Quantitative analysis of associations between DNA hypermethylation, hypomethylation, and DNMT RNA levels in ovarian tumors.

How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5'-gene region or promoter hypermethylation, satellite, or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the quantitative MethyLight assay, six of the 55 examined gene loci (LTB4R, MTHFR, CDH13, PGR, CDH1, and IGSF4) were significantly hypermethylated relative to the degree of malignancy (after adjustment for multiple comparisons; P < 0.001). Importantly, hypermethylation of these genes was associated with degree of malignancy independently of the association of satellite or global DNA hypomethylation with degree of malignancy. Cancer-related increases in methylation of only two studied genes, LTB4R and MTHFR, which were appreciably methylated even in control tissues, were associated with DNMT1 RNA levels. Cancer-linked satellite DNA hypomethylation was independent of RNA levels for all DNMT3B isoforms, despite the ICF syndrome-linked DNMT3B deficiency causing juxtacentromeric satellite DNA hypomethylation. Our results suggest that there is not a simple association of gene hypermethylation in cancer with altered DNMT RNA levels, and that this hypermethylation is neither the result nor the cause of satellite and global DNA hypomethylation.

DNA methylation profiles of female steroid hormone-driven human malignancies.

Tumor DNA contains valuable clues about the origin and pathogenesis of human cancers. Alterations in DNA methylation can lead to silencing of genes associated with distinct tumorigenic pathways. These pathway-specific DNA methylation changes help define tumor-specific DNA methylation profiles that can be used to further our understanding of tumor development, as well as provide tools for molecular diagnosis and early detection of cancer. Female sex hormones have been implicated in the etiology of several of the women's cancers including breast, endometrial, ovarian, and proximal colon cancers. We have reviewed the DNA methylation profiles of these cancers to determine whether the hormonal regulation of these cancers results in specific DNA methylation alterations. Although subsets of tumors in each of these four types of cancers were found to share some DNA methylation alterations, we did not find evidence for global hormone-specific DNA methylation alterations, suggesting that female sex hormones may participate in different tumorigenic pathways that are associated with distinct DNA methylation-based molecular signatures. One such pathway may include MLH1 methylation in the context of the CpG island methylator phenotype.

Influence of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on cell cycle progression and proliferation of cultured arterial smooth muscle cells.

8-(N,N-Diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a putative inhibitor of intracellular calcium mobilization, causes a dose-dependent inhibition of serum-induced proliferation of arterial smooth muscle cells in culture. Neither early rise in cytosolic calcium concentration nor induction of early induced cell cycle dependent genes (c-fos, ornithine decarboxylase) are inhibited after serum stimulation in presence of 100 microM TMB-8. In contrast, expression of thymidine kinase, a gene normally induced in late-G1 phase, is entirely inhibited by TMB-8. Taken together with flow cytometry studies, these results indicate that TMB-8 blocks cell cycle progression in mid- or late-G1 phase by a mechanism not directly related to early responses to serum stimulation since TMB-8 is also effective when introduced several hours after serum stimulation.

Les modalités de communication dans le comportement sexual de Calliphora vomitoria (Diptera: Calliphoridae): vers une interprétation de la parade.

During sexual behaviour, the body and the behaviour of the partner provide a pattern of distant or contact signals. In order to evaluate in which respect courtship behaviour and its success depend on the comparison between the male's expectancy (template) and its actual perception, we have presented to male flies partner bodies with modified odours or shapes or both from different sexes and ages. Our data demonstrated the simultaneous use of different sensory channels. They suggest two different processes which might be involved in the construction of the template. Firstly, it could result from sensory reafferences, with a shuttle between stimuli and responses. In that case, the mismatch between expectancy and actual perception might induce disturbances in the courtship behaviour. If the differences are too high, it could stop the courtship sequence which resumes at its beginning. Secondly, the template could result from a process of representation. Then, when expectancy does not fit the perceived image, courtship would not be disturbed and the male could extract a new significance from the network of stimuli provided by the partner's body.

Stability of dyadic dominance status and recognition of the opponent in male crickets Gryllus bimaculatus (Orthoptera: Gryllidae).

In dyads of adult male Gryllus bimaculatus, a dominance relationship is established at the first agonistic interaction between the two insects. The comparison of the aggressive behaviour displayed by adult males shows that field crickets act in an identical manner when confronted with an unfamiliar opponent or with one already encountered in a different environment. In contrast, analysis of five successive conflicts in a given environment shows that the dynamics of the interactions varies according to whether the opponents are always the same or one is changed at each encounter. The conflicts are solved more rapidly when the interactions involve the same opponents.

The role of prior agonistic experience in dominance relationships in male crickets Gryllus bimaculatus (Orthoptera: Gryllidae).

Experiments were carried out to study the effect of social conditioning (prior experience of dominance and submission) in dominance relationships between adult male Gryllus bimaculatus. The dominance status of a male cricket appears to be directly linked to its immediate prior agonistic experience. An experience of dominance increases the probability of victory and one of submission decreases it. The effect is maximum when one opponent has experienced dominance and one subordination. The aggressive behavior of males is significantly influenced by prior agonistic experience for 6 h and the effect disappears entirely after 24 h. The cost and benefit of a conflict appears to be dependent on the motivational state of each opponent, in turn modulated by the outcome of prior agonistic interactions.

Description du comportement sexuel de Calliphora vomitoria (Diptera: Calliphoridae) par une technique d'analyse de textes.

Description of the sexual behaviour of Calliphora vomitoria (Diptera: Calliphoridae) with a lexical analysis software package. The sexual behaviour of Calliphora vomitoria was described using a lexical analysis software package considering courtship as a series of words, without any arbitrary categorization. Normal as well as manipulated partners of both sexes were presented to the males. Results showed that normal courtship and copulation occurred when the wings were modified but present and correctly oriented, whereas perturbations were observed and copulation disappeared after inversion and various head modifications. Missing elements (head and wings) were less disruptive than their inversion. If the partner was supposed to be scanned from the head to the abdomen via the wings and if it was supposed to perceive the relative position of these different parts, it is obvious that the absence of an element did not act as an error message and courtship and copulation were preserved. On the other hand, inversion of the same elements seemed to induce wrong or inconsistent informations affecting courtship structure, probably because of sequence disturbance. The courtship preservation in front of every kind of manipulation leads us to question what image of the sexual partner is constructed.

Changes in cuticular hydrocarbon composition in relation to age and sexual behavior in the female Calliphora Vomitoria (Diptera).

Qualitative and quantitative changes in epicuticular hydrocarbon synthesis/release were observed during the first gonadotrophic cycle of Calliphora vomitoria. Up to 12 hours after eclosion, hydrocarbon synthesis/release is low, with little change in the relative proportions of the compounds detected. At 24 hours there is a marked increase in hydrocarbon synthesis/release, especially in the monomethylalkanes (both absolute and relative amounts). Between 48 and 72 hours, hydrocarbon synthesis stabilizes. At 83 hours, two hydrocarbon patterns can be distinguished, depending on the state of receptiveness of the females. The results show for the first time that differences in age, sexual attractiveness and receptivity are accompanied by characteristic changes in cuticular n-alkanes and mono-methylalkanes.

[Non verbal communication abilities in severe traumatic brain injury]. / Compétences de communication non verbale des traumatisés crâniens graves.

OBJECTIVE: Discursive abilities of severe brain injured patient are always impaired: loss of flexibility, lack of cohesion and coherence, often more elliptic. We know few about nonverbal competencies during discourse. The objective is to verify nonverbal abilities of these patients by pragmatic analysis. METHODS: Four men were examined more than 7 years after severe traumatic brain injury. Nonverbal Prutting and Kirchner Pragmatic Protocol (1987) were done allowing to a qualitative and quantitative measurement of paralinguistic behaviour: prosody and quality of speech, facial expression, posture, gaze, gesture. Two conditions were recorded: dual (descriptive discourse) and group (conversational discourse). Associated impairments such as cognitive and dysexecutive functioning were also investigated. RESULTS/DISCUSSION: Impoverishment (loss of ability) or impaired inadequacity was observed in all patients. Paralinguistic competences of conversational discourse was worse than descriptive one. Facial expression, gaze functioning, referential gesture were more often impaired. Maladjustment could be interpretated in reference with dysexecutive syndrome. CONCLUSION: In spite of the lack of information about the range of normal pragmatic behaviour, it seems that brain injured patients have shown poor nonverbal abilities during discourse. Rehabilitation training of communication skills would integrate this fact in order to improve interactivity and social relationship.

Sequencing and cardiac expression of natriuretic peptide receptors A and C in normal and heart failure pigs.

Pharmacological treatments able to activate natriuretic receptors (NPRs) and inhibit cardiac remodelling in heart failure (HF) patients, are currently under investigation. To better understand the therapeutic potential of the NPRs activation is necessary to dispose of experimental models devoid of confounding effects. The pig constitutes an animal model largely used but its genome is not completely sequenced. Aims of this study were to sequence NPR-A and NPR-C in Susscrofa and to evaluate ANP, BNP and NPRs mRNA expression in cardiac tissue of normal and HF minipigs in order to have a starting point for future studies devoted to check new potential drugs. Cardiac tissue was collected from adult male minipigs without (n=4) and with HF (n=5). Pig NPR-A (179bp) and NPR-C (203bp) mRNA were partially sequenced (GenBank n.: FJ518622, FJ518621). Compared to control, ANP and BNP gene expression resulted higher in all the cardiac chambers of HF heart. This increase is associated to a down-regulation of NPR-A and an up-regulation of NPR-C in HF. These sequences will provide a new tool to investigate the role of natriuretic peptides and of their receptors under physiological and pathological conditions and their response to therapeutic interventions.

CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.

BACKGROUND: The concept of CpG island methylator phenotype (CIMP) is not universally accepted. Even if specific clinicopathological features have been associated with CIMP, investigators often failed to demonstrate a bimodal distribution of the number of methylated markers, which would suggest CIMP as a distinct subtype of colorectal cancer. Previous studies primarily used methylation specific polymerase chain reaction which might detect biologically insignificant low levels of methylation. AIM: To demonstrate a distinct genetic profile of CIMP colorectal cancer using quantitative DNA methylation analysis that can distinguish high from low levels of DNA methylation. MATERIALS AND METHODS: We developed quantitative real time polymerase chain reaction (MethyLight) assays and measured DNA methylation (percentage of methylated reference) of five carefully selected loci (promoters of CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1) in 460 colorectal cancers from large prospective cohorts. RESULTS: There was a clear bimodal distribution of 80 microsatellite instability-high (MSI-H) tumours according to the number of methylated promoters, with no tumours showing 3/5 methylated loci. Thus we defined CIMP as having >or=4/5 methylated loci, and 17% (78) of the 460 tumours were classified as CIMP. CIMP was significantly associated with female sex, MSI, BRAF mutations, and wild-type KRAS. Both CIMP MSI-H tumours and CIMP microsatellite stable (MSS) tumours showed much higher frequencies of BRAF mutations (63% and 54%) than non-CIMP counterparts (non-CIMP MSI-H (0%, p<10(-5)) and non-CIMP MSS tumours (6.6%, p<10(-4)), respectively). CONCLUSION: CIMP is best characterised by quantitative DNA methylation analysis. CIMP is a distinct epigenotype of colorectal cancer and may be less frequent than previously reported.

[Influence of sexual maturation on cardiac activity and reactivity of Calliphora vomitoria. I. -- Cardiac activity (author's transl)]. / Influence de la maturité sexuelle sur l'activité et la réactivité cardiaques de Calliphora vomitoria (Diptère, calliphoridae) I. -- Contrôle de l'activité cardiaque

1. Calliphora cardiac rhythm shows a regular alternation of retrograde beating phases with high frequency (fast phases) and of anterograde beating phases with low frequency (slow phases). 2. Male cardiac activity exhibits only small variations during sexual maturation. 3. In females, the ovarian development induces a decrease of slow phase duration and a variation of anterograde beat frequency (it decreases the first five days and then increases). 4. Cardiac characteristics of allatectomized or ovariectomized animals are similar to those of young females with small ovaries. 5. These results reveal an important correlation between ovarian development and many cardiac characteristics. The most important physiological factor seems to be the increase in ovary size and/or its consequences (abdominal distention).

Induction of cell cycle-dependent genes during cell cycle progression of arterial smooth muscle cells in culture.

Serum stimulation of arterial smooth muscle cells in culture induces a progression through the cell cycle and cell proliferation. Most genes previously described as cell cycle-dependent in various cell types also demonstrate a cell cycle-dependent expression in arterial smooth muscle cells. As in other cell types, these genes can be classified into three groups according to their mode of expression: "immediate early" genes (c-fos, c-myc, ...), "delayed early" genes (2F1, ...), and "late-G1" genes (proliferating cell nuclear antigen, thymidine kinase, . . .). In addition to these previously described genes, three genes isolated from a cDNA library of stimulated smooth muscle cells have been demonstrated to be cell cycle-dependent: A21, the rat JE gene, and L51 can be classified as "immediate early" genes, while M11 represents a new member of the "delayed early" gene family.

Effects of angiotensins on cellular hypertrophy and c-fos expression in cultured arterial smooth muscle cells.

An increase in cell size and protein content was observed when quiescent arterial smooth muscle cells in culture were incubated with either angiotensin II or III. These effects were inhibited by the specific angiotensin type-1 receptor antagonist losartan (DuP753) but not by CGP42112A. In parallel, a transient and dose-dependent induction of c-fos was demonstrated not only with angiotensins II and III but also with angiotensin I. Both angiotensins II and III exerted their maximal effect at 1 microM, while angiotensin I needed a tenfold-higher concentration to exert an identical effect. As for hypertrophy, losartan also inhibits angiotensin-induced c-fos expression, suggesting that this gene may be involved into the hypertrophic process. Angiotensin-I-mediated c-fos induction is partially inhibited by the angiotensin-converting enzyme inhibitors captopril and trandolaprilate; given that an angiotensin-converting enzyme activity was detected in these smooth muscle cell cultures, these results suggest that angiotensin-I-induced c-fos expression is mediated in part via angiotensin-I conversion to angiotensin II, but also by other unidentified pathway(s). Angiotensin I could essentially induce smooth muscle cell hypertrophy by indirect mechanisms, while angiotensins II and III act directly on smooth muscle cells.

Cuticular hydrocarbons of Calliphora vomitoria (Diptera): relation to age and sex.

Changes in epicuticular hydrocarbon content and composition were examined from 3 to 120 hr postemergence in both sexes of Calliphora vomitoria. n-Alkanes and mono- and dimethylalkanes with 20 to 31 carbon atoms were the major hydrocarbons detected. Alkenes were only detected in the females. Males had fewer hydrocarbons (40 versus 49 for the females). In males this number remained constant from 3 to 120 hr, whereas in females the number increased progressively from 3 to 48 hr. The males had a greater total of hydrocarbons than the females (x6 versus x3.5). They had significantly more hydrocarbons by 48 hr, the time of sexual maturation, while in females, the hydrocarbon content increased between 6 and 24 hr postemergence, just before the onset of previtellogenesis. Hydrocarbon synthesis continued up to 120 hr in males, but remained constant in the females after 24 hr. Hydrocarbon composition differed in males and females and between the young (3 and 6 hr) and the older animals (24 to 120 hr). The young animals were characterized by the presence of monomethylalkanes with chain lengths over 25C and the older animals by monomethylalkanes with chain lengths less than 25C. Alkenes were found only in females. The males were characterized by the presence of di- and monomethylalkanes with 23, 24, and 26 carbon atoms.

Cell cycle dependent gene expression in quiescent stimulated and asynchronously cycling arterial smooth muscle cells in culture.

The expression of a set of cell cycle dependent (CCD) genes (c-fos, c-myc, ornithine decarboxylase (ODC), and thymidine kinase (TK)) was comparatively studied in cultured arterial smooth muscle cells (SMC) during exit from quiescence and exponential proliferation. These genes, which were not expressed in quiescent SMC, were chronologically induced after serum stimulation. c-fos mRNA were rapidly and transiently expressed very early in the G1 phase; c-myc and ODC peaked a few hours after serum stimulation and then remained at an intermediary level throughout the first cell cycle; TK mRNA and activity then appeared at the G1/S boundary and peak in G2/M phases. Except for c-fos, the other genes were also expressed in asynchronously cycling SMC (ACSMC); their expression was studied in elutriated subpopulations representative of cell cycle progression. c-fos mRNA were undetectable in any sorted subpopulations, even in the pure early G1 population. Despite a slight increase as the cell cycle advanced, c-myc and ODC genes were expressed throughout the ACSMC cell cycle. A faint TK activity was found in G1 subpopulations and increased in populations enriched in other phases; in contrast, TK mRNA remained highly expressed in all elutriated subpopulations. This study demonstrates significant modulations in CCD gene expression between quiescent stimulated and asynchronously cycling SMC in culture. This suggests that the events occurring during the emergence of SMC from quiescence are probably different from those in the G1 phase of ACSMC.

Increased proteolytic processing of protein tyrosine phosphatase mu in confluent vascular endothelial cells: the role of PC5, a member of the subtilisin family.

Cleavage and subsequent release of the extracellular domains of receptor protein tyrosine phosphatases (RPTP) occur at high cell density and may have an important role in regulating their activity. Because cleavage of RPTP occurs at a target motif (RXK/RR) recognized by a family of subtilisin/kexin-like endoproteases, we postulated that members of the subtilisin family may have an important role in this cleavage. We show in this report that the membrane-associated RPTPmu--both in its full 200-kDa form and as a 100-kDa cleavage product--is upregulated 4- and 7-fold, respectively, as human umbilical vein endothelial cells (HUVEC) approach confluence. To determine whether RPTPmu cleavage depended on PC5 (a subtilisin/kexin like endoprotease present in endothelial cells), we transfected COS cells with expression plasmids coding for RPTPmu and PC5 or the closely related protease PACE4. PC5, but not PACE4, cleaved RPTPmu, and RPTPmu cleavage was absent in COS cells transfected with an expression plasmid encoding a mutant PC5 whose active-site serine had been mutated to alanine. We also performed RNA blot analysis to determine whether PC5 expression was affected by confluence in HUVEC. PC5 mRNA levels were upregulated by more than 30-fold when confluence in HUVEC increased from 25% to 100%. These results indicate that PC5 may have an important role in mediating the cleavage of RPTPmu in response to contact inhibition in HUVEC.

Osteopontin overexpression is associated with arterial smooth muscle cell proliferation in vitro.

To isolate the genes involved in the cell cycle G1 phase progression of arterial smooth muscle cells (SMCs), a cDNA clone (M11) was previously selected by differential hybridization screening of a mid-G1 serum-stimulated SMC cDNA library. The delay of induction after mitogenic stimulation, time of expression, and need for new protein synthesis for full expression made it possible to classify this gene in the "delayed early" gene group. Determination of the partial M11 cDNA sequence showed full homology with the osteopontin gene (secreted phosphoprotein 1, 2ar), an Arg-Gly-Asp-containing extracellular matrix protein. Osteopontin mRNA was also detected in the aorta at levels as high as in the kidney but lower than in bone, two tissues in which it has been previously detected. In vitro analysis of osteopontin expression in serum-stimulated quiescent SMCs and asynchronously cycling SMCs demonstrated that osteopontin overexpression was associated with SMC proliferation. In view of our results, the high osteopontin expression observed by others in the injured carotid artery could be explained by the involvement of SMCs in the proliferative process. Taken together, these results suggest that osteopontin may play an important role in pathological processes that are associated with arterial SMC proliferation, such as atherosclerosis or restenosis.

Exogenous ATP induces a limited cell cycle progression of arterial smooth muscle cells.

Because exogenous ATP is suspected to influence the proliferative process, its effects on the cell cycle progression of arterial smooth muscle cells were studied by investigating changes in the mRNA steady-state level of cell cycle-dependent genes. Stimulation of cultured quiescent smooth muscle cells by exogenous ATP induced chronological activation not only of immediate-early but also of delayed-early cell cycle-dependent genes, which were usually expressed after a mitogenic stimulation. In contrast, ATP did not increase late G1 gene mRNA level, demonstrating that this nucleotide induces a limited cell cycle progression of arterial smooth muscle cells through the G1 phase but is not able by itself to induce crossing over the G1-S boundary and consequently DNA synthesis. An increase in c-fos mRNA level was also induced by ADP but not by AMP or adenosine. Moreover, 2-methylthioadenosine 5'-triphosphate but not alpha, beta-methyleneadenosine 5'-triphosphate mediated this kind of response. Taken together, these results demonstrate that extracellular ATP induces the limited progression of arterial smooth muscle cells through the G1 phase via its fixation on P2 gamma receptors.