Expression and prognostic relevance of activated extracellular-regulated kinases (ERK1/2) in breast cancer

Extracellular-regulated kinases (ERK1, ERK2) play important roles in the malignant behaviour of breast cancer cells in vitro. In our present study, 148 clinical breast cancer samples (120 cases with follow-up data) were studied for the expression of ERK1, ERK2 and their phosphorylated forms p-ERK1 and p-ERK2 by immunoblotting, and p-ERK1/2 expression in corresponding paraffin sections was analysed by immunohistochemistry. The results were correlated with established clinical and histological prognostic parameters, follow-up data and expression of seven cell-cycle regulatory proteins as well as MMP1, MMP9, PAI-1 and AP-1 transcription factors, which had been analysed before. High p-ERK1 expression as determined by immunoblots correlated significantly with a low frequency of recurrences and infrequent fatal outcome (P=0.007 and 0.008) and was an independent indicator of long relapse-free and overall survival in multivariate analysis. By immunohistochemistry, strong p-ERK staining in tumour cells was associated with early stages (P=0.020), negative nodal status (P=0.003) and long recurrence-free survival (P=0.017). In contrast, expression of the unphosphorylated kinases ERK1 and ERK2 was not associated with clinical and histological prognostic parameters, except a positive correlation with oestrogen receptor status. Comparison with the expression of formerly analysed cell-cycle- and invasion-associated proteins corroborates our conclusion that activation of ERK1 and ERK2 is not associated with enhanced proliferation and invasion of mammary carcinomas

Cervix carcinoma is associated with an up-regulation and nuclear localization of the dual-specificity protein phosphatase VHR

BACKGROUND: The 21-kDa Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase (encoded by the DUSP3 gene) plays a critical role in cell cycle progression and is itself regulated during the cell cycle. We have previously demonstrated using RNA interference that cells lacking VHR arrest in the G1 and G2 phases of the cell cycle and show signs of beginning of cell senescence. METHODS: In this report, we evaluated successfully the expression levels of VHR protein in 62 hysterectomy or conization specimens showing the various (pre) neoplastic cervical epithelial lesions and 35 additional cases of hysterectomy performed for non-cervical pathologies, from patients under 50 years of age. We used a tissue microarray and IHC technique to evaluate the expression of the VHR phosphatase. Immunofluorescence staining under confocal microscopy, Western blotting and RT-PCR methods were used to investigate the localization and expression levels of VHR. RESULTS: We report that VHR is upregulated in (pre) neoplastic lesions (squamous intraepithelial lesions; SILs) of the uterine cervix mainly in high grade SIL (H-SIL) compared to normal exocervix. In the invasive cancer, VHR is also highly expressed with nuclear localization in the majority of cells compared to normal tissue where VHR is always in the cytoplasm. We also report that this phosphatase is highly expressed in several cervix cancer cell lines such as HeLa, SiHa, CaSki, C33 and HT3 compared to primary keratinocytes. The immunofluorescence technique under confocal microscopy shows that VHR has a cytoplasmic localization in primary keratinocytes, while it localizes in both cytoplasm and nucleus of the cancer cell lines investigated. We report that the up-regulation of this phosphatase is mainly due to its post-translational stabilization in the cancer cell lines compared to primary keratinocytes rather than increases in the transcription of DUSP3 locus. CONCLUSION: These results together suggest that VHR can be considered as a new marker for cancer progression in cervix carcinoma and potential new target for anticancer therapy

Hypericum perforatum treatment: effect on behaviour and neurogenesis in a chronic stress model in mice

<p>Abstract</p> <p>Background</p> <p>Extracts of <it>Hypericum perforatum </it>(St. John's wort) have been traditionally recommended for a wide range of medical conditions, in particular mild-to-moderate depression. The present study was designed to investigate the effect of Hypericum perforatum treatment in a mouse model of anxiety/depressive-like behavior, induced by chronic corticosterone administration.</p> <p>Methods</p> <p>CD1 mice were submitted to 7 weeks corticosterone administration and then behavioral tests as Open Field (OF), Novelty-Suppressed Feeding (NSF), Forced Swim Test (FST) were performed. Cell proliferation in hippocampal dentate gyrus (DG) was investigated by both 5-bromo-2'-deoxyuridine (BrdU) and doublecortin (DCX) immunohistochemistry techniques and stereological procedure was used to quantify labeled cells. Golgi-impregnation method was used to evaluate changes in dendritic spines in DG. Hypericum perforatum (30 mg/Kg) has been administered for 3 weeks and then neural development in the adult hippocampus and behavioral changes have been examined.</p> <p>Results</p> <p>The anxiety/depressive-like state due to chronic corticosterone treatment was reversed by exogenous administration of Hypericum perforatum; the proliferation of progenitor cells in mice hippocampus was significantly reduced under chronic corticosterone treatment, whereas a long term treatment with Hypericum perforatum prevented the corticosterone-induced decrease in hippocampal cell proliferation. Corticosterone-treated mice exhibited a reduced spine density that was ameliorated by Hypericum perforatum administration.</p> <p>Conclusion</p> <p>These results provide evidence of morphological adaptations occurring in mature hippocampal neurons that might underlie resilient responses to chronic stress and contribute to the therapeutic effects of chronic Hypericum perforatum treatment.</p

Different patterns of Ca2+ signals are induced by low compared to high concentrations of P2Y agonists in microglia

Brain-resident macrophages (microglia) are key cellular elements in the preservation of tissue integrity. On the other hand, they can also contribute to the development of pathological events by causing an extensive and inappropriate inflammatory response. A growing number of reports indicate the involvement of nucleotides in the control of microglial functions. With this study on P2Y receptors in rat microglia, we want to contribute to the definition of their expression profile and to the characterisation of their signalling mechanisms leading to Ca2+ movements. Endogenous nucleotides, when applied at a concentration of 100 μM, elicited robust Ca2+ transients, thanks to a panel of metabotropic receptors comprising mainly P2Y2, P2Y6 and P2Y12 subtypes. The involvement of P2Y12 receptors in Ca2+ responses induced by adenine nucleotides was confirmed by the pharmacological and pertussis toxin sensitivity of the response induced by adenosine diphosphate (ADP). Beside the G protein involved, Gi and Gq respectively, adenine and uracil nucleotides differed also for induction by the latter of a capacitative Ca2+ plateau. Moreover, when applied at low (sub-micromolar) concentrations with a long-lasting challenge, uracil nucleotides elicited oscillatory Ca2+ changes with low frequency of occurrence (≤ 1 min−), sometimes superimposed to an extracellular Ca2+-dependent sustained Ca2+ rise. We conclude that different patterns of Ca2+ transients are induced by low (i.e., oscillatory Ca2+ activity) compared to high (i.e., fast release followed by sustained raise) concentrations of nucleotides, which can suggest different roles played by receptor stimulation depending not only on the type but also on the concentration of nucleotides

Elderly Japanese women with cervical carcinoma show higher proportions of both intermediate-risk human papillomavirus types and p53 mutations

The p53 mutation has been found only in 0–6% of cervical carcinomas. In light of recent studies demonstrating that mutation of p53 gene was found in over 20% of the patients with vulvar carcinoma a disease of elderly women and a known human papillomavirus (HPV)-related malignancy, we analysed mutation of the p53 gene in 46 women with cervical carcinomas at the age of 60 or more (mean; 71 years, range; 60–96 years). The presence of HPV and its type were analysed by polymerase chain reaction (PCR)-based assay using the consensus primers for L1 region. Mutation of the p53 gene was analysed by PCR-based single-strand conformation polymorphism and DNA sequencing technique. Point mutation of the p53 gene was detected in 5 out of 46 (11%) cervical carcinomas: 1 of 17 (6%) samples associated with high-risk HPVs (HPV 16 and HPV 18) and 4 of 27 samples (15%) with intermediate-risk HPVs (P = 0.36) whereas no mutation was found in 2 HPV negative cases. The mutated residues resided in the selective sequence known as a DNA-binding domain. The immunohistochemistry revealed the overexpression in cancer tissues positive for p53 mutation. All of the observed mutations of the p53 gene were transition type, suggesting that the mutation may be caused by endogenous mutagenesis. Although falling short of statistical significance reduces the strength of the conclusion, data presented here imply that p53 gene mutation, particularly along with intermediate-risk HPV types, may constitute one pathogenetic factor in cervical carcinoma affecting elderly women. © 1999 Cancer Research Campaig

The functional loss of the retinoblastoma tumour suppressor is a common event in basal-like and luminal B breast carcinomas

Introduction Breast cancers can be classified using whole genome expression into distinct subtypes that show differences in prognosis. One of these groups, the basal-like subtype, is poorly differentiated, highly metastatic, genomically unstable, and contains specific genetic alterations such as the loss of tumour protein 53 (TP53). The loss of the retinoblastoma tumour suppressor encoded by the RB1 locus is a well-characterised occurrence in many tumour types; however, its role in breast cancer is less clear with many reports demonstrating a loss of heterozygosity that does not correlate with a loss of RB1 protein expression. Methods We used gene expression analysis for tumour subtyping and polymorphic markers located at the RB1 locus to assess the frequency of loss of heterozygosity in 88 primary human breast carcinomas and their normal tissue genomic DNA samples. Results RB1 loss of heterozygosity was observed at an overall frequency of 39%, with a high frequency in basal-like (72%) and luminal B (62%) tumours. These tumours also concurrently showed low expression of RB1 mRNA. p16INK4a was highly expressed in basal-like tumours, presumably due to a previously reported feedback loop caused by RB1 loss. An RB1 loss of heterozygosity signature was developed and shown to be highly prognostic, and was potentially a predictive marker of response to neoadjuvant chemotherapy. Conclusions These results suggest that the functional loss of RB1 is common in basal-like tumours, which may play a key role in dictating their aggressive biology and unique therapeutic responses

Purinergic modulation of microglial cell activation

Microglial cells are resident macrophages in the brain and their activation is an important part of the brain immune response and the pathology of the major CNS diseases. Microglial activation is triggered by pathological signals and is characterized by morphological changes, proliferation, phagocytosis and the secretion of various cytokines and inflammatory mediators, which could be both destructive and protective for the nervous tissue. Purines are one of the most important mediators which regulate different aspects of microglial function. They could be released to the extracellular space from neurons, astrocytes and from the microglia itself, upon physiological neuronal activity and in response to pathological stimuli and cellular damage. Microglial activation is regulated by various subtypes of nucleotide (P2X, P2Y) and adenosine (A1, A2A and A3) receptors, which control ionic conductances, membrane potential, gene transcription, the production of inflammatory mediators and cell survival. Among them, the role of P2X7 receptors is especially well delineated, but P2X4, various P2Y, A1, A2A and A3 receptors also powerfully participate in the microglial response. The pathological role of microglial purine receptors has also been demonstrated in disease models; e.g., in ischemia, sclerosis multiplex and neuropathic pain. Due to their upregulation and selective activation under pathological conditions, they provide new avenues in the treatment of neurodegenerative and neuroinflammatory illnesses