Temozolomide induced c-Myc-mediated apoptosis via Akt signalling in MGMT expressing glioblastoma cells.

PURPOSE: We investigated the molecular mechanisms underlying the cytotoxic effect of Temozolomide (TMZ) in both O(6)-methylguanine-DNA methyl transferase (MGMT) depleted as well as undepleted glioblastoma cell lines. Since TMZ is used in clinics in combination with radiotherapy, we also studied the effects of TMZ in combination with ionising radiation (IR). METHODS: Cell colony-forming ability was measured using a clonogenic assay. Cell cycle analysis and apoptosis were evaluated by Flow Cytometry (FCM). Proteins involved in cell cycle control were detected by Western blot and co-immunoprecipitation assays. RESULTS: Our data showed that TMZ, independent of MGMT expression, inhibited glioblastoma cell growth via an irreversible G(2) block in MGMT depleted cells or the induction of apoptosis in MGMT normal expressing cells. When TMZ was administered in combination with IR, apoptosis was greater than observed with either agent separately. This TMZ-induced apoptosis in the MGMT expressing cells occurred through Akt/Glycogen-Synthase-Kinase-3ß (GSK3ß) signalling and was mediated by Myelocytomatosis (c-Myc) oncoprotein. Indeed, TMZ phosphorylated/activated Akt led to phosphorylation/inactivation of GSK3ß which resulted in the stabilisation of c-Myc protein and subsequent modulation of the c-Myc target genes involved in the apoptotic processes. CONCLUSION: C-Myc expression could be considered a good indicator of TMZ effectiveness.

Cord blood CD133 cells define an OV6-positive population that can be differentiated in vitro into engraftable bipotent hepatic progenitors.

Cell therapy represents the most promising alternative strategy for end-stage liver diseases and hepatic progenitors are the best candidates. We have identified a reservoir of immature hepatic precursors within human cord blood, which can derive engraftable bipotent progenitors. We isolated a stem cell subset CD133+/CD34+/OV6(low) expressing a surface-marker profile consistent with that of fetal liver cells. Upon induction of hepatic commitment by a medium containing cytokines and factors involved in vivo oval-cell activation, a heterogeneous cell population displaying characteristics of functional oval-cell-like bipotent hepatic progenitors was obtained. The cells expressed markers of hepatocytes and cholangiocytes and were highly enriched in OV6, c-Met, c-Kit, and Thy-1. They also displayed liver functional activity as glycogen storage, urea production, albumin secretion, and inducible CyP2B6 activity. When injected into liver-damaged severe-combined immunodeficient mice, induced bipotent hepatic progenitors appropriately engrafted livers of recipient animals, where they formed clusters of human-derived cells expressing human leucocyte antigen-class I, Hep-Par1, and OV6 antigens. Human-specific albumin, alpha-fetoprotein, and cytokeratin 19 were also expressed. In transplanted animals, AST serum levels showed a significative reduction with regard to controls. This human model for in vitro progenitor-cell activation may provide a powerful tool for elucidating the pathways and synergies that regulate this complex process and can represent a valuable source, exploitable for liver cell-based therapies and regenerative medicine.

Interleukin 12B (IL12B) genetic variation and pulmonary tuberculosis: a study of cohorts from The Gambia, Guinea-Bissau, United States and Argentina.

We examined whether polymorphisms in interleukin-12B (IL12B) associate with susceptibility to pulmonary tuberculosis (PTB) in two West African populations (from The Gambia and Guinea-Bissau) and in two independent populations from North and South America. Nine polymorphisms (seven SNPs, one insertion/deletion, one microsatellite) were analyzed in 321 PTB cases and 346 controls from Guinea-Bissau and 280 PTB cases and 286 controls from The Gambia. For replication we studied 281 case and 179 control African-American samples and 221 cases and 144 controls of European ancestry from the US and Argentina. First-stage single locus analyses revealed signals of association at IL12B 3' UTR SNP rs3212227 (unadjusted allelic p = 0.04; additive genotypic p = 0.05, OR = 0.78, 95% CI [0.61-0.99]) in Guinea-Bissau and rs11574790 (unadjusted allelic p = 0.05; additive genotypic p = 0.05, OR = 0.76, 95% CI [0.58-1.00]) in The Gambia. Association of rs3212227 was then replicated in African-Americans (rs3212227 allelic p = 0.002; additive genotypic p = 0.05, OR = 0.78, 95% CI [0.61-1.00]); most importantly, in the African-American cohort, multiple significant signals of association (seven of the nine polymorphisms tested) were detected throughout the gene. These data suggest that genetic variation in IL12B, a highly relevant candidate gene, is a risk factor for PTB in populations of African ancestry, although further studies will be required to confirm this association and identify the precise mechanism underlying it.

Thyroid hormones (T3 and T4): dual effect on human cancer cell proliferation.

Several findings suggest that the patient's hormonal context plays a crucial role in determining cancer outcome. The exact nature of thyroid hormone action on tumour growth has not been established yet, in fact contrasting data show thyroid hormones have a promotory or an inhibitory action on cancer cell proliferation depending on the case. We hypothesized that not only tissue specificity, but also specific mutations occurring during tumoral development in different thyroid hormone cellular targets are responsible for this dual effect. To test our hypothesis we analysed, by time-course and bromodeoxyuridine assay, thyroid hormone effects on the proliferation of six cancer cell lines originating from the same tissue or organ but carrying different mutations (in phospho-inositide 3 kinase or ß-catenin genes). The data obtained in this study show how mutations that affect the balance between degradation and stabilization of ß-catenin assume a remarkable importance in determining the cell-specific thyroid hormone effect on cell growth.

The thyroid hormone T3 improves function and survival of rat pancreatic islets during in vitro culture.

Ex vivo islet cell culture in the presence of stimulating factors prior to transplantation is considered a good strategy in contrast to the short conclusion of islets transplantation. Previously, we demonstrated how T3 can increase b-cell function via specific activation of Akt; therefore we hypothesized that thyroid hormone T3 can be considered a promising candidate for the in vitro expansion of islet cell mass. Rat pancreatic islets have been isolated by the collagenase digestion and cultured in the presence or not of the thyroid hormone T3 10⁻7 M. Islets viability has been evaluated by the use of two different dyes, one cell-permeable green fluorescent dye and propidium iodide, and by the analysis of core cell damage upcoming. Moreover, islets function has been evaluated by insulin secretion. The ability of b-cells to counteract apoptosis induced by streptozotocin has been analyzed by TUNEL assay. We demonstrated that treatment of primary cultures of rat pancreatic islets with T3 results in augmented ß-cell vitality with an increase of their functional properties. In addition, a sensible reduction of the core cell damage has been observed in the T3 treated islets, suggesting the preservation of the ß-cells integrity during the culture period. Nonetheless, the insulin secretion is sensibly augmented after T3 stimulation. The strong increment shown in Akt activation suggests the involvement of this pathway in the observed phenomena. In conclusion we indicate T3 as a good factor to improve ex vivo islets cell culture.

Mitotane sensitizes adrenocortical cancer cells to ionizing radiations by involvement of the cyclin B1/CDK complex in G2 arrest and mismatch repair enzymes modulation.

Mitotane inhibits steroid synthesis by an action on steroidogenic enzymes, as 11beta-hydroxylase and cholesterol side chain cleavage. It also has a cytotoxic effect on the adrenocortical cells and represents a primary drug used in the adrenocortical carcinoma (ACC). H295R and SW13 cell lines were treated with mitotane 10(-5) M and ionizing radiations (IR) in combination therapy, inducing an irreversible inhibition of cell growth in both adrenocortical cancer cells. As shown in a previous report, mitotane/IR combination treatment induced a cell accumulation in the G2 phase. Here, we report the radiosensitizing properties of mitotane in two different ACC cell lines. The drug reveals the effectiveness to enhance the cytotoxic effects of IR by attenuating DNA repair and interfering on the activation of mitosis promoting factor (MPF), mainly regulated by the degradation of cyclin B1 in the mitotic process. These events may explain the inappropriate activation of cdc2, implicated in G2/M phase arrest and probably induced by the mitotane and IR in the combined treatment. Indeed, treatment with purvalanol, a cdc2-inhibitor prevents cell cycle arrest, triggering the G2/M transition. The observation that mitotane and IR in combination treatment amplifies the activation level of cyclin B/cdc2 complexes contributing to cell cycle arrest, suggests that the MPF could function as a master signal for controlling the temporal order of different mitotic events. Moreover, we report that mitotane interferes in modulation of mismatch repair (MMR) enzymes, revealing radiosensitizing drug ability.

Liver stem cells and possible clinical applications.

The discovery of several sources of hepatic progenitors in extra-hepatic organs and tissues, both in animal models and in humans, supports opportunities to isolate, grow and expand them in vitro. Microenvironment factors involved in regulating proliferation and commitment of liver cell precursors have been identified and better characterization of liver stem cell pathobiology would greatly improve the understanding of liver differentiation/ regeneration processes, especially those leading to hepatocarcinogenesis. The goal of these researches has been to discover the most available, suitable and easy-to-use pluripotent stem cells (PSC) sources for cell-based therapies in genetic and acquired liver diseases, therapies which to date have required liver transplantation. This report reviews the efforts, so far, to either investigate the presence of PSC in hepatic and extra-hepatic districts or evaluate their capacity to differentiate in vitro and to restore in vivo liver function.

Design, Construction and Validation of Targeted BAC Array-Based CGH Test for Detecting the Most Commons Chromosomal Abnormalities.

We designed a targeted-array called GOLD (Gain or Loss Detection) Chip consisting of 900 FISH-mapped non-overlapping BAC clones spanning the whole genome to enhance the coverage of 66 unique human genomic regions involved in well known microdeletion/microduplication syndromes. The array has a 10 Mb backbone to guarantee the detection of the aneuploidies, and has an implemented resolution for telomeres, and for regions involved in common genomic diseases. In order to evaluate clinical diagnostic applicability of GOLDChip, analytical validity was carried-out via retrospective analysis of DNA isolated from a series of cytogenetically normal amniocytes and cytogenetically abnormal DNA obtained from cultured amniocytes, peripheral blood and/or cell lines. We recruited 47 DNA samples corresponding to pathologies with significant frequencies (Cri du Chat syndrome, Williams syndrome, Prader Willi/Angelman syndromes, Smith-Magenis syndrome, DiGeorge syndrome, Miller-Dieker syndrome, chromosomes 13, 18 and 21 trisomies). We set up an experimental protocol that allowed to identify chromosomal rearrangements in all the DNA samples analyzed. Our results provide evidence that our targeted BAC array can be used for the identification of the most common microdeletion syndromes and common aneuploidies.

Thyroid hormones induce cell proliferation and survival in ovarian granulosa cells COV434.

Numerous evidences indicate that thyroid hormones exert an important role in the regulation of the reproductive system in the adult female. Although a clear demonstration of the thyroid-ovarian interaction is still lacking, it is conceivable that thyroid hormones might have a direct role in ovarian physiology via receptors in granulosa cells. In this study we analyzed if thyroid hormone treatment could affect cell proliferation and survival of COV434 cells. To this aim cell growth experiments and cell cycle analyses by flow cytometry were performed. Secondly the T(3) survival action was tested by TUNEL assay and MD30 cleavage analysis. We showed that T(3), and not T(4), can protect ovarian granulosa cells COV434 from apoptosis, regulating cell cycle and growth in the same cells. The increase in cell growth resulted in an augmented percentage of the cells in the S phase and, in a reduction of the doubling time (18%). Subsequently apoptotic pathway induced by serum deprivation has been evaluated in the cells exposed or not to thyroid hormone treatment. The T(3) treatment was able to remarkably counteract the apoptotic process. Even at the ultrastructural level there was an evident protective effect of T(3) in the cells that, besides the maintenance of the original morphology and, the absence of basophilic cytoplasm, conserved normal junctional areas. Furthermore, the protective T(3) effect evaluated by FACS analysis in the presence of a PI3K inhibitor revealed, as also confirmed by Western Blot on pAkt, that the PI3K pathway is crucial in T(3) survival action.

The TRbeta1 is essential in mediating T3 action on Akt pathway in human pancreatic insulinoma cells.

Thyroid hormone action, widely recognized on cell proliferation and metabolism, has recently been related to the phosphoinositide 3 kinase (PI3K), an upstream regulator of the Akt kinase and the involvement of the thyroid hormone receptor beta1 has been hypothesized. The serine-threonine kinase Akt can regulate various substrates that drive cell mass proliferation and survival. Its action has also been characterized in pancreatic beta-cells. We previously demonstrated that Akt activity and its activation in the insulinoma cell line hCM could be considered a specific target of the non-genomic action of T3. In this study we analyzed the molecular pathways involved in the regulation of cell proliferation, survival, size, and protein synthesis by T3 in a stable TRbeta1 interfered insulinoma cell line, derived from the hCM, and evidenced a strong regulation of both physiological and molecular events by T3 mediated by the thyroid hormone receptor beta1. We showed that the thyroid receptor beta1 mediates the T3 regulation of the cdk4.cyc D1.p21(CIP1).p27(KIP1) complex formation and activity. In addition TRbeta1 is essential for the T3 upregulation of the Akt targets beta-catenin, p70S6K, and for the phosphorylation of Bad and mTOR. We demonstrated that the beta1 receptor mediates the T3 upregulation of protein synthesis and cell size, together with the cell proliferation and survival, playing a crucial role in the T3 regulation of the PI3K/Akt pathway.

A three-step pathway comprising PLZF/miR-146a/CXCR4 controls megakaryopoiesis.

MicroRNAs (miRNAs or miRs) regulate diverse normal and abnormal cell functions. We have identified a regulatory pathway in normal megakaryopoiesis, involving the PLZF transcription factor, miR-146a and the SDF-1 receptor CXCR4. In leukaemic cell lines PLZF overexpression downmodulated miR-146a and upregulated CXCR4 protein, whereas PLZF knockdown induced the opposite effects. In vitro assays showed that PLZF interacts with and inhibits the miR-146a promoter, and that miR-146a targets CXCR4 mRNA, impeding its translation. In megakaryopoietic cultures of CD34(+) progenitors, PLZF was upregulated, whereas miR-146a expression decreased and CXCR4 protein increased. MiR-146a overexpression and PLZF or CXCR4 silencing impaired megakaryocytic (Mk) proliferation, differentiation and maturation, as well as Mk colony formation. Mir-146a knockdown induced the opposite effects. Rescue experiments indicated that the effects of PLZF and miR-146a are mediated by miR-146a and CXCR4, respectively. Our data indicate that megakaryopoiesis is controlled by a cascade pathway, in which PLZF suppresses miR-146a transcription and thereby activates CXCR4 translation.

Vdelta2 T-lymphocyte responses in cord blood samples from Italy and Côte d'Ivoire.

Cord blood T lymphocytes are immature and their functional defect partially reflects a suboptimal level of costimulatory signals provided by neonatal antigen-presenting cells. Neonatal Vdelta2 T lymphocytes, a small component of cellular immunity involved in the response against bacteria, protozoa, virus-infected cells and tumours, are also considered to be immature. Cord blood Vdelta2 T lymphocytes are mostly naïve, proliferate poorly and do not produce cytokines in response to the model phosphoantigen isopentenyl pyrophosphate. We cultured cord blood mononuclear cells with the aminobisphosphonate Pamidronate or with live bacille Calmette-Guérin, and showed that both elicit a strong cord blood Vdelta2 T-cell proliferative response, inducing the expression of activation markers and promoting the differentiation from naïve to memory cells. Our results suggest that cord blood Vdelta2 T cells are not inherently unresponsive and can mount strong responses to aminobisphosphonates and mycobacteria. Neonatal Vdelta2 T lymphocytes may be important participants in responses to microbial infections early in life.

Atopic dermatitis and asthma.

Atopic dermatitis (AD), a chronic inflammatory skin disease, frequently associated with respiratory allergy, is one of the most common skin disorders observed in children. The prevalence of AD and other allergic diseases is increasing in industrialized countries, representing a major burden on health care cost. AD has been proposed as an "entry point" for subsequent allergic diseases, suggesting the possibility that effective management of AD could prevent the development of respiratory allergy or at least reduce the severity of asthma and allergic rhinitis. AD and asthma share a common genetic and pathogenic basis, and several longitudinal studies provided evidence for the atopic march from AD to allergic rhinitis and asthma. However, because only a few prospective studies starting at children's births and having a sufficiently long follow-up have been developed, little is known about the natural course of AD and the potential succession of atopic phenotypes in childhood. Finally, recent genetic and epidemiological data raised the question whether AD may either develop to asthma or be part of a syndrome consisting of both diseases.

MicroRNAs 17-5p-20a-106a control monocytopoiesis through AML1 targeting and M-CSF receptor upregulation.

We investigated the role of microRNAs (miRNA) 17-5p, 20a and 106a in monocytic differentiation and maturation. In unilineage monocytic culture generated by haematopoietic progenitor cells these miRNAs are downregulated, whereas the transcription factor acute myeloid leukaemia-1 (AML1; also known as Runt-related transcription factor 1, Runx1) is upregulated at protein but not mRNA level. As miRNAs 17-5p, 20a and 106a bind the AML1 mRNA 3'UTR, their decline may unblock AML1 translation. Accordingly, transfection with miRNA 17-5p-20a-106a suppresses AML1 protein expression, leading to M-CSF receptor (M-CSFR) downregulation, enhanced blast proliferation and inhibition of monocytic differentiation and maturation. Treatment with anti-miRNA 17-5p, 20a and 106a causes opposite effects. Knockdown of AML1 or M-CSFR by short interfering RNA (siRNA) mimics the action of the miRNA 17-5p-20a-106a, confirming that these miRNAs target AML1, which promotes M-CSFR transcription. In addition, AML1 binds the miRNA 17-5p-92 and 106a-92 cluster promoters and transcriptionally inhibits the expression of miRNA 17-5p-20a-106a. These studies indicate that monocytopoiesis is controlled by a circuitry involving sequentially miRNA 17-5p-20a-106a, AML1 and M-CSFR, whereby miRNA 17-5p-20a-106a function as a master gene complex interlinked with AML1 in a mutual negative feedback loop.

Role of a molecular variant of rat atrial natriuretic Peptide gene in vascular remodeling.

Previous studies in a hypertensive animal model of stroke and in humans showed that mutations of the atrial natriuretic peptide (ANP) gene are associated with increased risk of stroke. To elucidate the vascular disease mechanisms that result from structural modifications of the ANP gene, we investigated a coding mutation of the ANP gene in stroke-prone spontaneously hypertensive rats (SHRsp). This mutation leads to a Gly/Ser transposition in the prosegment of ANP. We found that presence of this mutation is associated with increased immunostaining of ANP in the wall of SHRsp cerebral vessels. The mutation causes a major inhibitory effect on endothelial cell proliferation, as assessed by thymidine incorporation, and on angiogenesis, as determined by an endothelial cell tube formation assay, in human umbilical vein endothelial cells (HUVEC) exposed to ANP/SHRsp. These in vitro findings show that the SHRsp-derived form of ANP has an inhibitory effect on vascular remodeling and they provide further support for a role of the ANP gene in the pathogenesis of cerebrovascular disease in the animal model.

3,3',5-Triiodo-L-thyronine inhibits ductal pancreatic adenocarcinoma proliferation improving the cytotoxic effect of chemotherapy.

The pancreatic adenocarcinoma is an aggressive and devastating disease, which is characterized by invasiveness, rapid progression, and profound resistance to actual treatments, including chemotherapy and radiotherapy. At the moment, surgical resection provides the best possibility for long-term survival, but is feasible only in the minority of patients, when advanced disease chemotherapy is considered, although the effects are modest. Several studies have shown that thyroid hormone, 3,3',5-triiodo-l-thyronine (T(3)) is able to promote or inhibit cell proliferation in a cell type-dependent manner. The aim of the present study is to investigate the ability of T(3) to reduce the cell growth of the human pancreatic duct cell lines chosen, and to increase the effect of chemotherapeutic drugs at conventional concentrations. Three human cell lines hPANC-1, Capan1, and HPAC have been used as experimental models to investigate the T(3) effects on pancreatic adenocarcinoma cell proliferation. The hPANC-1 and Capan1 cell proliferation was significantly reduced, while the hormone treatment was ineffective for HPAC cells. The T(3)-dependent cell growth inhibition was also confirmed by fluorescent activated cell sorting analysis and by cell cycle-related molecule analysis. A synergic effect of T(3) and chemotherapy was demonstrated by cell kinetic experiments performed at different times and by the traditional isobologram method. We have showed that thyroid hormone T(3) and its combination with low doses of gemcitabine (dFdCyd) and cisplatin (DDP) is able to potentiate the cytotoxic action of these chemotherapic drugs. Treatment with 5-fluorouracil was, instead, largely ineffective. In conclusion, our data support the hypothesis that T(3) and its combination with dFdCyd and DDP may act in a synergic way on adenopancreatic ductal cells.

Thyroid hormone receptor TRbeta1 mediates Akt activation by T3 in pancreatic beta cells.

It has recently been recognized that thyroid hormones may rapidly generate biological responses by non-genomic mechanisms that are unaffected by inhibitors of transcription and translation. The signal transduction pathways underlying these effects are just beginning to be defined. We demonstrated that thyroid hormone T3 rapidly induces Akt activation in pancreatic beta cells rRINm5F and hCM via thyroid hormone receptor (TR) beta1. The phosphorylation of Akt was T3 specific and dependent. Coimmunoprecipitation and colocalization experiments revealed that the phosphatidylinositol 3 kinase (PI3K) p85alpha subunit and the thyroid receptor beta1 were able to form a complex at the cytoplasmic level in both the cell lines, suggesting that a 'cytoplasmic TRbeta1' was implicated. Moreover, we evidenced that T3 treatment was able to induce kinase activity of the TRbeta1-associated PI3K. The silencing of TRbeta1 expression through RNAi confirmed this receptor to be crucial for the T3-induced activation of Akt. This action involved a T3-induced nuclear translocation of activated Akt, as demonstrated by confocal immunofluorescence. In summary, T3 is able to specifically activate Akt in the islet beta cells rRINm5F and hCM through the interaction between TRbeta1 and PI3K p85alpha, demonstrating the involvement of TRbeta1 in this novel T3 non-genomic action in islet beta cells.

Comparison of traditional and abbreviated salbutamol aerosol therapy using a new spacer mouth mask.

Abbreviated aerosol therapy has been suggested to increase compliance by delivering the same therapeutic dose, but more rapidly than traditional aerosol therapy. A new spacer mouth mask, which is recommended for use in abbreviated aerosol therapy, is now available in Italy. The aim of this study was to compare traditional and abbreviated salbutamol aerosol therapy in 30 asthmatic children using the new spacer mouth mask. Thirty asthmatic children (18 boys and 12 girls; aged 4-13 years) were evaluated during severe asthma attacks (forced expiratory volume at 1 second [FEV(1)] <60% of the predicted value) and randomly allocated to treatment with two different schedules of aerosol therapy. Aerosol therapy was administered to group A in the usual manner, with patients breathing in and out at tidal volume until the nebulizer bowl was empty. Group B received therapy with the new spacer mouth mask used in accordance with manufacturer's instructions, i.e., placing the mask tightly over the mouth and instructing the child to breathe in through the mouth and out through the nose a total of five times at tidal volume, keeping the mouth open. The amount of drug available to patients in group A was approximately 768 microg, whereas 176 microg was available to those in group B. The FEV(1) increased in all patients and there was no difference in the degree of improvement between the groups (p < 0.05). The results indicate equivalent bronchodilatation between abbreviated and traditional aerosol therapy but because abbreviated therapy takes less time, it may improve compliance.

Co-localization of susceptibility loci for psoriasis (PSORS4) and atopic dermatitis (ATOD2) on human chromosome 1q21.

Psoriasis (PS) is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation and altered differentiation. Atopic dermatitis (ATOD) is a chronic inflammatory, pruritic and eczematous disease frequently associated with respiratory atopy. These diseases are associated with distinct immunologic abnormalities and represent typical examples of complex diseases triggered by both genetic and environmental factors, as demonstrated by independent twin studies. Genome wide linkage studies have mapped susceptibility loci on several chromosomes (PSORS1-9; ATOD1-5). Four of them overlap on chromosomes 1q21, 3q21, 17q25 and 20p although ATOD is quite distinct from PS and these two diseases rarely occur together in the same patient. An association fine-mapping study has been performed to refine PSORS4 and ATOD2 susceptibility loci on chromosome 1q21 analyzing two independently collected cohorts of 128 PS and 120 ATOD trios. Genotype and haplotype analysis of PSORS4 and ATOD2 led us to detect significant p value for haplotypes defined by MIDDLE and ENDAL16 markers in both PS (p = 0.0000036) and ATOD (p = 0.0276), suggesting a strict co-localization within an interval of 42 kb. This genomic interval contains a single gene, LOR, encoding for loricrin. Polymorphic markers mapping in regulatory and coding regions did not show evidence of association in neither of the two diseases. However, expression profiles of LOR in skin biopsies have shown reduced levels in PS and increased levels in ATOD, suggesting the existence of a specific misregulation in LOR mRNA production.

Gemcitabine treatment of experimental C6 glioma: the effects on cell cycle and apoptotic rate.

Gemcitabine (dFdCyd) is a deoxycytidine analogue showing a broad spectrum of cytotoxic activity; additionally, at non-cytotoxic concentrations, it is a potent radiosensitiser. Its in vitro and in vivo effects were studied on C6 rat glioma. In vitro, dFdCyd induced an increase in S-phase cells at the end of treatment, with a reduction in G1 and G2 cell cycle-phase cells and relevant effects on the apoptotic rate. The in vivo effects of dFdCyd were studied on rats bearing intracranial C6 glioma. The drug was administered at a dose of 120 mg/Kg every 3 days x 4 doses. A significant effect on tumour growth was detected by longitudinal MRI analyses. Furthermore, the drug induced an inhibitory effect on tumour growth, 72 h after a 300 mg/Kg single dose. Analyses performed on tumour specimens showed relevant G1-phase accumulation and about 45% apoptotic cells. The present results justify further studies to determine the potential efficacy of dFdCyd in the treatment of malignant gliomas.