Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer.

CpG island hypermethylation and global genomic hypomethylation are common epigenetic features of cancer cells. Less attention has been focused on histone modifications in cancer cells. We characterized post-translational modifications to histone H4 in a comprehensive panel of normal tissues, cancer cell lines and primary tumors. Using immunodetection, high-performance capillary electrophoresis and mass spectrometry, we found that cancer cells had a loss of monoacetylated and trimethylated forms of histone H4. These changes appeared early and accumulated during the tumorigenic process, as we showed in a mouse model of multistage skin carcinogenesis. The losses occurred predominantly at the acetylated Lys16 and trimethylated Lys20 residues of histone H4 and were associated with the hypomethylation of DNA repetitive sequences, a well-known characteristic of cancer cells. Our data suggest that the global loss of monoacetylation and trimethylation of histone H4 is a common hallmark of human tumor cells.

Inactivation of the lamin A/C gene by CpG island promoter hypermethylation in hematologic malignancies, and its association with poor survival in nodal diffuse large B-cell lymphoma.

PURPOSE: Lamins support the nuclear envelope and provide anchorage sites for chromatin, but they are also involved in DNA synthesis, transcription, and apoptosis. Although the lack of expression of A-type lamins in lymphoma and leukemia has been reported, the mechanism was unknown. We investigated the possible role of CpG island hypermethylation in lamin A/C silencing and its prognostic relevance. PATIENTS AND METHODS: The promoter CpG island methylation status of the lamin A/C gene, encoding the A-type lamins, was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction in human cancer cell lines (n = 74; from 17 tumor types), and primary leukemias (n = 60) and lymphomas (n = 80). Lamin A/C expression was determined by reverse-transcription polymerase chain reaction, Western blot, immunohistochemistry, and immunofluorescence. RESULTS: seven (50%) of 14 leukemia- and five (42%) of 13 lymphoma cell lines. The presence of hypermethylation was associated with the loss of gene expression while a demethylating agent restored expression. In primary malignancies, lamin A/C hypermethylation was present in 18% (nine of 50) of acute lymphoblastic leukemias and 34% (14 of 41) of nodal diffuse large B-cell lymphomas. The presence of lamin A/C hypermethylation in nodal diffuse large B-cell lymphomas correlated strongly with a decrease in failure-free survival (Kaplan-Meier, P = .0001) and overall survival (Kaplan-Meier, P = .0005). CONCLUSION: Epigenetic silencing of the lamin A/C gene by CpG island promoter hypermethylation is responsible for the loss of expression of A-type lamins in leukemias and lymphomas. The finding that lamin A/C hypermethylation is associated with poor outcome in diffuse large B-cell lymphomas suggests important clinical implications.

Lymphoepithelioma-like carcinoma of the bladder: three cases with clinicopathological and p53 protein expression study.

Lymphoepithelioma-like carcinoma of the bladder is an uncommon neoplasm, of which 49 cases have been described in the English literature, none of which has been studied for p53 protein expression. We studied three muscle-infiltrating cases of this tumor using immunohistochemical, in situ hybridization and polymerase chain reaction (PCR) methods. The three cases were positive for epithelial markers and negative for lymphoid antigens in the tumoral syncytial areas. The intensive infiltrate of small cells was negative for epithelial and positive for lymphoid markers. This population was mainly made up of cytotoxic T-lymphocytes, positive for TIA-1. p53 protein was intensely positive in more than 90% of the epithelial component nuclei, being negative in the lymphoid cells. PCR study did not show mutations on p53. Both lymphocytes and epithelium were negative for Epstein-Barr virus markers, such as the latent membrane protein and EBER (Epstein-Barr-encoded RNA). The prognosis was very good after radiotherapy and chemotherapy treatment, preserving the bladder despite the muscle infiltration. The presence of an intense cytotoxic T-lymphocyte population may be related to this good prognosis. Both aspects, p53 protein status and T-lymphoid population, had never been studied before in bladder lymphoepithelioma-like carcinoma.

Prenatal exposure to methamphetamine in the rat: ontogeny of tyrosine hydroxylase mRNA expression in mesencephalic dopaminergic neurons.

Methamphetamine (Meth) is an illicit substance known to interfere with catecholaminergic systems and a popular recreational drug among young adult women, that is, in gestational age. Tyrosine hydroxylase (TH), the rate-limiting enzyme of the synthetic pathway of catecholamines, is a good marker to assess potential effects of Meth in catecholaminergic (particularly in dopaminergic) systems. In the rat, prolonged neonatal Meth exposure altered several dopaminergic markers (TH activity and gene expression) in substantia nigra pars compacta (SN) and in caudate-putamen (TH activity) when animals matured. However, it was never verified whether gestational exposure to Meth might compromise TH enzyme in the pups during the neonatal immature periods. The present study was designed to address this issue by analyzing TH gene expression, measured by in situ hybridization in SN and ventral tegmental area (VTA), dopaminergic areas that are well characterized as target areas for Meth, and in rats prenatally exposed to this psychostimulant. To this end, dated pregnant Wistar rat dams received 5 mg Meth hydrochloride/kg body weight/day. It was administered subcutaneously from gestational day 8 until 22. The control group was pair-fed and saline injected, using the same experimental protocol as for Meth-treated dams. On the day of birth (postnatal day 0, PND 0), litters were culled to 8 pups, sex-balanced whenever possible, and were followed until the day of sacrifice (PND 7, 14, or 30). Meth treatment differentially affected TH mRNA levels in VTA and SN, in an age- and gender-dependent manner. Thus, TH mRNA levels were decreased in the VTA of PND 7 and PND 14 females gestationally exposed to Meth; this effect was not evident in males or on PND 30. TH mRNA levels also tend to decrease in SN of PND 14 females gestationally exposed to Meth. Collectively, the present results indicated that gestational Meth exposure affects TH gene expression in the postnatal life, a phenomenon that appears to be transient, since it is no longer evident by the end of the first month of life in the rat.

Effects of perinatal exposure to delta 9-tetrahydrocannabinol on operant morphine-reinforced behavior.

The present study examined the effects of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) when administered during the perinatal period on morphine self-administration in adulthood. To this end, pregnant Wistar rats were daily exposed to Delta(9)-THC from the fifth day of gestation up to pup weaning, when they were separated by gender and left to mature to be used for analyses of operant food- and morphine-reinforced behavior in a progressive ratio (PR) schedule. We also analyzed dopaminergic activity (DOPAC/DA) in reward-related structures during specific phases of the behavioral study. In both reinforcement paradigms, food and morphine, females always reached higher patterns of self-administration than males, but this occurred for the two treatment groups, Delta(9)-THC or vehicle. These higher patterns measured in females corresponded with a higher DOPAC/DA in the nucleus accumbens prior to the onset of morphine self-administration in comparison to males. Interestingly, DOPAC/DA was lower in Delta(9)-THC-exposed females compared to oil-exposed females and similar to oil- and Delta(9)-THC-exposed males. In addition, Delta(9)-THC-exposed females also exhibited a reduction in DOPAC/DA in the ventral tegmental area, which did not exist in males. All these changes, however, disappeared after 15 days of morphine self-administration and they did not reappear after 15 additional days of extinction of this response. Our data suggest that females are more vulnerable than males in a PR schedule for operant food and morphine self-administration; perinatal Delta(9)-THC exposure is not a factor influencing this vulnerability. The neurochemical analysis revealed that the activity of limbic dopaminergic neurons prior to morphine self-administration was higher in females than males, as well as that the perinatal Delta(9)-THC treatment reduced the activity of these neurons only in females, although this had no influence on morphine vulnerability in these animals.

Functional signatures identified in B-cell non-Hodgkin lymphoma profiles.

Gene-expression profiling in B-cell lymphomas has provided crucial data on specific lymphoma types, which can contribute to the identification of essential lymphoma survival genes and pathways. In this study, the gene-expression profiling data of all major B-cell lymphoma types were analyzed by unsupervised clustering. The transcriptome classification so obtained, was explored using gene set enrichment analysis generating a heatmap for B-cell lymphoma that identifies common lymphoma survival mechanisms and potential therapeutic targets, recognizing sets of coregulated genes and functional pathways expressed in different lymphoma types. Some of the most relevant signatures (stroma, cell cycle, B-cell receptor (BCR)) are shared by multiple lymphoma types or subclasses. A specific attention was paid to the analysis of BCR and coregulated pathways, defining molecular heterogeneity within multiple B-cell lymphoma types.

Expression of the NF-kappaB targets BCL2 and BIRC5/Survivin characterizes small B-cell and aggressive B-cell lymphomas, respectively.

Nuclear factor kappa B (NF-kappaB) activation has been proposed as a cardinal feature of tumourigenesis, although the precise mechanism, frequency, relevance, and extent of NF-kappaB activation in lymphomas remain to be fully elucidated. In this study, expression profiling and tissue microarray studies of 209 and 323 non-Hodgkin's lymphomas (NHLs) respectively, including the most frequent sub-types of NHL, were employed to generate a hypothesis concerning the most common NF-kappaB targets in NHL. These analyses showed that NF-kappaB activation is a common phenomenon in NHL, resulting in the expression of distinct sets of NF-kappaB target genes, depending on the cell context. BCL2 and BIRC5/Survivin were identified as key NF-kappaB targets and their expression distinguished small and aggressive B-cell lymphomas, respectively. Interestingly, in the vast majority of B-cell lymphomas, the expression of these markers was mutually exclusive. A set of genes was identified whose expression correlates either with BIRC5/Survivin or with BCL2. BIRC5/Survivin expression, in contrast to BCL2, was associated with a signature of cell proliferation (overexpression of cell cycle control, DNA repair, and polymerase genes), which may contribute to the aggressive phenotype and poor prognosis of these lymphomas. Strikingly, mantle cell lymphoma and chronic lymphocytic leukaemia expressed highly elevated levels of BCL2 protein and mRNA, higher than that observed in reactive mantle zone cells or even in follicular lymphomas, where BCL2 expression is deregulated through the t(14;18) translocation. In parallel with this observation, BIRC5/Survivin expression was higher in Burkitt's lymphoma and diffuse large B-cell lymphoma than in non-tumoural germinal centre cells. In vitro studies confirmed that NF-kappaB activation contributes to the expression of both markers. In cell lines representing aggressive lymphomas, NF-kappaB inhibition resulted in a decrease in BIRC5/Survivin expression. Meanwhile, in chronic lymphocytic leukaemia (CLL)-derived lymphocytes, NF-kappaB inhibition resulted in a marked decrease in BCL2 expression.

Changes in prodynorphin and POMC gene expression in several brain regions of rat fetuses prenatally exposed to Delta(9)-tetrahydrocannabinol.

Recently, we demonstrated that prenatal Delta(9)-tetrahydrocannabinol (Delta(9)-THC) exposure alters proenkephalin mRNA levels in several brain regions of rat fetuses. In the present study, we analyzed mRNA levels of the two other opioid peptide precursors, prodynorphin and pro-opiomelanocortin (POMC), in several brain nuclei of rat fetuses which were exposed daily to Delta(9)-THC from day 5 of gestation. Prenatal Delta(9)-THC exposure altered POMC and prodynorphin mRNA levels in most of the brain areas studied at different fetal ages, but the effects were sex-dependent. Thus, POMC mRNA levels increased in Delta(9)-THC-exposed females, but decreased in Delta(9)-THC-exposed males at GD21 in the arcuate nucleus, cerebral cortex and habenular nuclei. POMC mRNA levels also increased in the arcuate nucleus and cerebral cortex of Delta(9)-THC-exposed fetuses at GD18. Prodynorphin mRNA levels were not altered by the prenatal Delta(9)-THC exposure in the striatum, cerebral cortex, hippocampus and hypothalamic structures of fetuses at GD16 and GD18, but a sexually dimorphic response was observed at GD21. Thus, prodynorphin mRNA levels increased in the cerebral cortex, hippocampus and paraventricular hypothalamic nucleus of Delta(9)-THC-exposed females, whereas no changes were observed in Delta(9)-THC-exposed males. In summary, Delta(9)-THC exposure altered the prenatal development of POMC and prodynorphin mRNA levels in several brain structures. Changes in POMC were similar to those reported previously for proenkephalin, increases in females but decreases in males, whereas changes in prodynorphin were only observed in females.

Alleviation of motor hyperactivity and neurochemical deficits by endocannabinoid uptake inhibition in a rat model of Huntington's disease.

Recent studies have demonstrated a loss of cannabinoid CB1 receptors in the postmortem basal ganglia of patients affected by Huntington's disease (HD) and in transgenic mouse models for this disease. These studies have led to the notion that substances that increase the endocannabinoid activity, such as receptor agonists or inhibitors of endocannabinoid uptake and/or metabolism, might be useful in the treatment of hyperkinetic symptoms of this disease. In the present study, we employed a rat model of HD generated by bilateral intrastriatal injections of 3-nitropropionic acid (3-NP), a toxin that selectively damages striatal GABAergic efferent neurons. These rats exhibited biphasic motor disturbances, with an early (1-2 weeks) hyperactivity followed by a late (3-4 weeks) motor depression. Analysis of GABA, dopamine, and their related enzymes, glutamic acid decarboxylase and tyrosine hydroxylase, in the basal ganglia proved marked decreases compatible with the motor hyperkinesia. In addition, mRNA levels for CB1 receptor, neuronal-specific enolase, proenkephalin, and substance P decreased in the caudate-putamen of 3-NP-injected rats. There were also reductions in CB1 receptor binding in the caudate putamen, the globus pallidus, and, to a lesser extent, the substantia nigra. By contrast, mRNA levels for tyrosine hydroxylase in the substantia nigra remained unaffected. Interestingly, the administration of AM404, an inhibitor of endocannabinoid uptake, to 3-NP-injected rats attenuated motor disturbances observed in the early phase of hyperactivity. Administration of AM404 also tended to induce recovery from the neurochemical deficits caused by the toxin in GABA and dopamine indices in the basal ganglia. In summary, morphological, behavioral, and biochemical changes observed in rats intrastriatally lesioned with 3-NP acid were compatible with a profound degeneration of striatal efferent GABAergic neurons, similar to that occurring in the brain of HD patients. As expected, a loss of CB1 receptors was evident in the basal ganglia of these rats. However, the administration of substances that increase endocannabinoid activity, by inhibiting the uptake process, allowed an activation of the remaining population of CB1 receptors, resulting in a significant improvement of motor disturbances and neurochemical deficits. These observations might be relevant to the treatment of hyperkinetic symptoms in HD, a human disorder with unsatisfactory symptomatic treatment for most patients.

Development of a real-time reverse transcription polymerase chain reaction assay for c-myc expression that allows the identification of a subset of c-myc+ diffuse large B-cell lymphoma.

Absence of a reliable method for determining the level of c-myc expression has impeded the analysis of its biological and clinical relevance in tumors. We have standardized the conditions for a real-time reverse transcription polymerase chain reaction analysis for c-myc expression, including the selection of an endogenous reference (18S rRNA), the adequate number of measurements for each sample (2 cDNA in triplicate), and suitable controls for determining inter- and intrarun variability (standard curve and calibrator). Subsequently, in a series of 56 non-Hodgkin's lymphomas, we analyzed the expression of c-myc mRNA, using real-time reverse transcription polymerase chain reaction, and of other functionally related proteins (bcl-6, p27, cyclin D3, and p53). As expected, all eight Burkitt's lymphoma cases analyzed had high levels of c-myc mRNA expression compared with that observed in reactive lymphoid tissue. There was a wider range of expression in diffuse large B-cell lymphoma, with 30% (15 of 48) of cases overexpressing c-myc. This overexpression was largely independent of c-myc translocations (4 of 5), as demonstrated by fluorescence in situ hybridization. In this large B-cell lymphoma series, a high level of c-myc expression predicted lower survival probability, irrespectively of the International Prognostic Index risk group classification. A slightly increased frequency of p53 inactivation was observed in the cases with c-myc overexpression, which suggests a growth advantage in lymphomas with concurrent deregulation of c-myc and p53. In addition, a moderate increase in bcl-6 protein expression was observed in the c-myc-positive cases, suggesting the existence of a complex interrelationship between these two genes. These findings suggest that c-myc may play a relevant role in the pathogenesis of a subset of large B-cell lymphoma and suggest the existence of additional regulatory mechanisms of c-myc expression to c-myc rearrangements.

Abnormal PcG protein expression in Hodgkin's lymphoma. Relation with E2F6 and NFkappaB transcription factors.

The Polycomb group (PcG) of proteins comprises a family of repressors of homeobox genes that play key roles in body formation, haematopoiesis and cell cycle control. In this study, a large-scale analysis of PcG protein expression (BMI1, MEL18, PH1, RNF2, RING1, and RYBP) was performed in 321 Hodgkin's lymphoma (HL) biopsies and in reactive lymphoid tissues using tissue microarrays. The relevance of PcG proteins in HL was also investigated by the simultaneous analysis of PcG and other proteins involved in the control of cell cycle, transcription machinery and lymphoid differentiation. The analysis revealed increased expression of a set of PcG proteins (particularly RYBP and BMI1) in tumour cells in comparison with reactive lymphoid tissue. One of the most striking findings was anomalous RYBP expression in 55% of classical HL cases associated with an unfavourable response to treatment and shorter survival. The data obtained in this study also show an association of PcG proteins with E2F6 and NFkappaB transcription factors. The statistical relationship between PcG and NFkappaB activation was further explored in HL-derived cell lines treated with curcumin, an NFkappaB inhibitor, and TNFalpha. Up- or downregulation of MEL18 was paralleled by loss or gain of activated NFkappaB, which suggests that NFkappaB may regulate expression of this protein. Investigation of the relationship between E2F6 and RING1 by immunofluorescence and confocal analysis, in HL cell lines and paraffin sections, revealed co-expression of both proteins in the same tumour cells. These results allow us to propose that the formation of transcription complexes with E2F6 may modify the functional status of PcG proteins in HSR cells.

Building an outcome predictor model for diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) patients are treated using relatively homogeneous protocols, irrespective of their biological and clinical variability. Here we have developed a protein-expression-based outcome predictor for DLBCL. Using tissue microarrays (TMAs), we have analyzed the expression of 52 selected molecules in a series of 152 DLBCLs. The study yielded relevant information concerning key biological aspects of this tumor, such as cell-cycle control and apoptosis. A biological predictor was built with a training group of 103 patients, and was validated with a blind set of 49 patients. The predictive model with 8 markers can identify the probability of failure for a given patient with 78% accuracy. After stratifying patients according to the predicted response under the logistic model, 92.3% patients below the 25 percentile were accurately predicted by this biological score as "failure-free" while 96.2% of those above the 75 percentile were correctly predicted as belonging to the "fatal or refractory disease" group. Combining this biological score and the International Prognostic Index (IPI) improves the capacity for predicting failure and survival. This predictor was then validated in the independent group. The protein-expression-based score complements the information obtained from the use of the IPI, allowing patients to be assigned to different risk categories.

Inactivation of the Lamin A/C gene by CpG island promoter hypermethylation in hematologic malignancies, and its association with poor survival in nodal diffuse large B-cell lymphoma

Purpose. Lamins support the nuclear envelope and provide anchorage sites for chromatin, but they are also involved in DNA synthesis, transcription, and apoptosis. Although the lack of expression of A-type lamins in lymphoma and leukemia has been reported, the mechanisms was unknown. We investigated the possible role of CpG island hypermethylation in lamin A/C silencing and its prognostic relevance. Patients and methods. The promoter CpG island methylation status of the lamin A/C gene, encoding the A-type lamins, was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction in human cancer cell lines (n=74; from 17 tumor types), and primery leukemias (n=60) and lymphomas (n=80). Lamin A/C expression was determined by reverse-transcription polymerase chain reaction. Western blot, immunochemistry, and immunofluorescence. Results. Lamin A/C promoter CpG island methylation was found in hematologic malignancies: seven (50 percent) of 14 leukemia- and five (42 percent) of 13 lymphoma cell lines. The presence of hypermethylation was associated with the loss of gene expression while a demethulating agent restored expression. In primary malignancies, lamin A/C hypermethylation was present in 18 percent (nine of 50) of acute lymphoblastic leukemias and 34 percent (14 of 41) of nodal diffuse large B-cell lymphomas. The presence of lamin A/C hypermethylation in nodal diffuse large B-cell lymphomas correlated strongly with a decrease in failure-free survival (Kaplan-Meier, P=.0001) and overall survival (Kaplan-Meier, P=.0005). Conclusion. Epigenetic silencing of the lamin A/C gene by CpG island promoter hypermethylation is responsible for the loss of expression of A-type lamins in leukemias and lymphomas. The finding that lamin A/C hypermethylation is associated with poor outcome in diffuse large B-cell lymphomas suggests important clinical implications