Loredana Albonici

bcl-2/bax mRNA expression ratio as prognostic factor in low-grade urinary bladder cancer

Apoptotic cell death represents an important mechanism for the precise regulation of cell numbers, and a defence mechanism against tumoral cells. bcl‐2 and bax genes are known to be involved in the control of apoptotic pathways; in particular, the ratio between bcl‐2 and bax represents a cell rheostat that is able to predict a cells response toward life or death to an apoptotic stimulus. In the present study we investigated the role of bcl‐2 and bax gene expression in a panel of 37 low‐grade tumours of the urinary bladder, and correlated the expression of these genes to the prognosis of patients in a follow‐up of more than one year. We found that levels of bax expression higher than bcl‐2 in bladder tumours well correlates to a better outcome for patients. Early relapses are much more frequently observed in those patients whose tumours express more bcl‐2 than bax mRNA. We conclude that the bcl‐2/bax expression ratio may be considered as a marker for disease progression in low grade bladder tumours, independently of clinical staging and histological grading.

Inflammatory cytokines stimulate vascular smooth muscle cells locomotion and growth by enhancing α5β1 integrin expression and function

The formation of atherosclerotic lesions requires the migration of vascular smooth muscle cells from the media into the intima of the artery and their proliferation. These events, which are preceded and accompanied by inflammation, are modulated by integrin receptors linking vascular smooth muscle cells to extracellular matrix molecules. Among them, fibronectin induces vascular smooth muscle cells to acquire the phenotype they show in the atherosclerotic plaque. Here we show that amounts of interleukin-1 beta, tumor necrosis factor alpha and interferon-gamma as possibly released by activated immune cells infiltrating atherosclerotic lesions, upregulate vascular smooth muscle cell expression of the alpha5beta1 integrin, a fibronectin receptor. This improves vascular smooth muscle cell capability of migrating toward soluble or anchored fibronectin and of adhering to immobilized fibronectin. The latter effect, in turn, augments vascular smooth muscle cell proliferative response to mitogens, as suggested by the increase of intracellular pH. Finally, the effects that inflammatory cytokines have on vascular smooth muscle cell locomotion and growth, are specifically blocked by anti-alpha5beta1 antibodies. As fibronectin and alpha5beta1 levels are augmented in vivo in the atherosclerotic plaques, these findings support the use of integrin antagonists as potential adjuvants in atherosclerosis treatment.

High Frequency of Human Papillomavirus Detection in Urinary Bladder Cancer

We investigated the presence of human papillomaviruses (HPVs) types 16 and 18 DNA in formalin-fixed, paraffin-embedded tissues from the urinary bladder (46 transitional carcinomas and 10 non-neoplastic normal urinary samples) to find a possible role for HPV types in urinary tract cancerogenesis. The analysis was performed using polymerase chain reaction followed by filter hybridization with oligonucleotide-specific probes. The HPV16 and/or HPV18 genomes were detected in 23 of 46 (50%) bladder carcinomas and in none of 10 (0%) non-neoplastic urinary samples. These results suggest that HPV16 and 18 may carry a risk for the development of malignancy in the urinary tract as it occurs in the anogenital regions.

Placenta Growth Factor Expression Has Prognostic Value in Malignant Pleural Mesothelioma

BACKGROUND Malignant pleural mesothelioma is highly aggressive and recurs rapidly despite radical multimodality treatment. Progression of mesothelioma is thought to be governed by various growth factors, including vascular endothelial growth factor (VEGF). Placenta growth factor (PlGF) belongs to the VEGF family, although no study has yet investigated its expression in mesothelioma. We hypothesized that PlGF is overexpressed in mesothelioma and could have prognostic value in patients treated by extrapleural pneumonectomy. METHODS We assessed by immunohistochemistry with semiquantitative classification (0 = no staining; 3 = strong staining), the expression levels of PlGF and its cognate receptors VEGF receptor 1, neuropilin-1, and neuropilin-2 in 27 patients with malignant pleural mesothelioma undergoing extrapleural pneumonectomy, in 14 patients with reactive mesothelium, and in 10 patients with normal mesothelium. RESULTS Whereas PlGF was not expressed in normal mesothelium, it was overexpressed (grade 3) more frequently in mesothelioma than in reactive mesothelium specimens (11 or 41% versus 1 or 7%, respectively, p = 0.03). Furthermore, in mesothelioma, VEGF receptor 1 and neuropilin-1 and -2 were overexpressed in 18 specimens (67%), 8 specimens (30%), and 9 specimens (33%), respectively. Mean survival after extrapleural pneumonectomy was 17 months. An inverse relationship was found between the degree of PlGF expression and survival in months (R = -0.45, p = 0.01). No correlation was found between tumor stage and survival (R = -0.33) and between tumor stage and PlGF expression (R = 0.07). CONCLUSIONS We have shown that PlGF can be overexpressed in malignant pleural mesothelioma. In addition, the finding of an inverse relationship between PlGF expression levels and survival suggests a pivotal role of this factor in the recurrence and progression of mesothelioma after extrapleural pneumonectomy.

Recombinant erythropoietin differently affects proliferation of mesothelioma cells but not sensitivity to cisplatin and pemetrexed

The combination of cisplatin and pemetrexed represents the newly established standard of care for patients with unresectable malignant mesothelioma (MM). However, this chemotherapy regimen appears to be associated with an increased prevalence of higher grade anemia as compared to treatment with cisplatin alone. Human recombinant erythropoietin (rHuEpo) is currently used for the treatment of anemia in cancer patients. Still, following the finding that the erythropoietin receptor (EpoR) is expressed by several tumor cells types and after the trials reporting that the recombinant cytokine can adversely affect tumor progression and patient survival, the clinical safety of rHuEpo administration to neoplastic patients has recently been questioned. The observation that the expression of EpoR, variably associated with the expression of the cognate ligand, is a common feature of MM cells prompted us to investigate whether treatment with rHuEpo could elicit proliferative and cytoprotective signals in EpoR-positive MM cell lines. Biochemical responsiveness of MM cells to rHuEpo was demonstrated by the time-course activation of both ERK1/2 and AKT following treatment with the recombinant cytokine. A moderately increased mitogenic activity was observed in two out of five MM cell lines treated with pharmacologically relevant concentrations of rHuEpo. On the other hand, the recombinant cytokine, administered either before or after cisplatin and pemetrexed, failed to interfere with the cytotoxic effects exerted by the chemotherapeutic drugs on the five MM cell lines. According to the presented findings, rHuEpo appears to have an overall limited impact on cell growth and no effect on MM sensitivity to chemotherapy.

Italian population data on the polymarker system and on the five short tandem repeat loci CSF1PO, TPOX, TH01, F13B, and vWA

A population study on five short tandem repeat (STR) loci and five sequence specific polymorphism loci was performed on unrelated Italian Caucasians. Separation and detection of the amplified STR fragments were carried out by high resolution vertical denaturing polyacrylamide gel electrophoresis (PAGE) and silver staining, respectively. The sequence specific loci were analyzed using the AmpliType PM Typing Kit (Perkin Elmer, Foster City, CA). All loci, except Gc (p = 0.031), meet Hardy-Wienberg expectations. In addition, there is no evidence for association of alleles between pairs of loci. The combined power of discrimination for the five STR loci is 0.9999862 and for the PM loci is 0.99503. The results suggest that these loci may be useful for human identification cases in Italy.

Fibroblast growth factor-2 transiently activates the p53 oncosuppressor protein in human primary vascular smooth muscle cells: implications for atherogenesis.

OBJECTIVE The development of atherosclerotic lesions associates with the proliferation of vascular smooth muscle cells (VSMC), and their migration from arterial tunica media to the intima. Fibroblast growth factor (FGF)-2 can trigger either phenomena, which are accompanied by the functional impairment of the p53 transcription factor. However, FGF-2 impact on p53 function in VSMC is largely unknown. METHODS AND RESULTS RT-PCR and Western blot analyses assayed FGF-2 effect on human primary VSMC expression of p53-induced molecules with a role in atherogenesis. Confocal microscopy evaluated whether FGF-2 could affect p53 distribution inside VSMC. Results indicate that VSMC exposure to FGF-2 at amounts stimulating the proliferation and migration of these cells promotes p53 phosphorylation and transient accumulation in VSMC nuclei. This is followed by an increase in the expression of the p53-induced thrombospondin (TSP)-1, a VSMC growth and motility factor, and human double minute 2 (HDM2), an antagonist of p53 transcriptional and growth suppressive activity. At later time points, in agreement with the increase of HDM2 and with the capability of this protein to export nuclear p53 to the cytoplasm, the content of p53 in VSMC nuclei is reduced, and the expression of the p53-targeted TSP-l and HDM2 is diminished. CONCLUSIONS Since FGF-2, p53, TSP-1, and HDM2 are expressed in human atherosclerotic lesions, the in vitro effects of FGF-2 described herein may be operative in vivo, providing a molecular mechanism for FGF-2 pro-atherogenic activity.

Placenta growth factor is a survival factor for human malignant mesothelioma cells.

Placenta growth factor (PlGF) is a key regulator of pathological angiogenesis and its overexpression has been linked to neoplastic progression. To assess whether PlGF could have a role in malignant mesothelioma (MM), we analyzed the expression of PlGF, VEGF, and their cognate receptors (VEGF-R1 and VEGF-R2) and co-receptors (neuropilin-1 and neuropilin-2) in MM cell lines as well as in resected MM tissues, hyperplastic/reactive mesothelium and normal mesothelium. MM cell cultures expressed both ligands and the associated receptors to a variable extent and released different amounts of PlGF. As assessed by immunohistochemistry, PlGF expression was switched on in hyperplastic/reactive compared to normal mesothelium. Moreover, 74 and 94% of MM tissues overexpressed PlGF and VEGF-R1, respectively (p<0.05 MM vs normal mesothelium). Administration of recombinant PlGF-2 did not elicit a significant stimulation of MM cell growth, while it was associated with a transient phosphorylation of Akt, suggesting that PlGF-2 could activate downstream effectors of proliferative and cytoprotective signals via VEGF-R1 in MM cells. Indeed, the administration of an anti-PlGF antibody was found to cause a significant reduction of MM cell survival. In conclusion, our data demonstrate that, by acting as a survival factor, PlGF can play a role which goes beyond the stimulation of angiogenesis in MM. This evidence could help the rational design of new therapeutic interventions for this aggressive tumor.

HMBA induces cell death and potentiates doxorubicin toxicity in malignant mesothelioma cells

PurposeMalignant pleural mesothelioma (MM), a rare tumor characterized by high local invasiveness and low metastatic efficiency, is poorly responsive to current therapeutic approaches. The aim of the present study was to evaluate the cytotoxic efficacy of the hybrid polar compound hexamethylene bisacetamide (HMBA), either as a single agent or in combination with the anthracycline doxorubicin (DOX), against MM cells.MethodsThe MM cell lines MM-B1 and MM-E1 were treated with HMBA, DOX or with combinations of the two drugs. Cell survival and death were assessed by the MTS assay and trypan blue staining/TUNEL, respectively. The interactions between drugs were evaluated by the method of Kern et al. Western blot analysis was used to investigate the expression of Bcl-2 family proteins.ResultsWhen administered alone, HMBA dose-dependently decreased the number of viable cells and increased the death rate of MM-B1 and MM-E1 cultures. Combinations of HMBA and DOX achieved a synergistic inhibition of MM cell survival, and the simultaneous administration of HMBA counteracted the resistance induced by DOX in MM-E1 cells. HMBA, used at cytostatic concentrations, reduced the ratio between antiapoptotic (Bcl-2, Bcl-XL) and proapoptotic (Bax) members of the Bcl-2 family of proteins, thus lowering the threshold for MM cell death commitment.ConclusionsHMBA has therapeutic potential in MM both as a single agent and through potentiation of DOX toxicity. These results support future investigations on the feasibility of intrapleural chemotherapy with this hybrid polar compound.

Detection of cellular heterogeneity by DNA ploidy, 17 chromosome, and p53 gene in primary carcinoma and metastasis in a case of ovarian cancer

An unusual case of a patient with ovarian carcinoma carrying the p53 point mutation in both metastases (omentum and lymph node), but not in the primary tumor, is described. The presence of a p53 single mutation (G:A) at the second base of codon 248 was examined by polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) analysis. This case was examined also by fluorescent in situ hybrization (FISH) analysis and flow cytometry (FCM) to obtain further information at the single cell level and to detect heterogeneity within a population of cells. FCM analysis evidenced the same multiple aneuploid cell subpopulations in primary and in metastatic samples showing the presence of a cellular heterogeneity. FISH analysis showed a disomic condition for the 17 chromosome in the primary and in one metastasis, while in the other metastasis a monosomic together with a disomic subpopulation was revealed. Our results confirm the independent clonal evolution of the metastasis. The late mutation event observed only in metastatic specimens suggests the hypothesis that in the primary tumor the wild-type gene either does not perform its control role for unknown genetic structural events or the p53 gene in this case does not play a critical role in carcinogenesis.