Biotransformation and cytotoxic properties of NO-donors on MCF7 and U251 cell lines.

Previous studies have shown a role for nitric oxide (NO) as a cytotoxic effector. In the present work, two chemically different NO-donors such as glyceryl trinitrate (GTN) and S-nitroso-N-acetylpenicillamine (SNAP) were evaluated for both NO release and cytostatic/cytotoxic properties. Nitrite accumulation in the supernatant of MCF-7 and U251 cell lines indicated a greater and quickly release of NO derived from SNAP. A time-course of hemoglobin absorption spectral changes showed a greater release of NO derived from GTN in presence of cells compared to the values observed in the media, confirming that the release of NO by GTN can be enzymatic and non-enzymatic. On the contrary, SNAP generated NO without contribution of cellular components and saturated oxyhemoglobin quickly, within 2 hours. Both NO-donors inhibited thymidine incorporation in a similar manner and dose-dependently in U251 cells, but not in MCF-7 cells, where SNAP at the highest tested dose of 1000 microM induced only a 33% cytostatic effect. About trypan blue exclusion test, after 24 h GTN and SNAP, releasing similar amounts of NO, showed comparable cytotoxic effects on U251 cells (50% dead cells), but not on MCF-7 cells, where GTN resulted more cytotoxic. From our data, the "in vitro" antitumoral activity of NO-donors seems to be related to the type of tumor cell lines, to the amount and duration of NO release.

Differential expression pattern of jun B and c-jun in the rat brain during the 24-h cycle.

Data from a previous report [3] demonstrated that the proto-oncogene c-fos mRNA expression undergoes basally a circadian fluctuation in the rat brain. The present study was designed to verify by means of Northern blot hybridization the eventual occurrence of a spontaneous oscillation in the expression of other two proto-oncogenes, jun B and c-jun, during 24 h. Rats were either entrained to a light-dark photoperiod or maintained under constant darkness or light. During the dark period, as well as the subjective night, the jun B mRNA levels in the cerebral cortex and striatum were 4-6 times higher than in the light hours or subjective day. No consistent oscillation was found in the c-jun mRNA expression during 24 h in any of the examined brain regions. These results suggest the possibility of different interactions of the c-fos, jun B and c-jun gene products throughout a 24-h period in discrete brain regions.

Cloning and functional expression of poly(ADP-ribose) polymerase cDNA from Sarcophaga peregrina.

A cDNA spanning the entire coding region for poly(ADP-ribose) polymerase (PARP) of Sarcophaga peregrina was isolated and the nucleotide sequence was determined. The longest open reading frame encodes a polypeptide of 996 amino acid residues with a molecular mass of 113,033 Da. The similarities to the human PARP in amino acid sequence were relatively low in the DNA-binding and auto-modification domains, but very high in the C-terminal catalytic domain: identity of amino acids is 34% in the N-terminal DNA-binding domain (residues 1-369), 27% in the auto-modification domain (residues 370-507), and 56% in the C-terminal NAD-binding domain (residues 508-996). Two zinc-fingers (C-X2-C-X28-H-X2-C and C-X2-C-X31-H-X2-C)2 and a basic region in the N-terminal DNA-binding domain recognized in other PARP are conserved. Downstream of the basic region, another cysteine-rich motif (C-X2-C-X13-C-X9-C), a putative zinc-finger, was found to be well conserved in the PARP of Sarcophaga, Drosophila and human. A leucine-zipper motif (L-X6-L-X6-L-X6-L) which was found in the auto-modification domain of Drosophila PARP, is disrupted in the Sarcophaga enzyme: the second leucine is replaced by proline, and the third leucine by valine. Full-length cDNA for Sarcophaga PARP was cloned into an expression plasmid and expressed in Escherichia coli. A lysate of E. coli cells containing expressed protein reacted with antibody against Sarcophaga PARP, and PARP activity was detected. Thus, we conclude that isolated cDNA encodes a functional Sarcophaga PARP cDNA.

c-fos mRNA is spontaneously induced in the rat brain during the activity period of the circadian cycle.

The basal expression of the proto-oncogene c-fos was studied by Northern blot analysis in different regions of the rat brain during 24 h. A striking spontaneous oscillation of c-fos mRNA expression was detected in animals kept in basal conditions with a 12 h light/12 h dark cycle. In these animals c-fos mRNA was just detectable during the rest hours (morning through afternoon), and was high during the activity hours (night). The periodicity of this oscillation persisted and became free-running when the animals were exposed for 6 consecutive days to constant light or darkness. It was thus demonstrated that the fluctuation of c-fos expression is circadian and is not created by the light-dark cycle, but the latter exerts a synchronizing effect. The oscillation of c-fos mRNA was modified by manipulations of the rest-activity cycle. In particular, the fluctuation observed in basal conditions was inverted, keeping the animals awake during the rest hours (diurnal) and allowing them to sleep in the activity period (nocturnal). These data indicated a close relationship between the oscillation of c-fos expression and the rest-activity cycle. Finally, electroencephalographic (EEG) monitoring was performed under behavioural control for 3 h before the animals were killed. These experiments confirmed that, irrespective of the time of day, the EEG pattern typical of a state of sleep (including both slow waves and paradoxical sleep) was associated with low or undetectable c-fos levels, whereas the protracted EEG desynchronization corresponding to wakefulness was associated with high c-fos expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in the steady-state levels of c-fos, c-jun and c-myc messenger RNA during mitogen-induced liver growth and compensatory regeneration.

The steady-state levels of c-fos, c-jun and c-myc messenger RNA were investigated in rat liver tissue after proliferative stimuli of different nature-namely, compensatory regeneration induced by partial hepatectomy or carbon tetrachloride administration-and direct hyperplasia induced by four different hepatomitogens: lead nitrate, ethylene dibromide, cyproterone acetate and nafenopin. We show here that whereas c-fos and c-jun expression increased soon after partial hepatectomy or carbon tetrachloride administration, an increased expression of c-jun in the absence of c-fos expression occurred during direct hyperplasia induced by lead nitrate and ethylene dibromide. When hyperplasia was induced by cyproterone acetate and nafenopin, the mitogenic response of the liver was not associated with an increased expression of c-jun or c-fos, despite the fact that the timing of the cell cycle was similar to that observed after partial hepatectomy. Finally, when c-myc expression was analyzed, it was found that proliferative conditions associated with an increased expression of this gene were characterized by an increased expression of c-jun. On the contrary, the hyperplasia induced by cyproterone acetate and nafenopin, which is characterized by a lack of increase in the expression of c-fos and c-jun, was also not associated with an increased c-myc expression. Similar results were obtained in these experiments with the mitogen nafenopin, a peroxisome proliferator. In fact, liver hyperplasia induced by this compound was not preceded or accompanied by an increased expression of c-fos and c-myc. This study suggests that depending on the nature of the proliferative stimulus, an increased expression of c-fos, c-jun and c-myc may not be necessary for in vivo induction of liver cell proliferation.

Induction of DNA polymerase beta during proliferation of mitogen-stimulated human lymphocytes.

On induction of proliferation of human peripheral blood mononuclear cells by phytohemagglutinin treatment, DNA polymerase beta activity increases markedly before and during DNA replication. The increase of enzymatic activity seems to be well correlated with the increase of DNA polymerase beta mRNA, which is induced by enhanced expression of the DNA polymerase beta gene. These data suggest that DNA polymerase beta is involved in DNA repair, which is linked to replicative DNA synthesis, or directly in replicative DNA synthesis in normal proliferating cells.

Regulation of poly(ADP-ribose) polymerase mRNA levels during compensatory and mitogen-induced growth of rat liver.

Poly(ADP-ribose) polymerase mRNA levels were studied in hepatic regeneration following partial hepatectomy, and in hyperplasia induced by the mitogen lead nitrate. A significant increase in the level of poly(ADP-ribose) polymerase mRNA was found 8 h after partial hepatectomy when no detectable increase of DNA synthesis could be observed; the level of poly(ADP-ribose) polymerase transcripts increased up to six-fold within 1-2 days. A similar increase of the level of poly(ADP-ribose) polymerase mRNA was found 24 h after treatment with lead nitrate. A twofold increase in poly(ADP-ribose) polymerase activity was observed 2 days after (a) partial hepatectomy and (b) lead nitrate treatment. From these results an important role of poly(ADP-ribose) polymerase in cell proliferation could be suggested.