Cancer etiology is influenced by alterations in protein synthesis that are not fully understood. In this study, we took a novel approach to investigate the role of the eukaryotic translation initiation factor eIF5A in human cervical cancers, where it is widely overexpressed. eIF5A contains the distinctive amino acid hypusine, which is formed by a posttranslational modification event requiring deoxyhypusine hydroxylase (DOHH), an enzyme that can be inhibited by the drugs ciclopirox and deferiprone. We found that proliferation of cervical cancer cells can be blocked by DOHH inhibition with either of these pharmacologic agents, as well as by RNA interference–mediated silencing of eIF5A, DOHH, or another enzyme in the hypusine pathway. Proteomic and RNA analyses in HeLa cervical cancer cells identified two groups of proteins in addition to eIF5A that were coordinately affected by ciclopirox and deferiprone. Group 1 proteins (Hsp27, NM23, and DJ-1) were downregulated at the translational level, whereas group 2 proteins (TrpRS and PRDX2) were upregulated at the mRNA level. Further investigations confirmed that eIF5A and DOHH are required for Hsp27 expression in cervical cancer cells and for regulation of its key target IκB and hence NF-κB. Our results argue that mature eIF5A controls a translational network of cancer-driving genes, termed the eIF5A regulon, at the levels of mRNA abundance and translation. In coordinating cell proliferation, the eIF5A regulon can be modulated by drugs such as ciclopirox or deferiprone, which might be repositioned to control cancer cell growth.

A recurrent paradigm in calcium signaling is the coordination of the target response of the calcium signal with activation of metabolic energy production to support that response. This occurs in many tissues, including cardiac and skeletal muscle where contractile activity and ATP production are coordinately regulated by the frequency and amplitude of calcium transients, endocrine and exocrine cells that use calcium to drive the secretory process, and hepatocytes where the downstream targets of calcium include both catabolic and anabolic processes. The primary mechanism by which calcium enhances the capacity for energy production is through calcium-dependent stimulation of mitochondrial oxidative metabolism, achieved by increasing NADH production and respiratory chain flux. Although this enhances energy supply, it also has the potential for deleterious consequences resulting from increased generation of reactive oxygen species (ROS). The negative consequences of calcium-dependent mitochondrial activation can be ameliorated when the underlying cytosolic calcium signals occur as brief calcium spikes or oscillations, with signal strength encoded through the spike frequency (frequency modulation). Frequency modulation increases signal fidelity, and reduces pathological effects of calcium, including excess mitochondrial ROS production and apoptotic or necrotic outcomes. The present article reviews these issues using data obtained in hepatocytes under physiologic and pathologic conditions.

Protein transporters present in cellular membranes can influence the intracellular concentration of a variety of drugs. Pefloxacin is a fluoroquinolone which is commonly used in systemic infections. Its transport mechanisms have never been described. In the present study, we demonstrated that pefloxacin uptake is carrier-mediated. Pefloxacin crosses the membrane in zwitterionic form. The transport seems to differ from that of other fluoroquinolones which use amino acid or organic anion transporters. Pefloxacin uptake is not influenced by the presence of hexose in the incubation medium. It does not use a nucleoside transport system to penetrate the cells but adenosine increases the uptake. The carrier is inhibited by verapamil at 60 min and is activated by a Ca2+-dependent system.

These results show that ICER specifically affects the tumorigenicity of prostate cancer cell without affecting their growth. Therefore, the manipulation of ICER expression could be used for the treatment of androgen-insensitive prostate tumors without causing undesirable toxicity to the cells.

Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis.

The damage to liver mitochondria is universally observed in both humans and animal models after excessive alcohol consumption. Acute alcohol treatment has been shown to stimulate calcium (Ca) release from internal stores in hepatocytes. The resultant increase in cytosolic Ca is expected to be accumulated by neighboring mitochondria, which could potentially lead to mitochondrial Ca overload and injury. Our data indicate that total and free mitochondrial matrix Ca levels are, indeed, elevated in hepatocytes isolated from alcohol-fed rats compared with their pair-fed control littermates. In permeabilized hepatocytes, the rates of mitochondrial Ca uptake were substantially increased after chronic alcohol feeding, whereas those of mitochondrial Ca efflux were decreased. The changes in mitochondrial Ca handling could be explained by an up-regulation of the mitochondrial Ca uniporter and loss of a cyclosporin A-sensitive Ca transport pathway. In intact cells, hormone-induced increases in mitochondrial Ca declined at slower rates leading to more prolonged elevations of matrix Ca in the alcohol-fed group compared with controls. Moreover, treatment with submaximal concentrations of Ca-mobilizing hormones markedly increased the levels of mitochondrial reactive oxygen species (ROS) in hepatocytes from alcohol-fed rats, but did not affect ROS levels in controls. The changes in mitochondrial Ca handling are expected to buffer and attenuate cytosolic Ca increases induced by acute alcohol exposure or hormone stimulation. However, these alterations in mitochondrial Ca handling may also lead to Ca overload during cytosolic Ca increases, which may stimulate the production of mitochondrial ROS, and thus contribute to alcohol-induced liver injury.

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Increases in both Ca2؉ and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca 2؉ signaling by the inositol 1,4,5-trisphosphateforming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mimicked by prolonged cyclic GMP elevation. Induction was without effect on Ca 2؉ signals in response to AlF 4 ؊ or inositol 1,4,5-trisphosphate, indicating that phospholipase C activation and release of Ca 2؉ from inositol 1,4,5-trisphosphate-sensitive Ca 2؉ stores were not targets for nitric oxide inhibition. Vasopressin receptor levels, however, were dramatically reduced in induced cultures. Our data provide a possible mechanism for hepatocyte dysfunction during chronic inflammation.