Margherita Greco

Increase in RNA and protein synthesis by mitochondria irradiated with helium-neon laser.

To gain further insight into the mechanism of cell photostimulation by laser light, both RNA and protein synthesis were measured in mitochondria irradiated with the low power continuous wave He-Ne laser (Energy dose: 5 Joules/cm2). Following mitochondrial irradiation, both the rate and amount of incorporation of alpha-[32P]UTP and L-[35S]methionine, used to monitor RNA and protein synthesis respectively, proved to increase. Electrophoretic analysis made of the synthesis products clearly shows that He-Ne laser irradiation stimulates the synthesis of all mitochondrial transcription and translation products.

The effect of rifampicin on mitochondrial RNA polymerase from rat liver

Mitochondria can carry out RNA synthesis by using their own DNA-dependent RNA polymerase. Properties of this enzyme, previously described, indicate that the rnitochondrial enzyme is different from the same nuclear enzyme [i, 2]. It would be interesting to find a similarity between the mitochondrial and bacterial RNA polymerase in analogy to that reported for the mitochondrial protein synthesis machinery [3]. In this respect the investigation of the sensitivity of the mitochondrial RNA polymerase towards rifamycin and its derivatives seems particularly useful since it has been shown that these antibiotics are strong inhibitors of RNA synthesis in bacteria but not in nuclei of animal cells [4]. Shmerling [5] measuring the 14C-ATP incorporation in the acid insoluble material of either intact or swollen mitochondria from rat liver has reported that RNA synthesis is completely inhibited by rifamycin in both kinds of mitochondrial preparations. On the other hand, Wintersberger et al. [6] have found that rifampicin a highly po(ent rifamycin derivative even at concentrations as high as 50/~g/ml has no effect on mitochondrial RNA polymerase from yeast or rat liver. We have thus investigated the effect of rifampicin on RNA synthesis in isolated mitochondria and nuclei as well as in the presence of the solubilized mitochondrial enzyme from rat liver.

Mitochondrial DNA depletion reduces PARP-1 levels and promotes progression of the neoplastic phenotype in prostate carcinoma

Mitochondrial dysfunction resulting from mitochondrial DNA (mtDNA) mutations and/or depletion has been correlated with cancer progression and drug resistance. To investigate the role of mtDNA in prostate cancer progression, we used LNCaP and PC-3 prostate carcinoma cells as experimental model. Compared to minimally invasive androgen-dependent LNCaP cells, highly invasive androgen-independent PC-3 cells, as well as androgen-independent DU145 and C4-2 cells, exhibited significantly reduced mtDNA content. In PC-3 cells, reduction of mtDNA was accompanied by decreased mitochondrial membrane potential (ΔΨm), increased migration onto the basement membrane protein laminin-1, reduced chemosensitivity to paclitaxel (IC50=110 nM vs. 22 nM) and decreased expression of poly(ADP-ribose) polymerase (PARP)-1. To investigate the relationship between mtDNA depletion and these phenotypic characteristics, we established mtDNA-depleted LNCaP cells [Rho(−)] by long-term exposure to ethidium bromide or treated wild-type LNCaP cells with a mitochondrial ionophore, carbonyl cyanide m-chlorophenylhydrazone. Both manipulations resulted in ΔΨm loss, acquisition of invasive cytology, increased motility onto laminin-1, reduced sensitivity to paclitaxel (IC50=~100 nM) and ~75% reduction in PARP-1 protein levels, resembling PC-3 cells. Overall, these results provide novel evidence demonstrating that mtDNA depletion in early prostate carcinoma may contribute to the acquisition of a more invasive phenotype that is less sensitive to paclitaxel-induced apoptosis.

Regulation of beta1C and beta1A integrin expression in prostate carcinoma cells.

β1C and β1A integrins are two splice variants of the human β1 integrin subfamily that act as an inhibitor and a stimulator of cell proliferation, respectively. In neoplastic prostate epithelium, both these variants are down-regulated at the mRNA level, but only β1C protein levels are reduced. We used an experimental model consisting of PNT1A, a normal immortalized prostate cell line, and LNCaP and PC-3, two prostate carcinoma cell lines, to investigate both the transcription/post-transcription and translation/post-translation processes of β1C and β1A. Transcriptional regulation played the key role for the reduction in β1C and β1A mRNA expression in cancer cells, as β1C and β1A mRNA half-lives were comparable in normal and cancer cells. β1C translation rate decreased in cancer cells in agreement with the decrease in mRNA levels, whereas β1A translation rate increased more than 2-fold, despite the reduction in mRNA levels. Both β1C and β1A proteins were degraded more rapidly in cancer than in normal cells, and pulse-chase experiments showed that intermediates and/or rates of β1C and β1A protein maturation differ in cancer versus normal cells. Inhibition of either calpain- or lysosomal-mediated proteolysis increased both β1C and β1A protein levels, the former in normal but not in cancer cells and the latter in both cell types, albeit at a higher extent in cancer than in normal cells. Interestingly, inhibition of the ubiquitin proteolytic pathway increased expression of ubiquitinated β1C protein without affecting β1A protein levels in cancer cells. These results show that transcriptional, translational, and post-translational processes, the last involving the ubiquitin proteolytic pathway, contribute to the selective loss of β1C integrin, a very efficient inhibitor of cell proliferation, in prostate malignant transformation.

Ultrastructural zonal heterogeneity of hepatocytes and mitochondria within the hepatic acinus during liver regeneration after partial hepatectomy.

Background information. Partial hepatectomy (70%) induces cell proliferation until the original mass of the liver is restored. In the first 24 h after partial hepatectomy, drastic changes in the metabolism of the remaining liver have been shown to occur. To evaluate changes in hepatocyte ultrastructure within the hepatic acinus during the liver regenerative process, we investigated, by light and electron microscopy observations on specimens taken 0 h, 24 h and 96 h after partial hepatectomy, the hepatocyte structure and ultrastructure in the periportal and pericentral area of the hepatic acinus, with a particular emphasis on mitochondria ultrastructure. Moreover, some biochemical events that could affect the mitochondria ultrastructure and function were investigated.

The effect of α-amanitin on RNA synthesis in rat liver mitochondria

In a previous paper [1 ] we have demonstrated that the mitochondrial RNA polymerase from rat liver is sensitive to rifampicin. Inhibition by rifampicin was dependent on the amount of the enzymic protein in the assay mixture suggesting that the inhibition occurs at the enzyme level as in bacterial systems. In this property mitochondrial RNA polymerase thus resembles the bacterial polymerase and differs from the nuclear one. On the other hand it is well known that c~-amanitin in eukariotic cells is a specific inhibitor of Mn2+-(NH4)2SO 4 activated nuclear RNA polymerase whereas it has no effect on nucleolar polymerase and on bacterial RNA polymerase [2, 3]. In this respect we thought it would be interesting to test the sensitivity of the mitochondrial enzyme towards a-amanitin in order to emphasize the dissimilarity between the nucleoplasmic and the mitochondrial RNA polymerase and at the same time the resemblance of the mitochondrial enzyme with the bacterial one. We have thus investigated the effect ofa-amanitin on RNA synthesis in isolated mitochondria as well as in the presence of a sotubilized preparation of mitochondrial RNA polymerase from rat liver. The results obtained demonstrate that the mitochondrial enzyme differs from the nucleoplasmic enzyme in sensitivity to ¢~-amanitin.

The in vitro-synthesized precursor and mature mitochondrial aspartate aminotransferase share the same import pathway in isolated mitochondria

Both the precursor and the mature form of mitochondrial aspartate aminotransferase were synthesized in a cell-free coupled transcription/translation system directed by the recombinant expression plasmid pOTS-pmAspAT and pOTS-mAspAT, respectively. Both newly synthesized forms of the protein were imported into isolated mitochondria, with the precursor correctly processed to the mature form. In both cases the import process showed resistance to externally added pronase and was abolished in mitochondria treated with the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Moreover the imported products showed the same intramitochondrial localization as judged by a subfractionation procedure. In both cases import was time dependent and was completed in about 15 min. Finally a competitive inhibition of the import of the precursor of aspartate aminotransferase was found due to externally added purified aspartate aminotransferase.

The genetic localization of presumptive mitochondrial messenger RNAs on rat-liver mitochondrial DNA.

Abstract High molecular weight mitochondrial (mt) RNAs were isolated from rat liver mitochondria and hybridized in the presence of excess competitor mt rRNA and/or mt tRNA to restriction fragments of mtDNA. The data reveals that there are a few areas of the mt-genome on which the complementary of these presumptive messenger RNAs is most pronounced. These areas are away from the parts of the genome which are coding for the mt rRNA or containing the D-loop.

On the effect of inhibitors of transcription and translation on RNA and protein synthesis by isolated rat liver mitochondria

Abstract The effect of the in vivo administration of some inhibitors of transcription and translation processes on the capacity of isolated rat liver mitochondria to synthesize RNA and protein has been studied. The in vivo treatment with cycloheximide, anisomycin and emetine inhibits the vitro synthesis of RNA and proteins, demonstrating that both mitochondrial processes are dependent on cytoplasmic protein synthesis. Of the three inhibitors used only emetine at higher doses acts directly on the mitochondrial proteinsynthesizing machinery because of the inhibition exerted in vitro at the level of mitochondrial ribsomes. The in vitro synthesis of RNA and protein by isolated mitochondria is sensitive to rifamycin SV that, however, has no effect when given in vivo, probably because the antibiotic is unable to reach the mitochondrial RNA polymerase molecule. The adminstration of α-amanitin produces an inhibition only at the level of mitochondrial protein synthesis, suggesting that mitochondrial transcription and translation can be independently controlled by extramitochondrial activities. Comparison of effects on mitochondrial protein synthesis during the course of adminstration of cycloheximide and α-amanitin suggests that the effects of α-amanitin is accounted for through its action on cytoplasmic protein synthesis.

Photomodulation of cellular and subcellular activities by He-Ne laser

In hepatocytes, proliferation of tetraploid and binuclear cells and an increase in cytosolic and mitochondrial protein synthesis are caused by He-Ne laser irradiation. To gain some insight into the mechanism of photomodulation of cellular and subcellular activities in isolated hepatocytes, intracellular mediators of cell photostimulation were investigated in intact cells and isolated mitochondria. Irradiation of isolated hepatocytes and isolated rat liver mitochondria was carried out with He-Ne laser (wavelength: 632.8 nm; fluence: 0.24 J cm-2; fluence rate: 12 mW cm-2). Changes in cytosolic [(Ca2+)c] and mitochondrial [(Ca2+)m] calcium concentration, and in mitochondrial (Δψm) and plasma (Δψc) membrane potential were, monitored using either colorimetric or fluorescent probes. C-fos expression was studied by Northern and immunoblotting analysis. As a result of irradiation, an increase in (Ca2+)c and a calcium-dependent increase in Δψc were found. The increase in (Ca2+)c, in turn, caused an increase in c-fos expression. Finally, an increase in (Ca2+)m, probably owing to the increase in Δψm, was found. Increase in (Ca2+)c, leading to activation of gene expression, and a general activation of mitochondrial metabolism, could play a major role in the mechanism of photostimulation of cellular activities by He-Ne laser.