Tumor Microenvironmental Cytokines Drive NSCLC Cell Aggressiveness and Drug-Resistance via YAP-Mediated Autophagy.

Dynamic reciprocity between cellular components of the tumor microenvironment and tumor cells occurs primarily through the interaction of soluble signals, i.e., cytokines produced by stromal cells to support cancer initiation and progression by regulating cell survival, differentiation and immune cell functionality, as well as cell migration and death. In the present study, we focused on the analysis of the functional response of non-small cell lung cancer cell lines elicited by the treatment with some crucial stromal factors which, at least in part, mimic the stimulus exerted in vivo on tumor cells by microenvironmental components. Our molecular and functional results highlight the role played by the autophagic machinery in the cellular response in terms of the invasive capacity, stemness and drug resistance of two non-small lung cancer cell lines treated with stromal cytokines, also highlighting the emerging role of the YAP pathway in the mutual and dynamic crosstalk between tumor cells and tumor microenvironment elements. The results of this study provide new insights into the YAP-mediated autophagic mechanism elicited by microenvironmental cytokines on non-small cell lung cancer cell lines and may suggest new potential strategies for future cancer therapeutic interventions.

The αMSH-Dependent PI3K Pathway Supports Energy Metabolism, via Glucose Uptake, in Melanoma Cells.

Stimulation of melanocytes and murine melanoma cells with αMSH plus the PI3K inhibitor LY294002 resulted in ROS increase, oxidative DNA damage, and pigment retention. We performed cellular and molecular biology assays (Western blot, FACS, immunofluorescence analysis, scratch assay) on murine and human melanoma cells. Treatment with αMSH plus LY294002 altered cortical actin architecture. Given that cytoskeleton integrity requires energy, we next evaluated ATP levels and we observed a drop in ATP after exposure to αMSH plus LY294002. To evaluate if the αMSH-activated PI3K pathway could modulate energy metabolism, we focused on glucose uptake by analyzing the expression of the Glut-1 glucose translocator. Compared with cells treated with αMSH alone, those exposed to combined treatment showed a reduction of Glut-1 on the plasma membrane. This metabolic alteration was associated with changes in mitochondrial mass. A significant decrease of the cell migratory potential was also observed. We demonstrated that the αMSH-dependent PI3K pathway acts as a regulator of energy metabolism via glucose uptake, influencing the actin cytoskeleton, which is involved in melanosome release and cell motility. Hence, these results could constitute the basis for innovative therapeutical strategies.

Microtubule-Based Mitochondrial Dynamics as a Valuable Therapeutic Target in Cancer.

Mitochondria constitute an ever-reorganizing dynamic network that plays a key role in several fundamental cellular functions, including the regulation of metabolism, energy production, calcium homeostasis, production of reactive oxygen species, and programmed cell death. Each of these activities can be found to be impaired in cancer cells. It has been reported that mitochondrial dynamics are actively involved in both tumorigenesis and metabolic plasticity, allowing cancer cells to adapt to unfavorable environmental conditions and, thus, contributing to tumor progression. The mitochondrial dynamics include fusion, fragmentation, intracellular trafficking responsible for redistributing the organelle within the cell, biogenesis, and mitophagy. Although the mitochondrial dynamics are driven by the cytoskeleton-particularly by the microtubules and the microtubule-associated motor proteins dynein and kinesin-the molecular mechanisms regulating these complex processes are not yet fully understood. More recently, an exchange of mitochondria between stromal and cancer cells has also been described. The advantage of mitochondrial transfer in tumor cells results in benefits to cell survival, proliferation, and spreading. Therefore, understanding the molecular mechanisms that regulate mitochondrial trafficking can potentially be important for identifying new molecular targets in cancer therapy to interfere specifically with tumor dissemination processes.

ePCL Electrospun Microfibrous Layers for Immune Assays: Sensitive ELISA for the Detection of Serum Antibodies Against HPV16 E7 Oncoprotein.

Human papillomavirus (HPV) type 16 is the etiologic agent of more than 50% anal/cervical cancers and about 20% oropharyngeal cancers. HPV16 E6 and E7 oncogenes favor the transformation and are essential for maintaining the transformed status. Serum anti-E6 and anti-E7 antibodies appear to have prognostic significance for HPV-associated cancers. However, most of the previous attempts to establish diagnostic tools based on serum detection of E6 and/or E7 antibodies have been unsuccessful, mainly due to the low accuracy of applied tests. This paper reports on a feasibility study to prove the possibility to easily immobilize HPV16 E7 onto electrospun substrates for application in diagnostic tools. In this study, poly(ε-caprolactone) electrospun scaffolds (called ePCL) are used to provide a microstructured substrate with a high surface-to-volume ratio, capable of binding E7 proteins when used for enzyme-linked immunosorbent assay (ELISA) tests. ePCL functionalized with E7 exhibited superior properties compared to standard polystyrene plates, increasing the detection signal from serum antibodies by 5-6 times. Analysis of the serum samples from mice immunized with HPV16 E7 DNA vaccine showed higher efficiency of this new anti-E7 ePCL-ELISA test vs control in E7-specific antibody detection. In addition, ePCL-E7-ELISA is prepared with a relatively low amount of antigen, decreasing the manufacturing costs.

Physical Interaction between HPV16E7 and the Actin-Binding Protein Gelsolin Regulates Epithelial-Mesenchymal Transition via HIPPO-YAP Axis.

Human papillomavirus 16 (HPV16) exhibits a strong oncogenic potential mainly in cervical, anogenital and oropharyngeal cancers. The E6 and E7 viral oncoproteins, acting via specific interactions with host cellular targets, are required for cell transformation and maintenance of the transformed phenotype as well. We previously demonstrated that HPV16E7 interacts with the actin-binding protein gelsolin, involved in cytoskeletal F-actin dynamics. Herein, we provide evidence that the E7/gelsolin interaction promotes the cytoskeleton rearrangement leading to epithelial-mesenchymal transition-linked morphological and transcriptional changes. E7-mediated cytoskeletal actin remodeling induces the HIPPO pathway by promoting the cytoplasmic retention of inactive P-YAP. These results suggest that YAP could play a role in the "de-differentiation" process underlying the acquisition of a more aggressive phenotype in HPV16-transformed cells. A deeper comprehension of the multifaceted mechanisms elicited by the HPV infection is vital for providing novel strategies to block the biological and clinical features of virus-related cancers.

The actin modulator hMENA regulates GAS6-AXL axis and pro-tumor cancer/stromal cell cooperation.

The dynamic interplay between cancer cells and cancer-associated fibroblasts (CAFs) is regulated by multiple signaling pathways, which can lead to cancer progression and therapy resistance. We have previously demonstrated that hMENA, a member of the actin regulatory protein of Ena/VASP family, and its tissue-specific isoforms influence a number of intracellular signaling pathways related to cancer progression. Here, we report a novel function of hMENA/hMENAΔv6 isoforms in tumor-promoting CAFs and in the modulation of pro-tumoral cancer cell/CAF crosstalk via GAS6/AXL axis regulation. LC-MS/MS proteomic analysis reveals that CAFs that overexpress hMENAΔv6 secrete the AXL ligand GAS6, favoring the invasiveness of AXL-expressing pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) cells. Reciprocally, hMENA/hMENAΔv6 regulates AXL expression in tumor cells, thus sustaining GAS6-AXL axis, reported as crucial in EMT, immune evasion, and drug resistance. Clinically, we found that a high hMENA/GAS6/AXL gene expression signature is associated with a poor prognosis in PDAC and NSCLC. We propose that hMENA contributes to cancer progression through paracrine tumor-stroma crosstalk, with far-reaching prognostic and therapeutic implications for NSCLC and PDAC.

Artichoke Polyphenols Sensitize Human Breast Cancer Cells to Chemotherapeutic Drugs via a ROS-Mediated Downregulation of Flap Endonuclease 1.

Combined treatment of several natural polyphenols and chemotherapeutic agents is more effective comparing to the drug alone in inhibiting cancer cell growth. Polyphenolic artichoke extracts (AEs) have been shown to have anticancer properties by triggering apoptosis or reactive oxygen species- (ROS-) mediated senescence when used at high or low doses, respectively. Our aim was to explore the chemosensitizing potential of AEs in order to enhance the efficacy of conventional chemotherapy in breast cancer cells. We employed breast cancer cell lines to assess the potential synergistic effect of a combined treatment of AEs/paclitaxel (PTX) or AEs/adriamycin (ADR) and to determine the underlying mechanisms correlated to this potential therapeutic approach. Our data shows that AEs/PTX reduced cell proliferation by increasing DNA damage response (DDR) mediated by Flap endonuclease 1 (FEN1) downregulation that results into enhanced breast cancer cell sensitivity to chemotherapeutic drugs. We demonstrated that ROS/Nrf2 and p-ERK pathways are two molecular mechanisms involved in the synergistic effect of AEs plus PTX treatment. To highlight the role of ROS herein, we report that the addition of antioxidant N-acetylcysteine (NAC) significantly decreased the antiproliferative effect of the combined treatment. A combined therapy could be able to reduce the dose of chemotherapeutic drugs, minimizing toxicity and side effects. Our results suggest the use of artichoke polyphenols as ROS-mediated sensitizers of chemotherapy paving the way for innovative and promising natural compound-based therapeutic strategies in oncology.

Polyphenols: Immunomodulatory and Therapeutic Implication in Colorectal Cancer.

Polyphenolic compounds, widely present in fruits, vegetables, and cereals, have potential benefits for human health and are protective agents against the development of chronic/degenerative diseases including cancer. More recently these bioactive molecules have been gaining great interest as anti-inflammatory and immunomodulatory agents, mainly in neoplasia where the pro-inflammatory context might promote carcinogenesis. Colorectal cancer (CRC) is considered a major public healthy issue, a leading cause of cancer mortality and morbidity worldwide. Epidemiological, pre-clinical and clinical investigations have consistently highlighted important relationships between large bowel inflammation, gut microbiota (GM), and colon carcinogenesis. Many experimental studies and clinical evidence suggest that polyphenols have a relevant role in CRC chemoprevention, exhibit cytotoxic capability vs. CRC cells and induce increased sensitization to chemo/radiotherapies. These effects are most likely related to the immunomodulatory properties of polyphenols able to modulate cytokine and chemokine production and activation of immune cells. In this review we summarize recent advancements on immunomodulatory activities of polyphenols and their ability to counteract the inflammatory tumor microenvironment. We focus on potential role of natural polyphenols in increasing the cell sensitivity to colon cancer therapies, highlighting the polyphenol-based combined treatments as innovative immunomodulatory strategies to inhibit the growth of CRC.

Lenvatinib for the treatment of renal cell carcinoma.

INTRODUCTION: Renal cell carcinoma (RCC) accounts for 2-3% of all solid tumors. Expression of the receptor for the vascular endothelial growth factor (VEGF) is one of the most common features of RCC. AREAS COVERED: Lenvatinib is a novel multi-kinase inhibitor that has been studied in several solid tumors. It has shown promising results in the treatment of RCC, especially when combined with everolimus, In this review, we summarize the available data of lenvatinib for the treatment of advanced/metastatic renal cell carcinoma. EXPERT OPINION: Lenvatinib in combination with everolimus has provided encouraging results in both clinical and laboratory investigations showing that blocking angiogenesis and the mTOR signalling pathway could be a remarkable approach for treating RCC. As an additive to this type of approach it would be interesting in future clinical settings testing also the combination of lenvatinib and everolimus with immune-therapy.

ω3 Polyunsaturated Fatty Acids as Immunomodulators in Colorectal Cancer: New Potential Role in Adjuvant Therapies.

Diet composition may affect the onset and progression of chronic degenerative diseases, including cancer, whose pathogenesis relies on inflammatory processes. Growing evidence indicates that diet and its components critically contribute to human health, affecting the immune system, secretion of adipokines, and metabolic pathways. Colorectal cancer (CRC) is one of the leading causes of death worldwide. Antineoplastic drugs are widely used for CRC treatment, but drug resistance and/or off-target toxicity limit their efficacy. Dietary ω3 polyunsaturated fatty acids (PUFA) have been gaining great interest in recent years as possible anti-inflammatory and anticancer agents, especially in areas such as the large bowel, where the pro-inflammatory context promotes virtually all steps of colon carcinogenesis. Growing epidemiological, experimental, and clinical evidence suggests that ω3 PUFA may play a role in several stages of CRC management exhibiting antineoplastic activity against human CRC cells, improving the efficacy of radiation and chemotherapy, ameliorating cancer-associated secondary complications, and preventing CRC recurrence. These effects are most likely related to the immunomodulatory activities of ω3 PUFA that are able to influence several aspects of the inflammatory process ranging from inflammasome activation, leukocyte recruitment, production of immune mediators to differentiation, and activation of immune cells. In this review, we will focus on the potential use of ω3 PUFA as adjuvant agents together with chemo/radiotherapy, highlighting the immunomodulatory effects most likely responsible for their beneficial effects in different stages of CRC management.

Interaction between the human papillomavirus 16 E7 oncoprotein and gelsolin ignites cancer cell motility and invasiveness.

The viral oncoprotein E7 from the "high-risk" Human Papillomavirus 16 (HPV16) strain is able, when expressed in human keratinocytes, to physically interact with the actin severing protein gelsolin (GSN). In a previous work it has been suggested that this protein-protein interaction can hinder GSN severing function, thus leading to actin network remodeling. In the present work we investigated the possible implications of this molecular interaction in cancer cell metastatic potential by analyzing two different human CC cell lines characterized by low or high expression levels of HPV16 DNA (SiHa and CaSki, respectively). In addition, a HPV-null CC cell line (C-33A), transfected in order to express the HPV16 E7 oncoprotein as well as two different deletion mutants, was also analyzed. We found that HPV16 E7 expression level was directly related with cervical cancer migration and invasion capabilities and that these HPV16 E7-related features were associated with Epithelial to Mesenchymal Transition (EMT) processes. These effects appeared as strictly attributable to the physical interaction of HPV16 E7 with GSN, since HPV16 E7 deletion mutants unable to bind to GSN were also unable to modify microfilament assembly dynamics and, therefore, cell movements and invasiveness. Altogether, these data profile the importance of the physical interaction between HPV16 E7 and GSN in the acquisition of the metastatic phenotype by CC cells, underscoring the role of HPV16 intracellular load as a risk factor in cancer.

Polyphenols as Modulator of Oxidative Stress in Cancer Disease: New Therapeutic Strategies.

Cancer onset and progression have been linked to oxidative stress by increasing DNA mutations or inducing DNA damage, genome instability, and cell proliferation and therefore antioxidant agents could interfere with carcinogenesis. It is well known that conventional radio-/chemotherapies influence tumour outcome through ROS modulation. Since these antitumour treatments have important side effects, the challenge is to develop new anticancer therapeutic strategies more effective and less toxic for patients. To this purpose, many natural polyphenols have emerged as very promising anticancer bioactive compounds. Beside their well-known antioxidant activities, several polyphenols target epigenetic processes involved in cancer development through the modulation of oxidative stress. An alternative strategy to the cytotoxic treatment is an approach leading to cytostasis through the induction of therapy-induced senescence. Many anticancer polyphenols cause cellular growth arrest through the induction of a ROS-dependent premature senescence and are considered promising antitumour therapeutic tools. Furthermore, one of the most innovative and interesting topics is the evaluation of efficacy of prooxidant therapies on cancer stem cells (CSCs). Several ROS inducers-polyphenols can impact CSCs metabolisms and self-renewal related pathways. Natural polyphenol roles, mainly in chemoprevention and cancer therapies, are described and discussed in the light of the current literature data.

Hepatitis C virus core protein modulates pRb2/p130 expression in human hepatocellular carcinoma cell lines through promoter methylation.

BACKGROUND: Hepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial. The nucleocapsid (core) protein of HCV has been shown to be directly implicated in cellular transformation and immortalization, enhancing the effect of oncogenes and decreasing the one of tumor suppressor genes, as RB1 and its protein product pRB. With the aim of identifying novel molecular mechanisms of hepatocyte transformation by HCV, we examined the effect of HCV core protein on the expression of the whole Retinoblastoma (RB) family of tumor and growth suppressor factors, i.e. pRb, p107 and pRb2/p130. METHODS: We used a model system consisting of the HuH-7, HCV-free, human hepatocellular carcinoma cell line and of the HuH-7-CORE cells derived from the former and constitutively expressing the HCV core protein. We determined pRb, p107 and pRb2/p130 protein and mRNA amount of the respective genes RB1, RBL1 and RBL2, RBL2 promoter activity and methylation as well as DNA methyltransferase 1 (DNMT1) and 3b (DNMT3b) expression level. The effect of pRb2/p130 over-expression on the HCV core-expressing HuH-7-CORE cells was also evaluated. RESULTS: We found that the HCV core protein expression down-regulated pRb2/p130 protein and mRNA levels in HuH-7-CORE cells by inducing promoter hyper-methylation with the concomitant up-regulation of DNMT1 and DNMT3b expression. When pRb2/p130 expression was artificially re-established in HuH-7-CORE cells, cell cycle analysis outlined an accumulation in the G0/G1 phase, as expected. CONCLUSIONS: HCV core appears indeed able to significantly down-regulate the expression and the function of two out of three RB family tumor and growth suppressor factors, i.e. pRb and pRb2/p130. The functional consequences at the level of cell cycle regulation, and possibly of more complex cell homeostatic processes, may represent a plausible molecular mechanism involved in liver transformation by HCV.

Long Term Exposure to Polyphenols of Artichoke (Cynara scolymus L.) Exerts Induction of Senescence Driven Growth Arrest in the MDA-MB231 Human Breast Cancer Cell Line.

Polyphenolic extracts from the edible part of artichoke (Cynara scolymus L.) have been shown to be potential chemopreventive and anticancer dietary compounds. High doses of polyphenolic extracts (AEs) induce apoptosis and decrease the invasive potential of the human breast cancer cell line, MDA-MB231. However, the molecular mechanism underlying AEs antiproliferative effects is not completely understood. We demonstrate that chronic and low doses of AEs treatment at sublethal concentrations suppress human breast cancer cell growth via a caspases-independent mechanism. Furthermore, AEs exposure induces a significant increase of senescence-associated ß-galactosidase (SA-ß-gal) staining and upregulation of tumour suppressor genes, p16(INK4a) and p21(Cip1/Waf1) in MDA-MB231 cells. AEs treatment leads to epigenetic alterations in cancer cells, modulating DNA hypomethylation and lysine acetylation levels in total proteins. Cell growth arrest correlates with increased reactive oxygen species (ROS) production in AEs treated breast cancer cells. Inhibition of ROS generation by N-acetylcysteine (NAC) attenuates the antiproliferative effect. These findings demonstrate that chronic AEs treatment inhibits breast cancer cell growth via the induction of premature senescence through epigenetic and ROS-mediated mechanisms. Our results suggest that artichoke polyphenols could be a promising dietary tool either in cancer chemoprevention or/and in cancer treatment as a nonconventional, adjuvant therapy.

Detection of phosphorylated insulin receptor in colorectal adenoma and adenocarcinoma: implications for prognosis and clinical outcome.

Colorectal carcinoma remains among the most frequent causes of cancer death. Besides the well-known genetic predisposition, a key role in colorectal adenoma and adenocarcinoma etio-pathogenesis, mainly in sporadic cases, is played by definite risk factors, such as obesity, type 2 diabetes, insulin resistance, hyper-insulinemia, and insulin therapy. These epidemiological data motivated us to determine, by means of immunohistochemistry, the amount of activated (phosphorylated) insulin receptor in archival samples from 22 colorectal adenoma and 117 adenocarcinoma patients, with the objective to estimate the role of this factor in colorectal epithelium transformation and cancer progression. Statistical analysis of the results clearly showed that positive staining for phosphorylated insulin receptor was significantly more frequent in adenomas than adenocarcinomas (P < 0.0001) and, within the adenocarcinoma cohort, it was more frequent in low-grade tumors (P = 0.005). In adenomas, staining was exclusively cytoplasmic, while in adenocarcinomas it was cytoplasmic and/or nuclear (P < 0.0001). Interestingly, disease-free survival in colorectal adenocarcinoma patients pointed out a significantly better prognosis for those bearing a positive staining for phosphorylated insulin receptor (P = 0.02). From these data, we can argue that activated insulin receptor plays a fundamental role at the early stages of tumorigenesis, where late stages could be characterized by a shift toward more active oncogenic drivers. Determining the amount of phosphorylated insulin receptor could thus represent a novel prognostic/predictive tool in colorectal adenocarcinoma patients.

Artichoke polyphenols induce apoptosis and decrease the invasive potential of the human breast cancer cell line MDA-MB231.

The human breast cancer cell line, estrogen receptor negative, MDA-MB231, was used to evaluate the antitumor effect of polyphenolic extracts from the edible part of artichokes (AEs). Treatment of cancer cells reduced cell viability and inhibited cell growth in a dose-dependent manner. Importantly, AEs did not have any effect on normal breast epithelial cell line, MCF10A. Chlorogenic acid (ChA), the most representative component of the polyphenolic fraction of artichoke, had no prominent effects on the cell death rate of MDA-MB231 cells. The addition of AEs to the cells, rather than ChA, triggered apoptosis via a mitochondrial and a death-receptor pathway, as shown by the activation of caspase-9 and caspase-8, respectively. Furthermore, an increase of the Bax:Bcl2 ratio and up-regulation of cyclin-dependent kinase inhibitor, p21(WAF1), crucial apoptotic players, were documented. According to our data on activation of caspase-9, a loss of mitochondrial transmembrane potential (Ψ(m)) was shown. Cell motility and invasion capabilities were remarkably inhibited by AEs-treatment in highly invasive MDA-MB231 cells. In addition, a significant decrease of proteolytic activity of metalloproteinase-2 protein (MMP-2), involved in degrading components of the extracellular matrix, was detected. Our findings indicate that AEs reduced cell viability, inhibited cell growth, triggered apoptotic mechanisms, and showed inhibitory properties against the invasive behavior of MDA-MB231 cancer cell line. Altogether, these data indicate the potential chemopreventive activity of artichoke polyphenolic extracts.

Management of Stage II testicular seminoma over a period of 40 years.

OBJECTIVES: To review the treatment, toxicity, and outcomes in patients with Stage II seminoma after orchidectomy. MATERIALS AND METHODS: A retrospective chart review of all patients with Stage II seminoma referred for initial treatment, from 1965 to 2005, was performed. Treatment approaches, toxicity, and outcomes were analyzed. RESULTS: A total of 106 patients (83 with Stage IIA, 19 with Stage IIB, and 4 with Stage IIC) were seen between 1965 and 2005. Median age at diagnosis was 36 years (range: 19-71). Median follow-up was 21 years (range: 1.2-42). Eighty-nine patients were treated with adjuvant radiotherapy alone; 13 patients received a combined treatment modality with chemotherapy and radiotherapy after orchidectomy, 4 patients were treated with chemotherapy alone. Generally the treatment was well tolerated, with the main toxicity occurring in patients treated with extended-field radiotherapy. The 5-year disease-specific survival was 96% for the entire group. The 5-year relapse-free survivals for Stages IIA, IIB, and IIC disease were 94%, 72.5%, and 75%, respectively. Fifteen patients developed a relapse and were managed by chemotherapy; 5 of them achieved complete remission and remain free from further recurrence at last follow-up, while 10 died of the disease. Second malignancies were diagnosed in 4 (3.7%) patients during the follow-up. CONCLUSIONS: In Stage IIA seminoma, radiotherapy continues to provide excellent results, as the majority of patients will be cured with this treatment alone. Radiotherapy or chemotherapy should be offered as an alternative to Stage IIB patients. Chemotherapy remains the treatment of choice for Stage IIC seminoma.

New technologies used in the study of human melanoma.

The amount of information on tumor biology has expanded enormously, essentially due to the completion of the human genome sequencing and to the application of new technologies that represent an exciting breakthrough in molecular analysis. Often these data spring from experimental procedures, such as a serial analysis of gene expression (SAGE) and DNA microarrays, which cannot be defined as hypothesis-driven: it may appear to be a "brute force" approach through which no information can be directly generated concerning the specific functions of selected genes in a definite context. However, interesting results are fruitfully generated, and thus it is important to consider the enormous potential these new technologies possess and to learn how to apply this novel form of knowledge in the emerging field of molecular medicine. This review, after a limited outline regarding several classic aspects of human cutaneous melanoma biology, genetics, and clinical approaches, will focus on the proficient use of up-to-date technologies in the study of the neoplastic disease and on their capability to provide effective support to conventional approaches in melanoma diagnosis, prognosis, and treatment.

Human papillomavirus-16 E7 interacts with Siva-1 and modulates apoptosis in HaCaT human immortalized keratinocytes.

The viral factor E7 plays a key role in the well-established association between "high-risk" Human Papillomavirus (HPV) infection and the development of epithelial malignant tumors, as uterine cervix and ano-genital cancer. To delve into the molecular mechanisms of HPV-mediated cell transformation, we searched for novel potential cellular targets of the HPV-16 E7 oncoprotein, by means of the yeast two-hybrid technique, identifying a protein-protein interaction between HPV-16 E7 and the pro-apoptotic cellular factor Siva-1. Using co-precipitation assays and the "PepSets" technique, we confirmed this physical interaction and mapped accurately, for both proteins, the amino acid residues involved. Additionally, we found that HPV-16 E7 competed in vitro with the binding of the Bcl-X(L) anti-apoptotic factor to Siva-1, an interaction that has a major inference in UV radiation-induced apoptosis. In HaCaT immortalized human keratinocytes, forced HPV-16 E7 expression by retroviral infection caused Siva-1 transcript up-regulation, detected by cDNA macroarray hybridization and real-time quantitative PCR, paralleled by an increased amount of protein. Confirming the anti-apoptotic role of HPV-16 E7 in the HaCaT cellular model, evaluated by nuclear morphology, we also found that Siva-1 expression produced a significant increase of the apoptotic rate in UV radiation-exposed HaCaT cells, and that this effect resulted explicitly counteracted by HPV-16 E7. Being apoptosis a key physiological process for the elimination of irreversibly injured cells, the anti-apoptotic role of HPV-16 E7, performed at least by its interference with Siva-1, can be considered an additional mechanism for the survival of damaged, potentially transforming, cell clones.

Unravelling the complexity of T cell abnormalities in common variable immunodeficiency.

We investigated several phenotypic and functional parameters of T cell-mediated immunity in a large series of common variable immunodeficiency (CVID) patients. We demonstrated that the vast majority of CVID patients presented multiple T cell abnormalities intimately related among them, the severity of which was reflected in a parallel loss of CD4+ naive T cells. A strong correlation between the number of CD4+ naive T cells and clinical features was observed, supporting the subgrouping of patients according to their number of naive CD4+ T lymphocytes. A reduced thymic output and disrupted CD4+ and CD8+ TCR repertoires paralleled the contraction of CD4+ naive T cell pools. The evaluation of activation markers and cytokine production indicated a strong T cell activation that was significantly related to the increased levels of T cell turnover and apoptosis. Finally, discrete genetic profiles could be demonstrated in groups of patients showing extremely diverse T cell subset composition and function. Naive CD4+ T cell levels were significantly associated with the switched memory B cell-based classification, although the concordance between the respective subgroups did not exceed 58.8%. In conclusion, our data highlight the key role played by the T cell compartment in the pathogenesis of CVID, pointing to the need to consider this aspect for classification of this disease.