Down-modulation of nuclear localisation and pro-fibrogenic effect of 4-hydroxy-2,3-nonenal by thiol- and carbonyl-reagents.

Among the oxidative breakdown products of omega-6 unsaturated fatty acids, the aldehyde 4-hydroxy-2,3-nonenal (HNE) is receiving increasing attention for its potential pathophysiological implication, which at least partly lies on the demonstrated ability to modulate gene expression of a number of genes. Here we show that a marked down-modulation of HNE nuclear localisation in cells of a macrophage line (J774-A1) can be afforded by treatment with sulfydryl and carbonyl reagents without significantly interfering with cell viability. As regards the addition of thiol-group reagents to the cell suspension, N-ethylmaleimide (NEM) led to a sustained decrease of HNE nuclear localisation, while 4-(chloromercuri)-benzene-sulfonic acid (PCMBS) gave a similar but more transient effect. Hydroxylamine (HYD), a carbonyl-group reagent, was also able to inhibit HNE nuclear localisation. The actual efficacy of the inhibitors used was then tested on the HNE-induced stimulation of transforming growth factor beta1 (TGFbeta1) production by J774-A1 cells. Indeed, the thiol reagents NEM and PCMBS, both markedly down-modulating HNE nuclear localisation, were able to inhibit HNE-induced increase of TGFbeta1 protein synthesis. The carbonyl reagent HYD was less effective on this respect, producing strong but incomplete protection against HNE-induced TGFbeta1 increase. Taken together, the results indicate that sulfydryl groups are involved in the process of HNE cellular internalisation, while both sulfydryl and carbonyl groups are involved in the process of HNE nuclear translocation, and consequently in the modulation of gene expression by the aldehyde. Further, an actual demonstration is provided that HNE-induced effect on gene regulation can be efficiently counteracted by suitable interference with HNE biochemistry.

Effect of the single major proteic fractions of the liver perchloric extract UK101 on the development of oral tumours in Syrian hamsters.

Squamous cell carcinoma of the oral cavity continues to be a major clinical problem, with about 100,000 new deaths each year worldwide. There is therefore a need to search for new tools to aid oral cancer treatment. We tested the inhibitory activity on chemical carcinogenesis of the three principal protein fractions of about 50, 14, and 8.5 kDa of the mixture UK101 derived from goat liver. These are composed principally of a glycoprotein rich in mannose residues, a protein with analogy to the heat shock protein family, and ubiquitin, respectively. The animal model employed was dimethylbenzanthracene-induced hamster cheek pouch carcinoma. Number of tumours per animal, tumour mass per animal, and proliferating cell nuclear antigen (PCNA) in non-tumour mucosa were quantified: the 14-kDa fraction was the most active; this was also confirmed by testing its corresponding recombinant material. The 50-kDa fraction was inactive, while the ubiquitin showed only low inhibitory activity. It is possible that the technique described and the results obtained could lead to an interesting clinical approach to the treatment of oral cancer.

Perchloric acid-soluble proteins from goat liver inhibit chemical carcinogenesis of Syrian hamster cheek-pouch carcinoma.

Chemically induced Syrian hamster cheek-pouch squamous cell carcinoma is very similar to the corresponding human tumour. This paper describes a blind study in which inhibition of dimethylbenzanthracene-induced cheek-pouch tumours by a goat liver extract denominated UK101 was investigated. Less than 40% of animals treated with UK101 developed tumours compared with 100% of the controls. Intermediate results (80%) were noted in a positive control group treated with Calmette-Guerin bacillus. Immunocytochemical testing of cheek-pouch mucosa by Mib5 showed significantly less proliferating cells in UK101 animals than in the controls. The effect of UK101 was completely reversed when dexamethasone was added in a third control group. A significant difference in complement-mediated cytotoxicity was noted in the sera of UK101-tested and control animals. These findings suggest that an immune mechanism is responsible for the inhibition of hamster cheek-pouch carcinoma by UK101.

Calcyclin gene expression modulation by medroxyprogesterone acetate.

Calcyclin is a cell-cycle-related gene corresponding to a calcium-binding protein whose expression is mainly controlled by platelet-derived growth factor. This paper illustrates medroxyprogesterone acetate (MPA) inhibition of endogenous calcyclin RNA expression of both estrogen-dependent human mammary carcinoma cells and estrogen-independent hamster fibroblasts. Transfection of fragments of the calcyclin promoter driving the chloramphenicol-acetyl-transferase (CAT) gene into hamster fibroblasts was used to evaluate the hormone sensitivity of different promoter regions by considering calcyclin expression at both the RNA and protein level, as evaluated by the CAT assay. A 164 bp promoter fragment showed a good activity that was inhibited by MPA, thereby confirming the results of the observation of endogenous calcyclin gene: smaller fragments, however, required cotransfection of progestin receptor to show full activity, with MPA displaying a stimulatory effect. These findings show that progestin modulation of calcyclin gene expression may be independent of progestin receptors, and that MPA has opposite effects on different promoter regions.

Enhancement of calcyclin gene RNA expression in squamous cell carcinoma of the oral mucosa, but not in benign lesions.

Oral cancer is a neoplasm with some known causes. Proliferation genes are significant among its few pathogenetic and prognostic factors. Calcyclin is a cell-cycle-related gene, the function of which is still unclear. Its expression and that of Haras and histone-H3 have been investigated in an assessment of their pathogenetic role in squamous cell carcinoma. RNA extracted from the pathological and normal mucosa of patients with squamous cell carcinoma (SCC) and benign lesions was reverse transcribed and amplified by the polymerase chain reaction (PCR). The expression of all three genes in the pathological mucosa was enhanced in SCC only. This suggests that they may be involved in its pathogenesis and provides another parameter for the differentiation of malignant and benign lesions.

L-histidine/medroxyprogesterone acetate interaction modulates human breast cancer cell growth and progestin receptor expression in vitro.

The effect of different L-histidine concentrations on human mammary tumour cell (CG5) proliferation was studied to test the hypothesis of a role of histidine in modulating sex steroid-regulated cell proliferation. Cell growth was only possible in the 10(-5) M and 10(-2) M range, while its inhibition by medroxyprogesterone acetate was confined to the 10(-4) M and 10(-3) M range. 10(-3) M L-histidine enhanced the effect of medroxyprogesterone acetate in reducing the number of cells in the S phase. The results show also that 10(-3) M L-histidine favours progestin diffusion into cells and increases progestin receptors density. The present data are in line with previous observations of the effect of histidine on the growth of experimental animal tumours, add evidence that histidine concentration influences the control of cell proliferation by sex steroids, and suggest a possible use of histidine in association with progestational drugs in the treatment of human neoplasia.

Correlation between pS2 protein positivity, steroid receptor status and other prognostic factors in breast cancer.

The cytosolic levels of pS2, an estrogen-regulated protein, were measured in 100 cases of primary breast cancer and related to several conventional histological and biochemical prognostic factors. The data were statistically analyzed on the basis of two different cutoff point for pS2: 4 and 11 ng/mg of cytosolic proteins. pS2 positivity (cutoff 11 ng/mg) was shown to be associated with small tumor size (p = 0.05), a higher differentiation grade (p = 0.007) and a smaller number of mitoses (p = 0.004), but not with menopausal status, lymph node involvement, cathepsin D levels, or proliferative activity determined by the monoclonal antibody Ki67. With the cutoff of 4 ng/mg, the statistical significance was confirmed only for the number of mitoses (p = 0.03), which was also the most closely related covariate in multivariate analysis (p = 0.008). As regards steroid receptor status, a significant difference was observed between pS2+ and pS2- cases (Chi-square = 8.9; p = 0.04, cutoff 4 ng/mg). in conclusion, pS2 positivity, being preferentially expressed in hormone-dependent cells and related to other well-known positive markers, may either indicate a good prognosis or predict responsiveness to endocrine treatment.

[Cryptococcosis in a female patient with angioimmunoblastic lymphadenopathy and dysproteinemia]. / Criptococcosi in una paziente affetta da linfoadenopatia angioimmunoblastica con disproteinemia.

A case of a 72-year-old woman affected by angioimmunoblastic lymphadenopathy with dysproteinemia is described. She was admitted to the hospital for serious cutaneous lesions and dementia. The patient had been treated with corticosteroids for the previous two years. Cryptococcosis was diagnosed by cutaneous biopsy. Antimycotic therapy together with corticosteroid withdrawal cured the cutaneous lesions and improved her psychiatric symptoms.

Supernatant of human umbilical vein endothelial cell culture can favour in vivo neutropoiesis.

Umbilical vein endothelial cells are known to be able to produce interleukins and colony stimulating factors. In the present work supernatant of human umbilical vein endothelial cell culture have been administered to neoplastic patients treated with chemotherapy to reduce the iatrogenic inhibition of hemopoiesis. While no undesired effect could be observed, neutrophil count was favourably influenced by endothelial cell supernatant administration. Such data can be considered useful in order to reduce collateral effect of antineoplastic therapy.

Identification of serum-responsive elements in the promoter of human calcyclin, a growth-regulated gene.

The calcyclin gene is growth regulated in a number of cell types from different animal species. We have identified in the promoter region of the human calcyclin gene the sequences responsible for its growth regulation by serum. A number of deletion mutants and synthetic nucleotides, all driving the chloroamphenicol transferase (CAT) gene, were constructed for this purpose. Serum-responsive elements can be localized in the last 42 bp immediately upstream from the CAP site.

Growth factor regulation of the promoter for calcyclin, a growth-regulated gene.

The steady-state levels of calcyclin mRNA are regulated by growth factors. Using deletion mutants of the 5'-flanking region and a linked reporter (the bacterial chloroamphenicol transferase gene), we have investigated the elements of the calcyclin gene's promoter that respond to growth factors. By a transient expression assay after transfection in BALB/c/3T3 cells, we have been able to show that the serum-inducible sequences are contained in a 164-base pair fragment just upstream of the cap site. This fragment also contains an enhancer, and responds to platelet-derived growth factor as well as to serum. The sequences from -1371 to -1194 upstream of the cap site contain an element which is negatively regulated by epidermal growth factor. These findings have been confirmed in hamster cell lines in which the deletion mutants of the calcyclin promoter controlled the expression of the cDNA for human thymidine kinase. These results indicate that, like in other growth-regulated genes the activity of the calcyclin promoter is modulated by both positive and negative elements. Even more intriguing, though, is the finding that some of these negative elements may be influenced by growth factors in the environment.

Structural and functional analysis of a growth-regulated gene, the human calcyclin.

Calcyclin was originally defined as a cDNA clone (2A9) whose cognate RNA is growth-regulated and whose sequence shows strong similarities to the sequences of the S-100 protein, a calcium-binding protein, as well as to a subunit of the major cellular substrate for tyrosine kinase. Using the full-length cDNA, we have now isolated from a human genomic library several phages containing calcyclin sequences. One of the phages, ch. 28-10, contains the entire calcyclin gene, plus extensive flanking sequences. The calcyclin gene is a unique copy gene and has 3 exons. The 5' flanking sequence has been characterized, both structurally and functionally. Besides a TATA box, it contains, in the region proximate to the cap site, GC boxes and a sequence with a strong homology to the enhancer core of the SV40 promoter. Other enhancer-like elements are found scattered in both the 5' and 3' flanking regions. The proximate 5' flanking region is very active in driving the transient expression of linked reporters in transfection experiments. Finally, the calcyclin gene has been localized to the long arm of human chromosome 1, near the ski oncogene.

Heparin neutralizes serum antiplasmin inhibition of peripheral blood leukocyte fibrinolytic activity.

Serum inhibition of peripheral blood fibrinolytic activity has been evaluated with the 125I-fibrin coated well method. The inhibitory activity was found in a 140,000 d serum fraction that contained alpha 2-antiplasmin. Addition of heparin to cell cultures at concentrations in the range of values obtained during anticoagulant therapy has been demonstrated to counteract such inhibitory activity. The phenomenon has been shown to be linked with plasminogen activation in the presence of fibrin, to lead to a weakening of antiplasmin activity. By clarifying an important aspect of the mechanism of heparin action, our findings support the view that heparin can be usefully employed in treating thrombotic syndromes, not only as an anticoagulant, but also as a "profibrinolytic" agent.

[Lymphocyte subpopulations in idiopathic dilatational cardiomyopathy]. / Sottopopolazioni linfocitarie nella cardiomiopatia dilatativa idiopatica.

Lymphocyte subsets have been examined in 23 patients affected by idiopathic dilated cardiomyopathy (IDCM). Patients were divided according to their functional class showing that compromised subjects exhibited high T-lymphocyte helper/suppressor ratio whereas the contrary was observed in the other patients. It has therefore suggested that IDCM is characterized by 2 distinct phases, each of them with different helper/suppressor ratio.

Interaction between leukocytes and serum plasminogen: an essential mechanism in peripheral blood fibrinolytic activity.

Intense fibrinolytic activity (FA) by granulocytes, mononuclear adherent cells, and natural killer (NK) cells was demonstrated by using 125I-fibrin-coated wells. This FA was greatly dependent on serum plasminogen, provided that serum inhibitors were destroyed by heating at 56 degrees C. Using monoclonal antibodies, it was also observed that these leukocytes produce urokinase and that this enzyme is secreted during adhesion by mononuclear adherent cells and during the cytotoxicity reaction against neoplastic cells by NK cells. Since cell intactness is necessary for leukocyte-serum interaction, stress is placed on the membrane function. We conclude that leukocytes are essential in peripheral blood FA and that transport of urokinase by circulating cells, inside their cytoplasm, could offset its lack of affinity for fibrin and thus avoid undesirable systemic effects.

Insulin modulates human mononuclear adherent cell fibrinolytic and phagocytic activities.

The effects of physiological concentrations of insulin on the fibrinolytic activity of peripheral blood leucocytes were investigated. Significant enhancement of the fibrinolytic activity of mononuclear adherent cells was observed, whereas lymphocytes and granulocytes were unaffected. Furthermore, insulin stimulated mononuclear adherent cells to phagocytize inert particles but not to secrete lysozyme. It is suggested that these results are attributable to the large number of insulin receptors present on the monocyte membrane, and that the regulatory mechanism of fibrinolysis and phagocytosis differs from that controlling the secretion of lysozyme.

[Effects of insulin on fibrinolytic and phagocytic activity of peritoneal macrophages in mice]. / Effetti dell'insulina sull'attività fibrinolitica e fagocitaria di macrofagi peritoneali di topo.

The effects of insulin on the in vitro fibrinolytic activity and on the ability to ingest inert latex particles of murine peritoneal macrophages were studied. The addition to the cell cultures of insulin at the concentration of 25 ng/ml significantly increased both fibrinolytic and phagocytic activities. These findings suggest that insulin may play an important role not only in the regulation of the cellular metabolic process but that also directly modulates specific cell functions.

[Phagocytic activity and cytochemical characterization of acute human myeloblastic leukemia cells]. / Attività fagocitaria e caratteristiche citochimiche in cellule di leucemie acute umane mieloblastiche.

The ability of leukemic cells to phagocytize in vitro inert latex particles was tested in a group of non-lymphoblastic acute leukemia causes classified according to the FAB criteria. Promyelocytic, myelomonocytic, and myeloblastic leukemias were found to possess the highest percentage of cells with phagocytic activity. The cytochemical characterization of the cells revealed that the majority of phagocytic elements were negative for alpha-naphtyl acetate staining and positive for the peroxidase reaction. These unexpected findings could be explained by the impaired differentiation process of leukemic cells.