Mitotic rate as an important prognostic factor in cutaneous malignant melanoma.

BACKGROUND: Recently, the quantification of mitoses in cutaneous melanoma has been discharged from the main prognostic variables of the TNM classification. OBJECTIVE: To investigate the prognostic value of the presence of mitoses in primary cutaneous melanoma and to establish the number of mitoses per mm2 that may have prognostic significance. METHODS: A retrospective observational study was performed on 141 patients treated for cutaneous melanoma, who were assessed by the same pathologist, and who had a minimum follow-up of 2 years. Clinical, epidemiological, histopathological and follow-up variables were gathered and compared with the number of mitoses to distinguish the significance of differences by means of univariate, multivariate, and survival analyses. RESULTS: The cut-off level related to a better sensitivity and specificity was 1.50 mitoses per mm2. The presence of two or more mitoses/mm2 showed a better relationship with prognostic variables and both the overall and disease-free survival than the presence of 1 or more mitoses/mm2. This happens especially in melanomas thicker than 0.8 mm and it could affect the staging in cases with Breslow between 1 and 2 mm. CONCLUSIONS: A mitotic rate of two or more mitoses per mm2 in cutaneous melanoma should be considered as a more accurate prognostic factor than one or more mitoses per mm2, particularly in tumors equal or greater than 0.8 mm in thickness.

Tumor suppressor SET9 guides the epigenetic plasticity of breast cancer cells and serves as an early-stage biomarker for predicting metastasis.

During the course of cancer progression, neoplastic cells undergo dynamic and reversible transitions between multiple phenotypic states, and this plasticity is enabled by underlying shifts in epigenetic regulation. Our results identified a negative feedback loop in which SET9 controls DNA methyltransferase-1 protein stability, which represses the transcriptional activity of the SET9 promoter in coordination with Snail. The modulation of SET9 expression in breast cancer cells revealed a connection with E2F1 and the silencing of SET9 was sufficient to complete an epigenetic program that favored epithelial-mesenchymal transition and the generation of cancer stem cells, indicating that SET9 plays a role in modulating breast cancer metastasis. SET9 expression levels were significantly higher in samples from patients with pathological complete remission than in samples from patients with disease recurrence, which indicates that SET9 acts as a tumor suppressor in breast cancer and that its expression may serve as a prognostic marker for malignancy.

Targeting the epigenetics of the DNA damage response in breast cancer.

Cancer is as much an epigenetic disease as it is a genetic disease, and epigenetic alterations in cancer often serve as potent surrogates for genetic mutations. Because the epigenetic factors involved in the DNA damage response are regulated by multiple elements, therapies to target specific components of the epigenetic machinery can be inefficient. In contrast, therapies aimed at inhibiting the methionine cycle can indirectly inhibit both DNA and protein methylation, and the wide variety of genes and pathways that are affected by these methylations make this global strategy very attractive. In the present study, we propose an adjuvant therapy that targets the epigenetics of the DNA damage response in breast cancer cells and that results in efficient apoptosis and a reduction in distant metastases in vivo. We observed that a combined therapy designed to uncouple adenosine metabolism using dipyridamole in the presence of a new synthetic antifolate, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-catechin, simultaneously and efficiently blocked both the folic cycle and the methionine cycle in breast cancer cells and sensitized these cells to radiotherapy. The treatment impeded the recruitment of 53BP1 and BRCA1 to the chromatin regions flanking DNA double-strand breaks and thereby avoided the DNA damage responses in breast cancer cells that were exposed to ionizing radiation. In addition, this hypomethylating therapy was also efficient in reducing the self-renewal capability of breast cancer-initiating cells and induced reversion of mesenchymal phenotypes in breast cancer cells.

Cholinergic system and cell proliferation.

The cholinergic system, comprising acetylcholine, the proteins responsible for acetylcholine synthesis and release, acetylcholine receptors and cholinesterases, is expressed by most human cell types. Acetylcholine is a neurotransmitter, but also a local signalling molecule which regulates basic cell functions, and cholinergic responses are involved in cell proliferation and apoptosis. So, activation of nicotinic and muscarinic receptors has a proliferative and anti-apoptotic effect in many cells. The content of choline acetyltransferase, acetylcholine receptors and cholinesterases is altered in many tumours, and cholinesterase content correlates with patient survival in some cancers. During apoptosis, acetylcholinesterase is induced and appears in the nuclei. Acetylcholinesterase participates in the regulation of cell proliferation and apoptosis through hydrolysis of acetylcholine and by other catalytic and non catalytic mechanisms, in a variant-specific manner. This review gathers information on the role of cholinergic system and specially acetylcholinesterase in cell proliferation and apoptosis.

Targeting the epigenetic machinery of cancer cells.

Cancer is characterized by uncontrolled cell growth and the acquisition of metastatic properties. In most cases, the activation of oncogenes and/or deactivation of tumour suppressor genes lead to uncontrolled cell cycle progression and inactivation of apoptotic mechanisms. Although the underlying mechanisms of carcinogenesis remain unknown, increasing evidence links aberrant regulation of methylation to tumourigenesis. In addition to the methylation of DNA and histones, methylation of nonhistone proteins, such as transcription factors, is also implicated in the biology and development of cancer. Because the metabolic cycling of methionine is a key pathway for many of these methylating reactions, strategies to target the epigenetic machinery of cancer cells could result in novel and efficient anticancer therapies. The application of these new epigenetic therapies could be of utility in the promotion of E2F1-dependent apoptosis in cancer cells, in avoiding metastatic pathways and/or in sensitizing tumour cells to radiotherapy.

The AChE membrane-binding tail PRiMA is down-regulated in muscle and nerve of mice with muscular dystrophy by merosin deficiency.

Since Duchenne muscular dystrophy was attributed to mutations in the dystrophin gene, more than 30 genes have been found to be causally related with muscular dystrophies, about half of them encoding proteins of the dystrophin-glycoprotein complex (DGC). Through laminin-2, the DGC bridges the muscle cytoskeleton and the extracellular matrix. Decreased levels of PRiMA-linked acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) have been observed in dystrophic muscle and nerve of dystrophin-deficient (mdx) and laminin-2 deficient (Lama2dy) mice. To help explain these observations, the relative content of AChE, BuChE and PRiMA mRNAs were compared in normal and Lama2dy mouse muscle and sciatic nerve. The 17-fold lower level of PRiMA mRNA in Lama2dy muscle explained the deficit in PRiMA-linked ChEs. This would increase acetylcholine availability and, eventually, the desensitization of nicotinic receptors. Abnormal development of the Schwann cells led to peripheral neuropathy in the Lama2dy mouse. Compared with normal nerve, dystrophic nerve displayed 4-fold less AChE-T mRNA, 3-fold more BuChE mRNA and 2.5-fold less PRiMA mRNA, which agreed with the lower AChE activity in dystrophic nerve, its increased BuChE activity and the specific drop in PRiMA-linked BuChE. The widely accepted role of glial cells as the source of BuChE, the observed dysmyelination of Lama2dy nerve and its increased BuChE activity support the idea that BuChE up-regulation is related with the aberrant differentiation of the Schwann cells.

The anti-inflammatory and anti-cancer properties of epigallocatechin-3-gallate are mediated by folate cycle disruption, adenosine release and NF-kappaB suppression.

OBJECTIVE: To understand the mechanism by which (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, exerts its anti-inflammatory action. METHODS: To check our hypothesis that the anti-inflammatory properties of EGCG could be related to its antifolate action and whether adenosine and its receptors are involved in EGCG action, we investigated the EGCG-induced suppression of NF-kappaB in Caco-2 cell monolayer, which acted as a model of the human intestinal epithelium. RESULTS: We demonstrate that the anti-inflammatory properties of EGCG are associated with its antifolate activity. By using a natural stable folate we were able to reverse the EGCG suppression of TNF-alpha-induced NF-kappaB activation, the phosphorylation and degradation of IkappaBalpha and the phosphorylation of Akt in this human colon carcinoma cell line. These suppressions were mediated by the release of adenosine following disruption of the folate cycle by EGCG. By binding to its specific receptors, adenosine can modulate the Akt and NF-kappaB pathway. Moreover, EGCG produces a significant increase in a specific adenosine receptor, which could explain the suppression of the constitutive activation of NF-kappaB in colon cancer cells. CONCLUSIONS: The data suggest that by modulating NF-kappaB activation, EGCG might not only combat inflammation, but also cancer.

Inhibition of Stenotrophomonas maltophilia dihydrofolate reductase by methotrexate: a single slow-binding process.

Although antifolates such as trimethoprim are used in the clinical treatment of Stenotrophomonas maltophilia infection, the dihydrofolate reductase (DHFR) of this microorganism is scarcely known because it has never been isolated. Here, we describe the purification of this enzyme and kinetically characterize its inhibition by methotrexate (MTX). Upon MTX treatment, time-dependent, slow-binding inhibition was observed due to the generation of a long-lived, slowly dissociating enzyme-NADPH-inhibitor complex. Kinetic analysis revealed a one-step inhibition mechanism (K(I) = 28.9 +/- 1.9 pM) with an association rate constant (k(i)) of 3.8 x 10(7) M(-1)s(-1). Possible mechanisms for MTX binding to S. maltophilia DHFR are discussed.

Analytical and clinical validation of a time-resolved immunofluorometric assay (TR-IFMA) for canine C-reactive protein in serum.

A time-resolved immunofluorometric assay (TR-IFMA) was developed for the determination of C-reactive protein (CRP) in canine serum. CRP was isolated from canine acute-phase serum by affinity chromatography on agarose coupled with phosphorylethanolamine. This isolated dog CRP was used as standard to calibrate the assay. Intra-assay and inter-assay coefficients of variation were in the ranges 5.3-7.1% and 4.8-13.3%, respectively. Accuracy, evaluated by adding 2 and 10 microg/ml of CRP to serum samples, provided recoveries of 99.9% and 106.8%. High correlation was found between CRP measurements by TR-IFMA and a by commercial enzyme-linked immunosorbent assay (R2 = 0.98). The limit of detection for the TR-IFMA method was 0.000067 microg/ml and the measurement of CRP in serial dilutions of acute-phase dog sera generated curves with the same slope as the one constructed with purified CRP. The TR-IFMA provides a precise, accurate and highly sensitive assay for CRP determination in dog samples. CRP levels in dogs with different diseases ranged between 10.2 and 210.7 microg/ml and were significantly higher than those observed in healthy dogs (< 7.1 microg/ml).

Very rare complementation between mitochondria carrying different mitochondrial DNA mutations points to intrinsic genetic autonomy of the organelles in cultured human cells.

In the present work, a large scale investigation was done regarding the capacity of cultured human cell lines (carrying in homoplasmic form either the mitochondrial tRNA(Lys) A8344G mutation associated with the myoclonic epilepsy and ragged red fiber (MERRF) encephalomyopathy or a frameshift mutation, isolated in vitro, in the gene for the ND4 subunit of NADH dehydrogenase) to undergo transcomplementation of their recessive mitochondrial DNA (mtDNA) mutations after cell fusion. The presence of appropriate nuclear drug resistance markers in the two cell lines allowed measurements of the frequency of cell fusion in glucose-containing medium, non-selective for respiratory capacity, whereas the frequency of transcomplementation of the two mtDNA mutations was determined by growing the same cell fusion mixture in galactose-containing medium, selective for respiratory competence. Transcomplementation of the two mutations was revealed by the re-establishment of normal mitochondrial protein synthesis and respiratory activity and by the relative rates synthesis of two isoforms of the ND3 subunit of NADH dehydrogenase. The results of several experiments showed a cell fusion frequency between 1.4 and 3.4% and an absolute transcomplementation frequency that varied between 1.2 x 10(-5) and 5.5 x 10(-4). Thus, only 0.3-1.6% of the fusion products exhibited transcomplementation of the two mutations. These rare transcomplementing clones were very sluggish in developing, grew very slowly thereafter, and showed a substantial rate of cell death (22-28%). The present results strongly support the conclusion that the capacity of mitochondria to fuse and mix their contents is not a general intrinsic property of these organelles in mammalian cells, although it may become activated in some developmental or physiological situations.

Increased butyrylcholinesterase levels in microsomal membranes of dystrophic Lama2dy mouse muscle.

The proportions and the glycosylation of butyrylcholinesterase (BuChE) forms in vesicles rich in sarcoplasmic reticulum from normal (NMV) and dystrophic (DMV) muscle were analyzed, using merosin-deficient dystrophic mice. BuChE activity in DMV was two- to threefold that in NMV. Globular amphiphilic G1A, G2A, and G4A and hydrophilic G4H BuChE forms were identified in NMV and DMV. The amount of G2A forms increased sevenfold in DMV, and the other forms increased about twofold. The higher BuChE level in DMV might reflect a maturational defect, with dystrophy preventing the down-regulation of BuChE with muscle development. About half of G1A, G2A, and G4H BuChE forms in NMV or DMV bound to Lens culinaris agglutinin (LCA), a higher fraction to wheat germ agglutinin (WGA), and little to Ricinus communis agglutinin (RCA). Most of the G4A forms in NMV or DMV bound to LCA or WGA; those from NMV failed to bind to RCA, whereas most of the variants in DMV bound to it, suggesting that the excess of tetramers in DMV is mainly RCA-reactive. The differential interaction of lectins with BuChE components from muscle microsomes, serum, and nerves confirmed that the microsomal BuChE was muscle-intrinsic. The results provide clues regarding the alterations that dystrophy produces in the biosynthesis of BuChE forms in muscle.

Glycosylation of cholinesterase forms in brain from normal and dystrophic Lama2dy mice.

Differences in the oligosaccharides attached to acetyl- (AChE) and butyrylcholinesterase (BuChE) forms in brain from control and merosin-deficient Lama2dy dystrophic mice were investigated by means of their interaction with agarose-immobilized lectins. Asymmetric AChE, hydrophilic and amphiphilic AChE and BuChE tetramers, and amphiphilic AChE and BuChE monomers were identified in brain. All ChE forms were strongly adsorbed to the lectins concanavalin A (Con A), Lens culinaris (LCA) and Triticum vulgaris (WGA), and poorly so to Ricinus communis agglutinin (RCA), suggesting that the oligosaccharides in AChE or BuChE subunits are similarly processed regardless of their state of polymerization. The lack of differences in the interaction of lectins with homologous AChE and BuChE forms in normal and dystrophic tissue indicates that, in contrast to ChEs forms in skeletal muscle, the dystrophic condition does not disturb the processing of the oligosaccarides of brain enzyme forms.

Glycosylation of acetylcholinesterase forms in microsomal membranes from normal and dystrophic Lama2dy mouse muscle.

The distribution and glycosylation of acetylcholinesterase (AChE) forms in vesicles derived from sarcoplasmic reticulum of normal muscle (NMV) were investigated and compared with those from dystrophic muscle vesicles (DMV). AChE activity was similar in NMV and DMV. Most of the AChE in NMV and half in DMV were released with Triton X-100. Asymmetric (A12) and globular hydrophilic and amphiphilic (G4H, G4A, G2A, and G1A) AChE species occurred in NMV and DMV, the lighter forms being predominant. The percentage of G4H and G4A decreased in DMV. A fraction of the AChE that could not be extracted with detergent was detached with collagenase. Most of the detergent-released A12 AChE from NMV and nearly half in DMV failed to bind to Ricinus communis agglutinin (RCA-I). Conversely, the collagenase-detached isoforms bound to RCA, revealing that asymmetric AChE associated with internal membranes or basal lamina differed in glycosylation. Moreover, nearly half of G4A AChE in DMV and a few in NMV bound to RCA. Most of the RCA-unreactive G4A forms in NMV come from sarcolemma. The results indicate that dystrophy induces minor changes in the distribution and glycosylation of AChE forms in internal membranes of muscle.

Molecular forms of acetyl- and butyrylcholinesterase in normal and dystrophic mouse brain.

In searching for possible differences in the composition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) forms in dystrophic brain, the distribution of various enzyme molecules in normal (NB) and dystrophic (DB) 129B6F1/J mouse brain has been investigated. The tissue was sequentially extracted with saline (S1) and with saline-Triton X-100 buffers (S2) to release soluble and membrane-bound cholinesterases. About 15% of the AChE and 35% of the BuChE activities in NB were recovered in S1, and the rest in S2. G4, G2, and G1 AChE and BuChE forms were identified in the soluble fractions obtained from NB and DB. The shift in sedimentation values of the separated AChE and BuChE species in sucrose gradients made with and without detergents revealed the occurrence of hydrophilic (H) and amphiphilic (A) variants of cholinesterases in the extracts. The amphiphilic properties of the several AChE and BuChE molecules were analyzed by Triton X-114 phase-partitioning and by phenyl-agarose chromatography. A12 (1%), G4A (72%), G4H (8%), and G2A + G1A (19%) AChE molecules, and G4A (34%), G4H (19%), and G2A + G1A (47%) BuChE forms, were identified in NB. The G4A AChE and BuChE isoforms differed in their interaction with Triton X-114 and with a hydrophobic matrix. Neither the extent of cholinesterase solubilization, nor the distribution of individual enzyme forms, was significantly altered in DB. The lack of specific differences in the distribution of AChE and BuChE forms between NB and DB suggests that the biosynthetic pathway leading to the various enzyme forms is altered in muscle but not in dystrophic mouse brain.

Amphiphilic properties of molecular forms of acetylcholinesterase in normal and dystrophic muscle.

Acetylcholinesterase (AChE) molecular forms were studied in normal (NM) and in dystrophic (DM) 129B6F1/J mouse muscle. Successive extractions of the tissue with saline and saline-Triton X-100 buffers yielded two soluble fractions, S1 and S2. Forty percent of the AChE in NM was measured in S1 and 60% in S2, and 65% and 35%, respectively, in extracts from DM. A12, A8, G4, G2, and G1 forms of AChE were found in S1 and S2 from NM and DM. A similar content of asymmetric molecules was noticed between NM and DM. G4 AChE was a minor species in DM, and G1 and G2 AChE were more abundant in DM than in NM. The amphiphilic properties of the several molecules were assessed by Triton X-114 phase-partitioning and hydrophobic chromatography. Thirty and 70% of the enzyme in a mixture of S1 and S2 partitioned in the detergent-rich and in the detergent-poor phases, respectively, whether the extracts were obtained from NM or DM. Asymmetric and G4 AChE predominated in the aqueous phase and G1 and G2 in the detergent phase. Ten and 25% of the enzyme in S1 from NM or DM, respectively, was adsorbed to the phenyl-agarose. Elution of the retained enzyme followed by sedimentation analysis revealed that a certain amount of asymmetric and most of the G1 and G2 forms were associated with the matrix. The content of amphiphilic asymmetric and light globular forms was notably higher in DM than in NM. The results suggest that dystrophic muscle produces a specific pattern of molecular forms of AChE.

G4 forms of acetylcholinesterase and butyrylcholinesterase in normal and dystrophic mouse muscle differ in their interaction with Ricinus communis agglutinin.

Differences in glycosylation between molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in muscle and serum of normal and dystrophic mice have been studied by means of their adsorption to immobilized lectins. Application of a two-step extraction procedure, first with saline buffer, and second with saline buffer and Triton X-100, brought into solution most of the muscle AChE and BuChE activities. The AChE activity was five times greater than that of BuChE in normal (NM) and dystrophic muscle (DM). The AChE activity in the serum of dystrophic mice was twice that measured in control animals, but the BuChE activity remained almost unchanged. Both AChE and BuChE in muscle and serum bound completely to concanavalin A (Con A) and Lens culinaris agglutinin (LCA). A12, A8 and G4 AChE, but not the light G2 and G1 AChE forms, in NM and DM were completely adsorbed to wheat germ agglutinin (WGA). Similarly, G4 BuChE, but not the G2 and G1 forms, were associated to WGA. A high proportion of G4 and G1 AChE and G4 BuChE forms in mouse serum were fixed to WGA. Asymmetric AChE in NM and DM reacted with Ricinus communis agglutinin (RCA) but the light AChE and BuChE forms in muscle and serum did not bind to the lectin. G4 AChE and G4 BuChE in NM were not recognized by RCA, but the isoforms in DM bound fully to the lectin. Serum G4 AChE from control or dystrophic mice did not react with RCA, but G4 BuChE was fixed to the lectin. Since RCA is specific for galactose, the results suggest that in dystrophic muscle galactose is incorporated early in G4 AChE and this affects the level of the functional tetramers destined for insertion in the plasma membrane.

Interaction of AChE with Lens culinaris agglutinin reveals differences in glycosylation of molecular forms in sarcoplasmic reticulum membrane subfractions.

Fractionation of muscle microsomes rich in sarcoplasmic reticulum (SR) by isopicnic centrifugation yielded three types of membranes. Heavy (HM), intermediate (IM), and light membranes (LM), with isopicnic points of 38, 33, and 25% w/w sucrose, were rich in terminal cisternae/triads, longitudinal SR, and T-tubules, respectively. All membrane subfractions displayed acetylcholinesterase (AChE) activity. About 60, 80, and 50% of total AChE in HM, IM, and LM was extracted with a Tris-saline-Triton buffer. AChE molecular forms of 4.5 S (G1), 10.5 S (G4), and 16 S (A12) were found in all membranes but their relative proportion varied among the several membranes. Asymmetric and tetrameric forms were partly sedimented with Lens culinaris agglutinin (LCA), but most of the monomeric AChE failed to interact with the lectin. However, some of the monomers, exclusively found in LM, reacted with LCA. The data suggest that monomeric AChE is classified in rough endoplasmic reticulum. A subset is destined to SR, a second one converted into oligomeric forms, and a third one is associated to external membrane after passing through the Golgi system.

Differential effects of ethanol on membrane-bound and soluble acetylcholinesterase from sarcoplasmic reticulum membranes.

The action of ethanol on the activity of membrane-bound and soluble acetylcholinesterase (AChE) in sarcoplasmic reticulum of skeletal muscle has been studied. Treatment of membranes with 2.5-12.5% v/v ethanol produced a slight stimulation of the AChE activity and inhibition at higher concentration. The enzyme remained associated with the membranes after these treatments. The enzyme solubilized with Triton X-100 was inhibited by ethanol in a time-independent manner. Isolated 16 S (A12), 10.5 S (G4) and 4.5 S (G1) forms of AChE were inhibited by ethanol to a similar extent. Samples were reversibly inhibited by ethanol, up to 12.5% v/v, and irreversibly at higher concentrations. Kinetic studies performed with isolated forms in the presence of 5-12.5% v/v ethanol showed that the solvent behaved as a competitive inhibitor of the asymmetric form but as a mixed inhibitor of the tetrameric and monomeric forms. The results show that the solvent interacts with active and/or regulatory sites of AChE from muscle microsomes.