Franco Ghezzo

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.

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 points for pS2: 4 and 11 ng/mg of cytosolic proteins. pS2 positivity (cutoff 11 ng/mg) was shown to be associated with smaller 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.

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

SummaryChemically 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–Guérin 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.