Class II-specific histone deacetylase inhibitors MC1568 and MC1575 suppress IL-8 expression in human melanoma cells.

Here, we explored the effects of the novel class II-specific histone deacetylase inhibitors (HDACis) MC1568 and MC1575 on interleukin-8 (IL-8) expression and cell proliferation in cutaneous melanoma cell line GR-M and uveal melanoma cell line OCM-3 upon stimulation with phorbol 12-myristate 13-acetate (PMA). We found that PMA upregulated IL-8 transcription via the AP-1 binding site and identified c-Jun as the transcription factor involved in this eventS. MC1568 and MC1575 inhibited IL-8 levels and cell proliferation in either unstimulated or PMA-stimulated melanoma cells. They acted by suppressing (i) c-Jun binding to the IL-8 promoter, (ii) recruitment of histones 3 and 4, RNA polymerase II and TFIIB to the c-Jun promoter, and (iii) c-Jun expression. Our findings provide new insights into mechanisms underlying anti-tumoral activities of class II-specific HDACis in human melanoma and suggest that they may constitute a novel therapeutic strategy for improving the treatment of this cancer.

FOXE1 is a target for aberrant methylation in cutaneous squamous cell carcinoma.

BACKGROUND: Several cancer-related genes are silenced by promoter hypermethylation in skin cancers. However, to date the somatic epigenetic events that occur in cutaneous squamous cell carcinoma (SCC) tumorigenesis have not been well defined. OBJECTIVES: To examine epigenetic abnormalities of FOXE1, a gene located on chromosome 9q22, a region frequently lost in SCC. METHODS: We investigated the methylation status of FOXE1 in 60 cases of cutaneous SCC by methylation-specific polymerase chain reaction, and comparatively examined mRNA and protein expression by real-time polymerase chain reaction and Western blot, respectively. RESULTS: We found a higher frequency of FOXE1 promoter hypermethylation in SCCs (55%), as compared with the adjacent uninvolved skin (12%) and blood control samples (9.5%). FOXE1 methylation was frequently seen in association with a complete absence of or downregulated gene expression. Treatment with the demethylating agent 5-Aza-2'-deoxycytidine resulted in profound reactivation of FOXE1 expression. CONCLUSIONS: These results indicate that FOXE1 is a crucial player in development of cutaneous SCC.

Investigation into FOXE1 genetic variations in cutaneous squamous cell carcinoma.

BACKGROUND: FOXE1 is a candidate tumour suppressor gene at human chromosome locus 9q22. This is a region frequently lost in squamous cell cancer. OBJECTIVES: To assess the influence of FOXE1 variations on genetic susceptibility to cutaneous squamous cell carcinoma (SCC). METHODS: We performed mutational analysis of FOXE1 in 320 DNA samples isolated from 60 SCC specimens, 60 adjacent histologically normal skin samples and 200 blood samples. RESULTS: No somatic mutations were evident. Instead the polyalanine tract showed marked variation in its length between samples from patients with SCC and normal controls. CONCLUSIONS: These results imply that another tumour suppressor gene at this locus may be more important than FOXE1 in skin carcinogenesis and suggest that variation in the FOXE1 polyalanine tract length predisposes to cutaneous SCC.

Serum levels of interleukin 1beta, interleukin 8 and tumour necrosis factor alpha as markers of gastric cancer.

Despite the efforts made, a serum marker reliable for the screening and follow-up of patients with gastric cancer has not yet been identified. The aim of this preliminary study was to test the role of pro-inflammatory cytokines interleukin 1beta, interleukin 8 and tumour necrosis factor alpha in patients with gastric cancer and in control groups. The statistical analysis of cytokines serum levels in the group with gastric cancer versus control groups has shown considerable differences (p < 0.001) in their mean rates. The results indicate that the cytokines interleukin 1beta, interleukin 8 and tumour necrosis factor alpha might perhaps act as diagnostic markers in patients with gastric cancer. Therefore, it is hypothesized that after more extended trials, their use in the screening and prognostic assessment of these patients could be a possibility.

Osteoprotegerin and bone mineral density in hemodiafiltration patients.

A newly identified cytokine, osteoprotegerin (OPG) appears to be involved in the regulation of bone remodeling. In vitro studies suggest that OPG, a soluble member of the TNF receptor family of proteins, inhibits osteoclastogenesis by interrupting the intercellular signaling between osteoblastic stromal cells and osteoclast progenitors. As patients with chronic renal failure (CRF) often have renal osteodystrophy (ROD), we investigated the role of osteoprotegerin (OPG) in ROD, and investigated whether there was any relationship between serum OPG, intact parathyroid (PTH) (iPTH), vitamin D, and trabecular bone. Serum OPG combined with iPTH might be a useful tool in the noninvasive diagnosis of ROD, at least in cases in which the range of PTH values compromises reliable diagnosis. Thirty-six patients on maintenance hemodiafiltration (HDF) and a control group of 36 age and sex matched healthy subjects with no known metabolic bone disease were studied. The following assays were made on serum: iPTH, osteocalcin (BGP), bone alkaline phosphatase, 25(OH)-cholecalciferol, calcium, phosphate, OPG, IGF-1, estradiol, and free testosterone. Serum Ca++, P, B-ALP, BGP, IGF-1, iPTH, and OPG levels were significantly higher in HDF patients than in controls, while DXA measurements and quantitative ultrasound (QUS) parameters were significantly lower. On grouping patients according to their mean OPG levels, we observed significantly lower serum IGF-1, vitamin D3 concentrations, and lumbar spine and hip bone mineral density in the high OPG groups. No correlation was found between OPG and bone turnover markers, whereas a negative correlation was found between serum OPG and IGF-1 levels (r=-0.64, p=0.032). Serum iPTH concentrations were positively correlated with bone alkaline phosphatase (B-ALP) (r=0.69, p=0.038) and BGP (r=0.92, p<0.001). The findings made suggest that an increase in OPG levels may be a compensatory response to elevated bone loss. The low bone mineral density (BMD) levels found in the high OPG group might have been due to the significant decrease in serum IGF-1 and vitamin D3 observed. In conclusion, the findings made in the present study demonstrate that increased OPG in hemodiafiltration patients is only partly due to decreased renal clearance. As it may partly reflect a compensatory response to increased bone loss, this parameter might be helpful in the identification of patients with a marked reduction in trabecular BMD.

Polyamines in nickel-titanium archwire-induced gingivitis in adolescents.

BACKGROUND: Polyamines spermine, spermidine, and putrescine are involved in a number of inflammatory diseases, but their role in the development of gingivitis and periodontitis has not been fully investigated. The goal of this investigation was to study the levels and the variations of these amines, and the main enzymes related to their metabolism, during archwire orthodontic treatment, a condition which may induce gingivitis. METHODS: Sixty patients (age range: 11 to 27 years) were examined for gingivitis occurring during nickel-titanium (Ni-Ti) archwire orthodontic treatment. Plaque and gingival indexes (PI, GI) as well as salivary polyamine metabolism before the archwire insertion (T0) and at 3 (T1), 6 (T2), and 12 (T3) months of treatment were measured. RESULTS: In patients in the age range of 14 to 17 years, spermine and spermidine, but not putrescine contents, as well as ornithine-decarboxylase (ODC) and S-adenosylmethionine-decarboxylase (SAMDC) activities, significantly rose at 3 months after insertion, without any change in periodontal parameters, and further increased at 6 months reaching the maximum at 12 months. GI increased later, from 6 to 12 months, while PI did not significantly change. Spermidine/spermine-N1-acetyltransferase (SSAT) activity remained unchanged from T0 to T3. On the contrary, in patients whose age was 11 to 13 or over 18 years, no significant variations in polyamine metabolism and periododontal parameters were observed at any examination time. CONCLUSION: These data support the hypothesis that salivary polyamines might be earlier indicators of gingivitis than the gingival index score in adolescents wearing archwire appliances.

Vitreous polyamines spermidine, putrescine, and spermine in human proliferative disorders of the retina.

BACKGROUND/AIMS: Many cytokines are involved in the pathogenesis of retinal proliferative diseases, but none has been shown to be related to a specific disorder. The aim of this study was to provide a selective marker of diabetes induced proliferative retinopathies. METHODS: 10 vitreous samples from 10 subjects affected by quiescent proliferative diabetic retinopathy (PDR), 20 vitreous samples from 20 subjects affected by active PDR, and 15 samples from 15 patients with proliferative vitreoretinopathy (PVR) were studied. Samples from 18 patients with a macular hole (n = 8) or pucker (n = 10) served as controls. Vitreous samples were obtained via pars plana vitrectomy. The polyamines spermidine, putrescine, and spermine, vascular endothelial growth factor (VEGF), interleukin 8 (IL-8), and transforming growth factor 1beta (TGF-1beta) were measured by high performance liquid chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA), and the correlation coefficients between the vitreous polyamine content and VEGF, IL-8, and TGF-1beta levels were determined. RESULTS: Spermidine and putrescine were expressed in normal vitreous, but spermine was not detectable. In all the test groups spermidine was 3-4 times higher than in control vitreous and putrescine was similarly lower. The spermine content was up to 15 times higher only in vitreous from patients affected by PDR. Correlation coefficients showed that the spermidine and putrescine level variations correlated with the VEGF and IL-8 content in the active PDR and PVR groups, but not in those with quiescent PDR patients, while spermine was correlated to these cytokines in PDR, but not in PVR groups. CONCLUSIONS: These data suggest a significant role for spermidine and putrescine as markers of proliferative diseases of the retina. The increase in spermine, restricted to diabetic states, may indicate that this polyamine is a unique and specific index of PDR.

Response to STI571 in chronic myelomonocytic leukemia with platelet derived growth factor beta receptor involvement: a new case report.

Based on its ability to inhibit the tyrosine kinase activity of ABL, as well as the c-kit and the Platelet Derived Growth Factor Receptor tyrosine kinases, the spectrum of diseases that may respond to STI571 is increasing. A recently recognized subgroup of myeloproliferative disorders/myelodysplastic syndromes (MPD/MDS) has a t(5;12)(q33;p13) with the activation of the gene for PDGFBR which encodes a receptor tyrosine kinase. Here, we present the case of a patient, with MPD/MDS, and eosinophilia, carrying a translocation t(5;12)(q33;p13) who achieved a complete remission following treatment with STI571, 400 mg daily. At the time of writing he still remains in complete remission with an excellent performance status. There is clearly a need for further studies of STI 571in MPD/MDS with chromosomal translocations involving PDGFBR to confirm this promising initial result.

Age-related salivary polyamine increase in adolescents wearing orthodontic Ni-Ti archwires.

Until now information about the influence of puberty on gingival tissue responses to Ni-Ti alloy haven't been available. Since our previous researches have demonstrated that Ni-Ti appliances have an influence on hyperplastic gingivopathy and data has pointed out a possible hormonal influence on the susceptibility of gingival tissue to mechanical stress, we have attempted to study the relationship between fertility hormones and the periodontal response to Ni-Ti appliances. Three groups, ranging from 6 to 17 years old, were tested for salivary polyamine concentrations and for fertility hormone levels 12 months after Ni-Ti application. Results obtained from Pearson's correlation coefficient between polyamine and sexual hormone concentrations, as well as gingival and plaque indexes, suggest that the adolescent gingival tissue undergoes an hyperplastic process after long-term use of Ni-Ti appliances in relation to the puberty age-restricted peak of fertility hormones.

Anion transport in normal erythrocytes, sickle red cells, and ghosts in relation to hemoglobins and magnesium.

"Band 3," an integral membrane protein of red blood cells, plays a relevant role in anionic transport. The C- and N-terminal portions of band 3 are cytoplasmatics, and the last is the link site for different glycolitic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, and hemoglobin. All or some of these interactions on the CDB3 protein could allow a subtle modulation of anion flux. The interaction among HbA, Mg(2+), and membrane proteins has been sufficiently investigated, but not the effect of Mg(2+) on pathological hemoglobin in relation to the influx of the SO(4)(2-). The aim of this study was to evaluate the involvement of hemoglobin S in sulfate transport. This has been measured with native and increased concentrations of Mg(2+), using normal erythrocytes containing HbA, sickle red cells containing HbS, or ghosts obtained from both erythrocytes and normal erythrocytes ghosts with HbS added. The magnitude of the SO(4)(2-) rate constant measured in normal red blood cells increased markedly when measured in the presence of varied Mg(2+) concentrations. The results show that a low increase of intracellular Mg(2+) concentrations exercises a different HbA modulation on band 3 protein and consequently higher anion transport activity. The same experiments carried out in sickle red cells showed that the SO(4)(2-) rate constant measured in the presence of native concentrations of Mg(2+) was normal, compared to normal red cells, and was not affected by any increase of intracellular Mg(2+). Our suppositions with regard to the importance exercised by the hemoglobin and the Mg(2+) on the SO(4)(2-) influx were confirmed by comparison of the data obtained through measuring SO(4)(2-) influx with native and increased concentrations of Mg(2+) in both normal and sickle red cell ghosts. Both revealed the same sensitivity to Mg(2+) due to withdrawal of hemoglobins. The incorporation of HbS in normal as well as in sickle red cell ghosts reduced the Mg(2+) response to sulfate influx in both the reconstituted ghosts. Our research demonstrated that the different effects exercised on the rate constants of SO(4)(2-) influx in normal (HbA) and sickle red cells (HbS) by the increased intracellular Mg(2+) could be ascribed to the physical-chemical influence exercised either on the hemoglobins or on the intracellular contents of erythrocytes.

Prostaglandin E2 signalling pathway in human T lymphocytes from healthy and conjunctiva basal cell carcinoma-bearing subjects.

Prostaglandin E-induced signal transduction pathways in human T cells from healthy and uveal melanoma-bearing subjects were studied. Transfection experiments showed that PGE2 was able to phosphorylate and activate the fusion trans-activator of the cAMP responsive element-binding protein (CREB). Phosphorylation was at least partially mediated by protein kinase A, as evidenced by the effects of specific kinase inhibitors. Western blotting experiments, which were performed to identify the CREB/ATF2 family members involved in the response to PGE2, revealed a modulation of proteins CREB1, CREB2 and ATF2 and phosphorylation of the 43 kDa form of CREB. Experiments of immunoprecipitation with CREB-binding protein (CBP) demonstrated that, after PGE2 treatment, all of the CREB/ATF isoforms studied, as well as the phosphorylated form of CREB (p-CREB), interacted with CBP. In basal conditions, T cells from patients with conjunctiva basal cell carcinoma showed the presence of p-CREB, which coimmunoprecipitated with CBP. CREB phosphorylation did not modify after PGE2 treatment whereas the p-CREB fraction bound to CBP increased in a delayed manner compared to normal subjects.

Estrogens do not modify MAP kinase-dependent nuclear signaling during stimulation of early G(1) progression in human breast cancer cells.

Estrogens are direct mitogens for hormone-responsive human breast cancercells, where they promote cell cycle progression and induce transcriptional activation of "immediate early" and cyclin genes. Nongenomic signaling by estrogens, including rapid changes of mitogen-activated protein(MAP) kinase and other signal-transduction-cascades activity, has been proposed to be essential for the mitogenic actions of these hormones and their nuclear receptors. Because regulation of gene transcription is considered a key step in cell cycle control by mitogenic protein kinase cascades, here we investigated the possibility that estrogen might induce the activation of extracellular signal-regulated kinase (Erk) 1/2-, c-Jun NH(2)-terminal kinase-, p38- or protein kinase A-responsive transcription factors in the cell nucleus during stimulation of early G(1) progression, a timing coincident with the maximum effects of these hormones on such enzyme activity. No significant changes in protein kinase-mediated transcription factor activity could be detected here after estrogen stimulation of either MCF-7 or ZR-75.1 cells. Furthermore, these steroids were able to induce activation of the human CCND1 gene promoter, accumulation of cyclin D1 and pRb phosphorylation, all key events in cell cycle stimulation by mitogens, even in the presence of Erk1/2 activation blockade by a MAP kinase-activating kinase (Mek)1/2 inhibitor. Thus, estrogens do not appear to convey significant protein kinase-dependent signaling to the cell nucleus during the early phases of human breast cancer cell stimulation. Furthermore, hormonal regulation of G(1) gene transcription can occur even without additional activation of the Mek-Erk1/2 pathway by estrogen receptors.

Determination of polyamines in human saliva by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic method for the determination of polyamines (spermine, spermidine and putrescine) in human saliva was developed. This method is based on pre-column derivatization with o-phthaldialdehyde (OPA). The derivatives were separated on a Nucleosil ODS column (250x4.6 mm I.D.; 5 microm). The gradient elution was performed with two mobile phases A (water) and B (methanol) at a flow rate of 0.8 ml/min. The column eluate was monitored by fluorescence detection (excitation, 360 nm; emission, 510 nm). The within- and between-assay coefficients of variation for all the compounds were below 5%. The detection limits for spermine, spermidine and putrescine were 0.04, 0.05 and 0.06 nmol/ml, respectively. The recovery was greater than 90%. Our analytical technique requires neither preliminary extraction with an organic solvent, nor long multi-step procedures. For saliva samples, this is a simple, rapid and highly reproducible method that can be easily applied to the routine determination of salivary polyamines, whose levels increase early in several pathological conditions.

Modified responses to PGE2 in polyamine biosynthesis by T lymphocytes of gastric- and conjunctiva basal cell-carcinoma patients.

The response to Prostaglandin (PG) E2 of T cells from gastric carcinoma (GC)- and conjunctiva-basal cell carcinoma (conjunctiva-BCC)-bearing patients has been studied in relation to polyamine metabolism. Polyamines are crucial co-factors in cell growth as well as differentiation and many works report that lymphocyte spermine (SP), spermidine (SPD) and putrescine (PUT) levels may be related to tumor proliferation. The present work aims to detect the basal and PGE2 induced concentrations of these polyamines and cAMP, ornithine decarboxylase (ODC), spermine N1-acetyltransferase (SAT) activities of T lymphocytes drawn from patients suffering from GC and conjunctiva-BCC since many carcinomas are characterized by high levels of PGE2. Data obtained from lymphocytes of neoplastic subjects were compared with those derived from PGE2-treated control lymphocytes. Results highlight a very significant increase of all the polyamine metabolites in PGE2-treated T cells from neoplastic patients in respect to the untreated and PGE2-treated control lymphocytes. Therefore, it is conceivable that the PGE2 content increase, often occurring during the epithelial tumour development, may contribute, through enhancement of polyamine metabolism, to tumor progression.

Effects of vitamin E and prostaglandin E2 on expression of CREB1 and CREB2 proteins by human T lymphocytes.

Both prostaglandins (PGs) and vitamin E are known to deeply affect immune responses. It is shown here that they both influence T cell-mediated immunity through reciprocal interference on the expression of cyclic-AMP responsive element binding (CREB) family proteins. CREB1 protein of human T lymphocytes was significantly modulated by a brief treatment of 5 to 10 min with PGE2. On the contrary, vitamin E appeared to be ineffective on the CREB1 behavior, while it abolished the PGE2-induced modulation of this protein. The CREB2 protein expression was also affected by PGE2 treatment, but a longer period of incubation (>20 min) was needed to observe these changes. Vitamin E showed a strong enhancing effect on CREB2 that was partially reversed by the subsequent treatment with PGE2. Our results support the idea that there is reciprocal interference between PGE2 and vitamin E on PGE2-induced signals in T lymphocytes. These data are in agreement with the reports concerning different cell systems and experimental conditions.

Glycolytic enzymes in polyamine-treated bovine retina.

The retina is characterized by glycolysis under aerobic conditions, mediated by lactate dehydrogenase isoenzyme-5 (LDH-5) as well as by the soluble isoenzyme of malate dehydrogenase. Bovine retina LDH and MDH isoenzymes and their activities were studied after polyamine treatment. Our results showed that LDH-5 isoenzyme presented the highest activity in untreated as well as in putrescine-treated retina. Decreased activity was present when the retina was treated with spermidine or spermine. It was demonstrated that retinic LDH-5 had a high affinity for lactate which enabled the isoenzyme to be more effective than the other LDH isoenzymes in the conversion of NADH to NAD. Therefore, the putrescine enhancing LDH-5 activity appeared to be capable of stimulating NAD-mediated rhodopsin regeneration. Putrescine induced a marked increase of both MDH isoenzymes--soluble (s-MDH) and mitochondrial (m-MDH), while spermine and spermidine mostly affected the soluble form of the enzyme. Putrescine induced a three-fold increase in s-MDH and m-MDH activities, while spermine and spermidine induced a four to five-fold increase in s-MDH. These results document the differential effects of polyamine treatment on LDH and MDH isoenzyme activities.

Polyamine metabolism in prostaglandin E2-treated human T lymphocytes.

The effects of Prostaglandin (PG) E2 treatment of human T lymphocytes on polyamine metabolism were investigated. PGE2 is known to inhibit lymphocyte proliferation, while polyamines play an important role in several biochemical processes leading to increased cell growth. Preincubation of T lymphocytes with PGE2 (10(-6) M) for 10 min was able to increase ornithine decarboxylase (ODC) activity and putrescine as well as spermine levels, while spermidine concentration was drastically reduced. After 30 and 60 min of treatment, a decrease in ODC activity and putrescine concentration was observed. On the contrary, the initial inhibition of spermine-N1-acetyltransferase (SAT) activity was followed by a progressive increase of this catabolic enzyme. These changes were related to modifications of cAMP concentrations. Our data may help clarify the mechanisms underlying the biphasic effect of PGE2, which ultimately leads to inhibition of cell proliferation.