homology to bacterial HtrA endoprotease, which acts as a chaperone at low temperatures and as a proteolytic enzyme that removes denatured or damaged substrates at elevated temperatures. The carboxyl terminus of Omi has extensive homology to a mammalian protein called L56 (human HtrA), but unlike L56, which is secreted, Omi is localized in the endoplasmic reticulum. Omi has several novel putative protein-protein interaction motifs, as well as a PDZ domain and a Src homology 3-binding domain. Omi mRNA is expressed ubiquitously, and the gene is localized on human chromosome 2p12. Omi interacts with Mxi2, an alternatively spliced form of the p38 stress-activated kinase. Omi protein, when made in a heterologous system, shows proteolytic activity against a nonspecific substrate β-casein. The proteolytic activity of Omi is markedly up-regulated in the mouse kidney following ischemia/reperfusion.

Human MRE11 is inactivated in mismatch repair‐deficient cancers

Mutations of the ATM and NBS1 genes are responsible for the inherited Ataxia‐Telangiectasia and Nijmegen Breakage Syndrome, both of which are associated with a predisposition to cancer. A related syndrome, the Ataxia‐Telangiectasia‐like disorder, is due to mutations of the MRE11 gene. However, the role of this gene in cancer development has not been established. Here we describe an often homozygous mutation of the poly(T)11 repeat within human MRE11 intron 4 that leads to aberrant splicing, impairment of wild‐type MRE11 expression and generation of a truncated protein. This mutation is present in mismatch repair‐deficient, but not proficient, colorectal cancer cell lines and primary tumours and is associated with reduced expression of the MRE11–NBS1–RAD50 complex, an impaired S‐phase checkpoint and abrogation of MRE11 and NBS1 ionizing radiation‐induced nuclear foci. Our findings identify MRE11 as a novel and major target for inactivation in mismatch repair‐defective cells and suggest its impairment may contribute to the development of colorectal cancer.

Angiogenesis inhibitors overcome tumor induced endothelial cell anergy.

We report here that tumor angiogenesis‐mediated endothelial cell (EC) anergy can be overcome by inhibitors of angiogenesis. We found previously that tumor growth, known to be dependent on angiogenesis, results in down‐regulation of endothelial adhesion molecules and tumor EC anergy to inflammatory signals. We hypothesized that counteracting angiogenesis induces re‐expression of adhesion molecules and normalizes responses to inflammatory cytokines. Here, we present data to show that the angiogenesis inhibitor platelet factor‐4 (PF4) is able to prevent basic fibroblast growth factor (bFGF)‐induced down‐regulation of intercellular adhesion molecule‐1 (ICAM‐1). Furthermore, PF4 restores ICAM‐1 expression following bFGF‐induced down‐regulation of ICAM‐1. This PF4 effect occurs at the protein level and the RNA level and it has functional impact on leukocyte adhesion. In addition, PF4 overcomes the tumor‐induced EC anergy to inflammatory signals such as tumor necrosis factor α (TNFα). Our findings may be the basis of new cancer therapies by combining anti‐angiogenic therapy and immunotherapy to decrease blood vessel formation and to increase the effectiveness of inflammatory reactions against tumors. Int. J. Cancer 80:315–319, 1999.

Effect of 4-Hydroxynonenal on c-myc Expression

The 4-hydroxynonenal aldehyde (HNE), a product of lipid peroxidation with high biological activity, inhibits cancerous growths in vivo and in vitro. The mechanism by which this aldehyde acts is not yet understood. The c-myc oncogene seems to be involved in the regulation of cellular multiplication and transformation. We evaluated the c-myc expression and the RNA, DNA and protein synthesis in K562 cells. These cells were incubated for 1 hour in presence of several aldehyde concentrations (range 5.10-7 to 10-4), then washed and kept for 20 hours in a growth medium until used. HNE inhibited both the nucleic acids and protein synthesis in a dose dependent manner, and c-myc expression was evaluated in the K562 cells after incubation with 10-4m or 10-6m HNE. HNE inhibited c-myc expression only at the highest dose. These preliminary results may suggest that the inhibition of c-myc expression is related to nucleic acid synthesis inhibition following HNE exposure.

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.

Angiotensin receptor blockers improve insulin signaling and prevent microvascular rarefaction in the skeletal muscle of spontaneously hypertensive rats

Objective Spontaneously hypertensive rats are an example of an animal model of genetic hypertension with insulin resistance. The aim of this study was to investigate insulin signaling in the heart and in the skeletal muscle of spontaneously hypertensive rats, as well as to evaluate the effects of renin–angiotensin system blockade. Design and Methods We investigated eight untreated spontaneously hypertensive rats of 12 weeks of age and eight age-matched normotensive Wistar–Kyoto controls. In addition, eight spontaneously hypertensive rats were treated for 8 weeks with the angiotensin receptor blocker olmesartan, and eight spontaneously hypertensive rats with the angiotensin-converting enzyme inhibitor enalapril. The heart and a skeletal muscle (quadriceps femoris) were promptly dissected and frozen. Insulin signaling was evaluated by Western blot analysis of involved proteins; in addition, microvessel density was indirectly evaluated by immunohistochemistry. Results Blood pressure values were normalized by both olmesartan and enalapril. In the heart, no statistically significant difference in the expression of proteins involved in insulin signaling was observed between untreated spontaneously hypertensive rats and Wistar–Kyoto controls. On the contrary, in the skeletal muscle of untreated spontaneously hypertensive rats, we noted a significant reduction of insulin receptors, of insulin-receptor substrate-1, and of phosphorylated-mammalian target of rapamycin. The treatment with olmesartan normalized insulin signaling, including expression of glucose transporter-4, whereas the treatment with enalapril was ineffective for the insulin receptor and less effective than olmesartan on the insulin-receptor substrate-1, phosphorylated-mammalian target of rapamycin and glucose transporter-4. There was a significant reduction in microvessel density in the skeletal muscle of spontaneously hypertensive rats compared with Wistar–Kyoto controls, and this was completely prevented by both olmesartan and enalapril. Conclusion These results suggest that changes in insulin signaling occur in the skeletal muscle but not in the heart of untreated spontaneously hypertensive rats. In the skeletal muscle, insulin signaling was restored by olmesartan, whereas enalapril was less effective. Effective antihypertensive treatment with olmesartan or enalapril was associated with prevention of microvascular rarefaction.

New mutations and protein variants of NBS1 are identified in cancer cell lines

Alterations of the NBS1 gene are responsible for Nijmegen breakage syndrome (NBS), which is characterized by chromosomal instability, radiosensitivity, and cancer predisposition. NBS1 protein (Nibrin) is part of a molecular complex (NBS1– MRE11A–RAD50) that is functionally involved in DNA double‐strand–break repair. Defects in recombination or in repair mechanisms at the level of DNA breakage can lead to chromosomal aberrations, genetic instability, as well as cancer predisposition syndromes (i.e., NBS, ataxia‐telangiectasia, Bloom syndrome). In this study, we examined 20 cancer cell lines to evaluate the potential involvement of NBS1 in tumoral pathogenesis. Three different mutations, generating truncated or aberrant NBS1 transcripts, were identified at the level of NBS1 mRNA. In addition, two shorter NBS1 protein variants were detected in two cell lines. These data suggest a possible involvement of NBS1 in tumor development.

High sensitivity of detection of TP53 somatic mutations by fluorescence-assisted mismatch analysis

We analyzed exons 5–9 of the TP53 gene in 41 breast cancers using direct sequencing, PCR‐SSCP (single‐strand conformation polymorphism), and fluorescence‐assisted mismatch analysis (FAMA), to test the level of specificity and sensitivity of each method. A major issue for the correct detection of TP53 somatic mutations in primary tumors is often represented by the large amount of normal DNA, which can cause excessive dilution of the mutant allele, with consequent possible false‐negative results. High sensitivity upon dilution of the mutant allele has been demonstrated for FAMA, a method based on the chemical cleavage of a mismatch within heteroduplex DNA molecules. Exons 5–9 of the TP53 gene were analyzed by FAMA using only two long bifluorescent fragments. Differences in sensitivity, accuracy, and specificity were observed among the above‐mentioned procedures. Thirteen of the 41 samples (31.7%) revealed TP53 genetic alterations by automated sequencing, 19 samples (46.3%) were positive for SSCP, whereas 14 samples (34%) showed variants detectable by FAMA. Seven samples were positive in SSCP, but negative in both FAMA and sequencing assays; however, 2 SSCP‐negative samples revealed evident signals by FAMA, indicating the presence of TP53 mutations. One of the latter samples showed the alteration by sequence analysis, whereas the other failed to reveal the mutation signal even by sequencing, as a consequence of the very small amount of the mutant allele resulting from the excess of contaminating normal DNA. Our results show that FAMA may represent a suitable and accurate assay for the routine diagnosis of TP53 somatic mutations in DNAs from solid tumor biopsies.

The angiogenic factor bFGF impairs leukocyte adhesion and rolling under flow conditions

Recirculation of leukocytes is mediated by the intricately regulated expression of adhesion molecules on both the vessel wall and leukocyte membranes. In the present paper it is demonstrated that tumor angiogenesis factors impair leukocyte rolling and adhesion under flow conditions. Three lines of evidence presented in this paper support this finding; (i) treatment of cultured endothelial cells (EC) with the angiogenic factor basic fibroblast growth factor (bFGF) results in decreased ICAM-1 expression and decreased numbers of adhering leukocytes under flow conditions. (ii) flow induced upregulation of endothelial ICAM-1 in the presence of bFGF does not yield ICAM-1 levels higher than on resting EC. (iii) bFGF decreases the TNFα mediated induction of E-selectin and ICAM-1 expression, resulting in decreased rolling and firm adhesion of leukocytes on the endothelial surface. For ICAM-1 it is demonstrated that bFGF inhibits TNFα induced levels of mRNA, and that this effects is transcriptionally regulated. These findings support our earlier described hypothesis that angiogenic factors are involved in the tumor derived escape mechanism from immune surveillance, since we demonstrate here that these mechanisms are operative under physiologic flow conditions.

The Murine p202 Protein, an IFN-Inducible Modulator of Transcription, Is Activated by the Mitogen Platelet-Derived Growth Factor

p202 is a murine interferon (IFN)-inducible protein belonging to a cluster of IFN-inducible genes (the 200 family) located in a segment of chromosome 1. It is a nuclear DNA-binding protein that is able to modulate transcription by interacting with a heterogeneous set of transcription factors, including NF-kappaB, (p50/p65), AP-1, c-fos, c-jun, and RB-1. The p202 protein is believed to attenuate cell growth/proliferation, mainly through the activation of IFN-stimulated of gene factor 3 (ISGF3), which binds IFN-stimulated response elements (ISRE) located in the promoters of type I IFN genes. In this report, we show that the p202 gene can also be induced by platelet-derived growth factor (PDGF), a mitogen known to drive G(0)-arrested cells toward reentry into the cell cycle. PDGF transiently enhances the steady-state mRNA level of p202 and increases the p202 protein level independently from IFN signaling, by acting at the transcriptional level on its promoter. The kinetics of p202 induction by PDGF are faster and more transient than those of IFN. These data identify p202 as a member of the IFN-inducible gene family that can be directly regulated by mitogenic stimuli.