Effects of miRNA-15 and miRNA-16 expression replacement in chronic lymphocytic leukemia: implication for therapy.

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

lncRNA profiling in early-stage chronic lymphocytic leukemia identifies transcriptional fingerprints with relevance in clinical outcome.

Long non-coding RNAs (lncRNAs) represent a novel class of functional RNA molecules with an important emerging role in cancer. To elucidate their potential pathogenetic role in chronic lymphocytic leukemia (CLL), a biologically and clinically heterogeneous neoplasia, we investigated lncRNAs expression in a prospective series of 217 early-stage Binet A CLL patients and 26 different subpopulations of normal B-cells, through a custom annotation pipeline of microarray data. Our study identified a 24-lncRNA-signature specifically deregulated in CLL compared with the normal B-cell counterpart. Importantly, this classifier was validated on an independent data set of CLL samples. Belonging to the lncRNA signature characterizing distinct molecular CLL subgroups, we identified lncRNAs recurrently associated with adverse prognostic markers, such as unmutated IGHV status, CD38 expression, 11q and 17p deletions, and NOTCH1 mutations. In addition, correlation analyses predicted a putative lncRNAs interplay with genes and miRNAs expression. Finally, we generated a 2-lncRNA independent risk model, based on lnc-IRF2-3 and lnc-KIAA1755-4 expression, able to distinguish three different prognostic groups in our series of early-stage patients. Overall, our study provides an important resource for future studies on the functions of lncRNAs in CLL, and contributes to the discovery of novel molecular markers with clinical relevance associated with the disease.

A phase II, single-arm, prospective study of bendamustine plus melphalan conditioning for second autologous stem cell transplantation in de novo multiple myeloma patients through a tandem transplant strategy.

This phase II trial evaluates, for the first time, the safety and efficacy of bendamustine plus high-dose melphalan (HDM) as a conditioning regimen before the second autologous stem cell transplantation (ASCT) in previously untreated multiple myeloma (MM) patients. In total, 32 ASCT patients received HDM (200 mg/m(2)) as conditioning for the first ASCT. After 3-6 months from the first ASCT, responding patients underwent a second ASCT following bendamustine (200 mg/m(2)) and HDM (140 mg/m(2)). High-dose chemotherapy and ASCT were performed with complete neutrophil and platelet recovery in all patients. The median number of days to neutrophil and platelet engraftment was 11 (range 9-15) and 12 (range 10-19), respectively. Only one subject experienced grade 3 diarrhea; the rate of mucositis and vomiting was significantly lower with the bendamustine plus HDM regimen compared with the HDM-only regimen (81.2 vs 96.9%, P=0.025 and 78.1 vs 100%, P=0.008). Overall response rate (ORR) was 81.2% after the first transplant, and 90.6% after the second, while complete response rates were 46.8 and 62.5%, respectively (P=0.016). Actuarial 2-year PFS and OS were 79% (95% confidence interval (CI), 60-98) and 97% (95% CI, 91-100), respectively. Bendamustine+HDM is feasible as the conditioning regimen for second ASCT in MM patients. The present study may pave the way for phase III studies specifically aimed at further investigating this combination strategy. The role of this combination in MM for conditioning regimen in a first or single ASCT setting should be also investigated.

N-(4-hydroxyphenyl)retinamide promotes apoptosis of resting and proliferating B-cell chronic lymphocytic leukemia cells and potentiates fludarabine and ABT-737 cytotoxicity.

The in vitro effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR, fenretinide) on primary B-cell chronic lymphocytic leukemia (CLL) cells from previously untreated CLL patients were investigated. 4HPR promoted the intrinsic apoptotic pathway by reactive oxygen species (ROS) generation and was accompanied by drop of Mcl-1 protein expression. The latter was not attributable to transcriptional downregulation but to protein degradation mediated by jun N-terminal kinase activation, and likely by NF-kB downregulation and Noxa upregulation. CLL cells stimulated in vitro with CD40L did not increase 4HPR chemoresistance if activation was accompanied by proliferation. Intra-patient analysis confirmed that the proliferating pool of CLL cells was more sensitive to the cytotoxic action of 4HPR than the activated but resting CLL subpopulation. The different 4HPR susceptibility of the two subpopulations was associated with higher Noxa expression in proliferating CLLs. Combination experiments revealed that 4HPR strongly potentiated ABT-737 cytotoxicity, especially in proliferating CLL cells that displayed amplified chemoresistance to ABT-737 alone. Synergic cytotoxicity was also demonstrated in combination with fludarabine, in both resting and stimulated CLL samples. This study entitles 4HPR to be assayed as a chemotherapeutic adjuvant for the treatment of CLL.

Liposomal amphotericin B in the treatment of visceral leishmaniasis in immunocompromised patients.

Leishmaniasis is a zoonosis caused by a protozoan of the Leishmania genus. First-line treatment for all forms is currently represented by the use of antimony derivatives, although toxic effects and the number of resistant strains in both immunocompromised and immunocompetent patients is increasing. Liposomal amphotericin B (L-AMB) is less toxic, more effective, and better tolerated, especially in human immunodeficiency virus-negative immunocompromised patients. We present 2 cases of transplanted patients affected by visceral leishmaniasis treated successfully with L-AMB.

A new membrane G protein-coupled receptor (GPR30) is involved in the cardiac effects of 17beta-estradiol in the male rat.

In the present study, we evaluated the transduction pathways involved in the cardiac effects elicited by 17beta-estradiol (E2) on the isolated, Langendorff perfused male Wistar rat heart. E2 and selective agonists for ERalpha and ERbeta induced a dose-dependent reduction of contractility which was blocked by the ER inhibitor ICI 182,780. Moreover, the potential involvement of the novel membrane estrogen receptor GPR30 in mediating estrogen activity was determined using the selective GPR30 ligand G-1. Notably, specific inhibitors of ERK, PI3K, PKA, and eNOS transduction pathways abolished the cardiac responses to E(2). Taken together, our data suggest that ERalpha and ERbeta along with several signaling cascades are involved in the action of E(2) on the male rat heart. Our results also point to a potential role of GPR30, however further evaluation is required in order to fully understand the contribution of the different estrogen receptors in mediating estrogen activity on cardiac performance.

Computational inference of replication and transcription activator regulator activity in herpesvirus from gene expression data.

One of the main aims of system biology is to understand the structure and dynamics of genomic systems. A computational approach, facilitated by new technologies for high-throughput quantitative experimental data, is put forward to investigate the regulatory system of dynamic interaction among genes in Kaposi's sarcoma-associated herpesvirus network after induction of lytic replication. A reconstruction of transcription factor activity and gene-regulatory kinetics using data from a time-course microarray experiment is proposed. The computational approach uses nonlinear differential equations. In particular, the quantitative Michaelis-Menten model of gene-regulatory kinetics is extended to allow for post-transcriptional modifications and synergic interactions between target genes and the Rta transcription factor. The kinetic method is developed within a Bayesian inferential framework using Markov chain Monte Carlo. The profile of the Rta transcriptional regulator, other post-transcriptional regulatory genes and gene-specific kinetic parameters are inferred from the gene expression data of the target genes. The method described here provides an example of a principled approach to handle a wide range of transcriptional network architectures and regulatory activation mechanisms to reconstruct the activity of several transcription factors and activation kinetic parameters in a single regulatory network.

IL4 gene delivery to the CNS recruits regulatory T cells and induces clinical recovery in mouse models of multiple sclerosis.

Central nervous system (CNS) delivery of anti-inflammatory cytokines, such as interleukin 4 (IL4), holds promise as treatment for multiple sclerosis (MS). We have previously shown that short-term herpes simplex virus type 1-mediated IL4 gene therapy is able to inhibit experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in mice and non-human primates. Here, we show that a single administration of an IL4-expressing helper-dependent adenoviral vector (HD-Ad) into the cerebrospinal fluid (CSF) circulation of immunocompetent mice allows persistent transduction of neuroepithelial cells and long-term (up to 5 months) CNS transgene expression without toxicity. Mice affected by chronic and relapsing EAE display clinical and neurophysiological recovery from the disease once injected with the IL4-expressing HD-Ad vector. The therapeutic effect is due to the ability of IL4 to increase, in inflamed CNS areas, chemokines (CCL1, CCL17 and CCL22) capable of recruiting regulatory T cells (CD4+CD69-CD25+Foxp3+) with suppressant functions. CSF delivery of HD-Ad vectors expressing anti-inflammatory molecules might represent a valuable therapeutic option for CNS inflammatory disorders.

Beta3-adrenoceptors modulate left ventricular relaxation in the rat heart via the NO-cGMP-PKG pathway.

AIMS: Using a model of isolated and Langendorff-perfused rat heart we analysed whether activation of beta3-adrenergic receptors (beta3-ARs) influences ventricular lusitropic performance. We also focused on the NOS/NO/cGMP/PKG cascade as the signal transduction mechanism. METHODS: Hearts were treated with increasing concentrations (from 10(-12) to 10(-6) m) of BRL(37344), a selective beta3-AR agonist, and cardiac performance was evaluated by analysing both lusitropic parameters and coronary motility. Cardiac preparations were also perfused with BRL(37344) in the presence of either isoproterenol (ISO) or nadolol, or pertussis toxin (PTx), or selective inhibitors of the NOS/NO/cGMP/PKG pathway. RESULTS: BRL(37344) caused a significant concentration-dependent reduction in (LVdP/dt)(min), a decrease in half time relaxation significant starting from 10(-12) m, and an increase in (LVdP/dt)(max)/(LVdP/dt)(min) ratio (T/-t). BRL(37344) abolished the ISO-mediated positive lusitropism. beta3-AR-dependent effects on relaxation were insensitive to beta(1)/beta2-AR inhibition by nadolol (100 nm), and were abolished by G(i/o) protein inhibition by PTx (0.01 nm). NO scavenging by haemoglobin (10 microm), and nitric oxide synthase (NOS) inhibition by NG-monomethyl-l-arginine (10 microm) revealed the involvement of NO signalling in BRL(37344) response. Pre-treatment with inhibitors of either soluble guanylate cyclase (ODQ; 10 microm) or PKG (KT(5823); 100 nm) abolished beta3-AR-dependent negative lusitropism. In contrast, anantin (10 nm), an inhibitor of particulate guanylate cyclase, did not modify the effect of BRL(37344) on relaxation. CONCLUSION: Taken together, our findings provide functional evidence for beta3-AR modulation of ventricular relaxation in the rat heart which involves PTx-sensitive inhibitory Gi protein and occurs via an NO-cGMP-PKG cascade. Whether the effects of beta3-AR stimulation on lusitropism are beneficial or detrimental remains to be established.

Absence of an intrathecal immune reaction to a helper-dependent adenoviral vector delivered into the cerebrospinal fluid of non-human primates.

Inflammation and immune reaction, or pre-existing immunity towards commonly used viral vectors for gene therapy severely impair long-term gene expression in the central nervous system (CNS), impeding the possibility to repeat the therapeutic intervention. Here, we show that injection of a helper-dependent adenoviral (HD-Ad) vector by lumbar puncture into the cerebrospinal fluid (CSF) of non-human primates allows long-term (three months) infection of neuroepithelial cells, also in monkeys bearing a pre-existing anti-adenoviral immunity. Intrathecal injection of the HD-Ad vector was not associated with any sign of systemic or local toxicity, nor by signs of a CNS-specific immune reaction towards the HD-Ad vector. Injection of HD-Ad vectors into the CSF circulation may thus represent a valuable approach for CNS gene therapy allowing for long-term expression and re-administration.

Antiestrogens upregulate estrogen receptor beta expression and inhibit adrenocortical H295R cell proliferation.

The molecular mechanisms involved in adrenocortical tumorigenesis are still not completely understood. In this study, using the H295R cell line as a model system, we investigated the role of estrogens and estrogen receptor (ER) alpha and ER beta in the growth regulation of adrenocortical tumors. We demonstrated that H295R cells are able to convert androgens to estrogens by a constitutive expression of active cytochrome P450 aromatase protein and express ER beta to a greater extent than ER alpha. Moreover, physiological concentrations of 17beta-estradiol (E2) determined an increase of thymidine incorporation, suggesting the presence of an autocrine mechanism in maintaining H295R cell proliferation. Evaluating the response to ER antagonists like 4-hydroxytamoxifen (OHT) and ICI 182 780 (ICI), we observed an up-regulation of ER beta and a dose-dependent inhibition of H295R cell proliferation. Whereas ICI determined the growth arrest of H295R cells, OHT induced morphological changes that were characteristic of apoptosis. According to the above-mentioned observations, OHT but not ICI clearly induced a marked expression of FasL and the cleavage of both caspase-8 and caspase-3. Interestingly, the apoptotic effects of OHT in H295R cells may be consequent to the enhanced levels of ER beta which stimulate the expression of FasL interacting with activating protein (AP)-1 sites located within its promoter sequence. In conclusion, we have demonstrated that H295R cells are able to transform androgens to estrogens that activate an autocrine mechanism, mediated by their own receptors, and contribute to regulate the proliferation of these cells. Moreover, this study points towards a role for ER beta as an important mediator of the repressive effects exerted by antiestrogens on H295R cells; however, further studies are needed to clarify its role in the control of adrenocortical cell proliferation and on the potential benefits of antiestrogens for treatment of adrenocortical cancer.

The red wine phenolics piceatannol and myricetin act as agonists for estrogen receptor alpha in human breast cancer cells.

Previous epidemiological reports have suggested that red wine intake is associated with beneficial health effects due to the ability of certain phytochemical components to exert estrogen-like activity. It has been also documented that estrogens induce the proliferation of hormone-dependent breast cancer cells by binding to and transactivating estrogen receptor (ER) alpha, which in turn interacts with responsive DNA sequences located within the promoter region of target genes. In order to provide further insight into the positive association between wine consumption and the incidence of breast carcinoma in postmenopausal women, we have evaluated the estrogenic properties of two abundant wine-derived compounds, named piceatannol (PIC) and myricetin (MYR), using as model systems the hormone-sensitive MCF7 and the endocrine-independent SKBR3 breast cancer cells. On the basis of our experimental evidence PIC and MYR may contribute to the estrogenicity of red wine since: (1) they transactivate endogenous ER alpha; (2) they activate the agonist-dependent activation function (AF) 2 of ER alpha and ER beta in the context of the Gal4 chimeric proteins; (3) they rapidly induce the nuclear immunodetection of ER alpha; (4) they regulate the expression of diverse estrogen target genes; (5) they compete with 17beta-estradiol for binding to ER alpha and ER beta; and--as a biological counterpart of the aforementioned abilities--(6) they exert stimulatory effects on the proliferation of MCF7 cells. Hence, the estrogenic activity of PIC and MYR might be considered at least as a potential factor in the association of red wine intake and breast tumors, particularly in postmenopausal women.

Xenoestrogens and the induction of proliferative effects in breast cancer cells via direct activation of oestrogen receptor alpha.

Environmental contamination with a variety of industrial products has been associated with developmental and reproductive abnormalities in wildlife species. Increasing evidence has suggested that bisphenol A (BPA) and 4-nonylphenol (NPH), two major endocrine-disrupting chemicals, might be responsible for adverse effects on humans as a consequence of ubiquitous use together with potential oestrogen-like activity. To provide insight into the oestrogen-like nature of BPA and NPH, their ability to activate a reporter gene construct via an oestrogen response element in the hormone-dependent breast cancer cell lines MCF7 and T47D was ascertained. Both compounds transactivated the endogenous oestrogen receptor (ER) alpha in a direct fashion since the anti-oestrogen 4-hydroxytamoxifen abolished the response. In addition, using steroid-receptor-negative HeLa cells engineered to express ERalpha and ERbeta and the hormone-binding domains of both ERalpha and ERbeta, BPA and NPH confirmed the direct transcriptional activity. Interestingly these properties were supported in MCF7 cells by the ability to autoregulate ERalpha expression as well as to induce its nuclear compartmentalization. We therefore evaluated by reverse transcriptase polymerase chain reaction the expression of oestrogen-controlled genes such as cathepsin D and TFF1 (formerly pS2), which were increased by both chemicals tested. The agonistic effects exhibited in all assays performed prompted the evaluation of a more complex biological response such as the proliferation of MCF7 and T47D cells. The same concentration of xenoestrogens eliciting substantial transcriptional activity significantly stimulated the proliferation of both breast cancer cell lines, although with a reduced effectiveness with respect to the natural hormone 17beta-oestradiol. The results indicate that the biological action of environmental oestrogen such as BPA and NPH should be taken into account for the potential impact on human disease-like hormone-dependent breast cancer. However, further studies are needed to clarify their bioavailability and metabolism as well as whether compound mixtures could produce noticeable effects by synergistic activity.

Acute leukemia following a previous malignancy: do acute lymphoid leukemia and acute myeloid leukemia have common risk factors?

INTRODUCTION: Within the framework of the GIMEMA Study Group, the characteristics of acute lymphoid leukemia and acute myeloid leukemia occurring in patients who have suffered a previous malignancy were studied. Assessment was also made of the clinical course, laboratory features and overall outcome of these conditions. MATERIALS AND METHODS: A four-year, multi-center retrospective study was conducted to evaluate the effect of treatment for previous hematological malignancy on the development of secondary leukemia. The study collected in the GIMEMA Archive of Adult Acute Leukemia 3934 new cases of acute leukemia (2964 AML, 901 ALL, 60 acute biphenotypic leukemia). Among these cases, data were evaluated from patients with a personal history of a previous malignancy, and included inquiring into demographic data, history of neoplastic diseases in the 1st degree relatives, type and treatment of the previous malignancy, latency until the development of a secondary acute leukemia diagnosis, laboratory features, treatment and outcome at the onset of secondary acute leukemia. RESULTS: Approximately 200 (5.1%) patients presented a previous malignancy. Twenty-one were affected by ALL and 179 by AML. The proportion of patients with secondary AML was higher than that of patients with secondary ALL (179/2964 vs 21/901, O.R. 2.69-95% C.I. 1.66-4.39, P<0.001). The median latency, from the onset of the previous malignancy to the development of secondary ALL was 27 months and to the development of secondary AML was 52 months (P<0.05). Furthermore, of patients who previously received chemotherapy more developed a second AML (66/127 sAML vs 5/21 sALL; O.R. 3.46-95% C.I. 1.10-11.56, P<0.01). CONCLUSION: In most cases, chemotherapy treatment for a previous malignancy can play a role in the development of secondary AML. In almost all cases of secondary ALL, the role of previous drugs does not appear to be relevant. On the basis of our analysis, performed systematically for the first time on a large adult series of acute leukemia, we conclude that in these patients a biological predisposition to cancer may be suspected.

Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector.

Adenovirus (Ad) and adeno-associated virus (AAV) have attractive and complementary properties that can be exploited for gene transfer purposes. Ad vectors are probably the most efficient vehicles to deliver foreign genes both in vitro and in vivo. AAV exhibits the unique ability to establish latency by efficiently integrating at a specific locus of human chromosome 19 (AAVS1). Two viral elements are necessary for the integration at AAVS1: Rep68/78 and the inverted terminal repeats (AAV-ITRs). In this study, we report the development of two helper-dependent adenoviral (HD) vectors, one carrying the Rep78 gene, the other an AAV-ITR-flanked transgene. Although Rep proteins have been demonstrated to interfere with Ad replication, HD Rep78 vector was successfully amplified on serial passages in 293CRE4 cells with a yield of 50-100 transducing units per cell. DNA integration at the AAVS1 site also was demonstrated in hepatoma cells coinfected with the HD-expressing Rep78 and with the second HD vector carrying a transgene flanked by AAV-ITRs. The high transduction efficiency, large cloning capacity, and high titer of the HD, combined with the site-specific integration machinery provided by AAV-derived components, make the Ad/AAV hybrid viruses a promising vehicle for gene therapy.

Acute promyelocytic leukaemia: epidemiology and risk factors. A report of the GIMEMA Italian archive of adult acute leukaemia. GIMEMA Cooperative Group.

Acute promyelocytic leukaemia (APL) exhibits peculiar epidemiological, clinical, cytogenetic and molecular features, compared to the other acute myeloid leukaemias (AML). Data on epidemiology and occupational risk factors for APL desumed from the GIMEMA archive are reported and compared with those of the other AML. An exploratory case-case study was designed on AML patients from 56 haematology centres in Italy. Overall, 4296 patients older than 15 yr with a new diagnosis of acute leukaemia were recorded between July 1992 and July 1997. Of these, 335 were classified as APL, and 2894 as other AML. The median age of APL patients was 43 compared to 59 yr for the other AML (p < 0.00001). In order to identify peculiar risk factors for APL development, different parameters were compared in the 2 groups. After adjusting by age no significant differences were observed with regard to education, lifetime prevalence of cancer among siblings and previous diseases in the patient's history. Occupational exposure as a possible risk factor for APL showed no increased risk compared to other AML among farmers, builders and leather workers. A significant association was found in electricians (OR=4.4, 95% CI=2.0-9.7) and a weak association was found in wood workers (OR=3.2, 95% CI=0.8-10.8). The proportion of APL with respect to other AML was significantly higher in the north east of Italy compared to the rest of the country (OR=1.7, 95% CI=1.3-2.2). These data confirm the younger age of APL patients compared to the other AML. A possible role of electromagnetic fields is suggested by the higher risk of APL in electrical workers and in the more industrialized areas of the country.

Site-specific integration in mammalian cells mediated by a new hybrid baculovirus-adeno-associated virus vector.

Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable cell lines. To prolong transgene expression, we have developed new hybrid vectors which associate key elements from adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectors contain a transgene cassette composed of the beta-galactosidase (beta-Gal) reporter gene and the hygromycin resistance (Hygr) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replication and integration in the host genome. Constructs were derived both with and without the AAV rep gene under the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrin promoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR-Hygr-beta-Gal sequence was obtained with insect cells only when the rep gene was placed in an antisense orientation to the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10- to 50-fold increase in the number of Hygr stable cell clones. Additionally, rep expression determined the localization of the transgene cassette in the aavs1 site in approximately 41% of cases as detected by both Southern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITR-flanked DNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts. These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNA constructs for ex vivo treatment of primary human cells.